Foodborne Disease Handbook, Volume 1: Bacterial Pathogens

Foodborne Disease Handbook Second Edition, Revised and Expanded Volume 1: Bacterial Pathogens

edited by

Y. H. Hui Science TechnologySystem West Sacramento, California

Merle D. Pierson Virginia PolytechnicInstitute and State University Blacksburg, Virginia

J. Richard Gorham Uniformed Services University of the Health Sciences Bethesda, Maryland

m M A R C E L

D E K K E R

MARCEL DEKKER, INC.

NEWYORK BASEL

ISBN: 0-8247-0337-5 This book is printed on acid-free paper.

Headquarters Marcel Dekker, Inc. 270 Madison Avenue. New York, NY 10016 tel: 212-696-9000; fax: 212-685-4540 Eastern Hemisphere Distribution Marcel Drkker AG Hutgasse 4. Postfach 812, CH4001 Basel, Switzerland tel: 4 1-6 1-261-8482; fax: 41 -6 1 -26 1-8896 World Wide Web http://www.dekker.com The publisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special Sales/Professional Marketing at the headquarters address above.

Copyright 0 2001 by Marcel Dekker, Inc. All Rights Reserved. Neither this book nor any part may be reproduced or transmitted in any form or by any means. electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Current printing (last digit): l 0 9 8 7 6 5 4 3 2 1

PRINTED IN THE UNITED STATES OF AMERICA

Introduction to the Handbook

The Foodboine Disease Handbook,Second Edition,Revised and Expanded, could not be appearing at a more auspicious time. Never before has the campaign for food safety been pursued so intensely on so many fronts in virtually every country around the world. This new edition reflects at least one of the many aspects of that intense and multifaceted campaign: namely, that research on food safety has been very productive in the years since the first edition appeared. The Hcmdbook is now presented in four volumes instead of the three of the 1994 edition. The four volumes are composed of 86 chapters, a 22% increase over the 67 chapters of the first edition. Much of the information in the first edition has been carried forward to this new edition because that information is still as reliable and pertinent as it was in 1994. This integration of the older data with the latest research findings gives the reader a secure scientific foundation on whichto base important decisions affecting the public's health. We are not so naive as to think that only scientific facts influence decisions affecting food safety. Political and economic factors and cotnpelling national interests may carry greater weight in the minds of decisionmakers than the scientific findings offered in this new edition. However, if persons in the higher levels of national governments and international agencies, such as the Codex Alinlentarius Commission, the World Trade Organization, the World Health Organization, and the Food and Agriculture Organization, who must bear the burden of decision-making need and are willing to entertain scientific findings, then the information in these four volumes will serve them well indeed. During the last decade of the previous century, we witnessed an unprecedentedly intense and varied program of research on food safety, as we have already noted. There are compelling forces driving these research efforts. The traditional food-associated pathogens, parasites, and toxins of forty years ago still continue to cause problems today, and newer or less well-known species and strains present extraordinary challenges to human health. These newer threats may be serious even for the immunocompetent, but for the immunocompromised they can be devastating. The relative numbers of the immunocompromised in the world population are increasing daily. We include here not just those affected by the humanimmunodeficiency virus (HIV), but also the elderly: the veryyoung; the recipients of radiation treatments, chemotherapy, and immunosuppressive drugs; paiii

Handbook iv

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Introduction

tients undergoing major invasive diagnostic or surgical procedures; and sufferers of debilitating diseases such as diabetes. To this daunting list of challenges must be added numerous instances of microbial resistance to antibiotics. Moreover, it is not yet clear how the great HACCP experiment will play out on the worldwide stage of food safety. Altruism and profit motivation have always made strange bedfellows in the food industry. It remains to be seen whether HACCP will succeed in wedding these two disparate tnotives into a unifying force for the benefit of all concerned-producers, manufacturers, retailers, and consumers. That HACCP shows great promise is thoroughly discussed in Volume 2, with an emphasis on sanitation in a public eating place. All the foregoing factors lend a sense of urgency to the task of rapidly identifying toxins, species, and strains of pathogens and parasites as etiologic agents, and of determining their roles in the epidemiology and epizootiology of disease outbreaks, which are described in detail throughout the Foodborne Disease Hmdbook. It is very fortunate for the consumer that there exists in the food industry a dedicated cadre of scientific specialists who scrutinize all aspects of food production and bring their expertise to bear on the potential hazards they know best. A good sampling of the kinds of work they do iscontained in these four new volumes of the Handbook. And the benefits of their research are obvious to the scientific specialist who wants to learn even more about food hazards, to the scientific generalist who is curious about everything and who will be delighted to find a good source of accurate, up-to-date information, and to consumers who care about what they eat. We are confident that these four volumes will provide competent, trustworthy, and timely information to inquiring readers, no matter what roles they may play in the global campaign to achieve food safety.

I

Y. H. Hui J. Richard Gorham Dnvid Kitts K. D. Murre11 Wai-Kit Nip Merle D. Piersorl Syed A. Sattnr R. A. Smith David G. Spoerke, Jr. Peggy S. Starzfield

Preface

The first volume of the Foodborne Disease Handbook, Second Edition, Revised m d Expanded, focuses on bacterial pathogens. Although great strides have been made in food sanitation in general and in the application of HACCP principles in particular, hardly a week goes by without a report in the media of some outbreak of foodborne bacterial disease. It is evident that there is a gap between the principles and facts recorded in the first volume of the Handbook and the application of these facts and principles to the protection of the public’s health. Outbreaks of foodborne bacterial disease do not happen spontaneously-the principle of cause-and-effect remains fully operational. Moreover, people do not usually get sick by consuming wholesome food. Further, those who produce, manufacture, retail, and serve food have no wish to make people sick. Thus, when a foodborne disease outbreak occurs, it suggests that some (probably preventable) circumstance or set of circumstances was allowed to occur that permitted the proliferation of bacteria or the production of their toxins. As HACCP principles emphasize, prevention is the key to averting foodborne bacterial disease outbreaks. The editors and contributors to this volume of the Foodborne Disease Harzdbook have worked diligently to ensure that readers representing all aspects of the food industry now have readily at hand all the facts and principles needed to prevent foodborne outbreaks of bacterial diseases.

Y. H. Hui Merle D. Piersol? J. Richard Gorhnm

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Contents

Introduction to the Harldbook Prefkce Contributors Contents of Other Volurzres

...

111

17

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I. Poison Centers 1. The Role of U.S. Poison Centers in Bacterial Exposures David G. Spoerke, Jr.

1

11. Bacterial Pathogens 2.

Bacterial Biota (Flora) in Foods James M. Jay

23

3. Aerornonas hydrophilu Carlos Abeyta, Jr., Sarrruel A. Palrmbo. arrd Gerard N. Stelma, Jr.

35

4. Update: Food Poisoning and Other Diseases Induced by Bacilluscereus Kathleen T. Rajkowski and James L. Smith

61

5. Brucella Shirley M. Halling and Edward J. Young

77

6. Campylobacter jejuni Don A. Franco a ~ Charles d E. Willinrns

83

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7.

Clostridium botulinum John W. Austh and Karen L. Dodds

107

8. Clostridium perfringens Dorothy M. Wrigley

139

9. Escherichia coli Mcrrguerite A. Neill, Phillip I. Tarr, David N. Tcrvlor, and Marcia Worf

169

10. Listeria nzonocytogenes Catherine W. Donnelly

213

11. Bacteriology of Salmonella Robin C. Anderson and Richcrrd L. Ziprin

247

12. Salmonellosis in Animals David J. Nisbet and Richard L. Ziprin

265

13. Human Salmonellosis: General Medical Aspects Richard L. Ziprin and Michael H. Hume

285

14. Shigella Anthony T. Maurelli and Keith A. Lmnpel

323

15. Staphylococcusaureus Scott E. Martin, Eric R. Mvers, and John J. Iandolo

345

16. Vibrio cholerae Charles A. Kaysner and June H. Wetherirlgton

383

17. Vibrio parahaenzolyticus Tuu-jyi Chai and John L. Pace

407

18. Vibrio vuln@cus Anders Dalsgaard, Lise H@i, DebiLinttous: and James D. Oliver

439

19. Yersinin Scott A. Minnich, Michael J. Smith, Steven D. Weagant, crnd Peter Feng

47 l

111. Disease Surveillance, Investigation, andIndicatorOrganisms 20.

Surveillance of Foodborne Disease Ewer1 C. D. Todd

515

Contents

ix

21. Investigating Foodborne Disease Dale L. Morse, Guthrie S. Birkhead, and Jack J. Guzewich

587

22. Indicator Organisms in Foods James M. Juy

645

Index

655

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Contributors

Carlos Abeyta, Jr.

U.S. Food and Drug Administration, Bothell, Washington

Robin C. Anderson Food Animal Protection Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, College Station, Texas John W. Austin Bureau of Microbial Hazards, Health Protection Branch, Health Canada, Ottawa, Ontario, Canada Guthrie S. Birkhead New York State Department of Health, Albany, New York Tuu-jyi Chai Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan, Republic of China Anders Dalsgaard Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark Karen L. Dodds Health Canada, Ottawa, Ontario, Canada Catherine W. Donnelly Department of Nutrition and Food Sciences, University of Vermont, Burlington, Vermont Peter Feng Division of Microbiological Studies, U.S. Food and Drug Administration, Washington, D.C. Don A. Franco

Animal Protein Producers Industry, Huntsville, Missouri

Jack J. Guzewich U.S. Food and Drug Administration, Washington, D.C. Shirley M. Halling U.S. Department of Agriculture, Ames, Iowa Lise HGi Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark Michael H. Hume Food Animal Protection Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, College Station, Texas JohnJ. Iandolo Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma xi

xii

James M. Jay

Contributors

University of Nevada Las Vegas, Las Vegas, Nevada

Charles A. Kaysner Seafood Products Research Center, U.S. Food and Drug Administration, Bothell, Washington Keith A. Lampel Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Washington, D.C. Debi Linkous

Burroughs Wellcome Research Fund, Raleigh, North Carolina

Scott E. Martin Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, Illinois Anthony T. Maurelli Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland Scott A. Minnich Department of Microbiology, University of Idaho, Moscow, Idaho Dale L. Morse Wadsworth Center, New York State Department of Health, Albany, New York Eric R. Myers Nalco Chemical Company, Naperville, Illinois Marguerite A. Neil1 Department of Medicine, Division of Infectious Disease, Brown University School of Medicine, Providence, Rhode Island David J. Nisbet Food and Feed Safety Research Unit, Food Animal Protection Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, College Station, Texas James D. Oliver University of North Carolina at Charlotte, Charlotte, North Carolina JohnL.Pace Biochenlistry Department, Advanced Medicine, Inc., South San Francisco, California Samuel A. Palumbo U.S. Department of Agriculture, Philadelphia, Pennsylvania KathleenT.Rajkowski Department of Food Safety, Agricultural Research Service, U.S. Department of Agriculture, Wyndmoor, Pennsylvania James L. Smith Microbial Food Safety Lab, Agricultural Research Service, U.S. Department of Agriculture, Wyndmoor, Pennsylvania Michael J. Smith Department of Microbiology, University of Idaho, Moscow, Idaho David G. Spoerke, Jr. Bristlecone Enterprises, Denver, Colorado Gerard N. Stelma, Jr. U.S. Environmental Protection Agency, Cincinnati, Ohio Phillip I. Tarr University of Washington and Children’s Hospital and Medical Center, Seattle, Washington David N. Taylor Walter Reed Army Institute of Research, Washington, D.C. Ewen C. D. Todd Health Protection Branch, Bureau of Microbial Hazards, Health Canada, Ottawa, Ontario, Canada Steven D. Weagant U.S. Food and Drug Administration, Bothell, Washington

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Contributors

Xiii

June H. Wetherington U S . Food and Drug Administration, Bothell, Washington Charles E. Williams Consultant, Arlington, Virginia Marcia Wolf Walter Reed Army Institute of Research, Washington, D.C. Dorothy M. Wrigley Mankato, Minnesota

Department of Biological Sciences, Minnesota State University,

Edward J. Young VA Medical Center and Baylor College of Medicine, Houston, Texas Richard L. Ziprin Food and Feed Safety Research Unit, Food Animal Protection Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, College Station, Texas

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Contents of Other Volumes

VOLUME 2: VIRUSES, PARASITES, PATHOGENS, AND HACCP I. PoisonCenters 1. The Role of Poison Centers in the United States David G. Spoerke, Jr.

11. Viruses 2. Hepatitis A and E Viruses Theresa L. Cron.leans, Michael 0. Fmol-ov, Omana V. Nainan, and Harold S. Margolis

3. Norwalk Virus and the Small Round Viruses Causing Foodborne Gastroenteritis Hazel Appleton 4. Rotavirus Syed A. Snttnr, V. S u s m Springthorpe, and Jason A. Tetro

5. Other Foodborne Viruses Syed A. Sattar and Jason A. Tetl-o 6. Detection of Human Enteric Viruses in Foods Lee-Ann Jaykus 7. Medical Management of Foodborne Viral Gastroenteritis and Hepatitis SuZarwe M. Mcrtsui and Rarnsey C. Cheung xv

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Contents of Other Volumes

8. Epidemiology of Foodborne Viral Infections Thornns M. Liithi

9. Environmental Considerations in Preventing the Foodborne Spread of Hepatitis A Syed A. Sattar and Sabah Bidawid

111. Parasites 10. Taeniasis and Cysticercosis Zbigniew S. Pawlowski and K. D. Murre11

11. Meatborne Helminth Infections: Trichinellosis William C.Campbell 12. Fish- and Invertebrate-Borne Helminths John H. Cross

13. Waterborne and Foodborne Protozoa Ronald Foyer

14. Medical Management Pm1 Prociv 15. Immunodiagnosis of Infections with Cestodes Bruno Gottstein

16. Immunodiagnosis: Nematodes H. Ray Gamble 17. Diagnosis of Toxoplasmosis Alan M. Johnson and J. P. Dubey 18. Seafood Parasites: Prevention, Inspection, and HACCP Ann M. Admm and Debra D. DeVlieger

IV. HACCP and the Foodservice Industries 19. Foodservice Operations: HACCP Principles 0. Peter S q d e r , Jr. 20. Foodservice Operations: HACCP Control Programs 0. Peter Snyder, Jr. Index

VOLUME 3: PLANT TOXICANTS I. PoisonCenters 1. U.S. Poison Centers for Exposures to Plant and Mushroom Toxins David G. Spoerke, Jr.

Contents of Other Volumes

11. Selected Plant Toxicants 2. Toxicology of Naturally Occurring Chemicals in Food Ross C. Beier and Herbert N. Nigg

3. Poisonous Higher Plants Doreen Grace Lang and R. A. Smith 4. Alkaloids R. A. Smith 5. Antinutritional Factors Related to Proteins and Amino Acids Iwin E. Liener 6. Glycosides Walter Majak and Miclznel H. Berm

7. Analytical Methodology for Plant Toxicants Alister David Muir

8. Medical Management and Plant Poisoning Robert H. Poppenga 9. Plant Toxicants and Livestock: Prevention and Management Michcrel H. R a l p h

111. Fungal Toxicants 10. Aspergillus Zojia Kozcrkiewicz 11. Claviceps and Related Fungi Gretchen A. Kuldau and Charles W. Bacon

12. Fusariunl Walter F. 0. Marmas 13. Penicillium Johr~I. Pitt 14. Foodborne Disease and Mycotoxin Epidemiology Sara Hale Herlly and F. Xcwier Bosch 15. Mycotoxicoses: The Effects of Interactions with Mycotoxins Heather A. Koshinsky, Adrieme Woytowich, and George G. Khachatourians

16. Analytical Methodology for Mycotoxins James K. Porter 17. Mycotoxin Analysis: Immunological Techniques Fur1 S. Chrr

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xviii

Volumes Contents of Other

18. Mushroom Biology: General Identification Features Dmid G. Spoerke, Jr. 19. Identification of Mushroom Poisoning (Mycetismus), Epidemiology, and Medical Management David G. Spoerke, Jr. 20. Fungi in Folk Medicine and Society Dnvid G. Syoerke, Jr.

VOLUME 4: SEAFOOD AND ENVIRONMENTAL TOXINS I. Poison Centers 1. Seafood and Environmental Toxicant Exposures: The Role of Poison Centers Dnvid G. Spoerke, Jr.

11. Seafood Toxins 2. Fish Toxins Bruce W. Halstencl 3. Other Poisonous Marine Animals Bruce W. Hulsteacl

4. Shellfish Chemical Poisoning Lyndon E. Llewellyn 5. Pathogens Transmitted by Seafood Russell P. Hemlig

6. Laboratory Methodology for Shellfish Toxins David Kitts 7. Ciguatera Fish Poisoning Yoshitsugi Hokema and Jocmne S. M. Yoshikuwa-Ebesu 8. Tetrodotoxin Joame S. M. Yoslzikablta-Ebesu,Yoslzitsugi Hokamn, nnd TUIIKIO Noguchi

9. Epidemiology of Seafood Poisoning Lorn E. Flerning, Dolores Kat:, Judy A. Bean, and Robertl! Hnnurlond

Contents of Other Volumes

10. The Medical Management of Seafood Poisoning Donm Glad Blythe, Eileen Hack, Giavarmi Washirzgtofl,and Lorn E. Fleming 11. The U.S. National Shellfish Sanitation Program Rebecca A. Reid a d Timothy D. Durmce

12. HACCP, Seafood, and the U.S. Food and Drug Administration Kim R. Yomg, Miguel Rodrigues Krrnzrst, arid George Per)? Hoskin 111. Environmental Toxins

13. Toxicology and Risk Assessment D o r ~ ~J.l lEcobichorz 14. Nutritional Toxicology David Kitts 15. Food Additives Lasclo P. Sornogyi

16. Analysis of Aquatic Contaminants Joe W. Kiceniuk 17. Agricultural Chemicals Debra L. Browning and Car1 K. Winter 18. Radioactivity in Food and Water Hank Kocol

19. Food Irradiation Hark Kocol 20. Drug Residues in Foods of Animal Origin Austin R. Long and Jose E. Roybal 21. Migratory Chemicals from Food Containers and Preparation Utensils Yvonne V. Y ~ r m 22. Food and Hard Foreign Objects: A Review J. Richard Gol-ham 23. Food, Filth, and Disease: A Review J. Richard Gorham 24. Food Filth and Analytical Methodology: A Synopsis J. Richard Gol-hnm

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1 The Role of U S . Poison Centers in Bacterial Exposures David G. Spoerke, Jr. Bristlecorle Enterprises, Denver, Colorado

I. Epidemiology A. B. C. D. E. F. G. H. I. J.

1

AAPCC 2 Who staffs a poison center'? 3 What types of calls are received? 4 How calls are handled 5 What references are used? 6 How poison centers are monitored for quality 6 Professional and public education programs 7 Related professional toxicology organizations 7 International affiliations 9 Toxicology and poison center Web sites 10

11. U.S. Poison Information Centers

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References 2 1

1.

EPIDEMIOLOGY

Epidemiological studies aid treatment facilities in determining risk factors, determining who becomes exposed, and establishing the probable outcomes with various treatments. A few organizations have attempted to gather such information and organize it into yearly reports. The American Association of Poison Control Centers and some federal agencies work toward obtaining epidemiological information, while the AAPCC has an active role in assisting with the treatment of exposures. Epidemiological studies assist government and industry in determining package safety, effective treatment measures, conditions of exposure, and frequency of exposure. Studies on bacterial exposures provide information on the type of people most commonly involved. (e.g., children, adults at home, outdoorsmen, industrial workers, or bluecollar workers). Studies can also tell us which bacterial species are most commonly involved. The symptoms first seen, the onset of symptoms, and the sequelae may also be determined and compared to current norms. l

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A.

AAPCC

1. What Are Poison Centers and the AAPCC? The group in the United States most concerned on a daily basis with potential poisonings due to household agents, industrial agents, and biologics (including plants and mushrooms) is the American Association of Poison Control Centers (AAPCC). This is an affiliation of local and regional centers that provides information concerning all aspects of poisoning and often refers patients to treatment centers. This group of affiliated centers is often supported by both government, private funds, and industrial sources. Poison centers were started in the late 1950s, with the first center thought to be in the Chicago area. The idea caught on quickly, and at the peak of the movement there were hundreds of centers throughout the United States. Unfortunately there were few or no standards regarding what might be called a poison center, the type of staff, hours of operation, or information resources. One center may have had a dedicated staff of doctors, pharmacists, and nurses trained specifically in handling poison cases; the next center may just have had a book on toxicology in the emergency room or hospital library. In 1993 the Health and Safety Code (Sec. 777.002) specified that a poison center must provide a 24-hour service for public and health care professionals and meet requirements established by the AAPCC. This action helped to standardize the activities and the staffs of the various poison centers. The federal government does not fund poison centers, even though for every dollar spent on poison centers there is a savings of $2 to $9 in unnecessary expenses (1,2). The federal agency responsible for the Poison Prevention Packaging Act is the U.S. Consumer Product Safety Commission (CPSC). The National Clearinghouse for Poison Control Centers initially collected data on poisonings, information on commercial product ingredients, and biological toxic agents. For several years the National Clearinghouse provided product and treatment information to the poison centers who handled day-to-day calls. At first, most poison centers were funded by the hospital in which they were located. As the centers grew in size and number of calls being handled, both city and state governments took on the responsibility of contributing funds. In recent years the local governments have found it very difficult to fund such operations and centers have had to look to private industry for additional funding. Government funding may take several forms, as a line item on a state’s budget, as a direct grant, or as moneys distributed on a percall basis. Some states with fewer residents may contract with a neighboring state to provide services to its residents. Some states are so populous that more than one center is funded by the state. Industrial funding also varies, sometimes as a grant, sometimes as payment for handling the company’s poison or drug information-related calls, sometimes as payment for collection of data regarding exposure to the company’s product. Every year the AAPCC issues a summary of all kinds of exposures. A few bacterial exposures are listed in this log, most of which have to do with food poisoning.

2. Regional Centers The number of listed centers has dropped significantly since its peak of more than 600. Many centers have been combined into regional organizations. These regional poison centers provide poison information and telephone management and consultation, collect pertinent data, and delivery professional and public education. Cooperation between regional poison centers and poison treatment facilities is crucial. The regional poison information center, in cooperation with local hospitals, should determine the treatment capabilities of

Poison Centers and Bacterial Exposure

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the treatment facilities of the region and identify and have a working relationship with their analytical toxicology laboratories, emergency departments, critical care wards, medical transportation systems, and extracorporeal elimination capabilities. This should be done for both adults and children. A “region” is usually determined by state authorities in conjunction with local health agencies and health care providers. Documentation of these state designations must be in writing unless a state chooses (in writing) not to designate any poison center or accepts a designation by other political or health jurisdictions. Regional poison information centers should serve a population base of greater than one million people and must receive at least 10,000 human exposure calls per year. The number of certified regional centers in the United States is now under 50. Certification as a regional center requires the following. 1. Maintenance of a 24 hours per day, 365 days per year service. 2. Providing service to both health care professionals and the public. 3. Having available at least one specialist in poison information in the center at all times. 4. Having a medical director or qualified designee, on call by telephone, at all times. 5. Service should be readily accessible by telephone from all areas within the region. 6. Comprehensive poison information resources and comprehensive toxicology information covering both general and specific aspects of acute and chronic poisoning should be available. 7. The center is required to have a list of on-call poison center specialty consultants. 8. Written operational guidelines, which provide a consistent approach to evaluation, follow-up, and management of toxic exposures should be obtained and maintained. These guidelines must be approved in writing by the medical director of the program. 9. There should be a staff of certified professionals manning the phones (at least one has to be a pharmacist or nurse with 2000 hours and 2000 cases of supervised experience). 10. There should be a 24-hour physician (Board Certified) consultation service. 11. The Regional Poison Center shall have an ongoing quality assurance program. 12. Other criteria, determined by the AAPCC, may be established with membership approval. 13. The regional poison information center must be an institutional member in good standing of the AAPCC. Many hospital emergency rooms still maintain a toxicology reference such as the POISINDEX@system to handle routine exposure cases, but rely on regional centers to handle most of the calls in their area.

B. Who Staffs a PoisonCenter? The staffing of poison centers varies considerably. The three professional groups most often involved are physicians, nurses, and pharmacists. Who answers the phones is somewhat dependent on the local labor pool, moneys available, and the types of calls being

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received. Others used have included students in medically related fields, toxicologists, and biologists. Persons responsible for answering the phones are either certified by the AAPCC or are in the process of obtaining certification. Passage of an extensive examination on toxicology is required for initial certification, with periodic recertification required. Regardless of who takes the initial call, a medical director and the other physician back-up are available. These physicians have specialized training or experience in toxicology and are able to provide in-depth consultations for health care professionals calling a center. 1. Medical Director A poison center medical director should be board certified in medical toxicology or in internal medicine, pediatrics, family medicine, or emergency medicine. The medical director should be able to demonstrate ongoing interest and expertise in toxicology as evidenced by publications, research, and meeting attendance. The medical director must have a medical staff appointment at a comprehensive poison treatment facility and must be involved in the management of poisoned patients. 2. Managing Director The managing director must be a registered nurse, pharmacist, or physician or hold a degree in a health science discipline. The individual should be certified by the American Board of Medical Toxicology (for physicians) or by the American Board of Applied Toxicology (for nonphysicians). They must be able to demonstrate ongoing interest and expertise in toxicology. 3. Specialistsin Poison Information These individuals must be registered nurses, pharmacists, or physicians or be currently certified by the AAPCC as a specialist in poison information. Specialists in poison information must complete a training program approved by the medical director and must be certified by the AAPCC as a specialist in poison information within two examination administrations of their initial eligibility. Specialists not currently certified by the Association must spend an annual average of no less than 16 hours per week in poison centerrelated activities. Specialists currently certified by the AAPCC must spend an annual average of no less than 8 hours per week. Other poison information providers must have sufficient background to understand and interpret standard poison information resources and to transmit that information understandably to both health professionals and the public. 4. Consultants In addition to physicians specializing in toxicology, most centers also have lists of experts in many other fields as well. Poison center specialty consultants should be qualified by training or experience to provide sophisticated toxicology or patient care information in their area(s) of expertise. An infectious disease consult would be appropriate for bacterial infections.

C.What

Types of Calls Are Received?

All types of calls are received by poison centers, most of which are handled immediately; others referred to more appropriate agencies. Which calls are referred depends on the center, its expertise, and the appropriateness of a referral. Below are lists of calls that generally fall into each group. Remember, there is considerable variation between poison

Poison Centers and Bacterial Exposure

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centers, and if there is any doubt, call the poison center and they will tell you if your case is more appropriately referred. Poison centers do best on calls regarding acute exposures. Complicated calls regarding exposure to several agents over a long period of time that produce nonspecific symptoms are often referred to other medical specialists, to the toxicologist associated with the center, or to an appropriate government agency. The poison center will often follow up on these cases to track the outcome and type of service given. Types of Culls Usually Accepted Drug identification Actual acute exposure to a drug or chemical Actual acute exposure to a biological agent (plants, mushrooms, various animals) Information regarding the toxic potential of an agent Possible food poisonings Types of Calls Often Referred Questions regarding treatment of a medical condition (not poisoning) Questions on common bacterial, viral, or parasitic infections General psychiatric questions Questions regarding proper disposal of household agents, such as batteries, bleach, insecticides Questions regarding use of insecticides (e.g., which insecticide to use, how to use it) unless related to a health issue, for example, a person allergic to pyrethrins wanting to know which product does not contain pryrethrins Records of all calls/cases handled by the center shall be kept in a form that is acceptable as a medical record. The regional poison information center should submit all its human exposure data to the Association’s National Data Collection System. The regional poison information center shall tabulate its experience for regional evaluation on at least an annual basis. It 1983 the AAPCC formed the Toxic Exposure Surveillance System (TESS) from the former National Data Collection System. Currently TESS contains nearly 16.2 million human poison exposure cases. Sixty-five poison centers, representing 181.3 million people, participate in the data collection. The information has various uses to both governmental agencies and industry, providing data for product reformulations, repackaging, recall, bans, injury potential, and epidemiology. The summation of each year’s surveillance is published in the Anlerican Jorlrnal sf Emergency Medicine in late summer or fall.

D. HowCallsAreHandled Most poison centers receive requests for information via the telephone. Calls come from both health care professionals and consumers. Only a few requests are received by mail or in person. These often involve medico-legal or complex cases. Most centers can be reached by a toll-free phone number in the areas they serve, as well as by a local number. Busy centers will have a single number that will ring on several lines. Calls are often direct referrals from the 911 system. In most cases poison center specialists are unable to determine the exact bacterial species, so it is difficult to give specific information. When the species is known (as in some food poisonings), specific information is available. Information about culture sensitivities, rates of infection, or potential sources is usually not available, and such calls are often referred to a regional epidemiologist or regional infectious disease agency.

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Poison information specialists listen to the caller, recording the history of the case on a standardized form developed by AAPCC. Basic information such as the agent involved, the amount of agent, time of ingestion, symptoms, previous treatment, and current condition are recorded, as well as patient information such as sex, age, phone number, who is with the patient, relevant medical history, and sometimes patient address. All information is considered a medical record, and is therefore confidential. The case is categorized (using various references) as (1) information only, no patient involved, (2) harmless and not requiring follow-up, (3) slightly toxic, no treatment necessary but a follow-up call is given, (4) potentially toxic, treatment given at home and followup given to case resolution, ( 5 ) potentially toxic, treatment may or may not be given at home, but it is necessary for the patient to be referred to a medical facility, or (6) emergency-an ambulance and/or paramedics are dispatched to the scene. Cases are usually followed until symptoms have resolved. In cases where the patient is referred to a health care facility, the receiving agency is notified. This history is relayed, toxic potential discussed, and suggestions for treatment given.

E. WhatReferencesAreUsed? References used also vary from center to center, but virtually all centers use a toxicology system called POISINDEX, which contains lists of products, their ingredients, and suggestions for treatment. The system is compiled using medical literature and medical specialists throughout the world. Only a few bacterial infections (such as food poisonings) are listed in this resource. After accessing the individual bacterial or food-poisoning entry in the database, the physician or poison information specialist is then referred to a treatment protocol that may be for a general class of agents. An unknown skin irritation or potential infection would deserve a consult with an infectious disease specialist. POISINDEX is available on microfiche, as a CD ROM, over a network, or on a mainframe. It is updated every 3 months. Various texts may also be used. It is very difficult to identify bacterial infections based on infortnation given over the phone, so often the assistance of an epidemiologist and infectious disease specialist is used. Some poison centers have more experience with certain types of poisonings than others, so often one center will consult another on an interesting case. These are often more complex cases or cases involving areas within both centers’ regions. A recent trend has been for various manufacturers not to provide product information to all centers via POISINDEX, but to contract with one poison center to provide for poison information services for the whole country. Product information is given to that center only, and cases throughout the country can only be handled by that one center.

F. How Poison Centers Are Monitored for Quality Most poison centers have a system of peer review in place. One person takes a call, another reviews it. Periodic spot review is done by supervisors and physician staff. General competence is assured by certification and re-certification via examination of physicians and poison information specialists.

Poison Centers and Bacterial Exposure

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G. ProfessionalandPublicEducationPrograms The regional poison information center is required to provide information to the health professionals throughout the region who care for poisoned patients on the management of poisoning. Public education progranx aimed at educating both children and adults about poisoning dangers and other necessary concepts related to poison control should be provided. In the past, several centers provided stickers or logos, such as Officer Ugh, Safety Sadie, and Mr. Yuck, that could be placed on or near potentially toxic substances. While the intent was to identify potentially toxic substances that children should keep away from, the practice has been much curtailed on the new assumption that in some cases the stickers actually attracted children to the products. In the spring of every year there is a poison prevention week during which national attention is focused on the problem of potentially toxic exposures. Many centers run special programs for the public, including lectures on prevention, potentially toxic agents in the home, potentially toxic biological agents, or general first aid methods. Although this week is an important time for poison centers, public and professional education is a yearround commitment. Physicians are involved in medical toxicology rounds, journal clubs, and lectures by specialty consultants. Health fairs, school programs, and various women’s clubs are used to educate the public. The extent of these activities is often determined by the amount of funding received from government, private organizations, and public donations.

H. RelatedProfessionalToxicologyOrganizations ACGIH American Conference of Governmental and Industrial Hygienists Address: Kemper Woods Center; Cincinnati, OH, 45240 Phone: 5 13-742-2020 FAX: 5 13-742-3355 ABAT American Board of Applied Toxicology Address: Truman Medical Center, West; 2301 Holmes St.: Kansas City, MO, 64 108 Phone: 8 16-556-3112 FAX: 8 16-881-6282 AACT American Association of Clinical Toxicologists Address: c/o Medical Toxicology Consultants; Four Columbia Drive; Suite 810: Tampa, FL, 33606 AAPCC American Association of Poison Control Centers Address: 3201 New Mexico Avenue NW: Washington, DC, 20016 Phone: 202-362-7217 FAX: 202-362-8377 ABEM American Board of Emergency Medicine Address: 300 Coolidge Road: East Lansing, MI, 48823 Phone: 5 17-332-4800 FAX: 5 17-333-2234 ACEP American College of Emergency Physicians (Toxicology Section) Address: P.O. Box 61991 1; Dallas, TX, 75261-991 1 Phone: 800-798- 1822 FAX: 214-580-2816

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HPS Hungarian Pharmacological Society Address: Centeral Research Insttitute for Chemistry; Hungarian Academy of Sciences; H-l525 Budapest; P.O. Box 17; Pusztaszeri ut 59-67, Hungary Phone: 36-1-135-21 12 ISOMT International Society of Occupational Medicine and Toxicology Address: USC School of Medicine; 222 Oceanview Ave., Suite 100; Los Angeles, CA, 90057 Phone: 213-365-4000 JSTS Japanese Society of Toxicological Sciences Address: Gakkai Center Building; 4-16, Yayoi 2-chome; Bunkyo-ku; Tokyo 113, Japan Phone: 3-3812-3093 FAX: 3-3812-3552 SOT Society of Toxicology Address: 1101 14th Street, Suite 1100; Washington, DC., 20005-5601 Phone: 202-37 1- 1393 FAX: 202-37 1-1090 e-mail: [email protected] SOTC Society of Toxicology of Canada Address: P.O. Box 517: Beaconsfield, Quebec; H9W 5V1, Canada Phone: 5 14-428-2676 FAX: 5 14-482-8648 STP Society of Toxicologic Pathologists Address: 875 Kings Highway, Suite 200; Woodbury, NJ, 08096-3172 Phone: 609-845-7220 FAX: 609-853-041 1 SSPT Swiss Society of Pharmacology and Toxicology Address: Peter Donatsch; Sandoz Phmna AG; Toxicologtie 88 1/130; CH4132 Muttenz, Switzerland Phone: 41-6 1-469-5371 FAX: 4 l -6 1-469-6565 WFCT World Federation of Associations of Clinical Toxicology Centers and Poison Control Centers Address: Centre Anti-Poisons; Hopital Edonard Herriot; 5 p1 d'Arsonva1; 69003 Lyon, France Phone: 33 72 54 80 22 FAX: 33 72 34 55 67 1.

International Affiliations

The AAPCC and its members attend various world conferences to learn of toxicology problems and new methods used by these agencies. An especially close relationship has formed between the American and Canadian poison center associations. Once a year the AAPCC and CAPCC hold a joint scientific meeting and invite speakers and other toxicology specialists from throughout the world to attend. Some international affiliated organizations are listed with the North American groups above.

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J. Toxicology and Poison Center Web Sites

Association of OccupationalandEnvironmentalClinics This group is dedicated to higher standards of patient-centered, multidisciplinary care emphasizing prevention and total health through information sharing, quality service, and collaborative research. Address: [email protected] FingerLakesRegionalPoisonCenter Address: [email protected] Medical/Clinical/Occupational Toxicology Professional Groups A list of primarily U.S. professional groups interested in toxicology, including a description of each group, their address, phone numbers, and contact names. Keyword: poison centers, toxicology Address: http://www.pitt.edu/-martint/pages/motoxorg.htr-t1 PoisonNet A mail list dedicated to sharing information, problem solving, and networking in the areas of poisoning, poison control centers, hazardous materials, and related topics. The list is intended for health care professionals, not the lay public. The moderators do not encourage responses to individual poisoning cases from the public. Keyword(sj: poisoning, poison control centers II. U.S. POISON INFORMATION CENTERS The Poison Control Center telephone numbers and addresses listed below are thought to be accurate as of the date of publication. Poison Control Center telephone numbers or addresses may change. The address and phone number of the Poison Control Center nearest you should be frequently checked. If the number listed does not reach the poison center, contact the nearest emergency service, such as 91 1 or local hospital emergency rooms. The author disclaims any liability resulting from or relating to any inaccuracies or changes in the phone numbers provided below. This information should NOT be used as a substitute for seeking professional medical diagnosis, treatment, and care. ("Indicates a Regional Center designated by the American Association of Poison Control Centers.)

ALABAMA Birmingham Regional Poison Control Center* Children's Hospital of Alabama 1600 Seventh Avenue, South Birmingham, AL 35233- 171 1 (800) 292-6678 (AL only) (205 933-4050

Tuscaloosrr Alabama Poison Control System, Inc. 408 A Paul Bryant Drive, East Tuscaloosa, AL 35401

(800) 462-0800 (AL only) (205) 345-0600

ALASKA Anchorage Anchorage Poison Center Providence Hospital P.O. Box 196603 3200 Providence Drive Anchorage, AK 995 19-6604 (800) 478-3193 (AK only)

Poison Centers and Bacterial Exposure FairbaA-s Fairbanks Poison Center Fairbanks Memorial Hospital 1650 Cowles St. Fairbanks, AK 99701 (907) 456-7 182

Los Angeles Los Angeles County University of Southern California Regional Poison Center" 1200 North State, Room 1107 Los Angeles, CA 90033 (800) 825-2722 (213) 222-3212

ARIZONA

Orange University of California Irvine Medical Center Regional Poison Center" 101 The City Drive, South Route 78 Orange, CA 92668-3298 (800) 544-4404 (CA only) (7 14) 634-5988

Phoenix Samaritan Regional Poison Center" Good Samaritan Medical Center l 130 East McDowell Road, Suite A-5 Phoenix, AZ 85006 (602) 253-3334 Tucson Arizona Poison and Drug Information Center" Arizona Health Sciences Center, Room 1156 1501 N. Campbell Ave. Tucson, AZ 85724 (800) 362-0101 (AZ Only) (602) 626-6016

Richnlond Chevron Emergency Information Center 15299 San Pablo Avenue P.O. Box 4054 Richmond, CA 94804-0054 (800) 457-2202 (510) 233-3737 or 3738 Sacmrnento Regional Poison Control Center" University of California at Davis Medical Center 23 15 Stockton Boulevard Rm HSF-124 Sacramento, CA 95817 (800) 342-3293 (northern CA only) (9 16) 734-3692

ARKANSAS Little Rock Arkansas Poison & Drug Information Center University of Arkansas College of Pharmacy 4301 West Markham, Slot 522 Little Rock, AR 77205 (800) 482-8948 (AR only) (501) 661-6161

CALIFORNIA

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Fresno Fresno Regional Poison Control Center:': Fresno Community Hospital & Medical Center 2823 Fresno Street Fresno, CA 93721 (800) 346-5922 (CA only) (209) 445-1222

Sal1 Diego San Diego Regional Poison Center" University of California at San Diego Medical Center 225 West Dickinson Street San Diego. CA 92013-8925 (800) 876-4766 (.CA only) (619) 543-6000

Sail Francisco San Francisco Bay Area Poison Center" San Francisco General Hospital 1001 Potrero Avenue Rm 1E86 San Francisco, CA 94122 (800) 523-2222 (315) 476-6600

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12 Sun Jose Regional Poison Center Santa Clara Valley Medical Center 751 South Bascom Avenue San Jose, CA 95128 (800) 662-9886, 9887 (CA only) (408) 299-5112, 5113, 5114

COLORADO Denver Rocky Mountain Poison Center* 1010 Yosemite Circle Denver. CO 80230 (800) 332-3073 (CO only) (303) 629-1 123

CONNECTICUT Furmington Connecticut Poison Control Center University of Connecticut Health Center 263 Farmington Avenue Farmington, CT 06030 (800) 343-2722 (CT only) (203) 679-3456

DELAWARE Wilmington Poison Information Center Medical Center of Delaware Wilmington Hospital 501 West 14th Street Wilmington, DE 19899 (302) 655-3389

DISTRICT OF COLUMBIA Wushingtorz National Capital Poison Center* Georgetown University Hospital 3800 Reservoir Road, North West Washington, DC 20007 (202) 625-3333

FLORIDA Jucksonville Florida Poison Information Center University Medical Center 655 West Eighth Street Jacksonville, FL 32209 (904) 549-4465 or 764-7667 Tduhussee Tallahassee Memorial Regional Medical Center 1300 Miccosukk Road Tallahassee, FL 32308 (904) 68 1-54 1 1

Tampa Tampa Poison Information Center* Tampa General Hospital Davis Islands P.O. Box 1289 Tampa, FL 33601 (800) 282-3171 (FL only) (813) 253-4444

GEORGIA Atluntu Georgia Regional Poison Control Center* Cerady Memorial Hospital 80 Butler Street South East Box 26066 Atlanta, GA 30335-3801 (800) 282-5846 (GA only) (404) 616-9000 Macon Regional Poison Control Center Medical Center of Central Georgia 777 Hemlock Street Macon, GA 3 1208 (912) 744-1 146, 1100or 1427 Sclvunnnlz Savannah Regional Poison Control Center Memorial Medical Center Inc. 4700 Waters Avenue Savannah, GA 31403 (912) 355-5228 or 356-5228

Poison Centers and Bacterial Exposure HAWAII Honolulu Kapiolani Women’s and Children’s Medical Center 1319 Punahou Street Honolulu, HI 96826 (800) 362-3585, 3586 (HI only) (808) 941-4411

IDAHO Boise Idaho Poison Center St. Alphonsus Regional Medical Center 1055 North Curtis Road Boise, ID 83706 (800) 632-8000 (ID only) (208) 378-2707

ILLINOIS Chicago Chicago and NE Illinois Regional Poison Control Center Rush Presbyterian-St. Luke’s Medical Center 1653 West Congress Parkway Chicago, IL 60612 (800) 942-5969 (Northeast IL only) (312) 942-5969

Normal Bromenn Hospital Poison Center Virginia at Franklin Normal, IL 61761 (309) 454-6666 Springfield Center and Southern Illinois Poison Resource Center St. John’s Hospital 800 East Carpenter Street Springfield, IL 62769 (800) 252-2022 (IL only) (217) 753-3330 Urbana National Animal Poison Control Center

13 University of Illinois Department of Veterinary Biosciences 2001 South Lincoln Avenue, 1220 VMBSB Urbana, IL 61801 (800) 548-2423 (subscribers only) (217) 333-2053

INDIANA Indianapolis Indiana Poison Center* Methodist Hospital 1701 North Senate Boulevard Indianapolis, IN 46202-1367 (800) 382-9097 (317) 929-2323

IOWA Des Moines Variety Club Drug and Poison Information Center Iowa Methodist Medical Center 1200 Pleasant Street Des Moines, IA 50309 (800) 362-2327 (5 15) 24 1-6254

Iowa City University of Iowa Hospitals and Clinics 200 Hawkins Drive Iowa City, IA 52246 (800) 272-6477 or (800) 362-2327 (IA only) (319) 356-2922 Sioux City St. Luke’s Poison Center St. Luke’s Regional Medical Center 2720 Stone Park Boulevard Sioux City, IA 51 104 (800) 352-2222 (IA, NE, SD) (712) 277-2222

KANSAS Kansas City Mid America Poison Center

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Kansas University Medical Center 39th and Rainbow Boulevard Room B-400 Kansas City, KS 66160-7231 (800) 332-6633 (KS only) (913) 588-6633 Topeka Stormont Vail Regional Medical Center Emergency Department 1500 West 10th Topeka, KS 66604 (913) 354-6100

Wichita Wesley Medical Center 550 North Hillside Avenue Wichita, KS 67214 (316) 688-2222

KENTUCKY Ft. Tllor?m Northern Kentucky Poison Information Center St. Luke Hospital 85 North Grand Avenue Ft. Thomas, KY 41075 (513) 872-51 11 Louislille Kentucky Poison Control Center of Kosair Children’s Hospital 3 15 East Broadway P.O. Box 35070 Louisville, KY 40232 (800) 722-5725 (KY only) (502) 589-8222

LOUISIANA Houma Terrebonne General Medical Center Drug and Poison Infommation Center 936 East Main Street Hourna. LA 70360 (504) 873-4069

Monroe Louisiana Drug and Poison Information Center

Northeast Louisiana University School of Pharmacy. Sugar Hall Monroe, LA 7 1209-6430 (800) 256-9823 (LA only) (3 18) 362-5393

MAINE Portland Maine Poison Control Center Maine Medical Center 22 Bramhall Stree t Portland. ME 04102 (800) 442-6305 (ME only) (207) 87 1-2950

MARYLAND Baltimore Maryland Poison Center* University of Maryland School of Pharmacy 20 North Pine Street Baltimore, MD 21201 (800) 492-2414 (MD only) (410) 528-7701

MASSACHUSETTS Bostoit Massachusetts Poison Control System* The Children’s Hospital 300 Longwood Avenue Boston, MA 031 15 (800) 682-9211 (MA only) (617) 232-2120 or 735-6607

MICHIGAN Ahian Bixby Hospital Poison Center Emma L. Bixby Hospital 8 1 Riverside 8 Avenue Adrian, MI 49331 (5 17) 263-2412

Poison Centers and Bacterial Exposure Detroit Poison Control Center Children's Hospital of Michigan 3901 Beaubien Boulevard Detroit, MI 48201 Outside metropolitan Detroit; (800) 4626642 (MI only) (3 13) 745-57 11 G r a d Rapids Blodgett Regional Poison Center 1840 Wealthy Street, South East Grand Rapids, MI 49506 Within MI: (SOO) 632-2727

Kalmmcoo Bronson Poison Information Center 252 East Love11 Street Kalamazoo, MI 49007 (800) 442-41 12 616 (MI only) (6 16) 34 1-6409

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Hattiesburg Forrest General Hospital 400 S. 28th Avenue Hattiesburg, MS 39402 (601) 288-3235

MISSOURI Kansas City Poison Control Center Children's Mercy Hospital 2401 Gillham Road Kansas City, MO 64108-9898 (8 16) 234-3000 or 234-3430 St. Louis Regional Poison Center* Cardinal Glennon Children's Hospital 1465 South Grand Boulevard St. Louis, MO 63104 (800) 392-9 111 (MO only) (800) 366-8888 (MO, west IL) (314) 772-5200

MINNESOTA MONTANA Minrleapolis Hennepin Regional Poison Center* 701 Park Avenue South Minneapolis, MN 554 15 (612) 347-3144 (612) 347-3141 (Petline) St. Paul Minnesota1 Regional Poison Center* St. Paul-Ramsey Medical Center 640 Jackson Street St. Paul, MN 55101 (800) 222-1222 (MN only) (612) 221-2113

MISSISSIPPI

Jackson University of Mississippi Medical Center 2500 North State Street Jackson, MS 39216 (601) 354-7660

Dernver Rocky Mountain Poison and Drug Center Denver, CO 80204 (800) 525-5042 (MT only)

NEBRASKA Omaha The Poison Center* Children's Memorial Hospital 8301 Dodge Street Omaha, NE 681 14 (800) 955-9119 (WY, NE) (402) 390-5400, 5555

NEVADA Las Vegas Humana Hospital-Sunrise* 3186 Maryland Parkway Las Vegas, NV 89109 (800) 446-6179 (NV only)

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Reno Washoe Medical Center 77 Pringle Way Reno, NV 89520 (702) 328-4144

NEW HAMPSHIRE Lebanon New Hampshire Poison Center Dartmouth-Hitchcock Medical Center 1 Medical Center Drive Lebanon, NH 03756 (800) 562-8236 (NH only) (603) 650-5000

NEW JERSEY Newark New Jersey Poison Information and Education Systems" 201 Lyons Avenue Newark, NJ 071 12 (800) 962-1253 (NJ only) (973) 923-0764 Plzillipsburg Warren Hospital Poison Control Center 185 Rosberg Street Phillipsburg, NJ 08865 (800) 962-1253 (908) 859-6768

NEW MEXICO Albuquerque New Mexico Poison and Drug Information Center* University of New Mexico Albuquerque, NM 87 131 (800) 432-6866 (NM only) (505) 843-251 1

NEW YORK Buffalo Western New York Poison Control Center Children's Hospital of Buffalo 219 Bryant Street Buffalo, NY 14222 (800) 888-7655 (NY only) (7 16) 878-7654 Mineola Long Island Regional Poison Control Center" Winthrop University Hospital 259 First Street Mineola, NY 1 1501 (516) 542-2323, 2324, 2325 New York City New York City Poison Control Center* 455 First Avenue, Room 123 New York, NY 10016 (212) 340-4494 (213) 764-7667 Nyack Hudson Valley Regional Poison Center Nyack Hospital 160 North Midland Avenue Nyack, NY 10920 (800) 336-6997 (NY only) (914) 353-1000 Rochester Finger Lakes Regional Poison Control Center University of Rochester Medical Center 601 Elmwood Avenue Rochester, NY 14642 (800) 333-0542 (NY only) (716) 275-5151 Syracuse Central New York Poison Control Center SUNY Health Science Center 750 E. Adams Street Syracuse, NY 13210 (800) 252-5655 (3 15)476-4766

NORTH CAROLINA Ashville Western North Carolina Poison Control Center

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Poison Centers and Bacterial Exposure Memorial Mission Hospital 509 Biltmore Avenue Ashville, NC 28801 (800) 542-4225 (NC only) (704) 255-4490 or 258-9907 Charlotte Carolinas Poison Center Carolinas Medical Center 100 Blythe Boulevard Charlotte, NC 28232-2861 (800) 848-6946 (704) 355-4000 Durham Duke Regional Poison Control Center P.O. Box 3007 Durham, NC 277 I O (800) 672-1697 (NC only) (919) 684-8111 Greensboro Triad Poison Center Moses H. Cone Memorial Hospital 1200 North Elm Street Greensboro, NC 2740 1- 1020 (800) 953-4001 (NC only) (919) 574-8105 Hickon Catawba Memorial Hospital Poison Control Center 810 Fairgrove Church Road, South East Hickory, NC 28602 (704) 322-6649

NORTH DAKOTA Fargo North Dakota Poison Center St. Luke’s Hospital 720 North 4th Street Fargo, ND 58 122 (800) 732-2200 (ND only) (701) 234-5575

OHIO Akron Akron Regional Poison Center

17 281 Locust Street Akron, OH 44308 (800) 362-9922 (OH only) (2 16) 379-8562 Canton Stark County Poison Control Center Timken Mercy Medical Center 1320 Tinlken Mercy Drive, North West Canton, OH 44667 (800) 722-8662 (OH only) (216) 489-1304 Cincinnati South West Ohio Regional Poison Control System and Cincinnati Drug and Poison Information Center* University of Cincinnati College of Medicine 231 Bethesda Avenue ML #l44 Cincinnati, OH 45267-0144 (800) 872-5 11 1(Southwest OH only) (513) 558-51 11 Cleveland Greater Cleveland Poison Control Center 2074 Abington Road Cleveland, OH 44106 (2 16)23 1-4455 Col~u~b~w Central Ohio Poison Center* 700 Children’s Drive Columbus. OH 43205 (800) 682-7625 (OH only) (614) 228-1323 Dayton West Ohio Regional Poison And Drug Information Center Children’s Medical Center One Children’s Plaza Dayton, OH 45404- 18 15 (800) 762-0727 (OH only) (5 13) 222-2227

Lorain County Poison Control Center Lorain Community Hospital 3700 Kolbe Road Lorain, OH 44053 (800) 821-8972 (OH only) (216) 282-2220

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Sandusky Firelands Community Hospital Poison Information Center 1101 Decatur Street Sandusky. OH 44870 (419) 626-7423

J

PENNSYLVANIA

Toledo Poison Information Center of Northwest Ohio Medical College of Ohio Hospital 3000 Arlington Avenue Toledo, OH 49614 (800) 589-3897 (OH only) (419) 381-3897

Hershey Central Pennsylvania Poison Center* Milton Hershey Medical Center Pennsylvania State University P.O. Box 850 Hershey, PA 17033 (800) 521-61 10 (717) 531-6111

Youngstown Mahoning Valley Poison Center St. Elizabeth Hospital Medical Center 1044 Belmont Avenue Youngstown, OH 44501 (800) 426-2348 (OH only) (2 16) 746-2222

Lrrncnster Poison Control Center St. Joseph Hospital and Health Care Center 250 College Avenue Lancaster, PA 17604 (717) 299-4546

Znnesville Bethesda Poison Control Center Bethesda Hospital 2951 Maple Ave Zanesville, OH 4370 1 (800) 686-4221 (OH only) (614) 454-4221

OKLAHOMA

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(800) 452-7165 (OR only) (503) 494-8968

Oklalzom City Oklahoma Poison Control Center Children's Memorial Hospital 940 Northeast 13th Street Oklahoma City, OK 73104 (800) 522-4611 (OK only) (405) 27 1-5454

OREGON Portland Oregon Poison Center Oregon Health Sciences University 3 181 South West Sam Jackson Park Road Portland, OR 97201

Philadelphia Philadelphia Poison Control Center" One Children's Center 34th and Civic Center Boulevard Philadelphia, PA 19104 (215) 386-2100 Pittsbtqh Pittsburgh Poison Center* One Children's Place 3705 Fifth Avenue at DeSoto Street Pittsburgh, PA 15213 (4 12) 68 1-6669 Willinrmport The Williamsport Hospital Poison Control Center 777 Rural Avenue Williamsport, PA 17701 (717) 321-2000

RHODE ISLAND Providence Rhode Island Poison Center* 593 Eddy Street Providence, RI 02903 (401) 444-5727

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Poison Centers and Bacterial Exposure SOUTH CAROLINA Chrrrlotte Carolinas Poison Center Carolinas Medical Center 1000 Blythe Boulevard Charlotte. NC 28232-2861 (800) 848-6946

Columbirt Palmetto Poison Center University of South Carolina College of Pharmacy Columbia, SC 29208 (800) 922-1 117 (SC only) (803) 765-7359

SOUTH DAKOTA Aberdeen Poison Control Center St. Luke's Midland Regional Medical Center 305 S. State Street Aberdeen, SD 57401 (800) 592-1889 (SD. MN, ND. WY) (605) 622-5678

Rapid City Rapid City Regional Poison Control Center 835 Faimont Boulevard P.O. Box 6000 Rapid City, SD 57709 (605) 341-3333

Sioux Falls McKennan Poison Center McKennan Hospital 800 East 21st Street P.O. Box 5045 Sioux Falls, SD 571 17-5045 (800) 952-0123 (SD only) (800) 843-0505 (IA, MN, NE) (605) 336-3894

TENNESSEE Knoxville Knoxville Poison Control Center

University of Tennessee Memorial Research Center and Hospital 1924 Alcoa Highway Knoxville, TN 37920 (6 15) 544-9400 hlerrrphis Southern Poison Center, Inc. Lebanheur Children's Medical Center 848 Adams Avenue Memphis, TN 38103-2821 (901) 528-6048

Nashville Middle Tennessee Regional Poison Center, Inc. 501 Oxford House 1161 21st Avenue South B-101VUII Nashville, TN 37232-4632 (800) 288-9999 (TN only) (615) 322-6435

TEXAS Conroe Montgomery County Poison Information Center Medical Center Hospital 504 Medical Center Blvd.

TX 77304 (409) 539-7700 Dallas North Central Texas Poison Center* Parkland Memorial Hospital 5201 Harry Hines Boulevard P.O. Box 35926 Dallas, TX 75235 (.goo) 441 -0040 (TX only) (214) 590-5000 El Pnso El Paso Poison Control Center Thomas General Hospital 48 15 Alameda Avenue El Paso, TX 79905 (915) 533-1244

Galveston Texas State Poison Control Center University of Texas Medical Branch 8th and Mechanic Street

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20 Galveston, TX 77550-2780 (800) 392-8548 (TX only) (7 13) 654-1701 (Houston) (409) 765-1420 (Galveston)

Lubbock Methodist Hospital Poison Control 3615 19th Street Lubbock, TX 7941 3 (806) 793-4366

WASHINGTON Seattle Washington Poison Center P.O. Box 5371 Seattle, WA 98105-0371 (800) 732-6985 (within WA) (206) 526-2121

WEST VIRGINIA UTAH Salt Lake City Utah Poison Control Center* Intermountain Regional Poison Control Center 410 Chipeta Way, Suite 230 Salt Lake City, UT 84108 (800) 456-7707 (UT only) (801) 581-2151

Charleston West Virginia Poison Center’# West Virginia University 3 110MacCorkle Avenue, South East Charleston, WV 25304 (304) 348-4211 (800) 632-3625 (WV only) Parkersburg St. Joseph’s Hospital Center 19th Street and Murdoch Avenue Parkersburg, WV 26101 (304) 424-4222

VERMONT WISCONSIN Burlington Vermont Poison Center Medical Center Hospital of Vermont 1 1 1 Colchester Avenue Burlington, VT 05401 (802) 658-3456

VIRGINIA Charlottesville Blue Ridge Poison Center* University of Virginia Health Sciences Center Box 67 Charlottesville, VA 22901 (800) 451-1428 (VA only) (804) 924-5543 Richmond Virginia Poison Center Virginia Commonwealth University MCV Station Box 522 Richmond, VA 23298-0522 (800) 552-6337 (VA only) (804) 786-9123

Madison Regional Poison Control Center University of Wisconsin Hospital 600 Highland Avenue Madison, W1 53792 (608) 262-3702 Milwaukee Poison Center of Eastern Wisconsin Children’s Hospital of Wisconsin 9000 West Wisconsin Avenue P.O. Box 1997 Milwaukee, W1 53201 (414) 266-2222

WYOMING Omaha The Poison Center* Children’s Memorial Hospital 8301 Dodge Street Omaha, NE 681 14 (800) 955-9119 (WY, NE) (402) 390-5400, 5555

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REFERENCES 1. Harrison, D. L., Draugalis, J. R., Slack, M. K.. and Langly, P. C. (1996). Cost effectiveness of Regional Poison Control Centers. Arch. Intern. Med. 156:2601-2608. 2. CPSC. CPSC Chairman Ann Brown Suggests Information Technology Studyto Support Work of Poison Centers. News Release #94-047, Tuesday March 15, 1994.

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2 Bacterial Biota (Flora) in Foods James M. Jay Uitirlersityoj-Nevada Las Vegas. Las Vegm, NeIlacIa

I. Introduction 23 A. Soils 24 B. Waters 24 C. Human and animal sources 11. CommonFoodborneGenera A. Gram positives B. Gram negatives 28

24 24

24

111. DetectionandEnumeration 28 A. Choice of incubationtimeandtemperature B. Choice of culture medium 28 C. Selective and differential media 39 D. Ratio of numbers 29 IV. PrevalenceinFoods 29 A. Ready-to-use vegetables B. Spices 30 C. Meat products 31 References

1.

28

29

32

INTRODUCTION

Because natural foods come from either a soil or water environment, they may beexpected to contain at least some of the bacteria that are common to these environments unless steps have beentaken to effect their destruction. In addition, some, especially human pathogens, enter the food supply from infected animals and human handlers. Fortunately, only a relatively small percentage of known soil bacteria can be found on foods of plant or animal origin. In the case of foods from fresh and ocean waters, a higher percentage of the bacterial biota from these environments may be associated with such foods due in large part to a less diverse biota in contrast to that of soils.

23

24

Jay

A. Soils The number of bacteria in the top layer of a rich farm soil is typically around one billion per gram. Numbers differ for soils under active cultivation or where cover crops have been plowed under in contrast to those that have not been disturbed for several years. In the former, the bacterial biota is dominated by zymogeneous types that are active degraders of simple plant constituents, and they consist of both gram-positive and gram-negative heterotrophic genera. These bacteria are typically on the surface of plants when they are plowed under, and they are the ones most likely to be found on plant-based foods. Foodborne pathogens such as the proteolytic strains of Clostridium botulinum and Bacillus cereus are soil bacteria. The bacterial biota of stable soils is dominated by the autochthonous or indigenous types that consist of humus feeders. This group is dominated by gram positives such as the mycobacteria and nocardioforms, and they are slow growers. Also, stable soils are populated by autotrophic bacteria that are antagonized by utilizable organic matter. While any or all of these types may appear transiently on plant products, they lack the capacity to adhere, and importantly, they are slow growers or are outcompeted by the zymogeneous biota noted above.

B. Waters The bacterial biota of aquatic-based foods is less diverse than that of soils because of the general lack of humus feeders and chemoautotrophic types in the water column. In the case of fresh waters, the nonphotosynthetic biota is quite similar to that of the surrounding soils due to rain run-off. The significant difference between the biota of fresh and sea waters is that the latter consists essentially only of gram-negative bacteria. Further, except for marine coastal zones, where numbers of nonphotosynthetic bacteria may be high, the biota of the open oceans is quite low. The salinity and lower temperatures of marine waters allow for the existence and growth of halophilic bacteria such as Photobacterium spp. Also, true psychrophilic bacteria are found along with larger populations of psychrotrophic types. Foodborne pathogens such as Vibrio cholerae and Vibrio paralzaemolyticus are found in marine and estuarine waters.

C. Human andAnimalSources Although soil and water may be viewed as the primary and original habitats of all bacteria, some species have lost the capacity to grow and multiply in either of these environments (e.g., whooping cough and syphilis agents). Others maintain the capacity to live in these environments while acquiring the capacity to live as parasites or conlmensuals on human or animal hosts (e.g., the two Vibrio spp. noted above). The largest number of foodborne pathogens are most often found in or on human and animal hosts. Shigella spp. are found only in humans, whereas Salmonella spp. are found in humans and other vertebrate animals.

II. COMMON FOODBORNEGENERA A.

Gram Positives

The gram-positive bacteria most often recovered from foods are summarized in Table 1. The 40 genera listed represent those that may be recovered from fresh, fermented, and

Biota

Bacterial Foods

(Flora) in

25

Table 1 The Most Common Genera of Gram-positive Bacteria Found in Fresh and Processed Foods ~

Group/genus Spore-formers Alicyclobacillus Aneurinibacillus Bacillus Brevibacillus Clostridium Paenibacillus Sporolactobacilhrs Lactics Carnobacterium Enterococcus Lactococcus Lactobacillus Luctospltaem Leuconostoc Oenococcus Pediococcus Streptococcus Tetragenococcus Vagococcro Weissella Corynefomx & related Arthrobacter Colvnebacteriurrr Bifidobncterium Brevibacterium Caseobacter Cellulonlorms Microbacterium Propionibacterium Miscellaneous groups Aerococclrs Brochothrix Deirlococcus Ensipe1othri.x Halobacterium Hdococcus Kocuria Listeria Micrococcus Mycobacterium Plnnococc1rs Rubrobacter Staphylococclts

Morphology

~~

~

~~~

~

Common sources/conunents

R R R R R R R

Canned fruits, juices Same as Bacillus Soils, air, utensils Same as Bacillus Soil, air, utensils Same as Bacillus Soil, chicken feed

R C C R C C C C C C C R

Meats, poultry, vegetation Feces, water, vegetation Raw milk Gastrointestinal tract, vegetation Rumen, anaerobic sludge Vegetation, sugar refineries Grapes, wines Vegetation Raw milk Pickling brines Water, fish, feces Processed meats, vegetation

R R R R R R R R

Soils (rare in foods) Decaying organic matter Raw milk, feces Certain cheeses Meat carcasses Vegetation Vegetation Vegetation, cheeses

C R C R R C C R C R C R C

Raw milk Processed meats Extremely radiation resistant Cattle, raw milk Salt water Salt water Same as Micrococcus Vegetation, zoonotic transmission Air, dust, utensils, handlers Raw milk of infected dairy herds Sea water, seafoods (a halophile) Extremely radiation resistant Nasals/Sl;in of handlers; hides

26

Jay

processed foods. In no single edible food product would one find all of the bacteria listed in Table 1. Fresh foods such as ground meats may be expected to contain 10-20 of the genera noted. The gram positives are listed under three groups based on morphological and physiological features. The endospore-forming bacteria are widespread in nature, and some of these may be found in any fresh food product. As soil organisms, their primary entry into foods is via dust and soil contamination of ingredients and utensils. The genus Alicyclobacillus is of significance as a cause of spoilage of canned fruits and juices. Foodborne pathogens are found in the genera Bacillus and Clostridium. The lactic acid bacteria are very common in nature on plants and plant products, especially fruits. They are responsible for a number of fermented food products including sauerkraut, pickles, and cheeses. Although common on vegetation, the enterococci are also common in the gastrointestinal tract of mammals. The presence and numbers of enterococci in human feces led to their use as indicators of fecal pollution of waters and also to their use as indicators of sanitary quality of some fresh foods. In general, the lactics are as a group the most beneficial of all bacteria to humans relative to the food supply. In addition to the fermented foods they produce, their production of bacteriocins and their overall antagonism of foodborne pathogens have led to their designation and use as “protective cultures” (1). On the other hand, some lactics have been incrinlinated in human infections, with the lactobacilli, pediococci, and enterococci being most often reported (2,3). Coryneform bacteria are a somewhat loosely defined group of organisms that are common in soils. The term “coryneform” refers to a microscopic picture of young cells that display a club or wedge shape and undergo postsnapping division that leads to palisade formations. The cells of older cultures of coryneforms shorten and appear as coccoids, and the genera do not produce spores. As originally defined, they consisted of four genera: Arthrobacter, Cellulonzor~as,Co~vnebacterirrm,and Microbacterirrm (4). The other coryneform and coryneform-like genera in Table 1 share microscopic features similar to the four genera noted. Some of the coryneform genera are involved in food fermentations (e.g., Brevibncterizm and Propiorzibcrcterium), and the genus Bijidobacterium is very important in the gastrointestinal tract of infants. Some bifido strains are used as food starter cultures (5). Because of their association more with human feces than with those of other animals, the bidifobacteria have been suggested as indicator organisms for human fecal pollution of waters (for review, see Ref. 6). The miscellaneous group in Table 1 consists of 13 genera of bacteria that are phylogenetically diverse. The genus Brochotlzrix is significant on processed meats, especially those stored under CO., atmospheres. More information on the gram-positive biota of meats can be obtained from Holzapfel (7). The deinococci and rubrobacteria are important only in irradiated foods because of their extreme radiation resistance. The micrococci are widespread in nature, and some species along with Kocuricr may be found in a number of food products. Significant foodborne pathogens are found among the staphylococci, and they are discussed further in a later chapter. Listeria nzonocytogerzes causes listeriosis in animals and humans and is closely related to Ensipelothri.~rhusioynthiae, which causes erysipeloid in animals. Mycobncterimz pcrratlrberc.clrlosisis of concern in pasteurized milk as the possible cause of Crohn’s disease.

Bacterial Biota (Flora) in Foods

27

Table 2 Most Common Gram-Negative Bacteria Found on Fresh and Processed Foods Group/genus

Morphology

Enterobacteriaceae Arizona Cedecea Citrobacter Edwardsiella Emir1 in Enterobacter Escherichicl Hajk ia Kluyvera Klebsiella Morganella Obsermbacterium Parltoea Proteus Providencin Salmonella Serratia Shigella Yersinia Miscellaneous Acetobacter Acirzetobacter Aerontorras Alcaligenes Alteromonrts Arcobacter Bacteroides Burkholderia Brucella Caityylobncter Chrorlzobacteritlill Deiilobacter Desu~otomaculurrl Flavobucteriunl Glucoizobucter Megasphaera Moraxella Pectinatus Photobacterium Plesiornonas Pseudonlonas Psycltrobncter Ralstonin Shewanella Vibrio Xnnthonronas Zymophilrrs ~~

~~

~~

C = Coccus; R = rod; S = spiral.

Common sources/comments

R R R R R R R R R R R R R R R R R R R

Feces, water Feces, water Vegetation, water, feces Feces, water Vegetation. feces, water Vegetation, water Feces, water Water, feces, meats Feces Vegetation, feces, water Feces, vegetation, water Beer wort Feces, spoiled meats Feces, water, fresh foods Similar to Proteus Poultry, feeds, other animals Vegetation, waters, feces Human feces Water, fresh meats, feces

R R R R R R R R R R R R R R R C R R R R R R R R S R R

Fresh apples, cider mills Water, vegetation, refrig. foods Water, feces. seafoods Water, vegetation, feces Marine waters. seafoods Hogs, cattle, sheep, vegetation Feces, polluted waters (anaerobic) Plant pathogens, vegetation Cows, sheep, hogs; raw milk Raw poultry, milk, bovines Vegetation, raw milk Extremely radiation resistant Canneries, canned foods Vegetation Grapes, honey bees, beerdwines Spoiled beer Soil, water, refrig. foods Spoiled beer Sea water, spoiled fish Water, feces Soil, water, refrig. fresh foods Meat, fish, poultry Tomato wilt disease Rancid butter, marine/fresh waters Waters, seafoods, feces Contain numerous plant pathogens In beer yeasts

28

Jay

B. Gram Negatives Members of the family Enterobacteriaceae constitute the largest phylogenetic group of foodborne gram-negative bacteria, and 19 genera are listed in Table 2. Any or all of these organisms may be found in animal feces under appropriate conditions, hence the common designation “enterics.” All of the salmonellae and shigellae are human pathogens, and some species/strains of Escherichia, Klebsiella, and Yersinia are human pathogens. The four coliform genera are among the enterics (Citrobacter,Enterobacter, Escherichia, and Klebsiella). The genera Citrobcrcter,Enterobacter, and Emjinia are well-known plant inhabitants. Common as food spoilage organisms are some Citrobacter, Hafizia, Puntoetr, Proteus, and Serrcrtia species since they contain psychrotrophic strains. The miscellaneous group in Table 2 includes some that cause human illness (Brucella, Campylobncter, and Vibrio) and others that are well-known plant pathogens (Pseudomonas, Ralstonia, and Xmthomonas). The bacteria that are most conspicuous on refrigerator-spoiled fresh foods such as ground meats are in the genera Acinetobacter, Alcaligenes, Alteronzonns, Moraxella, Psychrobncter, and Pseudornonas, with the latter genus being the single most important of the psychrotrophic spoilage bacteria. More information on the foodborne bacterial pathogens can be obtained from the ICMSF monograph (8) and from the specific chapters that follow in this volume.

111.

DETECTION ANDENUMERATION

On the surface, the detection and enumeration of bacteria in foods could be carried out in the same way as for nonfood products. However, the experiences of many over the years indicate that it is not that simple. Factors that set a food analysis apart from that of, say, a blood or spinal fluid analysis are discussed in the following sections.

A.

Choice of Incubation Time and Temperature

A typical fresh food product contains some bacteria that grow well between 4 and 7°C and some that can grow between 42 and 46°C. Those that grow at the lower temperatures generally require longer incubation times. When colony-forming units (CFU) are to be enumerated, the long incubation can lead to overgrowth of the slow growers by some of the fast growers and spreaders such as Bacillus and Proteus spp. All too often, individuals who are not experienced food microbiologists incubate plates at 37°C and expect results in 18-24 hours. With the exception of Canlpylobacter spp., all foodborne bacteria of significance grow well at 30-32”C, and incubation time should be extended to 48 hours.

B. Choice of Culture Medium Because of the high heterogeneity of the foodborne bacterial biota, electing the proper culture medium is not easy. Those without experience with foodborne bacteria tend to use media that are too rich or complex. Such media tend to allow for overgrowth of some bacteria during prolonged incubations. Before carrying out bacterial determinations on foods, it is strongly recommended that a standard reference method be used: the two recommended reference works are the FDA Bacteriological Amlytical Mar2ual (commonly referred to as BAM) (9) and the Compendium of Methods-for the Microbiological

Bacterial Foods Biota (Flora) in

29

Examination of Foods, referred to as the Compendium (10). Both works contain recommended culture methods for all bacteria of significance in foods in addition to some bioassay, molecular, and genetic methods of analysis for cells and/or their products.

C.SelectiveandDifferentialMedia The use of a selective plating medium such as Macconkey agar to recover gram-negative bacteria from, say, spinal fluid is distinctly different from its use to recover gram-negative organisms from a food product such as fresh ground beef. In the case of spinal fluid, the bacterial biota is neither as heterogeneous nor as large in quantity as for ground beef. Further, when low dilutions of meat homogenates are planted onto the surface of selective media, meat particles and constituents are known to exert a neutralizing effect on the selective agents. The small food particles can serve as microniches for the growth of some bacteria that would otherwise be inhibited by the selective agent.

D. Ratio of Numbers Recovering bacterial pathogens from foods is often like trying to find the proverbial needle in a haystack. When human stool cultures are examined for salmonellae where salmonellosis is suspected, typically these organisms are at levels of 10s-108 CFU/g of stool, although lower numbers are sometimes seen. In this case, the ratio of salmonellae to nonsalmonellae is such as to allow for direct enumeration and isolation using appropriate selective media. The ratio of salmonellae to the background bacterial biota of a product such as ground beef is such that direct enumeration and isolation are all but impossible since the usually low numbers of salmonellae are typically overwhelmed by the much larger background biota. Thus, nonselective enrichments are necessary, and they are widely used in the food microbiology laboratory. The foregoing deals with the determination of viable numbers of bacteria, variously referred to as aerobic plate count (APC) or standard plate count (SPC) of organisms per gram or CFU. Rapid nonculture methods have been developed for all significant foodborne pathogens, and the references noted above are excellent sources. Although these methods have proven to beinvaluable, there continues to be a valuable place for the classical culture methods. Nonculture methods typically detect both viable and nonviable cells, and when it is desirable to know if viable cells are present, a culture method is required. The impact of molecular and other nonculture detection methods on bacteriological assessments of foods has been reviewed and discussed by Feng (1 l).

IV. PREVALENCE IN FOODS A.

Ready-to-UseVegetables

In consideration of the high incidence and prevalence of bacteria in the soils and waters that basic food products grow in, it should not besurprising to find relatively high numbers on products that have not been subjected to bactericidal treatments. The prevalence of

30

Jay

Table 3 Log,, Mean APC/g of a Variety of Ready-to-Use Vegetable Products from Four Different Countries Product

N

Means

Location

Ref.

Salad mix Parsley. fresh Parsely, frozen Lettuce Salad mix Carrot sticks Celery Chopped lettuce Cole slaw Radishes Bean sprouts Lettuce (hydroponic)

2 11 14 24 3 15 4 4 1 1 2 34

7.85 6.67 5.26 7.82. 7.92 5.60 4.50 52 0 7 .OO 6.04 7.76 7.35

England Germany Germany Italy Italy United States United States United States United States United States United States United States

12 13 13 14 15 16 16 16 17 17 18 19

One sample/month over a 23-month period.

bacteria in a variety of ready-to-use vegetable products is presented in Table 3, and it can be seen that numbers between 1 and 10 million/g are common. Additional ready-to-use vegetables are presented in Table 4 where on day 0 the APC means/g were between 10,000 and 1 million. However, after these products were held for 4 days at 4"C, the APCs of most increased to > 1 milliodg (20). Typically, the bacterial biota of products of this type consists of organisms on the plant products during their growth (e.g., many of the lactic genera and gram-negative bacteria such as Envinin and Enterobacter). Also, a significant part of the biota would be expected to come from handlers, cutting and processing equipment, storage containers, and the air.

Spices B. Numbers of bacteria per gram of retail-store spices are known to be high, and some examples are presented in Table 5 of spices examined in Austria (21). Of the 10 products presented, rosemary had the lowest APC, with a mean of 4.83 log/g, and black pepper Table 4 Log,,, Aerobic Plate Counts/g on Fresh-Cut, Ready-to-Use Vegetables Stored at 4"Ca Vegetables Chopped lettuce Salad mix Cauliflower florets Sliced celery Cole slaw mix Carrot sticks Broccoli florets Green peppers

APC day 0

APC day 4

4.85 5.35 4.82 5.67 5.13 5.13 5.58 5.99

5.63 6.05 5.45 6.59 6.95 6.27 6.59 7.22

The products had a recommended shelf life of 7 days at 4°C. Source: Ref. 20. a

Bacterial Biota (Flora) in Foods

31

Table 5 Log,,, Numbers/g of Microorganisms Found in Some Spices in Vienna, Austria Allspice Caraway Chili powder China spice curry Ginger Nutmeg Black pepper Rosemary Thyme

6.89 6.04 5.91 7.42. 6.79 7.00 5.53 7.34 4.83 6.7 1

Source: Ref. 21

and China spice were the highest, with 7.34 and 7.42/g, respectively. Typically, products of this type contain large numbers of molds, especially black pepper. Among the bacteria, sporeformers are usually abundant as well as gram-positive cocci such as the micrococci. Gram-negative bacteria are either absent or quite low in numbers in products of this type if they have been properly dried and packaged. Some spices (e.g., rosemary and thyme) are also known to possess some antibacterial properties.

C. Meat Products The bacterial biota of fresh ground meats is reflective of carcass contamination during slaughtering, processing, and storage. Since such products are kept at refrigerator temperatures, it is not surprising to find large numbers of psychrotrophic bacteria. Mean log,, APCs/g of ground beef tested in 1914 and in 1994 are presented in Table 6. Retail store fresh ground beefmay be expected to contain >lo5 CFU/g. This is true for the data presented in Table 6 prior to 1994 where the USDA reported a mean of only 3.90 CFU/g for563 nationwide samples of fresh ground beef (29). This surprisingly low numTable 6 Examples of Log,, Aerobic Plate Count (APC) Numbers of Bacteria/g of Fresh Ground Beef Year

Number

APC mean

Ref.

1914 1936 1957 1964 1975

44 41 96 26 5 5 140 140 563

7.23 6.49 7.5 1 6.72 5.30J 7.95b 6.30 6.81 3.90

22 23 24 25 26 26 27 28 29

1976 1977 1994 ~~~~~

On day 0. After 5 days at 4.5"C.

32

Jay

ber could be a reflection of the downward trend for appearance of bacteria in such products due in part to more attention being paid to sanitation throughout the production cycle or in part to the methodology employed in gathering these data. For example, the mean of 3.90 is the result of APC determinations being made at 35°C for 24 hours. For fresh meat products, this number would be higher if incubation was between 25 and 32°C for at least 48 hours. It was shown by Rogers and McCleskey (24) that the APC of retail store ground beef was considerably higher when plates were incubated at 7°C for 7 days than when incubated for 48hours at 30°C. From Table 6 it may be noted that >2.5 log cycle increase occurred after a 5-day incubation at 4.5"C in the meats sampled by Goepfert and Kim (26). Regarding the history of our knowledge of bacteria in fresh ground meats, the earliest proposed APC standard for acceptable product was made by Marxer in 1903 (30), and the limit was 106/g. The numbers in Table 6 for samples tested in 1914 and 1936 are reflective of this general level. The only U.S. state ever to adopt bacteriological standards for ground beef was Oregon, and it allowed up to 5 X 106/g (see Ref. 31). The Oregon law was in effect in 1973-1977. The APC limit in the Oregon standard is a reflection of what was considered safe and achievable under good production practices during that time. With APCs of approximately 106/g, fresh ground beef was rarely the source of foodborne outbreaks in the United States prior to the late 1980s. Whether the increased incidence of foodborne outbreaks from ground meats are related to lower numbers of background organisms is of possible concern, and it has been addressed (32). Of the genera of bacteria listed in Tables l and 2, most investigators have found around 20 in beef, poultry, fish, and sausage products. Ayres (33) identified 19 genera from refrigerated beef and 30 genera from the surfaces of beef, sausage, fish, and chicken (34). In a study of pork sausage, Sulzbacher and McLean (35) identified 16 genera. In a Canadian study, 19 genera were identified from fresh ground beef (36). In a study of the number of genera in fresh and spoiled ground beef, nine genera were identified from 69 isolates from the beef when fresh, and following the frank refrigerator spoilage of samples from the same batch, only four genera were found, with 16 of the 19 isolates being pseudomonads (37).

REFERENCES 1. Holzapfel. W. H., Geisen, R., and Schillinger, U. (1995). Biological preservation of foods with reference to protective cultures, bacteriocins and food-grade enzymes.Znt. J. Food Microbiol.. 24:343-362. 2. Aguirre, M., and Collins. M. D., (1993). Lactic acid bacteriaand human clinical infection. J. Appl. Bacteriol., 75:95-107. 3. Jett, B. D., Huycke,M. M., and Gilmore. M.S . (1994). Virulenceof enterococci. Clin. Microbiol. Rev., 7:462-478. 4. Jones, D. (1975). A numerical taxonomic study of coryneform and related bacteria. J. Gen. Microbiol., 8752-96. 5. Hughes, D. B., and Hoover, D. G. (1991). Bifidobacteria: Their potential for use in American dairy products. Food Teclznol., 45(4):74, 76, 78-80. 82. 6. Jay, J. M. (2000). Modern Food Microbiology, 6th ed., Aspen Publishers, Gaithersburg,MD. 7. Holzapfel, W. H. (1998). The gram-positive bacteria associated with meat and meat products. (1998). In The Microbiology of Meat and Poultn (A. Davies and R. Board, eds.). Aspen Publishers, Gaithersburg, MD, 1998.

Bacterial Biofa (Flora) in Foods

33

8. ICMSF. (1996). Microorganisms in Foods 5. Microbiological specifications of Food Pnthogem, Aspen Publishers, Gaithersburg, MD. 9. (1995). FDA Bacteriological Analytical Manual, 8th ed. AOAC Int., Gaithersburg, MD. 10. Vanderzant, C., and Splittstoesser, D. F., eds. (1992).Cornpendiurn of Methods for the Microbiological Examination of Foods. American Public Health Association, Washington,DC. 11. Feng, P. (1997). Impact of molecular biology on the detection of foodborne pathogens.Mol. Biotechnol., 7:267-278. 12. Brocklehurst, T.F., Zaman-Wong, C.M., and Lund, B. M. (1987). A note on the microbiology of retail packs of prepared salad vegetables. J. Appl. Bacteriol., 63:409-415. 13. Kaferstein, F. K. (1976). The microflora of parsley. J. Milk Food Technol., 39:837-840. 14. Ercolani, G.L. (1976). Bacteriological quality assessment of fresh marketed lettuce and fennel. Appl. Environ. Microbiol., 31:847-852. (1976). 15. Vescovo, M., Orsi,C., Scolari, G., and Torriani, S . (1995). Inhibitory effectof selected lactic acid bacteria onmicroflora associated with ready-to-eat vegetables.Lett. Appl. Microbiol., 21: 121-125. 16. Garg, S . , Churey, J. J., and Splittstoesser, D. F. (1990). Effect of processing conditions on the microflora of fresh-cut vegetables. J. Food Prot., 53:701-703. 17. Rafil, F., Holland, M.A., Hill, W. E., and Cerniglia, C. E. (1995). Survivalof Shigellaflerneri on vegetables and detection by polymerase chain reaction. J. Food Prot., 58:727-732. 18. Jinneman, K. C., Trost, P. A., Hill, W. E., Weagant, S . D., Bryant, J. L., Kaysner, C. A., and Wekell, M. M.(1995). Comparisonof template preparation methods from foods for amplification of Escherichia coli 0157 Shiga-like toxins type I and I1 DNA by multiplex polymerase chain reaction. J. Food Prot., 58:722-726. 19. Riser, E. C., Grabowski, J., and Glenn, E. P. (1984). Microbiology of hydroponically-grown lettuce. J. Food Prot., 47:765-769. 20. Odumeru, J. A., Mitchell, S . J., Alves, D. M., Lynch,J. A., Yee, A. J., Wang, S . L., Styliadis, S., and Farber,J. M. (1997). Assessment of the microbiological qualityof ready-to-use vegetables for health-care food services. J. Food Prot., 60:954-960. 21. Kneifel, W., and Berger, E. (1994). Microbiologicalcriteria of random samples of spices and herbs retailed on the Austrian market. J. Food Prot., 57393-901. 22. Weinzirl, L., and Newton, E. B. (1914). Bacteriological analyses of hamburger steak with reference to sanitary standards. Am. J. Public Health, 4:413-416. 23. Elford, W. C. (1936). Bacterial limitations in ground fresh meat. Am. J. Public Health, 26: 1204-1206. 24. Rogers, E. R., and McCleskey, C. S . (1957). Bacteriological quality of ground beef in retail markets. Food Technol., 11:318-320. 25. Jay, J. M. (1964). Beef microbial quality determinedby extract-release volume (ERV).Food Technol.. 18(10):133-137. 26. Goepfert, J. M., and Kim, H. U. (1975). Behavior of selected foodborne pathogens in raw ground beef. J. Milk Food Technol., 38:449-452. 27. Westhoff, D., and Feldstein, F. (1976). Bacteriological analysisof ground beef. J. Milk Food Prot., 39:401-404. 28. Foster, J. F., Fowler. J. L., and Ladiges, W. C. (1977). A bacteriological surveyof raw ground beef. J. Food Prot., 40:790-794. 29. U.S. Department of Agriculture, Food Safety and Inspection Service. (1996). Nationwide federal plant raw ground beef microbiological survey,August 1993-March 1994, USDA, Washington, DC. und der Haltbarkeit des Fleisches 30. Marxer, A. (1903). Beitrag zur Frage des Bakteriengehaltes bei gewohnlicher Aufbewahrung. For-tsckr. Vet.-Hyg., 1:328. 31. Carl, K. E., (1975). Oregon’s experience with microbiological standards for meat. J. Milk Food Technol., 38:483-486. 32. Jay, J. M. (1997). Do background microorganisms play a role in the safety of fresh foods? Trends Food Sci. Teclmol., 8:421-424.

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33. Ayres, J. C. (1960). Temperature relationshipsand some other characteristicsof the microbial flora developing on refrigerated beef. Food Res.. 25:l-18. 34. Ayres, J. C. (1960). The relationship of organisms of the genus Psezdonzorzns to the spoilage of meat, poultry and eggs. J. Appl. Bacteriol. 23:471-486. 35. Sulzbacher, W. L., and McLean, R. A. (1951). The bacterialflora of fresh pork sausage.Food Techno].,5:7-8. 36. Lefebvre, N., Thibault, C., and Charbonneau, R. (1992). Improvementof shelf-life and wholesomeness of ground beef by irradiation. 1. Microbial aspects. Meat Sci., 32203-213. 37. Jay, J. M. (1967). Nature, characteristics, and proteolytic properties of beef spoilage bacteria at low and high temperatures. Appl. Microbial.. 15:943-944.

Aeromonas hydrophila Carlos Abeyta, Jr. U.S. Food and Drug Administration, Borhell, Wadzington

Samuel A. Palumbo U.S. Department of Agriculture, Philadelphia, Pennsylvania

Gerard N. Stelma, Jr. U.S. Emironmentrrl Protection Agewcy, Cincinnati, Ohio

I. Introduction 36 11. Classification and Characteristics 37

A. Classification B. Characteristics

37 40

111. Pathogenicity 40 A. Aeromonas infections B. Enterotoxins 41 C. Other virulence factors

40 43

IV. Control 44

A. Low temperature 44 44 B. Modified atmosphere C. pH and NaCl 45 D. Nitrite 45 E. Bacteriocins 46 46 F. Water activity 46 G. Chemical food additives H. Multiple barrier approach 47 I. Inactivation 47 V. Detection and Isolation 50 A. General considerations 50 B. Procedures 50 C. Sampling, enrichment, and isolation procedures References 52

50

Abeyta et al.

36

I.

INTRODUCTION

Aeromonas hydrophila translated simply means a "water-loving, gas-producing' ' bacterium. It was first reported by Zimmerman (1) and then Sandrelli (2), who isolated the organism and demonstrated its pathogenicity in frogs. Since that time, Aeromoncrs species have been isolated from other aquatic animals such as finfish, shellfish, crustaceans, and amphibians (3-5). This group of organisms is pathogenic to many aquatic species and causes hemorrhagic septicemia (red sore disease) in many fresh water pond cultured and wild native fish (6,7). It is well established that Aeronzonas is a component of the intestinal flora of healthy fish (8,9) and is widely distributed in nature. A. hydrophila is commonly found in watersheds (5,10,11). The prevalence and distribution of Aeronzonns spp. in fresh water habitats and the marine environment are well documented. It appears that Aeronzonas spp. may not be truly indigenous to the marine environment but may have a transient existence after entering salt water via rivers or sewage inputs. Aeromonas that are shed in sewage can multiply in sewage lines to significant numbers prior to discharge into receiving waters (12). Aeromonas is also present in terrestrial warm-blooded animals (13), including humans (14), and have been associated with three types of human illnesses; extraintestinal, wound, and gastrointestinal infections (14-20). Extraintestinal diseases are the most common, with a mortality rate as high as 61% in septicemic patients that are immunosuppressed. Wound infections involving Aeromonas usually are linked with injuries incurred during recreation or other activities in the aquatic environment (21-23). For gastrointestinal infections, a common source of A. hydrophila outbreaks is such water supplies as natural mineral springs, marine water environments, chlorinated and unchlorinated domestic supplies, and watersheds polluted by sewage effluents (24,25). There are statistical relationships between Aerolnonas species in water sources and health risks. However, there is no firm documentation of serious health effects after exposure to domestic waters containing Aeromonas (24). A serious health risk may be present for immunosuppressed patients with underlying malignancies after exposure to domestic and recreational waters containing Aeromonns species (14,26,27). The presence of Aeromonas in the food chain is well documented. This organism can be readily isolated from seafoods, foods of terrestrial animal origin such as meats, dairy products, poultry, and vegetables (28-34). Its presence in the food chain is of great concern because of its capability of growth at refrigerated temperatures (35). Seafood products are the most common food source of Aeromorzas. Both finfish and shellfish are known reservoirs of this bacterium. In 1986, A. hydrophila was the causative agent of an oysterborne outbreak in Florida (36). An attack rate of 100% (N = 7) was reported. A. hydrophila was isolated from the remaining uneaten oysters and from stool specimens from patients. In cases of foodborne bacterial illnesses in which shellfish are implicated, laboratories have included Aeronzonns in the general screening for causative microorganisms. Controversy concerning pathogenicity still remains. At present there are great difficulties assessing the regulatory significance of Aeromonas species in foods (37). From a clinical standpoint, the organism is no doubt of great concern to immunocompromised patients with underlying malignancies. Foods containing high levels of Aeromonns species destined to these individuals should be regarded hazardous. On the other hand, the role of foods containing high levels of Aeromonas spp. destined for healthy individuals is

Aeromonas hydrophila

37

uncertain. Although Aeromonas has been implicated in cases of gastrointestinal illness from foods, the exact mechanism by which Aerornonas species cause disease is not understood fully. Confusion concerning the assessment of pathogenicity remains unsolved. This was made evident in humanfeeding studies of virulent viable strains of Aeromonas species (high doses ranging from lo4 to lolo) involving 57 volunteers; only 2 developed mild diarrhea (38). One explanation for the failure of these strains to elicit diarrhea was presented. Kirov et al. observed the inability of Aerornonas species to adhere to the intestinal mucosa (39). This observation was noted in the shift of pilated environmental isolates toward nonpilated forms once the intestinal tract was infected. The nonpilated forms isolated from stools would be noninfective when used in challenge studies. The objectives of this chapter are to review the available information concerning this emerging recognizable pathogen. The reader is given information needed to assess further epidemiology, pathogenicity, management, detection, and isolation.

II. CLASSIFICATION AND CHARACTERISTICS A.

Classification

The genus Aeromonas was first proposed by Kluyver and Van Niel in 1936, as described by Popoff in Bergey’s Manual of Systematic Bacteriology (40). Various investigators divided the motile aeromonads into species including A. hydrophila, A. punctnda, A. formicans, A.liquefuciens, A. anaerogenes,A. proteolytica, and A. cnviae andA. sobria (41,42). The complexity of the problem was shown by Popoff et al., who found that A. hydrophila could be divided into distinct groups based on their divergence by DNA/DNA hybridization (43). The mesophilic A. hydroplzilngroup is collectively referred to as motile aeromonads. The genus Aeromonas consists of two well-separated groups of organisms: (a) the psychrophilic nonmotile aeromonads known as Aeromonas sahonicidn subspecies (pathogenic to fish but not to humans) and (b) the mesophilic motile aeromonads (the A. hydrophila group), which are divided into the species A. hydrophila, A. sobria, and A. caviae. The A. hydrophila group is associated with human illnesses. Most recent, a fourth motile Aeromonas spp., A. veronii, has been added to this list (44); it is distinguished from the other motile aeromonads by a positive ornithine decarboxylase test. Motile aeromonads belong to the family Vibrionaceae. Separation of the genus Aeromonas from related members of the family can be accomplished by biochemical reactions (Table 1). Aeromonas species are easily confused with group F Vibrios (V. fluvialis). Vibrios and motile aeromonads can be differentiated by their phenotypical reactions: salt requirement for growth and 0/129 sensitivity. Motile aeromonads are facultative anaerobic, gram-negative straight rods with rounded ends, measuring approximately 0.3- 1.O pm in diameter and 1.O-3.5 pm in length. They are motile by means of a single polar flagellum in liquid medium, and their metabolism is both respiratory and fermentative. They break down carbohydrates to acid or acid and gas (CO? and H?). Nitrate is reduced to nitrite, and oxidase and catalase are produced. Optimum growth temperatures range from 22 to 28°C. NaCl tolerance ranges from 0 to 4%, and tolerance to pH ranges from 5.2 to 9.8. They produce exoenzymes such as amylase, protease, phospholipase, and DNase and are resistant to vibriostatic agent 01129 (2,4-diamino-6,7-diisopropylpteridine). The mol% G + C of the DNA is 57-63 (Bd, T,,,).

Abeyfa et a/.

38

Table 1 Differentiation of the Genus Aeromonas from Other Genera of the Family Vibrionaceae Test Plesiowonas

Vibrio

Inhibition by 0/129 10 P8 150 P8 Na' requirement for growth Oxidase Gas from glucose Fermentation of inositol Fermentation of mannitol Ornithine decarboxylasea Growth on thiosulfate-citrate-bile salt-sucrose agar

Aeronzonns R

R S

R R

S

+ +

-

+ + +

-

+ + +

-

-

-

+ D +

-

-

R. Resistant: S . sensitive; D, differs among biotypes. verorlii is positive (+).

Table 2 Differential Phenotypic Characteristics salmorlicidu

of Motile Aeronlonas Species and Aeromonas b

Motile Characteristics

Aerorttonns spp.

1

,..I

.

. L

A. snlnlonicida

"

Motility Oxidase Ornithine decarboxylasea Arginine dihydrolase Indole production Starch, gelatin, DNA and RNA hydrolysis Citrate (Simmons') Citrate (Christensen's) ONPG Brown water-soluble pigment Growth without NaCl Fermentation of sucrose Fermentation of maltose, galactose, and trehalose Fermentation of cellobiose, lactose. and sorbitol Fermentation of glycerol, dulcitol, rhamnose, inositol, xylose, raffinose, and adonitol Rods in singles and pairs Coccobacilli in pairs, chains, and clumps Growth in nutrient broth at 37°C Aeronzonas varonii is ODC Differs among strains. c Aberrant strains occur. Source: Ref. 30.

i

+ + + + + db d

+ +

+ +"

+ -

+ + + -

d

+ + +c

d

-

-

-

+

-

-

+

+ -

+.

t

I

Aeromonas hydrophila 39

Abeyta et al.

40 Table 4 DifferentiationAmong the Aeromonas Izydrophia Group ~

~~

~~

~

Biochemical test“

A. hydrophila

Motility Esculin hydrolysis Growth in KCN L-Arginine utilization L-Lysine utilization L-Arabinose utilization Fermentation of salicin Fermentation of sucrose Fermentation of mannitol Breakdown of inositol Acetoin from glucose (Voges-Proskauer) Gas from glucose Indole production Oxidase P-Hemolysis H2S from cysteine

A. sobria

A. cavine

+

+ + + + + + + +

+b

+ + + + + + + + -

-

+

+

+ + -

+, Typically positive; -, typically negative. Source: Ref. 45. B. Characteristics The differential characteristics of motile aeromonads from nonmotile Aeromonns species are presented in Table 2. The common characteristic that distinguishes the A. hydrophila group (motile aeromonads) from A. snlnlonicicla subspecies of course is motility, but other similarities include lack of pigmentation, growth in nutrient broth at 37”C, colonial morphology, and monotrichous flagellation in liquid medium. In contrast, A. sdnzonicida does not grow at 37°C (optimum growth is at 22-25°C) and are nonmotile. Aeromonads can be easily grown in most routinely bacteriological selective and differential media (Table 3). On nonselective media such as trypticase soy agar, they are difficult to distinguish morphologically among members of the Enterobacteriaceae. The leading factors to consider in choosing the optimal culture medium is the type of sample matrix and the selective agent that will eliminate other competing organisms. Speciation of the motile aeromonads has been proposed by numerous investigators (Table 4). Biochemical reactions that are important for speciating among motile Aeromonns are based on esculin hydrolysis, growth in potassium cyanide (KCN) broth, salicin fermentation at 26”C, gas from glucose, and H2S from cysteine but not from thiosulfate.

111.

PATHOGENICITY

A. Aeromonas Infections Gastroenteritis is the most common foodborne illness attributed to the A. hydrophila group. About three quarters of Aerornorzns gastroenteritis cases are “choleralike,” characterized by watery stools and a mild fever; vomiting may also occur in children under 2 years of age. The other one quarter of cases are “dysenterylike,’ ’ characterized by blood and mucus

Aeromonas

41

in the stools (17). Aeromonns-associated diarrhea is normally mild and self-limiting (19); however, severe cases of both types of diarrhea have been observed (46-50). Aeromonads also have been implicated as the cause of localized wound infections, pneumonia, and such disseminating infections as bacteremia or septicemia and meningitis (16,18,19,5155). Although disseminating Aeromorlns infections may originate in infected wounds (16), the patient's gastrointestinal tract is considered to be the source of Aeromonns species in those infections (56). Therefore, disseminating infections should also be considered as potentially foodborne. One of several controversial issues regarding Aeromonas pathogenicity is the question of whether the aeromonads are all opportunistic pathogens, capable only of attacking hosts with impaired body defenses, or whether some of these organisms are sufficiently virulent to pose a threat to normal hosts as well. The fact that Aeromonas species are associated most frequently with diarrhea in the very young or in older adults (17,57-59) and the unusually high rate of isolation of aeromonads from patients with hematological malignancies (60) suggest that theyare opportunists. On the other hand, there is accumulating evidence that Aeromoms spp. also infect normal adults. George et al. identified Aeromonas isolates as the primary infectious agents responsible for gastroenteritis in a number of adult patients with no underlying disorders (20), and several literature reports describe severe cases of bothcholeralike (46,6 1) and dysenterylike (48) diarrhea in individuals who were otherwise healthy. Surprisingly, even bacteremia (51) and septicemia (53), which are usually associated with immunocompromised hosts (14,56), have been observed in immunocompetent young adults. It can be argued that immunocompetent individuals infected by aeromonads were affected by unrecognized predisposing conditions; however, it is quite likely that some highly virulent strains exist among the members of this diverse genus, which consists of at least 9 validated and/or proposed species (62) and 13 known hybridization groups that are extremely heterogeneous in biochemical structural and genetic properties (63). B. Enterotoxins The most controversial issue related to Aeromonns pathogenicity concerns the relative roles of various putative enterotoxins in causing diarrhea. There are reports of both heatstable (56"C, 10 min) and heat-labile cytotonic enterotoxins and cytotoxic enterotoxins, some related and others unrelated to cholera toxin. The controversy is not over the variety, which is not unusual for a diverse group of organisms, of enterotoxins reported, but over the fact that researchers who reported evidence for one of these toxins usually were unable to observe any of the others. The various putative enterotoxins produced by Aeromonas species are described below. Ljungh et al. reported partially purifying an enterotoxin with a molecular weight of 15 kD that was stable after treatment at 56°C for 10 minutes (64,65). This molecule, which was serologically unrelated to cholera toxin (CT), caused fluid accumulation in the permanently ligated rabbit ileal loop (RIL), rounding of Y-l cells without death, and stimulation of cyclic AMP (adenosine monophosphate) synthesis. Further evidence for a heat-stable cytotonic enterotoxin was provided by Chakraborty et al., who reported cloning the cytotonic enterotoxin gene into Escherichia coli (66). Culture filtrates of the clone caused elongation of Chinese hamster ovary (CHO) cells and fluid accumulation in the RIL after treatment at 56°C for 20 minutes. These activities also were unrelated to CT. Both of these groups reported that the P-hemolysin was inactive in the RIL.

42

Abeyfa et al.

Potomski et al. (67) reported using affinity chromatography to isolate an A. sobria toxin that cross-reacted with CT, caused rounding of Y-l cells and fluid accumulation in both RILs and infant mice after heating at 56°C for 20 minutes. All of these activities were reportedly neutralized by antiserum to CT. Schultz and McCardell (68) also reported rounding of Y-l cells and stimulation of cyclic AMP synthesis by Aeronzorzns culture filtrates that were heated at 56°C for 20 minutes. These activities were partially neutralized by antiserum to CT. They also reported that DNA from strains producing the cytotonic enterotoxin reacted with one or more synthetic oligonucleotide probes coding for CT. Chopra and Houston (69) reported purifying a cytotonic enterotoxin that caused fluid accumulation in the RIL, stimulated cyclic AMP synthesis, and caused elongation of C H 0 cells. The purified toxin had a molecular weight of 44 kDa, was free of hemolytic and cytotoxic activities, and was not cross-reactive with antiserum to CT. The biological activities of the purified toxin were heat labile at 56°C. The first evidence for a cytotoxic enterotoxin was provided by Cumberbatch et al. (70), who reported a correlation between cytotoxic and RIL activities. All of their cytotoxic isolates also were hemolytic. They found no evidence for a separate cytotonic activity in any of their isolates. Turnbull et al. (71) and Burke et al. (72) also observed a strong correlation between enterotoxin production and hemolytic activity. Asao et al. provided the first direct evidence for a cytotoxic enterotoxin (73). They purified a hemolysin from A. hydrophila strain AH-l to electro-phoretic homogeneity and observed that it was cytotoxic to Vero cells and had enterotoxic activity in both the RIL and suckling mouse assays. The hemolysin had a molecular weight of 50 kDa and was heat labile at 56°C for 5 minutes. Stelma et al. (74) showed that the Asao hemolysin was P-hemolysin and that antiserum to the purified hemolysin completely neutralized the RIL activities of filtrates of P-hemolytic Aeronzonas isolates, indicating that P-hemolysin alone can cause the changes in intestinal permeability associated with diarrhea. These researcher also used polyclonal antiserum to show serological cross-reactions between the hemolysin purified from the Japanese strain, AH- 1, and hemolysins produced by isolates from diverse geographic origins. Several additional studies provided evidence that the Aeromonns P-hemolysins are a family of molecules related to the "aerolysin" originally described by Bernheimer and Avigad (75). Rose et al. (76,77) purified a 52 kDa protein toxin that possessed hemolytic, cytotoxic, and enterotoxic activities as well as serological cross-reactivities to both CT and the Asao (AH-l) hemolysin. The biological activities of their toxin were neutralized by homologous antiserum and antiserum against the AH-l hemolysin but not by antiserum against cholera toxin. Asao et al. (78) demonstrated that the hemolysin produced by A. hydrophila CA1 1, a U.S. Gulf Coast isolate, was related immunologically to AH-l hemolysin but also possessed unique antigenic determinants. Millership et al. (79), Kozaki et al. (80), and Stelma et al. (81) provided evidence that hemolysins from A. sobt-in and A. veronii also possessed enterotoxic activities and were serologically related to AH-l hemolysin. Evidence from several surveys suggests that the cytotoxic enterotoxins are the most common Aerornorzas enterotoxins. Cumberbatch et al. found no cytotonic activity in the filtrates of 96 isolates (70). Likewise, Johnson and Lior found no cytotonic activity in the filtrates of 73 isolates (82), and Seidler et al. found cytotonic activity in the filtrates of only 20 of 330 isolates (6%) (83). Stelma et al. found evidence for cytotonic activity in one of 24 isolates (74), but this was later shown to be caused by sublethal doses of partially denatured cytotoxic enterotoxin after heating at 56°C (84).

Aeromonas hydrophila

43

The absence of significant enterotoxic activity in the P-hemolysin purified by Ljungh et al. (64) may be due to differences in the purification procedures. Ljungh et al. used a six-step procedure that recovered only 0.6% of the original hemolytic units (64). In contrast, Asao et al. used a two-step procedure that recovered 65% of the original hemolytic units and probably yielded a product closer to the native molecule in all of its properties (73). The failure of the E. coZi clones carrying the A. hydrophila hemolysin gene to cause fluid accumulation in the RIL (66) was later shown to be due to the inability of the E. coli to release the hemolysin from the cells (85). Although the heat-labile cytotoxic enterotoxins appear to be the mostcotnmon Aeromonas enterotoxins, the relative roles of these toxins and the cytotonic e,nterotoxins in Aerornonas diarrhea are not known. Determination of the relative roles of these toxins will require the development of better animal models and either the use of strains that produce only one toxin or the use of transposon mutagenesis to inactivate various toxins one at a time. It has been agreed generally that A. cctvirre isolates were noncytotoxigenic and were not enteric pathogens (17,86). However, Namdari and Bottone recently reported detecting a heat-stable cytotoxin in culture filtrates of isolates grown in double-strength trypticase soy broth (TSB) (87). They, like earlier investigators, did not detect enterotoxin in filtrates of A. caviae grown in single-strength TSB. Several recent studies have also linked A. caviae to diarrhea in very young children and individuals aged 50 years or more (8890).

C. OtherVirulenceFactors The negative results of the human feeding study performed by Morgan et al. provided evidence that strains producing enterotoxin active in the RIL are not always diarrheagenic and that multiple virulence factors are involved (38). The strains used in that study did not possess either adhesion factors for colonization of the intestine or ability to invade tissues. Several studies have provided evidence that some aeromonads possess adhesion factors that correlate with the possession of pili (39,91,92). The most interesting study was that of Kirov et al. (39), who observed that environmental enterotoxigenic isolates possessed nutnerous pili, which appeared to be lost once infection was established. This suggests that in future studies environmental isolates may be more appropriate for human feedings than clinical isolates. The relationship between virulence factors and ability of Aeronzor~zsstrains to cause disseminating infections has not been established. Lethality tests with both normal mice and mice immunosuppressed by x-irradiation have focused primarily on relating virulence to biotype or phenospecies (93,94). The results of those studies and one of invasiveness to mammalian cells (95) indicated that A. hydrophila and A. sobria were inherently more virulent than A. caviae, but with significant strain-to-strain variation within a species. One property linked to virulence is a surface array protein (S layer) commonly found in human isolates from extraintestinal infections. The role of the S layer in human and animal infections is not clear, but it appears to be substantially different from the role of the S layer of A. salmonicida in fishdisease (96,97). Other properties of motile aeromonads that have been linked to virulence in other bacterial species include the ability to invade mammalian cells (95,98), resistance to serum bactericidal effects (99,100), production of a siderophore capable of removing iron from transferrin (101,102), a mechanism for utilization of iron from heme compounds (102), and production of proteases (103). Loss of

et

44

Abeyta

al.

viability after growth in broth containing 0.5% glucose (suicide phenomenon) has been associated with both enteropathogenicity and virulence (104), but the significance of this phenomenon is not known. The relative significance of each of these putative virulence characteristics cannot be determined until appropriate animal models are developed, preferably models in which the animals are compromised in a way that mimics the condition of the susceptible human host. i

IV. CONTROL The presence, survival, and growth of bacteria in foods can be controlled by the applying the three Ks: keep them out, keep them from growing, and/or kill them. The A. hydrophila group occurs widely in the aquatic environment (105), and this undoubtedly represents the major source of these bacteria in foods. Their widespread occurrence in virtually all fresh and processed foods surveyed attests to their ubiquitous presence in foods (105). It would thus appear that it is difficult if not impossible to keep them out of foods. Based on this, control of their levels would then depend on extrinsic and intrinsic factors such as low temperature, modified atmosphere, pH/acid, salt and water activity, and the use of food preservatives to keep them from growing and on measures such as high temperatures (55°C and above), irradiation, and low pH, especially when adjusted with organic acids, to kill them.

A.

Low Temperature

Low-temperature holding (5°C) or refrigeration of fresh and processed foods has traditionally been relied on by the food industry to prevent the growth of foodborne pathogens. However, A. hydrophila, along with other foodborne pathogens, has been observed to grow at holding temperatures typically used for fresh and processed foods (106). It has long been observed that A. hydrophila can grow at 5°C or below (35,107,108). Palumbo et al. (32) determined that naturally occurring A. hydrophila could grow competitively in various retail foods of animal origin during storage for one week at 5°C. Callister and Agger (34) made a similar observation for fresh vegetables held at 5°C. Using ground pork inoculated with A. hydrophila, Palumbo (109) verified the low-temperature competitive growth of this bacterium in a food and determined that additional factors such as NaC1, vacuum packaging, and pH can also affect its growth. Since A. hvdrophila can readily and competitively grow in foods held at 5"C, factors other than low refrigeration assume greater importance in controlling its growth in foods.

B. Modified Atmosphere Members of the family Vibrionaceae are facultative anaerobes (42). Broth studies (1 10) and surveys of red meats stored under various atmospheres (30,3 1,35,111-113) have verified this. Based on broth studies, Palumbo et al. (1 10) determined that overall A. hydrophila grew as well anaerobically as it did aerobically. The observations from the red meat storage surveys indicated that the meats' microflora and/or storage temperature rather than the packaging atmosphere appears to control the numbers and types of the bacteria that develop and are detected. When conditions favor the development of Pseudomonas

.

.

I~

Aeromonas hydrophila

45

species or lactic acid bacteria, these bacteria will dominate the Aeromonas species (31,109,113).

C. pH andNaCl After temperature, pH and NaCl are the next most important food parameters controlling the growth of most bacteria in foods. As a typical gram-negative bacterium, A. hydrophila is fairly sensitive to acid or low pH (<5.5) (35,114) and high NaCl (>4.5%) (35); in addition, as shown in Table 5, the limiting pH and NaCl levels are temperature dependent. While the observations in Table 5 were generated in brain-heart infusion (BHI) broth, similar responses were seen during studies of the bacterium in ground pork in which pH and NaCl interacted to restrict the growth of A. hydrophila at lower levels than with either factor individually (109). Using a multifactorial approach, Palumbo et al. (110,115) studied the individual effects and interactions of temperatures, pH, NaC1, and sodium nitrite on the growth kinetics of the bacterium in BHI broth. In these two studies, all factors interacted to increase lag and generation times as pH decreased and the NaCl level increased. For most studies described, HC1 was the acidulant; however, Palumbo and Williams (1 14) used different acidulants and observed differences in the response of A. hydrophila based on the acidulant used, with acetic and lactic acids being the most restrictive and H2S04and HC1 being the least so. Overall, the order of effectiveness for the acids tested are (listed from most restrictive to the most permissive): acetic, lactic, tartaric, citric, H2S04,and HCl. Their activity appeared to be related to their pK,s. D. Nitrite Sodium nitrite in combination with NaCl acts as the curing agent in cured meat products. Its continued permitted use in cured meat products is based on its anticlostridial activity. While members of the A. hydrophila group generally cannot grow in cured meats, they can be isolated from these products (1 16,117). The growth-inhibiting mechanism is sodium nitrite combined with their brine content, pH (about 6.0), vacuum packaging, and low storage temperatures (1 10). These conditions provide an environment in which A.

Table 5 Influence of Temperature on pH and NaCl Limits for A. hydrophila K144 Grown Aerobically in Brain-Heart Infusion Broth Limits of Temperature (C)

28

4

PH G at 5.5 NG at 4.5 NG at 5.5

G. Growth: NG, no growth. Highest level tested. Source: Ref. 35.

NaCl (9%) G at 4 3 G at 3.5 NG at 4.5

46

Abeyfa et al.

Izydrophiln cannot grow readily or compete with the normal flora of lactobacilli, micrococci, and yeasts found in these products.

E. Bacteriocins Bacteriocins are polypeptide antimicrobial compounds produced by various strains of lactic acid bacteria (LAB). There is increasing interest today in the “natural” preservation of foods, and the use of lactic acid cultures and/or the bacteriocin(s) isolated from them have been studied for their effectiveness against spoilage and foodborne pathogens. Lewus et al. (1 18) reported on a study in culture broth, which investigated the activity of various bacteriocin-producing LAB against A. hydrophila. They observed that LAB isolated from meats as well as known LAB cultures would inhibit the bacterium. Santos et al. (1 19) observed that the bacteriocin(s) of a dairy starter culture inhibited A. hydrophila in both skim and ewe’s milk. However, in studies by Kalchayanand et al. (120), neither nisin (4000 activity units/mL) nor pediocin (4000 activity units/mL) was individually effective against the bacterium in broth, but when combined with a preliminary heating or freezing treatment (stresses to damage the cell membrane and cell wall), A. hydrophila became sensitive. At this time, bacteriocins would appear to have some potential uses against this bacterium, but conditions for their optimal activity and effectiveness need to be developed.

F. WaterActivity Water activity (a,) is ameasure of the amount of free water available for microbial growth. Since all microbes need free water for growth, any food substance that binds water can control microbial growth. Some common food substances that can bind water include various salts including NaCl (its activity is discussed above), sugars and carbohydrates, and amino acid and proteins. In perhaps the only study of its kind currently available, Santos et al. (121) studied the effect of a, (adjusted with NaC1, glycerol, and polyethylene glycol) on the growth of three strains (food isolates) of A. hyc/rophiln at 28, 10, and 33°C. The minimum a, for growth varied with strain, temperature, and type of humectant, with NaCl being the most inhibitory and glycerol being the least at comparable a,$s.

G. ChemicalFoodAdditives Various miscellaneous chemicals (many are GRAS [generally regarded as safe]) are added to foods, and in addition to their other functions, they can control the growth of foodborne pathogens, including A. lzydoyhilu. Some of these include essential oils, liquid smoke, food preservatives, and polyphosphates. Stecchini et al. (122) studied the effects of the essential oils of clove, coriander, nutmeg, and pepper in botha model system and noncured cooked pork and observed that the bacterium was inhibited. Liquid smoke prepared from several species of wood also inhibited A . h,dr-ophi/a (123). When the food preservatives methyl p-hydroxybenzoate and potassium sorbate were tested against four psychrotrophic foodborne bacteria, A . hydrophila was the most sensitive (134). When Venugapal et al. (125) testedthe food preservatives butylated hydroxyanisole, propylhydroxy parabenzoate, and sodium tripolyphosphate against A. hydrophila, they observed that protease secretion was more sensitive than growth. When Palumbo et al. (126) tested the food polyphosphates Sodaphos, Hexaphos, sodium pyrophosphate, and sodium tripolyphosphate against the bacterium individually in culture broth, they observed only small changes

4

I

Aeromonas hydrophila

47

in growth kinetics (lag and generation times). However, when combined with 3.5% NaCl, the number of viable A. hydrophila declined to an undetectable level. This effect was also noted when the combination was tested in ground pork.

H. MultipleBarrierApproach As is apparent from the above discussion on the influence of specific factors in controlling the growth of A. hydrophila, most factors (NaCl [expressed both as a percent and as a,], sodium nitrite, and pH), at the levels generally encountered in foods, individually cannot restrict the growth of the bacterium in foods. Research from this laboratory has indicated that multiple barriers (factors) or the multifactorial approach (two or more factors at less than maximum inhibitory levels) can successfully be used to inhibit foodborne pathogens such as Listeria ~~lonocytogenes (127), Shigella fiexl1eri (128), Yersinia enterocolitica (129), E. coli 0157:H7 (130), and A. h,,drOphih (110,115). From these studies, predictive models or equations have been developed to allow description of how changes in such culture (food) parameters as temperature, pH, NaCl level, and atmosphere can alter a bacterium's growth kinetics (e.g., lag and generation times). Often, small changes in a single parameter can bring about dramatic increases in lag and generation times and a decreased hazard frotn that particular pathogen. These equations have been incorporated into a user-friendly, Windows-based computer software program (Pathogen Modeling Program, version 5.1, available from the Microbial Food Safety Research Unit (Eastern Regional Research Center [ERRC], U. S. Department of Agriculture, 600 E. Mermaid Lane, Wyndmoor, PA 19038 or from the ERRC website (WWW.ARSERRC.GOV). I. Inactivation There are several food-processing operations that can inactivate bacteria and other microorganisms. These include heating (cooking), irradiation, sanitizing and disinfecting, and acidification. In addition, treatments such as the lactoperoxidase system have been shown to inactivate bacteria in foods. 1. Heating Heating is one of the primary treatments for the destruction of pathogenic and spoilage bacteria in foods. As withother bacteria, the thermal resistance of A. hydrophila is affected by factors such as growth temperature, age of the culture, and heating menstruum [buffer or different foods] (13 1,132). Nishikawa et al. (133) determined that A. hydrophila was more heat sensitive than Salmonella typhiwzuiurn and E. coli 0157:H7 when heated in either hamburger or egg yolk. Condon et al. (13 1) calculated a DS5"C= 0.17 minute and a Z = 5.1 1°C for a single strain when heated in buffer. Using both clinical and food strains, Palumbo et al. (132) determined DlsJcof 5.2 and 4.3 minutes, respectively, when heated in saline or raw milk and a Z = 6.2"C. These few studies suggest that this bacterium's thermal resistance is similar to that of other gram-negative bacteria found in foods and the bacterium should be inactivated by the heat treatments given many food products during their normal processing

2. Irradiation Irradiation along with heating represents a means of putting energy into a system (culture broth or food) to inactivate microorganisms. Though there are very few studies on the

Abeyta et al.

48

radiation resistance of A. hydrophila (134,135), it does appear to be relatively radiation sensitive and pasteurizing doses aimed at eliminating other foodborne pathogens such as SuZmoneZZa and E. coli 0157 :H7 should also eliminate A. hydrophila (136). 3. Chlorine and Other Sanitizers Cattabiani evaluated the influence of various food plant sanitizers (disinfectants), including chlorine, on four strains of A. hydrophila (137). The results of his study are presented in Table 6; in general, the bacterium appears to have susceptibilities similar to other gramnegative bacteria found in foods. Knochel studied the chlorine resistance of motile Aeromonas species using two different methods and observed that Aeromonas species were more susceptible to chlorine that other gram-negative bacteria such as E. coli, Klebsiella species, and Pseudomonas aeruginosa (138). As can be seen from the data in Table 6 , A. hydrophila is inactivated readily by chlorine at the levels used for the treatment of drinking water. However, the bacterium can be isolated from chlorinated water supplies (24,139,140), even when the test for E. coli is negative (24,140). The recovery of the organism from chlorinated water may be explained as posttreatment recontamination, the presence of unusually large numbers of A. hydrophila, or the presence of organic matter that can inactivate the added chlorine. An alternate explanation may be offered: A. hydrophila is known to be injured by sanitizer treatments (141) and, when selective media are used to isolate the bacterium after treatment, the bacterium is not recovered and thought to be absent. Thus, the presence of the

,

Table 6 Sensitivity of A. hydrophila to Disinfectants Time of exposure at 25°C (min) Concentration

5

10

+ + + +

-

-

3+/l3+/1-

1+/31+/3-

-

-

-

3+/1-

2+/2-

1

Compound Sodium hypochlorite

Quaternary ammonium compound

Iodoform

2-Chlorophenol

Glutaraldehyde

5 PPm 2.5 ppm 1.25 ppm 0.625 ppm 0.31 ppm 1:12,500 1:25,000 1:50,000 1:100,000 10 PPm 5 PPIn 1 PP" 0.2% 0.1% 0.05% 0.125% 0.0625% 0.03 % 1

-, Sensitive. reduction of four or more log cycles in viable count; Source: Ref. 137.

-

+ + 3+/1+ + + + -

3+/1-

+

+. resistant,

+

+ + 1+/3+ + -

+

Aeromonas hydrophila

49

bacterium in such foods as poultry carcasses and vegetables may represent contamination via the potable water supply. 4. Organic Acids In the study by Palumbo and Williams (1 14) cited above in the section on pH and NaC1, the influence of organic acids in combination with temperature and NaCl on the growth kinetics (lag and generation times) of A. hydrophila was investigated. They also observed that certain combinations were lethal to the bacterium, These findings are presented in Fig. 1. Again, the form of the acidulant was important, with acetic and lactic acids being the most toxic and the two inorganic acids being the least toxic. While these findings suggest that A. hydrophila should not be a problem in various fermented and pickled foods, actual experimental studies and food surveys gave mixed observations. Aytac and Ozbas (142) determined that A. hydrophila inoculated into yogurt mix decreased to undetectable during the fermentation; however, Knochel and Jeppesen (117) were able to isolate the bacterium from 10% of the mayonnaise-based salads, often at levels of >105/g.

5. Lactoperoxidase System The enzyme lactoperoxidase (LP) in the presence of thiocyanate and hydrogen peroxide can produce an active antimicrobial system in milk; this system is bactericidal for gramnegative bacteria including A, hydrophila. Santos et al. (143) observed that A. hydrophila decreased to undetected in broth, skim milk, and ewes’ milk and indicated that the LP system, when used in combination with low temperatures, could be useful in controlling the presence of this bacterium in fluid dairy products. Santos et al. (144) determined that the LP system could effectively reduce the levels of A. hydrophila during the manufacture of the Spanish fresh sheep’s cheese Villalon. It would appear that the LP system could provide an adjunct to good manufacturing practices in controlling A. hydrophila in dairy foods in general.

4.4 4.0

S

p

3.6

W

,$ 3.2

2

2.8

S 2.4

8 2.0

l4

1.6

1.2 0

50

100

150

200

250

300

Hours at 5°C Fig. 1 Effect of different acids (HC1, sulfuric, tartaric, citric, acetic, and lactic) on the decline in viable count of A. hydrophila in BH1 broth at pH 5.0 and 5°C (0.5% NaCl). Dashed line-lower limit of detection (log,, = 1.33).

Abeyta et al.

50

V.

DETECTIONANDISOLATION

A.

GeneralConsiderations

In handling of suspect samples, immediate analysis upon arrival in the laboratory is preferred. Motile aeromonads have the capability of proliferating at refrigeration temperatures. If samples are to be analyzed within a few days, store at refrigeration temperatures. Samples kept longer than one week should be stored at -20 or -72°C. An enrichment procedure is necessary when analyzing frozen foods and foods that contained injured aeromonads or low levels of motile aeromonads.

B. Procedures Procedures suggested for isolating and enumerating motile aeromonads from foods are found in the Bacteriological AnalyticalMarzual (BAM) (45). These procedures have been used in Food and Drug Administration laboratories for the analysis of various foods samples. Since the publication of these methods, other procedures have been developed and are recommended in conjunction with BAM methodology for isolation and enumeration of motile aeromonads. These procedures are discussed in the next section.

C. Sampling,Enrichment,andIsolationProcedures The following procedures are based on analysis of a 25 mL or 50 g analytical unit at 1:9 (sample/diluent) ratio. For samples containing less that 25 g, add enough diluent to maintain a 1:9 ratio. For samples requiring enrichment techniques, the following procedures should be followed. Aseptically weigh a 25 g sample into a sterile wide-mouthed, screw-cap jar (500 mL) or other appropriate container (i.e., stomacher bags). Add 225 mL of sterile trypticase soy broth with ampicillin (TSBA) and blend for 2 minutes Loosen jar cap about 1/4 turn and incubate 24 t 2 hours at 35 ? 2°C. After incubation of enrichment broth for 18-24 hours at35"C, transfer 3 mm loopfuls of inoculum onto Macconkey agar (MAC), peptone-beef extract-glycogen (PBG) agar, and Yersinia selective agar (YSA) base supplemented with cefsulodin and novobiocin as described in BAM to yield isolated colonies. A recent recommendation is to use starch ampicillin (SA) agar instead of PBG and YSA. For enumerating aeromonads in TSBA, inoculate a 3- or 5- tube most probable number (MPN) series containing TSBA from serial dilutions. Incubate broth tubes for 18-24 hours at 35°C. Follow procedures as described above for isolation of aeromonads. For samples not requiring an enrichment procedure, aseptically weigh 25 g of sample into an appropriate container and add 225 mL of sterile 0.1% peptone water. Make appropriate serial dilutions and surface plate 0.1 mL portions onto selective media, as described below, with a sterile bent glass rods to distribute the entire inoculum evenly over the surface of the media. Starch-ampicillin (SA) agar developed by Palumbo et al. (32) is recommended for direct surface plating of samples. Starch hydrolysis was selected since this enzyme activity is largely restricted to Aerornorzas and Vibrio species. The use of ampicillin effectively suppresses growth of coliforms and members of the family Enterobacteriaceae. SA has been used effectively for red meats, chicken, raw milk, seafoods, and fresh vegetables. Most recent, marine environmental samples including shellstock oysters, sediment, and marine waters were evaluated by SA direct plating for enumeration of motile aeromonads (see Table 7). Results indicated that direct plating with SA was

Aeromonas

51

Table 7 Evaluation of Starch-Ampicillin Agar in Recovering Motile Aeromonads from Shellfish-Growing Waters in Humbolt Bay, Eureka, California Media Sample lo)

MCA/TSBA ( MPN)a

SAA/TSBA (MPN)b

MCA & SAA/TSBA (CFU)' (MPN)'

SAA

Oysters Water Sediment

1.17'(60)' 2.17 (90) 3.69 (100)

1.40 (70) 2.23 (90) 3.76 (100)

1.47 (70) 2.34 (90) 4.11 (100)

0.15 (10) 0.91 (70) 3.37 (100)

(I2 =

Most probable number(MPN) determinations in tryptic soy brothwith ampicillin (TSBA) plated onto MacConkey agar (MCA). h Plated onto starch-ampicillin agar (SAA). c Combined MCA and SAA. Colony forming unit determinations in direct plating onto SAA. e Log,(, perg or mL. Percent positive of motile aerornonads detected.

ENRICHMENT OR DIRECT PLATING

c SELECTIVE AGARS .1 OXIDASE (

c

A . HYDROPHlL 5.

+) MEDIUM(AHM)

SALT TOLERANCE OR VlBRlOSTATlC 01129

c DIFFERENTIATION AND CONFIRMATION .1 CONVENTIONAL TEST OR RAPID DIAGNOSTIC TEST (i.e., API) Hemolysin Gas from glucose H,S from cysteine Esculin hydrolyis Acetoin from glucose KCN Arginine Arabinose Salicin Sucrose

Fig. 2 Schematic diagram for the isolation of motile aeromonads. (From Ref. 45.)

52

Abeyta et al.

effective with sediment samples, but not for shellstock oysters and marine water, in which the enrichment procedure showed an advantage over SA direct plating. Identification and confirmation of motile aeromonads can be accomplished by following the schematic diagram of Fig. 2. Aeromonas hydrophila medium (AHM) described by Kaper et al. (145) is useful for rapid presumptive identification and differentiation from coliforms and enterics. AHM is a single tube medium testing for fermentation of mannitol and inositol, ornithine decarboxylation, indole production, motility, and H,S production from sodium thiosulfate and cysteine. Studies by Abeyta et al. found this medium useful in identifying environmental marine isolates (1 1). The efficacy of the multitest screening AHM was determined. Of the 1396 strains positive for oxidase, 76% (1,065) gave typical reactions in AHM. Of the 1065 isolates, 95% were confirmed as motile aeromonads. Other rapid test systems such as API 20E, VITEK, and MICRO-ID are useful in rapid identification of typical motile aeromonads, however, these systems have their limitations. For example A. hydrophila and V.JEuvialisare related closely biochemically. Such additional tests as salt tolerance are recommended before a final identification can be reached.

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33. 34.

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37. 38.

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79. Millership, S. E., Barer, M. R., Mulla, R. J., and Maneck, S. (1992). Enterotoxic effects of Aeromonas sobria hemolysin in a rat jejunal perfusion system identified by a specific neutralization with a monoclonal antibody. J. Gen. Microbiol., 138:261-267. 80. Kozaki, S., Asao, T., Kamata, Y., and Sakaguchi, G. (1989). Characterization of Aeromonas sobria hemolysin by use of monoclonal antibodies against Aeromonas hydrophila hemolysins. J. Clin. Microbiol., 27:1782-1786. 81. Stelma, G. N.. Jr., Johnson, C. H., and Spaulding, P. L. (1988). Experimental evidence for enteropathogenicity in Aeromonns veronii. Can. J. Microbiol., 34:877-880. 82. Johnson, W. M., and Lior, H. (1981). Cytotoxicity and suckling mouse reactivity of Aeronzonas hydrophila isolated from human sources. Can. J. Microbiol., 27:1019-1027. 83. Seidler, R. J., Allen, D. A., Lockman, H.,Colwell, R. R., Joseph,S. W., and Daily. 0. P. (1980).Isolation enumeration and characterization of Aeronzonas from polluted waters encountered in diving operations. Appl. Environ. Microbiol., 39:lOlO-1018. Kaylor, L. O., and Johnson, C. H. ( 1986). 84. Bunning, V. K., Crawford, R. G., Stelma, G. N., Jr., Melanogenesis in murine B 16 cellsexposed to Aerontonas hydrophilu cytotoxic entertoxin. Can. J. Microbiol., 32:815-819. 85. Chakraborty, T., Huhle, B., Bergbauer, H., and Goebel, W. (1986). Cloning expression and mapping of the Aeronlonas hydrophila aerolysin gene determinant in Escherichiacoli K12. J. Bncteriol., 167:368-374. 86. Janda, J. M., Reitano, M., and Bottone, E. J. (1984). Biotyping of Aeromonas isolates as correlate to delineating a species-associated disease spectrum. J. Clin. Microbiol.. 19:4447. 87. Namdari, H.,and Bottone, E. J. (1990). Cytotoxin and enterotoxin production as factors delineating enteropathogenicity of Aerontonas caviae. J. Clin. Microbiol., 28: 1796-1798. 88. Kuijper, E. J., Zanen, H. C. and Peeters. M. F. (1987). Aerontorzas-associated diarrhea in the Netherlands. Ann. Intern. Med., 106:640-641. 89. Kuijper, E. J., and Peeters, M. F. (1991). Bacteriologicaland clinical aspects of Aeromonasassociated diarrhea in the Netherlands. Experientia, 47:432-434. 90. Namdari, H., and Bottone, E. J. (1990). Microbiologic and clinical evidence supporting the role of Aerornonns caviae as a pediatric enteric pathogen. J. Clin. Microbiol., 28:837-840. 91. Clark, R. B., Knoop, F. C., Padgitt, P. J., Hu, D. H., Wong, J. D., and Janda, J. M. (1989). Attachment of mesophilic aeromonads to cultured mammalian cells. Curr. Microbiol., 19: 97- 102. 92. Hokama, A., Honma, Y., and Nakasone, N. (1990). Pili of an Aerontonas hydrophila strain as a possible colonization factor. Microbiol. Intmz~nol.,34:901-915. 93. Brenden, R. A., and Huizinga, H. W. (1986). Susceptibilityof normal and X-irradiated animals to Aeromonas hydrophila infections. Curr. Microbiol., 13:129-132. 94. Janda, J. M., Clark, R. B., and Brenden, R. (1985). Virulence of Aeromonas species as assessed through mouse lethality studies. Curr. Microbiol., 12:163-168. 95. Watson, I. M., Robinson, J. O., Burke, V., and Gracey, M. (1985). Invasiveness ofrleromonas spp in relation to biotype virulence factors and clinical features. J. Clin. Microbiol., 22:4851. 96. Janda, J. M., Oshiro, L. S., Abbott, S. L., and Duffey, P. S. (1987). Virulence markers of mesophilic aeromonads: Association of the autoagglutination phenomenon with mouse pathogenicity and the presence of a peripheral cell-associated layer.Irlfect. Inmun., 55:30703077. 97. Kokka, R.P.. Vedros, N. A., and Janda, J.M. (1991). Characterization of classic and atypical serogroup 0 : l l Aerornonns: Evidence that the surface array protein is not directly involved in mouse pathogenicity. Microb. Patlzog., 10:71-79. 98. Pazzoglia, G., Sack, R. B., Bourgeois, A. L., Froehlich, J., and Eckstein, J. (1990). Diarrhea and intestinal invasiveness of Aerontonas strainsin the removable intestinal tie rabbit model. Ilfect. Immun., 58:1924-1931.

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99. Janda, J. M., Brenden, R., and Bottone, E. J. (1984). Differential susceptibility to human serum by Aeromonas spp. Curr. Microbiol.. 11:325-328. 100. Palumbo, S. A., Bencivengo, M. M., Del Corral, F., Williams, A. C., and Buchanan, R. L. (1989). Characterization of the Aeromonas hydrophila group isolated from retail foods of animal origin. J. Clin. Microbiol., 27:854-859. 101. Barghouthi, S., Young, R., Olson, M. 0. J., Arceneaux, J. E. L., Clem, L. W., and Byers, B. R. (1989). Amonobactin, anovel tryptophan- or phenylalamine-containingphenolate siderophore in Aeromonas hydrophila. J. Bacteriol., 171:1811-1816. 102. Massad, G., Arceneaux, J. E. L., and Byers, B. R. (1991). Acquisition of iron from host sources by mesophilic Aeromonas species. J. Gen. Microbiol., 137:237-241. and distribution of extracellu103. Leung, K.-Y.,and Stevenson, R. M. W. (1988). Characteristics lar proteases from Aeromonas hydrophila. J. Gen. Microbiol., 134:151- 160. 104. Namdari, H., and Bottone, E.J. (1988). Correlationof the suicide phenomenon in Aerolnonas species with virulence and enteropathogenicity. J. Clin. Microbiol., 262615-2619. 105. Palumbo, S . , Abeyta, C., Jr., and Stelma, G. N., Jr. (1992). Aeromonas hydrophila Group. In Comperlditun of Methods for the Microbiological Examinationof Foods, 3rd ed., APHA, Washington, D.C., pp. 497-515. 106. Palumbo, S. A. (1986). Is refrigeration enough to restrain foodborne pathogens? J. Food Prot., 49:1003-1009. 107. Eddy, B. P., and Kitchell, A. G. (1959). Cold-tolerant fermentative gram-negative organisms from meat and other sources. J. Appl. Bacteriol., 2257-63. and temperature characteristics 108. Rouf, M. A., andRigney, M.M. (1971). Growth temperatures of Aeronzonas. Appl. Microbiol., 22:503-506. 109. Palumbo, S . A. (1988). The growth of Aeromonns hydrophila K144 in ground pork at 5°C. Int. J. Food Microbiol., 7:41-48. 110. Palumbo, S. A., Williams, A. C., Buchanan, R. L., and Phillips, J. G. (1992). Model for the anaerobic growth of Aeromonas hydrophila K144. J. Food Prot. 55:260-265. 111. Gill, C. O., and Reichel, M. P. (1989). Growthof cold-tolerant pathogensYersinia enterocolitica, Aeromonns hydrophila and Listeria monocytogenes on high-pH beef packaged under vacuum or carbon dioxide. Food Microbiol., 6:223-230. 112. Lee, B. H., Simard, R. E., and Holley, R. A. (1985). Effectof temperature and storage duration on the microflora, physicochemical and sensory changesor vacuum- or nitrogen-packed pork. Meat Sci., 13:99-112. 113. Grau, F. H., Eustace, I. J.. and Bill, B. A. (1985). Microbial flora of lamb carcasses stored at 0°C in packs flushed with nitrogen or filled with carbon dioxide. J. Food Sci., 50:482485, 491. 114. Palumbo, S. A., and Williams, A. C. (1992). Growth of Aeronlonas hydrophila as affected by organic acids. J. Food Sci., 57:233-235. 115. Palumbo, S. A., Williams, A. C., Buchanan, R. L., and Phillips, J. G. (1991). Model for the aerobic growth of Aerontonas hydrophila K144. J. Food Prot., 54:429-435. 116. Gobat, P.-F., and Jemmi, T. (1993). Distribution of mesophilic Aeromonas species in raw and ready-to-eat fish and meat products in Switzerland. Znt. J. Food Microbiol., 20:117120. 117. Knochel, S., and Jeppesen, C. (1990). Distributionand characteristics of Aeromonas in food and drinking water in Denmark. Int. J. Food Microbiol., 10:317-322. 118. Lewus, C. B., Kaiser, A., and Montville, T. J. (1991). inhibitionof foodborne bacterial pathogens by bacteriocins fromlactic acid bacteria isolated from meat. Appl. Environ. Microbiol., 57:1683-1688. 119. Santos. J. A., Lopez-Diaz, T. M., Garcia-Fernandez,M. C., Garcia-Lopez, M.-L., and Otero, A. (1996). Effectof a lactic starter culture on the growth and protease activityof Aeromonas hydroihila. J. Appl. Bncteriol., 80:13-18. 120. Kalchayanand, N., Hanlin, M. B., and Ray, B. (1992). Sublethal injurymakes gram-negative

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from bottle waters and domestic water supplies in Saudi Arabia. J. Food Prof., 49:471476. Cattabiani, F., and Brindani, F. (1988). Valutazione del Danneggiamento di Tip0 Structurale da Disinfecttanti in Aeronzonas, Vibrio, e Plesiomonas. Arch. Vet. Ital., 39:245-253. Aytac, S. A., and Ozbas, Z. Y. (1994). Survey of the growth and survival of Yersinia enterocolitica and Aerontonas hydrophila in yogurt. Milchwissenschafi, 49:321-324. Santos, J. A., Gonzalez. C., Garcia-Lopez, M.-L., Garcia-Fernandez, M.-C., Otero, A. (1994). Antibacterial activity of the lactoperoxidase system against Aerorrronns hydrophila in broth, skim milk and ewes' milk. Lett. Appl. Micr-obiol., 19:161-164. Santos, J. A., Lopez-Diaz, T. M., Garcia-Fernandez, M. C., Garcia-Lopez, M. L., and Otero, A. (1995). Antibacterial effect of the lactoperoxidase system against Aeronzorzns hydrophila and psychrotrophs during the manufacturing of the Spanish sheep fresh cheese Villalon. Milchwissenschnj?, 50:690-692. Kaper, J. B., Lockman, H., and Colwell, R. R. (1981). Aer-ortzoitns hydrophila, ecology and toxigenicity of isolates from an estuary. J. Appl. Bacteriol.. 50:359-377. Joseph, S. W., Janda, M., and Carnahan, A. (1988). Isolation, enumeration and identification of Aeromonas sp. J. Food Safety, 9:23-25. McCoy, R. H., and Pilcher, K. S. (1974). Peptone beef extract glycogen agar, a selective and differential Aeronzonns medium. J. Fish. Res. Board Can.. 3 1:1153- 1155. Havelaar, A. H., During, M.. and Versteegh, J. F. M. (1987). Ampicillin-dextrin agar medium for the enumeration of Aero.omonns species in water by membrane filtration. J. Appl. Bncteriol.. 62:279-287. Von Graevenitz, A., and Bucher, C. (1983). Evaluation of differential and selective media for isolation of Aeronlonas and Plesiomonas sp. from human feces. J. Clin. Microbiol., 17: 16-21. Janda, J. M., Dixon, A., Raucher, B., Clark, R. B., and Bottone, E. J. (1984). Value of blood agar for primary isolation and clinical implication of simultaneous isolation of Aeromonas hydrophila and Aerornonas ctlvine from a patient with gastroenteritis. Clin. Microbiol., 20: 1221-1222. Schubert, R. H. W. (1977). Uber den Nachweis von Plesionlonns shigelloides Habs and Schubert, 1962, und ein Elektivmedium, den Inositol-Brillantgriin-Gallesalz-agar. Ernst-Rodenwaldt-Arch., 4:97- 103. Schubert, R. H. W. (1967). Das vorkommen der Aeromonaden in oberirdische Gewassern. Arch. Hyg., 1501688-708. Roland, F. P., Salt-starch-xylose-lysine deoxycholate agar. A single medium for the isolation of sodium and non-sodium dependent enteric gram negative bacilli. Med. Microbiol. Irnmunol., 1631241-249. Rogol, M., Sechter, I., Grinberg, L., and Gerichter, C. B. (1979). Pril-xylose-ampicillin agar, a new selective medium for the isolation of Aeronlonas hydrophila. J. Med. Microbiol., 12: 229-23 1. Shread, P., Donovan, F., and Lee, J. (1981). A survey of the incidence of Aeromonas in human feces. Soc. Gen. Microbiol. Quart., 3:184. Altorfer, R., Alwegg, M., Zollinger-Iten, J.. and von Graevenitz, A. (1985). Growth of Aeromonas spp. on cefsulodin-irgasan-novobiocin agar selective for Yersinia enterocolitica. J. Clirt. Microbiol., 22:478-480. Hoban, D., Forsyth, W., Gratton, G., and William, T. (1981). Detection of Aeromonas hq" drophila from diarrhea stools using Macconkey Tween 80 agar. Abst. Ann. Mtg. Ant. Soc. Microbiol. C4. Hoban, D. (1983). Survey of diarrheal illness associated with Aeronzonas hyd?-oplzilain Manitoba. Abst. Ann. Mtg. Am. Soc. Microbiol. C151. Shotts. E. B., Jr., and Rinder, R. (1973). Medium for the isolation of Aeronzonas hydrophila. Appl. Microbiol., 26:550-553.

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4 Update: Food Poisoning and Other Diseases induced by Bacillus cereus Kathleen T. Rajkowski and James L. Smith U.S. Department of Agriculture, Wyndnoor, Pennsylvania *

I. Introduction 61 11. Foodborne Bucillus cereus Outbreaks62

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IV. V. VI. VII. VIII.

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A. Guidelinesforidentifying B. cereus outbreaks63 B. B.cereus foodborneincidentsreported in 1992-199763 Characteristics of the Food Poisoning Toxins Produced by Bacillus cereus A. Emetic toxin 65 B. Diarrheic toxin(s) 66 Nongastrointestinal Disease Induced by Bacillus cereus Infection 67 Occurrence inFood 67 Growth and Survival 70 Inhibition and Inactivation7 1 Isolation, Identifications and Characterization 7 1 FutureResearchNeeds72 References 73

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INTRODUCTION

The endospores of the genus Bacillus are ubiquitous in the environment and are capable of surviving adverse conditions. To date, Norris et al. (1) provide the most comprehensive list of conditions and environments from which Bucillus spores can be isolated. Since the spores are air-, soil-, and/or waterborne, it is only natural that various Bacillus spp. are isolated from foods and areas where food is produced. Since its first isolation in 1948

* Mention of brand or firm name does not constitute an endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned. 61

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associated with a foodborne disease outbreak, Bacillus cereus is considered the most common pathogen in the genus. Toxins produced by the vegetative cells lead to gastrointestinal upset and/or vomiting. In the United States, B. cereus foodborne diseases are a relatively minor concern: however, they are still a major concern worldwide. Recent reviews on various aspects of B. cereus and its disease potential have been published by Drobniewski (2), Fermanian et al. (3), Granum ( 4 3 , Granum and Lund (6), and Schultz and Smith (7). This chapter will summarize the reports of the more recent B. cerem foodborne outbreaks, toxin characterization, nongastrointestinal diseases caused by B. cereus infection, and the psychrotropic characteristics of B. cereus strains.

II. FOODBORNEBACILLUSCEREUS OUTBREAKS Data from the surveillance for foodborne disease outbreaks in the United States indicate that B. cereus is not a major concern. For the 10-year period 1983-1992, there were a total of 1396 bacterial foodborne outbreaks, with only 37 of those outbreaks due to B. cereus. Also, during that time period, there were a total of 83,5 10 cases of bacterial foodborne diseases, with 694 cases occurring in the B. cer-eus outbreaks. Thus, B. cereus was the cause of 2.7% of the bacterial foodborne outbreaks and 0.83% of the outbreaks cases. Death does not appear to be a consequence of foodborne B. cereus outbreaks (8,9). While foodborne disease due to B. cereus is not commonly reported, it is probable that the disease is underreported because most cases are sporadic. The ingestion of contaminated Chinese food and fried rice was responsible for almost half (45.9%) of the B. cereus foodborne outbreaks in the United States. Most outbreaks (67.6%) occulred during the months of May to October (8,9). During the 10-year reporting period, 43.2% of the B. cereus outbreaks occurred in commercial food establishments (cafeterias, delicatessens, restaurants) and 21.6% occurred in homes. In 28 of the 37 outbreaks, the factors (or errors) contributing to B. cereus outbreaks were reported. In 27 of 28 outbreaks (96.4%), improper holding temperature of the food was a contributing factor. The use of contaminated equipment, inadequate cooking, poor personal hygiene, and obtaining food from an unsafe source were contributing errors in 17.9, 17.9, 14.3, and 3.6%, respectively, of foodborne B. cereus outbreaks (8,9). Wilson et al. (10) indicated that Bacillus food poisoning has increased in Northern Ireland. During the 1 9 8 0 ~only ~ one Bncillus-induced foodborne incident was reported; however, nine incidents occurred in 1991 and four in 1992. B. cereus was isolated in most incidents. Bacillus subtilis was isolated in four incidents, however, B. cereus also was isolated from the food in two of those incidents. The foods involved were generally rice and/or chicken and in 10 of 14 incidents, the food was eaten in or taken out from Chinese restaurants. Clinically, the symptoms in almost all of the cases involved in the incidents were predominately vomiting and nausea with an incubation period of 1-6 hours. Thus, it appears that the emetic toxin was responsible for the majority of incidents occurring in Northern Ireland (10). Most of the incidents occurred during the months of April to August. It is probable that inadequate temperature control of cooked rice was the food-processing error that contributed to most of the Bncillrrs-induced food poisoning in Northern Ireland. The mean number of foodborne outbreaks in Taiwan for the period 1987-1993 was 81 (range: 57-93); however, in 1994 the number of outbreaks reported totaled 102 (1 1). A bacterial agent was identified in 74 of the 1994 outbreaks. Eleven of the outbreaks

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were due to B. cereus (14.9%) exceeded only by Vibrio parahaemolyticus (56.7%) and Staphylococcus aureus (20.3%). Snln?onella spp. accounted for only 8.1% of the foodborne outbreaks of bacterial origin (l 1). Food poisoning incidents involving B. cereus are common in the Netherlands. For the four year period 1991-1994, there were 124 incidents with 789 cases of foodborne disease with a bacterial etiology. B. cereus was responsible for 40 of 124 (32.3%) incidents with 172 of 789 (21.7%) cases. Chinese-Indonesian food accounted for 17 of 40 (42%) of the B. cereus-induced food poisoning incidents (12). Salmonella spp. caused fewer incidents (31/124) than B. cereus; however, there were more cases (290/789) with Salmonelln.

A.

Guidelinesfor Identifying B. cereus Foodborne Outbreaks

Two or more persons must become ill for an incident to be called a foodborne disease outbreak (9). In order to confirm that an outbreak is due to B. cereus, organisms must be isolated from the stool of two or more ill persons and not from the stool of controls; alternatively, an outbreak can be confirmed by isolation of 2 10’ B. cereleMs organisms from epidemiologically implicated food (provided that the food specimen has been properly handled). The incubation period for illness induced by emetic toxin is 1-6 hours. Vomiting is the major symptom seen in patients; some patients have diarrhea, but fever is uncommon. The incubation period for persons ingesting diarrheal toxin is 6-24 hours, with patients having diarrhea and abdominal cramps. A few patients show vomiting; fever is uncommon (9).

B. B. cereus Foodborne Incidents Reported in 1992-1 997 1. Two individuals developed gastrointestinal symptoms after eating spaghetti with homemade pesto. Both emetic and diarrheic toxin were involved in this incident. A large batch of the food had been prepared 4 days before the incident and although stored refrigerated, the food had been permitted to remain at room temperature for lengthy periods during the 4-day period. The preparation of a large batch of food which was intermittently subjected to temperature abuse over a 4-day period contributed to the outbreak (13). The outbreak was interesting because one patient died of fulminant liver failure induced by emetic toxin. 2. B. cereus was present in vegetable pakora (4.4 X lo5 CFU/g) and fried rice (2.0 X lo7 CFU/g) in two foodborne incidents involving five people who had eaten in an Indian restaurant in Scotland. All isolates belonged to the same enterotoxigenic serotype. There were a number of processing errors: rice was prepared too far in advance and in greater quantities than needed for a given safe time period. The cooked rice was not properly cooled and was at improper holding temperatures for long periods. The presence of B. cereus in foods other than rice indicated cross-contamination, and a high coliform count in foods indicated handling by person(s) with poor personal hygiene (14).

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3. At a college sport dayin Thailand, 470 individuals were ill with vomiting, nausea, and abdominal pain; approximately half ofthe individuals reported diarrhea. The ingestion of cream-filled eclairs was significantly associated with illness. The mean incubation period was 3.2 hours, which suggested a preformed toxin in thefood. Initial laboratory investigation indicated presence of B. cereus in the eclairs. A few days later, a different laboratory reported the presence of enterotoxin-producing S. aureus. A dual etiology for the outbreak may have been possible; however, before the eclairs had been sent to second laboratory, they had been at room temperature for 12 hours. In addition, the phage types of S. nureus isolated from the cooks were not similar to the phage type isolated from eclairs. Therefore, it is probable that the outbreak was due to B. cereus emetic toxin. The failure to refrigerate the eclairs after preparation and during serving led to the outbreak (15). 4. A stew ingested by 142 competitors at a Norwegian Ski Championship was the source of B. cereus-induced diarrhea. B. cereus was present in the stew at 2 X los CFU/g. Theincubation time was 12-36 hours, and most patients recovered within 24 hours. The kitchen in which the food was prepared was contaminated profusely with B. cereus. The problem was eliminated by thorough cleansing and decontamination of the kitchen with hypochlorite (16). 5. An outbreak of diarrheic foodborne disease involved 139 individuals attending a university field day barbecue in South Carolina. B. cereus was isolated from barbecued pork at levels > lo5 CFU/g. At least half of the pork was cooked 2 days in advance of the barbecue and held unrefrigerated for 18 hours before boning. The boned barbecued pork was then stacked 20 cm deep in metal trays and refrigerated. On the day of the barbecue, the pork was heated; however, the heating unit was not working properly. A hot temperature was not maintained during transportation to the site and while serving the pork to attendees. Preparation of the pork barbecue too far in advance and failure to maintain safe storage and serving temperatures were the food-processing errors that led to the outbreak (17). 6. Emetic toxin from B. cereus was responsible for food poisoning in 14 individuals (12 children and 2 adults) from two child care centers located in Virginia. The ill individuals had eaten chicken fried rice at a catered luncheon prepared by a local restaurant. Leftover chicken fried rice contained B. cereus at > lo6 cfu/g. Improper cooling of the cooked rice and improper holding temperatures contributed to the outbreak (18). 7. A catered wedding reception in California was the site of an outbreak of B. cereus food poisoning due to diarrheic toxin. The outbreak involved 55 individuals, with Cornish hen as the suspect food. The birds contained B. cereus at a level of 1.4 X lo6 CFU/g. There were several errors committed by the caterer which played a role in the outbreak. He was licensed to serve only 29 restaurant customers, yet on this occasion he was serving over 300 customers at two different locations on the same day. It was probable that the Cornish hens were not properly thawed, resulting in undercooked birds. In addition, the cooked Cornish hens were transported and held in an unrefrigerated van for several hours. That the caterer lacked facilities for large-scale food preparation was the chief reason for this outbreak (19).

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Symptoms found in the above outbreaks included diarrhea without vomiting or vomiting that was sometimes followed by diarrhea. The diarrheal toxin does not appear to induce vomiting, but on occasion the emetic toxin appears to 'give both vomiting and diarrhea. It would appear that under some conditions, emetic toxin-producing strains may also produce a diarrheal-type toxin. In the outbreaks listed above, the error in food preparation appeared to be improper holding of the food at temperatures high enough to allow growth of B. cereus. Prompt cooling of the food with refrigerated storage would have prevented the outbreaks. An interesting outbreak of B. cereus intoxication occurred in Germany, which involved Rottweiler puppies; B. cereus-contaminated milk powder was the food agent. Puppies fed formula containing milk powder (in addition to receiving mother's milk) became ill with vomiting and diarrhea. Five puppies, whose mother was not producing milk, were fed exclusively on formula containing the contaminated milk powder and died. B. cereus was present at a level of 2.7-3.5 > lo4 CFU/g in the milk powder. The presence of the organism in the powdered milk and symptoms suffered by the puppies indicated that this outbreak was due to intoxication by B. cereus (20).

111.

CHARACTERISTICSOFTHE FOOD POISONING TOXINS PRODUCED BY Bacillus cereus

A. Emetic Toxin Emetic toxin is preformed in foods, i.e., B. cereus at levels of 105-108 CFU/g of food produce toxin, which causes vomiting when the food is eaten. Symptoms-vomiting, nausea and malaise-appear within 0.5 to 5 hours. Duration of illness is generally 6-24 hours (5,6). Foods implicated in B. cereus emetic food poisoning include fried and cooked rice, pasta, noodles, and pastry. Agata et al. (21) found a strong association of emetic toxin production with the H1 serovar phenotype of B. cereus. Isolates of B. cereus that produce enterotoxin activity or hydrolyzed starch did not produce emetic toxin. The structure of the emetic toxin has been elucidated, and the compound has been named cereulide. Cereulide is a 36-membered cyclic dodecadepsipeptide with the sequence of cycb(D-O-Leu-D-Ala-L-o-val-L-Val)3with the formula, C57H9601SN6 and a molecular weight of l 19l .6. Cereulide is a potassium ionophore closely related to valinomycin (22-24). Cereulide induces vomiting in Suncus murinus (house musk shrew) and rhesus monkeys (25,26). The emetic toxin is not antigenic, does not act in the rabbit or mouse ileal loop test, produces vacuolation in HEp-2 cells but is not cytotoxic, is stable for 90 minute to 121"C, is stable to pH 2.0 and pH 11, is not hemolytic, and does not induce vascular permeability in the skin of the rabbit (5,26). Cereulide induces vomiting when it stimulates the vagus nerve by binding to the 5-HT3receptor. Using the S. murinus emetic model, Agata et al. (25) found that vagotomy eliminated the emetic action of cereulide. The 5-HT3receptor antagonist, ondansetron, also abolished cereulide-induced vomiting in Suncus. In addition, cereulide is a mitochondrial poison. Using rat liver mitochondria, Mahler et al. (13) showed that emetic toxin inhibited electron transport and uncoupled oxidative phosphorylation.

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Nothing is known about the synthesis of cereulide. It is probable that the cyclic peptide is enzymatically synthesized from its component amino acids and is not a product of a gene.

B. Diarrheic Toxin(s) The toxin(s) responsible for the diarrheal syndrome are formed in the small intestine of the host. Unlike the emetic toxin, which is preformed in food, any preformed diarrheic toxin, due to its loss of activity at pH 3 and its sensitivity to trypsin, would be destroyed as it passes through the gastrointestinal system (27). The infectious dose ranges from lo5 to lo7 organisms per host. Symptoms include abdominal pain, watery diarrhea, and occasionally nausea, which generally appear within 8-16 hours after ingestion of contaminated food. Duration of illness is generally 12-24 hours (6). Foods that are frequently implicated in B. cereus diarrheic food poisoning include meat products, soups, vegetables, puddings, sauces, milk and milk products. Enterotoxin activity is decreased or lost if thetoxin is subjected to a pH of 3, trypsin, chymotrypsin, pepsin, or heating at 80°C for 10 minute (4,27). Baker and Griffiths (27) demonstrated that the immunological activity of the enterotoxin was approximately four times more stable when heated in milk as compared to broth. However, since diarrheic activity was not tested, it is not clear that the toxin still retained the ability to cause disease. Granum and Lund (28) studied 85 strains of B. cerem isolated from dairy products and found that 50 (58.8%) were enterotoxigenic, 13 (15.3%) were psychrophilic, and 6 (7. l %) produced toxin at 6°C. The ability to grow and produce enterotoxin at refrigerated temperatures indicates that refrigeration may not be enough to protect consumers from B. cereus food poisoning. Hemolysin BL (HBL), a tripartite entity consisting of three protein components, possesses hemolytic, cytotoxic, dermonecrotic, and vascular permeability activity (29,30). In addition, HBL induces fluid accumulation in rabbit ileal loops, thereby indicating that it is an enterotoxin. HBL consists of three protein components: L? (46 kDa), L, (38 kDa), and B (37 kDa), and maximal expression of all HBL activities requires all three protein entities (30). Heinrichs et al. (31) and Suwan et al. (32) have cloned the genes for HBL (hbZC, hblD, IzblA, hZaB), determined the nucleotide sequence of the genes, and deduced the amino acid sequences of the three protein components. Lund and Granum (33,34) characterized a nonhemolytic enterotoxin complex from strains of B. cereus responsible for outbreaks of diarrheal food poisoning. The nonhemolytic enterotoxin (NHE) consisted of three protein moieties: B (105 m a ) , L2 (54 kDa), and L, (39 m a ) , and all components were needed for maximum cytotoxicity. Apparently, NHE was not tested in the ileal loop assay. The sequencing of NHE is underway (34). Diarrhea-inducing strains of B. cereus may produce either HBL or NHE or both (34). It would appear that there are at least two tripartite enterotoxins, HBL and NHE, in B. cereus. The role of “enterotoxin T” as an agent of diarrhea in B. cereus foodborne illness is not clear. Enterotoxin T consists of a single protein that is cytotoxic, positive in the mouse ileal loop assay and possesses vascular permeability activity (35). Enterotoxin T is a product of the gene bceT, which has been cloned (35). Granum and Lund (34) state that there is no real evidence that enterotoxin T causes food poisoning. Two commercial kits are available for the detection of B. cereus enterotoxins: the Oxoid BCET-RPLA and Tecra ELISA kits. The Oxoid kit measures L? of HBL, whereas t

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Bacillus cereus

67

the other kit measures L: of NHE: thus, the kits do not assay the same enterotoxin (34). But it would appear that both kits are necessary to detect enterotoxin production in food poisoning due to B. cereus. Beecher and Wong (36) developed a simple and inexpensive method for detecting HBL-producing strains of B. cereus. Their assay is based on a distinctive discontinuous hemolytic pattern produced by HBL-producing strains on blood agar plates.

IV. NONGASTROINTESTINALDISEASEINDUCED Bacillus cereus INFECTION

BY

In addition to causing classical food poisoning symptoms affecting the gastrointestinal system, B. cereus may cause disease in other areas of the body. Nongastrointestinal diseases that may result from B. cereus infections include (a) local infections such as ocular and wound infections, (b) bacteremia and septicemia, (c) central nervous system infections, (d) respiratory tract infections, and (e) endocarditis (2). Details from a number of case reports (1995-1997) of B. cereus-induced nongastrointestinal diseases are given in Table 1. Drobniewski (2) has a table listing cases of B. cereus-induced ocular infections that occurred during the period 1951-1990; another table lists other nongastrointestinal B. cereus infections that occurred during 1965-1992 (2). A table of clinical features that appeared in cases of B. cereus infections occurring in patients with leukemia during 19711995 is presented by Akiyama et al. (41). The cases of nongastrointestinal B. cereus infections presented in Table 1 and in the tables of Drobniecwski (2) and Akiyama et al. (41) indicate that individuals immunocompromised either by illness or medication are at particular risk for disease induced by this organism. Using both in vitro and in vivo methods, Beecher et al. (45) demonstrated that the B. cereus HBL enterotoxin had retinal toxicity and may be involved in endophthalmitis (inflammation of the inner eye). B. cereus is one of the major infective agents responsible for blindness in humans (45). Antibody neutralization of HBL factor in cell-free crude concentrated and dialyzed culture filtrates eliminated approximately 50% of the in vitro retinal toxicity. Purified HBL wasless toxic than culture filtrates containing the same level of HBL. These results indicate that B. cereus ocular virulence may be multifactorial and requires other toxic moieties in addition to HBL (45). Fulminant liver failure ending in death was apparently induced by the B. cereus emetic toxin cereulide (13). Purified emetic toxin from the disease causing strain inhibited electron transport and uncoupled oxidative phosphorylation in rat liver mitochondria (13). Many liver failure syndromes have been linked to mitochondrial injury and disruption of mitochondrial activity (40). Thus, cereulide acting as a mitochondrial poison led to liver failure in the patient described by Mahler et al. (13). The apparent correlation of nongastrointestinal disease syndromes with B. cereus diarrheic and emetic toxins warrants further study.

V.

OCCURRENCE IN FOOD

Some food sources from which B. cereus has been isolated are summarized in Table 2. In a more recent report, Hatakka (58) examined the microbiological quality of airline hot

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Table 2 Survey of Food Products from Which Bacillus cereus was Isolated and % Incidence Food product

5% Incidence

Rice and rice products Boiled rice Spices Milk products Chicken and meat products Buffalo meat Ready-to-serve moist food Powdered infant formula Nonfat dry milk Skim milk powder Buesco Blanco cheese soft hard Various food samples from a Spanish retail market Seed samples (alfalfa, mung bean, wheat or seed mixtures) Poultry products Pork carcasses Beef carcasses Chicken carcasses Raw beef Pork Chicken Ham Sausage Uncooked hamburger Cooked hamburger Meat product additives Tempeh samples

28.5 100 30 100 80 35 83 75 100 10.3 69 63 14.7 cells 82.4 spores 67.3

6.9 7 11 0 7.9 4.4 7.2 3.8 14.9 45.5 23.7 39.1 16

Country

Ref.

India

47

India United States

48 49

Japan

50

Venezuela

51

Spain

52

United States

53

England Sweden

54 55

Japan

56

Netherlands

57

meals over a 3-year period and found that 3% of the meals contained pathogenic bacteria, of which B. cereus was the most common pathogen. Using the fatty acid profiles for the identification of 229 B. cereus from environmental sources (dairy farm to processing plant), Lin et al. (59) determined that B. cereus spores in raw milk were the major source of B. cereus in pasteurized milk based on the comparison of the profiles from environmental and pasteurized milk isolates. The report of teGiffel and Beumer (60) confirmed that the presence of psychrotrophic B. cereus spores in raw milk resulted in contaminated pasteurized milk products. The vegetative cells in raw milk are rapidly killed during pasteurization at 65OC, whereas the endospores of B. cereus are heat-resistant (61). Properly pasteurized dairy products, when stored at recommended refrigerated conditions (maximum 7°C) and used within the expiration date usually cause no problems for healthy adults. More information can be found in the bulletins of the International Dairy Federation devoted to B. cereus in milk and milk products (62,63).

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VI.GROWTH

AND SURVIVAL

The Agricultural Research Service of the U.S. Department of Agriculture has developed a growth model for B. cereus, which can be found on the Internet at http:// www.arserrc.gov/mfs. The Pathogen Modeling Program Version 5.1 forWindows can be entered through the home page. The growth characteristics of B. cereus were reviewed by Schultz and Smith (7). The incidence of B. cereus in raw and processed foods, especially when held at low temperatures, has received considerable attention. The research data indicate that few foods are free of the organism and that growth and toxin fortnation can occur at psychrotropic temperatures (28). Bergann (64), reporting on the growth temperature requirements of 50 B. cereus strains, showed that more than half of the strains grew at 10°C, six at 8"C, and one at 6°C. Garcia-Armesto and Sutherland (65), after characterizing both psychrotropic and nlesophilic Bacillus species isolated from milk, found one B. cereus isolate that was clearly psychrotropic (grew at 6.5"C but not at 40°C in 2 days). The cold adaptation response was demonstrated by Foegeding and Berry (66) after determining the growth characteristics of 27 B. cereus clinical and food isolates. This cold adaptation was also reported by Christiansson et al. (67). They found that some strains of B. cereus were acclimated to grow at 8"C, and when the relationship between cytotoxicity and growth of these isolates was compared, most became cytotoxic at approximately lo* c f d n i l . The dairy isolates that produced cytotoxicity in milk at 8°C under aerated conditions also gave positive vascular permeability reaction with rabbits, which is indicative of B. cereus diarrheal toxin (67). In the study by Granum et al. (28), they found of the 85 B. cereus strains isolated from dairy products, 6 were both psychrotropic and enterotoxigenic and exhibited good growth at 6°C. While B. cereus is commonly found in milk and other dairy products and milk is a suitable substrate for toxin production under aerated conditions even at low temperatures, the incidence of B. cereus food poisoning in milk is low probably due to insufficient aeration during normal storage (67). Van Netten et al. (68) found that B. cereus isolates involved in two foodborne outbreaks of diarrheal illness were able to grow and produce toxin at 4-7°C. Strains of B. cereus of the emetic type, which were isolated during an outbreak involving pasteurized milk, were able to grow at 4-7"C, but enterotoxin production was not detected using the Oxoid test kit for diarrheal toxin. It is quite probable that the emetic toxin can beproduced at low incubation temperatures, although they did not test for it (68). Fermanian et al. (69) reported that a foodborne and clinical isolate grew at 10°C and that both isolates were toxin positive using the Oxoid kit. The presence of psychrotropic B. cereus strains in dried infant foods is a concern. Rowan and Anderson (70) reported that of the B. cereus isolated from reconstituted milkbased infant fornlulas (MIF), 1,4, and 16 strains grew at 4, 6, and 8"C, respectively, after 15 days. They were tested for diarrhea enterotoxin production, and 9 of the 21 isolates produced enterotoxin. The authors recommended brief refrigeration (4°C) storage of the reconstituted MIF forthis product to remain safe. They also tested the effect of maltodextrin on the growth and synthesis of the diarrheal enterotoxin in MIF and found that supplementing with 20.1% maltodextrin supported both growth and diarrheal toxin production when incubated for 14 hours or more at 25°C (71). Jaquette and Beuchat (72) reported that psychrotrophic B. cereus can survive and grow in reconstituted infant rice cereal. They recommended immediate consumption or holding the food for <48 hours at 58°C (72).

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VII.INHIBITIONANDINACTIVATION Schultz and Smith (7) reviewed the inactivation and growth inhibition of B. cereus. The inhibition by the addition of 50 IU of nisin to skim milk resulted in a decrease in numbers for 9 of the 12 psychrotropic B. cereus strains tested by Dufrenne et al. (73). They also reported that 250 IU decreased the bacterial numbers of all strains tested. Beuchat et al. (74) studied the effect of nisin and temperature on the growth of and enterotoxin production by psychrotropic B. cereus in beef gravy. They reported that as little as 1 pg of nisin/ mL may be effective in inhibiting or retarding growth of B. cereus and production of the diarrheal enterotoxin by both the vegetative cells and spores in beef gravy at 8°C. Jaquette and Beuchat (75) showed a correlation between pH, nisin, and temperature on growth and survival of B. cereus. Using a concentration of as low as 1 pg of nisin/tnL the effect was more pronounced in inhibiting the growth of B. cereus as the temperature decreased from 15 to 8 or 5°C and at more acidic conditions (pH 5.53). Not only does the pH affect the growth of B. cereus, but there are reports that as the pH of sporulation media decreases there is a drop in sporulation accompanied by a decreased D-value of the produced spores (76). In addition as the media pH decreases from 7.0 to 4.0, a decrease in D-value was reported by Mazas et al. (77). Mahakarnchanakul and Beuchat (78) studied the effect of growth temperature and NaCl concentration on the thermotolerance of a psychrotropic and mesophilic B. cereus. After a downshift in growth temperature from 30 to 10°C followed by incubation for 3 to 9 days, an increase in thermotolerance was observed for the psychrotropic strain when heated in BHI broth containing 2 or 4% NaCl compared to the thermotolerance in the broth containing 0.5% NaC1. They attribute the thermotolerance to the production of proteins with molecular weights of 63, 40 and 29 kDa for the psychrotropic strain. Gmnum (4) also suggested that changes in tetnperature may influence the ability of B. cereus to grow in minimally processed foods during distribution and storage.

VIII.ISOLATION,IDENTIFICATION, AND CHARACTERIZATION In their 1992 review, van Netten and Kramer (79) discussed the use of media for the isolation and enumeration of B. cereus in foods, and an earlier review (80) discussed the use of various media for isolation of B. cereus occurring in foods and clinical specimens. The selective mannitol-egg yolk-polymyxin (MYP) agar is recommended in the methods for isolation of B. cereus (81,82) ; whereas, either MYP or KG agars are, suggested for use by Harmon et al. (83). Regardless of which selective agar is used for isolation, confirmational identification still relies on the biochemical profiles (81,82,83). A rapid Bacillus identification system is available utilizing the API system (Analytab Products, Plainview, New York) which can be used to confirm isolates of B. cereus from selective agars (84). Since the review by Schultz and Smith (7) there have been nofurther updated reports on the use of the 23 flagella (H) antigens for serological typing of the B. cereus strains involved in food poisoning, and this typing method is still an effective way to distinguish between strains (85). The production, assay, and/or pathological effects of the various toxins produced by B. cereus have been reviewed by Granum (4), Granum et al. (28), Granum and Lund (6), Schultz and Smith (7), Gilbert and Kramer (86), Turnbull (87j, Kramer and Gilbert

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(80), Shinagawa (85), and Jackson (88). Anderson et al. (89) developed a rapid bioassay for detection of B. cereus emetic toxin based on the loss of motility of boar spermatozoa upon 24 hours of exposure to extracts of emetic B. cereus strains or contaminated food. The detection limit for food was 3 g of rice containing 106-107cfu of emetic B. cereus per gram. Currently there is no commercial available kit for the detection of the emetic toxin (cereulide) (5). A reversed passive latex agglutination test (Oxoid BCET-RPLA) (6) and an enzyme-linked immunosorbent assay (ELISA) (TECRATM BDE vusual immunoassay) (90) are available to test for the diarrheal toxin. The differentiation and genetic studies used to identify B. cereus from other Bacillus species was reviewed by Schultz and Smith (7). A phylogenetic tree of the group 1 Bacillus was constructed by Ivanov (91) using the G + C content in DNA. They showed a statistically reliable correlation between the evolutionary distances of 16s rRNA sequences (Ei) and parameter Pi having a coefficient of correlation between Ei and Pi equaling 0.97 for representatives of group 1, which include B. nrzthracis, B. cereus, B. circulnns, B.jinnus, B. Zautus, and B. subtilis. Based on the 16s rDNA and the 23s rDNA, Lechner et al. (92) propose that the psychrotolerant species of the B. cereus group be considered a new species that can grow at 4-7°C but not at 43°C be called Bacillus weiherzstephnnensis sp. nov. Nilsson et al. (93) developed a RAPD-PCR method for large-scale typing of B. cereus. The procedure was highly discriminatory for B. cereus strains and was found to give reproducible classification of RAPD fingerprints for five reference strains. Francis et al. (94) reported on the development of a single PCR assay using a major cold shock protein to discriminate between psychrotropic and mesophilic B. cereus strains. Koo et al. (95) obtained a recombinant antibody by reverse transcription-PCR that had B. cereus spore-binding ability. A rapid PCR-based DNA test for the enterotoxic B. cereus was reported by Mantynen and Lindstrom (96). Lin et al. (97) differentiated the B. cereus group (cereus, mycoides, and tlzuringiensis) using Fourier transform infrared spectroscopy. The absorbance peaks between 1800 and 1500 cm” for B. cereus were different from the other Bacillus studied. Carlson et al. (98) also concluded that B. cereus and B. thuringiensis should be considered one species after analyzing the pulsed-field gel electrophoresis and the multilocus enzyme electrophoresis profiles. The profiles were indistinguishable. Helgason et al. (99) concluded after analyzing the multilocus enzyme electrophoresis profiles of 154 B. cereus/ B. thuringiemis isolates from soil samples that a high degree of recombination occurred. Reports have implicated B. thuringiensis in gastrointestinal disease (2). Both B. cereus and B. thuringiensis were isolated from a gastroenteritis outbreak (100). All B. cereus and B. thurir~giensisisolates showed cytotoxic effects characteristic of enterotoxin-producing B. cereus.

IX. FUTURERESEARCHNEEDS More research is needed in the following areas: 1. There is a need to determine if the tripartite enterotoxins, HBL and NHE, are the only diarrheal toxins produced by food poisoning strains of B. cereus or if there are other toxins such as “enterotoxin T,” which is a monoprotein moiety. 2. Studies are needed to determine the intestinal receptor site(s) of the tripartite enterotoxins and how these toxins induce diarrhea.

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3. How B. cereus synthesizes cereulide (emetic toxin) is unknown; it is probably produced enzymatically, but there is no information available at the present time. 4. Two studies (13,44) indicate that HBL and cereulide play a role in the induction of B. cereus nongastrointestinal diseases. More studies are needed to confirm these observations and the potential role of the toxins in pathogenesis of the nongastrointestinal diseases. 5. Ribsomal operon ribotyping is of interest and results of study in this area may prove to be a useful tool in differentiation of the various bacilli.

REFERENCES 1. Norris, J. R., Berleley, R. C. W., Logan, N. A., and O’Donnell, A. G. (1981). The genera Bacillus and Sporolactobacillus. In The ProkaQlotes, Vol. 2 (M. P. Starro, H. Stolp, H. G. Truper, A. Balows, and H. G. Schlegel, eds.), Springer-Verlag,New York, pp. 1711-1742. 2. Drobniewski, F. A. (1993). Clin. Microbiol. Rev., 6:324-338. 3. Fermanian, C., Fremy, J.-M., and Lahellec, C. (1993). J. Rap. Meth. Autont. Microbiol., 2: 83-134. 4. Granum, P. E. (1994). J. Appl. Bacteriol., 76:61S-66S. 5. Granum, P. E. (1997). Bacillus cereus. In Food Microbiology: Fundanzentals and Frontiers (M. P. Doyle, L. R. Beuchat and T. J. Montville, eds.), ASM Press.Washington, DC, pp. 327-336. 6. Granum, P. E., and Lund, T. (1997). FEMS Microbiol. Lett., 157:223-228. 7. Schultz, F. J., and Smith, J. L. (1994). Bacillus: Recent advances in Bacillus cereus food poisoning research. InFoodborne Disease Handbook: Diseases Caused by Bacteria, Vol. 1 (Y. H. Hui, J. R. Gorlkam, K. D. Murrell, and D. 0 Cliver, eds.), Marcel Dekker, Inc., New York, pp. 29-62. 8. Bean, N. H., Griffin, P. M., Goulding, J. S., and Ivey, C. B. (1990). MMWR, 39, #SS-l. 9. Bean, N. H., Goulding, J. S., Lao, C., and Angujio, F. J. (1996). MMWR, 45, #SS-6. 10. Wilson, I. G., Wilson, T. S., and Kramer, J. M. (1993). Lancet, 342:928. 11. Pan, T. M., Chiou, C. S., Hus, S . Y., Huang, H. C., Wang, T. K., Chiu, S. I., Yea, H. L., and Lee, C. L. (1994). J. Fornzos. Med. Assoc., 95:417-420. 12. Simone, E., Goosen, M., Noterman, S. H. W., and Bergdorff, M. W. (1997). J. Food Prot., 60~442-446. 13. Mahler, H., Pasi, A., Kramer, J. M., Schulte, P., Scoging, A. C., BCir, W., and Krihenbuhl, S. (1997). N. Eng. J. Med., 336: 1142-1 148. 14. Dickson, A., and Morgan, M. (1995).Scottish Centre Infect. Environ. Health Weekly Report 29(5):7. 15. Thaikruea, L., Pataraarechachai, J., Savanpunyalert, P., and Maluponjiragul, U.(1995). Sotheast, Asian J. Trop. nted. Public Health, 26:78-85. 16. Granum, P. E., Naestvold A., and Gundersby, K. N. (1997). FEMS Microbiol. Lett., 157: 223-228. 17. Luby, S., Jones, J., Dowda, H., Kramer J., and Horan, J. (1993). J. Infect. Dis., 167:14521455. 18. Centers for Disease Control and Prevention. (1993). MMWR, 43:177-178. 19. Slaten, D. D., Oropeza, R. I., and Werner, S. B. (1992). Pub. Health Repts., 107:477-480. 20. Gareis, M., and Walz, A. (1994). Tiertirztl. Untschau., 49:319-322. 21. Agata, N., Ohta M., and Mori, M. (1996). Curr. Microbiol., 33:67-69. 22. Agata, N., Mori, M.. Hota, M., Swan, S., Ohtani I., and Isobe, M. (1994). FEMS Microbiol. Lett., 121:31-34.

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23. Isobe, M., Ishikawa, T., Suwan, S., Agata N., and Ohta, M. (1995). Bioorg. Med. Clzem. Lett., 5:2855-2558. 24. Ryan, P. A., Macmillan J. D., and Zilinskas, B. A. (1997). J. Bncteriol., 179:2251-2556. 25. Agata, N., Ohta, M., Mori, M., and Isobe, M. (1995). FEMS Microbiol. Lett., 129:17-20. 26. Sinagawa, K., Konama, H., Sekita, H., and Sugii, S. (1995). FEMS Microbiol. Lett.. 130: 87-90. 27. Baker, J. M., and Griffiths, M. W. (1995). J. Food Prof., 58:443-445. 28. Granum, P. E., and Lund, T. (1997). FEMS Microbiol. Lett., 157:223-228. 29. Beecher, D. J., and Wong, A. C. L. (1994). Appl. Environ. Microbiol., 60: 1646-1651. 30. Beecher, D. J.. Schoeni, J. L., and Wong, A. C. L. (1995). Infect. Irrtnrun., 63:4423-4428. 31. Heinrichs, J. H., Beecher, D. J., Macmillan, J. D., and Zilinskas, B. A. (1993). J. Bucteriol., 175:6760-6766. 32. Suwan, S., Isobe, M., Ohtani, I., Agata, N., Mori, M., and Ohta, M. (1995). J. Clzem. Soc. Perkin Truns., 1:765-775. 33. Lund, T., and Granum, P. E. (1996). FEMS Microbiol. Lett., 141:151-156. 34. Lund, T., and Granum, P. E. (1997). Microbiology, 143:3329-3336. 35. Agata, N., Ohta, M., Arakawa, Y., and Mori, M. (1995). Microbiology, 141:983-988. 36. Beecher, D. J., and Wong, A. C. L. (1994). Appl. Environ. Microbiol., 60:1646-1651. 37. Sato, K., Ichiyama, S., Ohmura. M., Takaski, M., Agata, N., Ohta, M., and Nakashima, N. (1998). J. Infect., 36:247-248. 38. Berner, R., Heinen, F., Pelz, K., Van Velthoven, V., Sauer, M., and Rorintheberg, R. (1997). Neuropediutrics, 28:333-334. 39. Meredith, F. T., Fowler, V. G., Gautier, M., Corey G. R., and Reller, L. B. (1997). Scund. J. Zrgect. Dis., 29:528-529. I., Akiyama, N., Mitani, K., Hirai,H.. Yazaki, Y., and Machi40. Motoi, N., Ishida, T., Nakano, nami, R. (1997). Acta Neuropatllol.. 93:301-305. 41. Akiyama, N., Mitani, K., Tanaka, Y., Hanazono, Y., Motoi, N., Zarkovic, M., Tange, T., Hirai, H., and Yazaki, Y. (1997). Intern. Med., 36:221-226. 42. Miller, J. M., Hair, J. G., Herbert, M., Herbert, L., and Roberts, F. J., (1997). J. Clin. Microbiol., 35:504-507. 43. Marley, E. F., Saini, N. K., Venkatraman, C., and Orenstein, J. M. (1995). Soutlz. Med. J.. 88:969-972. 44. Strittmatter, M., Hamann, G., Sahin, U., Feiden, W., Kohl, K., and Schinlrigk, K. (1995). Newenarzt, 66:785-788. 45. Beecher, D. J., Pulidok, J. S., Barney N. P. and Wong, A. C. L. (1995). h$ect. Zmmun., 63: 632-639. 46. Schafer, D. F., and Sorrel M. F. (1997). N. Eng. J. Med., 336:1173-1174. 47. Kamat, A. S., Nerkard, D. P., and Nair, P. M. (1998). J. Food Sufeq, 10:31-41. 48. Bachhil, V. N., and Jaiswal, T. N. (1998). J. Food Sci. Techrrol., 25:371-372. 49. Harmon, S. M., and Kautter, D. A. (1987). J. Food Prof., 54:372-374. 50. Suzuki, A., Kawanishi, T., Takayama, S., Haruta, M., Simizu, Y., Ogiwara, H., and Jinbo, K. (1984). J. Food Hygienic Soc. Jpn, 25: 106-1 11. 51. Arispe, I., and Westhoff, D. (1984). J. Food Prot., 47:27-35. 52. Mosso, A., Garcia-arribas, L., Cuena, J. A., and De la Rosa, C. (1989). J. Food Prot., 52: 184- 188. 53. Harmon, S. M., Kautter, D. A.. and Solomon, H. M. (1987). J. Food Prot., 50:62-65. 54. Sooltan, J. R. A., Mead, G. C., and Norris, A. P.(1987). Food Microbiol., 4:347-351. 55. Ternstrom, A., and Molin, G. (1987). J. Food Prot. 50:141-146. 56. Konuma, H., Shinagawa, D., Tokumaru, M., Onoue Y., Konno, S., Fujino, N., Shigehisa, T., Kurata, H., Kurata, Y., Kuwabara, Y., and Lopes, C. A. M. (1988). J. Food Prot., 51: 324-326. 57. Samson, R. A., Van Kooij, J. A., and De Boer, E. (1987). J. Food Prot., 50:92-94.

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cereus 75

58. Hatakka. M. (1998). J. Food Prot., 61:1052-1056. 59. Lin, S., Schraft, H., Odumeru. J. A., and Griffiths, M. W. (1998). Int. J. Food Microbiol., 43:159-171. 60. teGiffel, M. C., and Beumer, R. R. (1998). Tijdschr. Diergeneeskd., 123528-632. 61. Holsinger, V. H., Rajkowski, K. T., and Stabel, J. R. (1997). Rev. Sci. Tech. Ofs Int. Epiz., 16:441-451. 62. (1992). Internrctional Dui?? Federatiow Bulletin No. 275, Brussels. 63. (1993). Inter~ationcrlDui?? Federation Bulletin No. 287, Brussels. 64. Bergann, T. (1989). Mlz. Vet.-Med., 4423-25. 65. Garcia-Armesto, M. R., and Sutherland, A. D. (1997). J. D a i p Res., 64261-270. 66. Foegeding, P. M., and Berry, E. D. (1997). J. Food Prot., 60: 1256-1358. 67. Christiansson, A., Naidu, A. S., Nilsson. I., Wadstrom, T., and Pettersson, H-E. (1989).Appl. Enl*irort.Alicrobiol., 55:2595-2600. 68. van Netten, P., van de Moosdijk, A., van Hoensel, P., Mossel, D. A. A., and Perales, I. (1990). J. Appl. Bucteriol., 69:73-79. 69. Fernlanian, C., Lapeyre, C., Fremy, J. M., and Claisse, M. (1997). J. Doily Res.. 64551 559. 70. Rowan, N. J., and Anderson, J. G. (1998). Lett. Appl. Microbiol., 26:161-165. 71. Rowan, N. J.. and Anderson, J. G. (1997). Appl. Environ. Microbiol., 63: 1182-1 184. 72. Jaquette, C. B., and Beuchat L. R. (1998). J. Food Prot., 61:1629-1635. 73. Dufrenne, J., Bijwaard, M., te Giffel, M., Beumer, R., Notermans, S. (~1995).hzt. FoodMicrobiol.. 27:175-183. 74. Beuchat, L. R., Claverno, M. R. S., and Jaquette, C. B. (1997). Appl. Emiron. Microbiol., 6311953-1958. 75. Jaquette. C. B., and Beuchat, L. R. (1998). J. Food Prot., 61563-570. and Martin, R. (.1997). Lett. Appl. Micro76. Mazas, M., Lbpez, M., Gonzilez, I., Bernardo, A., biol., 25:331-334. 77. Mazas, M., Lbpez, M., Gonzilez, I., Gonzilez, J.. Bernardo, A., and Martin, R. (1998). J. Food Safen,, 18:25-36. 78. Mahakarnchanakul. W., and Beuchat, L. R. (1999). J. Food Prot., 6257-64. 79. van Netten, P., and Kramer, J. M. (1992). Int. J. Food Microbiol., 17:85-99. 80. Kramer. J. M., and Gilbert, R.J. (1989). Bacillus c e r e ~ and s other Bacillus species. InFoodborne bacterial pathogens (M. P. Doyle, ed.), Marcel Dekker, Inc., New York, pp 21-70. 81. Rhodehamel E. J., and Harmon, S. M. (1998). Bacillus cereus. In FDA BrrcteriologicalAltalytical Manzlal, 8th ed., AOAC International, Gaithersburg, Maryland. 82. AOAC OfficialMethods of Analysis. (1998). Bacillus cerezis. In Foods-AOAC OfJicicrl Method 980.31, Vol. 1 (P. Cunniff, ed.), AOAC International, Gaithersburg, Maryland. 83. Harmon, S. M., Goepfert,J. M., and Bennett, R. W. (1991).Bacillus cereus. In Compendium of Methods for the Microbiological E.yamination of Food (C. Vanderzant and D. F. Splittstoesser, eds.),American Public Health Assoc.. Washington, DC. 84. Logan, N. A. and Berkeley, R. C. W. (1984). J. Gen Microbiol.. 130:1871-1 883. 85. Shinagawa, K. (1990). Int. J. Food Microbiol., 10:125-142. 86. Gilbert, R. J. and Kramer, J. M. (1984). Biochenz. Soc. Trans., 12:198-200. 87. Turnbull, P. C. B. (1986). Bacillzcs cereus toxins. In Plrnnnacology of Bacterid Toxins (F. Dorner and J. Drews, eds.), Pergamon Press, New York. 88. Jackson, S. G. (1991). J. Assoc. 08 Anal. Chenz., 74:704-706. 89. Andersson.M.A., Mikkola, R., Helin, J., Andersson, M. C., and Salkinoja-Salonen, M. (1998). Appl Emiron. Microbiol., 64:1338-1343. 90. Tan, A., Heaton, S., Farr, L. and Bates, J. (1997). J. Appl. Microbiol., 82677-682. 91. Ivanov, V. N. (1998). Mikrobiol Z., 60:75-82. 92. Lechner, S., Mayr, R., Francis, K. P.. Pruss, B. M., Kaplan. T., Weissner-Gunkel,E., Stewart, G. S., and Scherer, S. (1998). Irtt. J. Syst. Bacteriol., 48:1373-1382.

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93. Nilsson, J., Sevesson, B., Ekelund, K. and Christiansson, A. (1998). Lett. Appl. Microbiol., 271168-172. 94. Francis, K. P., Mayr, R., von Stetten, F., Stewart, G. S., Scherer, S. (1998). Appl. Emiron. Microbiol., 64:3525-3529. 95. Koo, K., Foegeding, P. M., and Swaisgood, H. E. (1998). Appl. Enl-irort. Microbiol., 64: 2490-2496. 96. Mantynen, V. and Lindstrom, K. (1998). Appl. Emiron. Microbiol., 64:1634-1639. 97. Lin, S. F., Schraft, H. and Griffiths, M. W. (1998). J. Food Prot., 61:921-923. 98. Carlson, C.R., Caugant. D. A. and Kolsto, A-B. (1994). Appl. Environ. Microbiol., 60:17191725. 99. Helgason, E., Caugant, D. A., Lecadet, M"., Chen, Y.. Mahillon, J., Lovgen, A., Hegna, I., Kvaloy, K. and KolstG, A-B. (1998). Cz4rr. Microbiol.. 37:80-87. 100. Jackson, S. G., Goodbrand,R. B., Ahmed, R. and Kasatiya, S . (1995). Lett. Appl. Microbiol., 21~103-105.

L

5 Brucella Shirley M. Halling U S . Depurtment of Agriculture, Ames, Iowa

Edward J. Young VA Medical Center and Baylor College of Medicine, Houston, Texas

I. Introduction 77 11. Etiological Agent 78

A. Taxonomy 78 B. Hardiness 79 111. Epidemiology and Foods Involved 79 A. Foodborne brucellosis 80 B. Travel 80 References 81

1.

INTRODUCTION

Brucellosis is a zoonosis caused by species of the bacterial genus Brucella. Humans are always an accidental host, commonly contracting the disease by direct contact with infected animals or by ingesting contaminated dairy products. Only three of the six species of Brucella are important human pathogens: B. abortus, B. melitensis, and B. suis; these three species, respectively, have a natural host preference for the following food animals: cattle, goats and sheep, and swine (1). Brucella canis, principally a pathogen for dogs, rarely causes human infection and is not associated with the consumption of food (2). The risk of contracting brucellosis is highest among farmers, ranchers, abattoir workers, and veterinarians. Clinical and research microbiologists handling Brucella are also at increased risk of infection. In contrast, human brucellosis linked to the consumption of such foods as goat’s milk cheese is not occupation related and often occurs without direct animal contact. Brucellosis exists worldwide and is prevalent especially in countries where programs to eradicate the disease in domestic animals are inadequate. In the United States, programs to control bovine brucellosis have resulted in a dramatic decline in human brucellosis caused by B. abortus (3). Likewise, B. melitensis has been eradicated from native goats 77

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and sheep since 1972. Brucella melitensis is the most prevalent species worldwide, but cases of human infection with this species in the United States generally are linked to importation of contaminated goat's milk cheese from Mexico (4,5). Eradication of B. szris has beenless successful; however, human infection with this species is primarily a problem within the meat-packing industry (6). The true incidence of human brucellosis in many countries is unknown owing in part to the lack of accurate reporting mechanisms. Brucellosis is enzootic throughout the Mediterranean basin and in countries of the Arabian peninsula. In Kuwait, for example, the incidence of human brucellosis reached epidetnic proportions in 1985 (68.9 cases per 100,000 population) (7). With the introduction of brucellosis control measures, the incidence of human infection fell to 20.1 cases per 100,000population by 1988. Unfortunately, efforts to control the disease were hampered by the war with Iraq, and the incidence of human disease no doubt will rise again. Brucellosis in animals can result in economic losses estimated in the millions of dollars annually. Animal production is affected by abortions and infertility. The impact of brucellosis in humans is lost labor, morbidity, and occasional mortality.

II. ETIOLOGICALAGENT Bmcellr species are small, nonmotile, non-spore-forming, gram-negative coccobacilli measuring 0.5-1.5 pm by 0.5-0.7 pm. They grow aerobically at 37OC, although many strains of B. abortus require an atmosphere of 5%-10% CO, for primary isolation (8). Brucellae can be grown on simple defined media containing only a few amino acids and vitamins (for a review of nutritional requirements see Ref. 9). The optimal pH for the growth of blucella in vitro is 6.6-7.4. Brucellae grow rather slowly, especially on primary isolation. Therefore, clinical laboratories should be alerted to maintain cultures for at least 28 days before declaring them sterile. The useof lysis-centrifugation techniques may shorten the time required for recovery of Brwcellcr from clinical specimens (10). Brucella colonies are nonpigmented and nonhemolytic on blood agar. They are approximately 23 mm in diameter, and they appear concave, moist, and glistening. Rough mutants arise spontaneously, especially in liquid medium supplemented with glucose. BrucelZa strains are always catalase positive, but oxidase activity varies. Urease activity is extremely variable, with B. c m i s and B. suis showing stronger reactions than B. abortus or B. melitensis. Proteolytic activity is weak, and gelatin, milk, and serum proteins are not digested. Growth of brucellae is inhibited by certain dyes, a technique that has been used to differentiate species and biovars.

A.

Taxonomy

The oxidative utilization of certain amino acids, urea cycle components, and carbohydrates has been used for taxonomic purposes, although this largely has been replaced by specific phage lysis patterns (1 ). 1 Six Brzrcellll species currently are recognized; however, genomic analysis reveals a high degree of genetic relatedness (12). The exact phylogenetic position of Brucella species remains unclear, however, genomic studies indicate genotypic similarities with the genera Agrobacteriunz and Rhizobium, which are parasites of plants (13).

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B. Hardiness The brucellae are vegetative bacteria, and, lacking spores, they are susceptible to inactivation by such environmental factors as extremes of temperature, drying, and ultraviolet irradiation. Nevertheless, brucellae can survive in moist soil, manure, animal carcasses, and some foods for prolonged periods of time. Pasteurization by the holder method or by the short-term, high-temperature method effectively kills Brllcella organisms. Commonly used disinfectants, used at appropriate concentrations, also effectively inactivate brucellae. Brucellae can be found in contaminated foods for variable periods of time (for a review, see Ref. 14). Factors affecting the survival of Brucella in food include the type of product, the moisture content, pH, age, and the presence of other microorganisms. Brucellae have been shown to remain viable in butter and ice cream for up to a month and in soft cheeses for several months. Although brucellae can survive for months after inoculation into sterile milk, they die within 10 days in naturally contaminated milk or in milk that has soured and become acidic. In contrast to dairy products, meat and meat products are less common sources of brucellosis. Nevertheless, raw meat, organs, and blood can contain brucellae and are potential vehicles for the transmission of brucellosis (15).

111.

EPIDEMIOLOGYANDFOODSINVOLVED

Brzrcelln nzelitensis was isolated firstin 1886 by David Bruce onthe island of Malta from splenic tissue of servicemen who had died from Malta or Mediterranean fever. The epidemiology of brucellosis, however, remained obscure until 1905, when Zammit, working with the Mediterranean Fever Commission, identified antibodies to Brucella in the serum of native goats. Subsequently, the bacteria were isolated from blood and milk of seemingly healthy animals, and ingestion of unpasteurized goat‘s milk was shown to be the source of human brucellosis. Although goats and sheep are the major reservoirs of B. nrelitensis, other food animals, especially camels, are suspected to be the source of brucellosis in some countries. In Oman, for example, serological surveys among animals in the south of the country where brucellosis is prevalent revealed that 8% of camels had antibodies to Brucella (16). Since camel’s milk traditionally is ingested raw and boiling is said to spoil its flavor, it seems likely that camels are an important source of human brucellosis. In 1897, Bernhard L. F. Bang, a Danish veterinarian and physician, first isolated B. abortus from cattle suffering from contagious abortion. It was not until 1918, however, that the relatedness of B. abortus and B. n~elitensiswas recognized by the American bacteriologist Alice Evans. For some time thereafter, the question of whether or not milk from infected cattle could transmit brucellosis to humans was debated. Eventually, cases of human brucellosis associated with the ingestion of raw cow’s milk were documented, and pasteurization was shown to protect the milk supply. Brucella suis was isolated first by J. E. Traum in 1914 from an aborted swine: however, initial studies misidentified it as a biovar of B. abortus. In fact, the first bacteriologically proven case of human brucellosis occurring in the United States was caused by B. suis, but it was not until the isolate was reexamined years later that its true identity was recognized (17). The transmission of B. suis to humans is mainly by direct contact with diseased animals, especially during slaughter and butchering. Transmission by contaminated aero-

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1

sols has been implicated in outbreaks of human brucellosis caused by B. suis in abattoirs (18). In various parts of the United States, feral swine are infected with B. suis, posing a threat to hunters who may be unaware of the disease (19,20). In addition, B. suis biovar 4 is prevalent among caribou and reindeer in the tundra regions of Alaska, Canada, and northern Siberia, and cases of brucellosis among native peoples of these regions have been traced to the ingestion of undercooked meat and bone marrow (21). With the exception of rare cases involving caribou, the role of Brucella-infected wildlife as a source of human infection is controversial. More than 40 species of wild animals have been shown to be susceptible to Brucella (22), but, with few exceptions, the role of wildlife and their ectoparasites in the epidemiology of brucellosis remains unknown. Herds of bison and elk in the Yellowstone National Park are known to be infected with B. abortus, and under experimental conditions the disease has been shown to spread from wild animals to cattle (23). However, proven incidences of such transmission occurring in nature are rare.

A.

FoodborneBrucellosis

Studies by the Mediterranean Fever Commission in 1906 showed that brucellosis could be transmitted to uninfected goats and monkeys by feeding them B. melitensis organisms or milk from infected animals. The role of unpasteurized milk as a source of brucellosis was demonstrated further when prohibition of goat's milk by the military in Malta resulted in a dramatic decline in brucellosis cases among servicemen. Later outbreaks of human brucellosis were traced to the consumption of unpasteurized milk from cows infected with B. abortus; however, this species appeared to be associated less commonly with food than B. melitensis (24). The reason for this is apparent when one considers the studies of Morales-Otero, who showed that B. abortus produced disease more often when inoculated into the abraded skin of volunteers than when it was ingested (25). These findings also are consistent with the observation that the infectivity of intestinal pathogens varies according to their resistance to the bactericidal action of gastric juice. According to this hierarchy, B. abortus is killed more rapidly than B. melitensis, which in turn is killed more rapidly than Salmorzella typhi and ShigellaJEexneri (26). It also has been suggested that patients using antacids for peptic ulcer disease may be at increased risk of oral infection with Brucella (27). The gastric barrier is only one of multiple factors that determine whether infection results from exposure to enteric pathogens (28). The relative virulence of the organism, the infecting inoculum, the immune status of the host, and the route of inoculation are but a few of the factors that determine whether a pathogen will invade and become established in the tissues.

!

B. Travel With the increase in tourism throughout the world, the field ofmedicine has become firmly established (29). The risk to travelers of such infections as diarrhea, malaria, hepatitis, typhoid fever, and sexually transmitted diseases is well recognized. The risk of contracting brucellosis is perhaps lower, but no less serious (30). This risk is especially high when tourists visit countries where brucellosis is endemic and when they ingest traditional foods

F.,

. .". -

..... "

a.._

Brucella

81

(31). In recent years, outbreaks of human brucellosis in the United States have been traced to the importation of dairy products from brucellosis-endemic areas (32,33).

REFERENCES 1. Meyer, M. E. (1990). Current concepts in the taxonomy of the genus Brucella. In Animal Brzlcellosis (K. Nielsen and J. R. Duncan, eds.). CRC Press, Boca Raton, Florida, pp. 1-17. 2. Carmichael, L. E. (1976). Canine brucellosis: An annotated review with selected cautionary comments. Theriogenology, 6: 105-1 16. the U.S. Ann. Sclavo, 3. Brown, G. (1977). The historyof the brucellosis eradication program in 19:20-34. 4. Young, E. J., and Suvannoparrat, U. (1975). Brucellosis outbreak attributed to ingestion of unpasteurized goat cheese. Arch. Intern. Med., 135:240-243. 5. Taylor, J. P., and Perdue, J. N. (1989). The changing epidemiology of human brucellosis in Texas, 1977-1986. Am. J. Epidentiol., 130:160-165. 6. Buchanan, T.M., Hendricks, S. L., Patton, C. M., and Feldman, R. A. (1974). Brucellosis in the United States, 1960-1972: An abattoir-associated disease.111. Epidemiology and evidence for acquired immunity. Medicine, 53:427-439. and Marafie, A. A.(1988). Thenature of brucello7. Mousa, A. R.M., Elhag,K. M., Khogali, M., sis in Kuwait: Study of 379 cases. Rev. Infect. Dis., 10:211-217. 8. Huddleson, I. F. (1921). The importance of an increased carbon dioxide tension in growing Bact. abortus (Bang). Cornel1 Vet., 11:210-215. 9. Gerhardt, P. (1958). The nutrition of brucellae. Bact. Rev., 22:81-97. 10. Kolman, S . , Maayan, M. C., Gotesman, G., Rozenszajn, L. A., Wolach, B.,and Lang, R. (1990). Comparison of the bactec and lysis concentration methods for recovery of Brucella species from clinical specimens. Eur. J. Clin. Microbiol. Infect. Dis., 10:647-648. Brucellosis: Clinicaland Labora11. Corbel, M.J. (1989). Microbiologyof the genus Brucella. In tory Aspects (E. J. Young and M. J. Corbel, eds.), CRC Press, Boca Raton, F L , pp. 53-72. 12. Corbel, M. J. (1988). International Committeeon Systematic Bacteriology Subcommittee on the Taxonomyof Brucella, Report of meeting, September5 , 1986, Manchester, England(editorial). Int. J. System. Bacteriol., 38:450-452. of 16s rRNA from 13. Dorsch, M., Moreno,E., and Stackebrandt, E. (1989). Nucleotide sequence Brzlcella abortus. Nucleic Acids Res., 17:1765. 14. International Dairy FederatiodFederation Internationale de Latiterie. (1 988). Behavior of pathogens in cheese. Document 122, Federation, Brussels, Belgium. 15. Sadler, W. W. (1960). Present evidence on the role of meat in the epidemiology of human brucellosis. Am. J. Public Health, 50504-514. 16. Ismaily, S. I. N., Harby, H. A. M., and Nicoletti, P. (1988). Prevalenceof Brucella antibodies in four animal species in the Sultanate of Oman. Troy. Animal Herrltlz Prod., 20:269-270. 17. Huddleson, I. F. (1943). Brucellosis in Man and Animals. Commonwealth Fund, New York. 18. Kaufman, A. F., Fox, M. D., Boyce, J. M., Anderson, D. C., Potter, M. E., and Mortone, W. J. (1980). Airborne spread of brucellosis. Ann. NY Acad. Sci., 353:105-114. 19. Becker, H.N., Belden, R. C., Breault, T., Burridge, M. J., Frankenberger, W.B., and Nicoletti, P. (1978). Brucellosis in feral swine in Florida. J. Am. Vet. Med. Assoc., 173:1 181-1 182. 20. Zygmont, S. M., Nettles, V. F., Shotts, E. B., Carmen, W. A., and Blackburn, B. 0. (1982). Brucellosis in wild swine: A serologic and bacteriologic survey in the southeastern United States and Hawaii. J. Am. Vet. Med. Assoc., 181:1285-1287. 21. Chan, J., Baxter, C., and Wennman, W. M. (1989). Brucellosis in an Inuit child probably related to caribou meat consumption. Scand. J. Infect. Dis., 21:337-338.

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22. Rementsova, M. M. (1987). Brucellosis in Wild Animals. Amerind, New Delhi, p. 41. 23. Davis, D. S. (1990). Role of wildlife in transmitting brucellosis. In Ad~lancesin Brucellosis Research (L. G. Adams, ed.), Texas A&M University Press. College Station, p. 371. 24. Nelson, C. B., and Giblin, M. (1950). Milk-borne brucellosis in Minnesota.Minnesota Med., 331981-982. 25. Morales-Otero, P. (1933). Further attempts at experimental infection of man with a bovine strain of Brucella abortus. J. Infect. Dis., 52:54-59. 26. Garrod, L. P. (1937). The susceptibilityof different bacteria to destruction in the stomach. J. Pathol. Bacteriol., 45:473-474. 27. Steffen, R. (1977). Antacids-A risk factor in travellers brucellosis? Scarzd. J. Irzfect. Dis., 9~311-312. 28. Antacids and brucellosis (1978). Br. Med. J., 1:739-741. and Schar, M. (1987). Health prob29. Steffen, R., Rickenbach. M., Wilhelm, U., Helminger, A., lems after travel to developing countries. J. Irzfect. Dis.. 156:84-91. 30. Young, E. J. (1989). Brucellosis in travelersand Brucella that travels. In Travel Medicine (R. Steffen, H. 0. Lobel, J. Haworth, and D. J. Bradley, eds.), Springer-Verlag, NewYork, pp. 375-377. of travelers to 31. Arnow, P. M., Smaron. M., and Ormiste, V. (1984). Brucellosis in a group Spain. JAMA, 25 1505-507. 32. Eckman, M. R. (1975). Brucellosis linked to Mexican cheese. JAMA, 232:636-637. 33. Thapar, M. K., Young, E. J. (1986). Urban outbreak of goat cheese brucellosis.Pediatr. btfect. Dis.. 5:640-643.

6 Campylobacter jejuni Don A. Franco Anirml Protein Producers Industry, Huntsville, Missotwi

Charles E. Williams Consultant, Arlington. Virginia

I.

Campylobacter Species 83 A. Isolation, classification, and zoonotic aspects 84 B. Morphology, growth, and comparison with other bacteria

11. EpidemiologyandEpizootiology

85

86

Animal A. reservoirs 86 B. Agricultural animal reservoirs 86 89 C. Other animal reservoirs D. Human reservoirs 89 E. Environmental reservoirs 90 111. Occurrence and Survival in Foods of Animal Origin IV. Modes ofTransmission 92 V. Pathogenicity-Clinical Manifestations

90

93

VI. Economic Impactand Other Costs 94 VII.PreventionandControl VIII.

Summary and Conclusions 99 References

1.

96

99

CAMPYLOBACTER SPECIES

Se.vera1 Cmnpylobncter species are capable of causing foodborne disease in humans, but C.jejurzi and C. coZi appear to be the most prominent of the clinical isolates throughout the world. In 1983, scientists from the U.S. Centers for Disease Control and Prevention (CDC) conducted an epidemiological case/control study on an American university campus where students had been stricken with enteritis caused by C.jejuni. They found that 43% of those who had contracted the disease missed classes, and some 15% were hospitalized 83

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(1). Poultry was identified as the most frequent vehicle of disease transmission. Even the football team was affected; the training diet had been changed to provide a higher ratio of poultry to red meat, and a number of the players became ill. Poor food handling was thought to be an important factor in the majority of the cases (2; R. V. Tauxe, personal communication, 1991). Although the high percentage of cases (70%) in which poultry was implicated is considered unusual-raw dairy products or contaminated water are more often the vehicles of transmission-the fact that young adults were victims of the disease is‘not. CumpyZobncter resembles other enteric pathogens in the rate at which it strikes infants and the elderly, but the organism is unique in that young adults between the ages of roughly 20 and 30 years, and males more than females, are infected at a high rate. The infection, in other words, is not simply opportunistic. The reasons for this pattern are still to be discovered, as are the solutions to such other mysteries as the sporadic nature of cases, the relatively low and invariant infectious dose (the number of organisms required to induce an infection), and the seasonal dropoff of cases from November to January. These and other curiosities can be regarded as academic, however, because the methods of preventing infection by Ccmpylobncter are the same as those for ScdrnoneZZa and other pathogens commonly found in raw foods of animal origin.

A.

Isolation, Classification, andZoonoticAspects

Cnmpylobacter, first identified as animal pathogens about 90 years ago, became recognized widely in the 1970s for causing human illness (3-5). Two veterinarians, McFadyean and Stockman, discovered the organisms in 1909 while investigating the cause of epizootic abortion in ewes (6). In 1913, the same researchers demonstrated that the organisms could be observed in infectious abortions, but World War I prevented dissemination of their work to the public and to health workers (6). For more than 40 years the organism was thought to be exclusively an animal pathogen. During his 1919 investigation of infectious abortions in U.S. cattle, Theobald Smith isolated (along with the “Bacillus of Bang”) a bacterium he described as a spirillum (7). After completing his study, Smith became acquainted with the work of McFadyean and Stockman and inferred that he and they had been studying the same bacteria. He subsequently confirmed his finding with Taylor, and Vibrio-fetuswas proposed as the name of the organism (7). In 1931, Jones et al. ascribed outbreaks of winter dysentery in calves to a bacterium they called Vibrio jejuni(8). Doyle in 1944 attributed swine dysentery to a similar organism (9). In 1946, Levy reported an outbreak of acute diarrhea in humans associated with milkborne organisms that resembled Vibrio jejuni (10). In 1947, Vinzent isolated Vibrio fetus from the blood of three pregnant women, two of whom aborted (1 1). Despite these findings, however, strains affecting humans were not studied extensively until 1957, when King distinguished two groups of organisms isolated from human blood cultures (12). One group corresponded closely to V. fetus,as it then was described; the other group King termed “related vibrios.” In 1963, Sebald and Viron found that these two groups of Vibrio species differed biochemically and serologically from the classical cholera and halophilic Vibrios since their DNA base-pair ratio (G + C mol%) differed from that of the other Vibrio species (13,14). On the basis of these findings, Sebald and Viron proposed that these species be

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removed from the genus Vibrio and that they be called Campylobacter, meaning curved rod (14). In 1972, Butzler et al. isolated Canzpylobacter jejuni from 5% of Belgian children with diarrhea (15). These findings were confirmed later by Skirrow (16). Workers since have reported similar findings, and in some laboratories Campylobacter isolations have outnumbered those of SnlmoneZla and Shigella combined (5,17). As recently as 35 years ago, pathogens were isolated from the stools of fewer than 25% of persons with acute diarrhea. Today, the causative agent is identified in as many as 65-85% of acute diarrhea cases (18). Cnnzpylobacterjejuni is one of the most frequently identified organisms. Methods developed over the years for isolating the organism include species identification, serotyping, biotyping, and phage typing and such molecular typing techniques as pulse-field gel electrophoresis, random amplified polymorphic DNA, and ribotyping. Serogroup I1 was the most common of human isolates in a recent Canadian study of enteritis cases (20). Since its first isolation from human diarrhea stools in 1971, C. jejuni has become recognized as one of the leading causes of gastroenteritis (2). Finch and Riley conservatively estimated that more than 16,000 people were infected by Campylobncter in 1982 (19). They also determined that diarrhea caused by Campylobacter was as common as Salmonella and more common than Shigella. Since most laboratories did not culture for the organisms or report it, the results are significant. Comparative estimates for the United States in 1982 show that C. jejuni infections occurred at a rate approximately equal to that for hepatitis B and at a rate 10 times that for influenza (21). Between 1980 and 1982, meat and poultry were the vehicles of 4 of 23 foodborne outbreaks of campylobacteriosis reported to the CDC-a rate second only to unpasteurized milk (22). Between 1988 and 1992, 2 of 29 foodborne outbreaks of campylobacteriosis reported to the CDC were attributable to meat as the vehicle, while milk was identified as the vehicle in 5 outbreaks (23). A 1984 case-control study by the Communicable Disease Control Section of the Seattle-King County Department of Public Health reported that chicken consumption was the predominant risk factor associated with C. jejuni enteritis; this association was supported by microbiological isolations (54).

B. Morphology,Growth,andComparisonwith Other Bacteria CnmpyZobncter species (25-28) are small, non-spore-forming, gram-negative bacteria with a characteristic curved, S-shaped, or spiral morphology. The cells may vary from 0.5 to 8 pm in width. The cells are highly motile, with a characteristic rapid, darting, corkscrew-like motility. They have a single unsheathed polar flagellum at one or both ends of the cell. Virtually all members of the genus are oxygen sensitive and can grow only under conditions of reduced oxygen tension that vary from almost anaerobic to microaerobic for the different species. An aerotolerant Campylobacter group has also been described (28). Ccmpylobacter species grow best in an atmosphere containing 5-10% oxygen and thus are considered microaerophilic. Although most will not grow under aerobic or anearobic conditions, C. jejuni will grow in candle jars, simplifying isolation. All Campylobacter species grow at 37°C; however, C. jejuni grows best at 42°C (29). Because C. jejuni is a very common pathogen in humans, many laboratories use incubation at 42°C for optimal isolation; however, not all species can be detected at this temperature.

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CcrrnpyZobacter species tend to multiply more slowly than the usual enteric flora. Thus, they cannot be isolated from fecal specimens without the use of selective techniques. The most common isolation methods currently involve the use of antibiotic-containing media. Three such media, Skirrow’s, Butzler’s, and Campy-BAP, or variations of them, are in wide use (39). Butzler’s medium and Campy-BAP contain cephalothin, which inhibits C. fetus and several other Campylobncter species, but they are best suited for isolating C. jejurl i . Cnrrrpylobncter species can be distinguished from other microorganisms by several standard criteria and frotn one another through biochemical testing (30). Visible colonies usually appear on the plating media within 24-48 hours. Occasionally, growth takes place after 72-96 hours of incubation. The serotypic diversity of C. jejuni is broad; the organism is similar in this respect to other bacteria with an ecological niche in the gastrointestinal tract of mammals. More than 60 different serotypes based on somatic (0)antigen and 50 different serotypes based on heat-labile antigens (capsular and flagellar) have been identified (31). Advances in molecular and DNA technology have shown that Cnmpylobactel. forms a distinct group of eubacteria with Helicobacter and Arcobncter (32,33). Campylobncter jejuni is sensitive to drying or freezing temperatures, characteristics that limit its transmission. The organism, however, survives in milk or water kept at 4°C for several weeks. The standard concentrations of chlorine for water disinfection and pasteurization effectively destroy the organism (3 1).

II. EPIDEMIOLOGYANDEPIZOOTIOLOGY Campylobacteriosis is a worldwide disease of animals that can be transmitted secondarily to humans. Although important aspects of the transmission of the organism remain unexplained, considerable progress has been made in the understanding of both the reservoirs and the prevalence of the infection.

A. AnimalReservoirs Zoonoses, diseases transmitted from animals to humans, are remarkable not only for their frequent occurrence but because they almost always are unsuspected and unrecognized. This is as true for Cnmpylobacter as for other zoonotic pathogens. CnmpyZobacterspecies are commonly found as commensals of the gastrointestinal tract of wild or domesticated cattle, sheep, swine, goats, dogs, cats, rodents, and all classes of poultry. Extensive reports in the scientific literature demonstrate that the reservoirs of organisms in the animals cause infection and disease in humans. Animals or animal products have been identified conclusively as sources of infection in several outbreaks, and many of the Cnmnpylobncter serotypes that cause disease in humans have been isolated from animals (34). B. AgriculturalAnimalReservoirs 1. Cattle C. jejuni appears to be a normal commensal of all classes of bovines. The prevailing evidence supports transmission from adult cattle to calves, but not between adults. It is

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possible at any given time for several different serotypes to be present in a herd (34). Carcasses may become contaminated with intestinal contents during slaughter. In one outbreak, 525 specimens were obtained from 100 slaughtered beef cattle and examined for the presence of C. jejurzi and C. coli by direct plating and enrichment techniques. Of the 100 animals, 50 were positive for C. jejmi and 1 was positive for C. coli. The distribution pattern of positively infected animals, in decreasing order, was steers (57%), bulls (40%), heifers (40%), and cows (22%). Of those animals infected, significantly higher isolation rates were obtained from gall bladders (34%), large intestines (36%), and small intestines (32%) than from liver (12%) or lymph nodes (1.4%) (35). Similar rates have been reported in subsequent studies (61). In Finland, fecal samples from 200 cattle showed an incidence of 5.5% for C. jejmi (36). Similarly, feces collected from 90 cattle in Sweden showed positive isolates from 17 (19%) (37). In a milking herd, the presence of the organism does not necessarily lead to condemnation of the milk because C. jejzjuni is destroyed by pasteurization. A community outbreak of gastroenteritis in Vermont was traced to consumption of unpasteurized milk produced at a commercial dairy. Two different testing schemes showed a C. jeju~ziisolate from a sick patient and an isolate from a diseased cow to be of the same serotype (38).

2. Swine Carcass contamination by Curzpylobactel- species appears more common in swine carcasses than in cattle, sheep, or goats. The danger of intestinal spillage is greater with the procedures used in dressing swine carcasses than with those used in dressing the carcasses of ruminants. A Canadian study of cooler-ready hog carcasses to evaluate the degree of contamination of the carcasses and the slaughter environment showed that the largest number of Cmpylobncter isolates, mostly C. coli, came from fecal samples-hence, from the animals themselves (39j. Isolates of C. jejmi from 65 animals in Zaire (29% of the total number examined) showed the highest isolation rates in swine-42 positives (44%) of 95 animals examined. In swine, the youngest animals, weanling pigs less than 2 months old, were positive more frequently than the older ones: there were 13 positives out of 19 weanlings examined and 25 positives out of 72 adult animals examined (40). Points have been identified in the slaughtering and dressing process where contamination by Cumpyllobncter- and other organisms may be limited only by strict observance of sanitary procedures (41). The postslaughter chilling process tends to suppress Ccunpylobncter. replication on the carcasses. This suppression apparently is not caused by low temperatures, but is due to the drying effect of the forced ventilation used in the abattoir cooling rooms. C~~vrzpylobacter species appear to be sensitive to drying (42). A Canadian study of 118 isolates from hogs showed 1 15 positive for Cumnpylobncter-coli serotype (43). Samples collected from 300 normal slaughtered hogs during epidemiological studies performed at six different slaughterhouses were examined for the presence of C. -fetus jejuni. Of these samples, 182, or 60.796, were positive (44).

3. Sheep The relationship between sheep and Ccmpylobctcter has been documented since as early as 1909, when the genus was implicated in ovine abortion. C. jejjmni is an intestinal commensal inmany sheep herds, and surveys have shown the organism to be a common isolate. However, C. jejuni has not been found in sheep as often as in other food animals.

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Isolations of Campylobacter species were made from 16 of 197 (8.1%) sheep fecal samples examined in Norway (45). Stomach contents and heart blood were collected from 10% of lambs from 25 farms for survey of Carnpylobucter fetus. C. fetus infection was found in 102 of the 762 lambs examined (46).

4. Poultry Campylobncter species frequently are present in the intestinal flora of commercially raised birds and such wild birds as pigeons, sea gulls, crows, and ravens. High percentages of C. jejuni isolations in pigeons, obtained in a Spanish study, suggest a need for further study of this possible source of human infection (50). Isolations have been made from poultry early in the growing process, but many flocks escape infection. A Canadian study has shown that C. jejuni could be isolated from all of 108 chicken samples (43). Reports vary on the incidence of positive isolates. While researching the association of Carnpylobacterjejuni with laying hens, Doyle reported peak rates of C. jejurzi isolations of about 25% in October and in late April to early May (47). A Dutch study of broiler chickens found the highest rate of Campylobacter and Sulmorzelln contamination from June to September and the lowest in March and a positive correlation between Canzpylobrrcter and Salmonella colonization within flocks of broiler chickens (51 ). Potential routes of entry of organisms into a flock include infection of newborn chicks from older birds, contamination of feed and water, and wildor game birds. Infection of poultry is often without clinical manifestations. Contamination or recontamination can affect bacterial loads on poultry either in the growing process or at the processing plant. The Contamination ofpoultry houses by carrier flocks has been shown to lead to contamination of subsequent generations of poultry if cleaning and disinfection between removal of market-ready birds and introduction of new flocks is ineffective. When the birds were brought to slaughter, contamination was spread to the processing equipment. Ineffective sanitation at the end of the day contributed to the contamination of birds the following day (48). During the slaughtering process, C. jejurzi from the intestinal contents of processed birds spreads to the carcasses. There also appears to be an inverse relationship between the amount of intestinal carriage and contamination. In one study, C. jejzrni was found to be widespread in each of 15 poultry plants evaluated, but on some days the organism was not isolated from any samples. Slaughtering of heavily contaminated flocks may result in a contamination rate of up to 100% for end products and seems to be unrelated to the type of slaughtering process (49). A total of 138 samples from two California chickenprocessing plants was collected from four groups of birds (7-12 weeks old) in 11 lots over six mornings. Isolation rates for all sites for three scald-water temperatures described were 66.7% (60"C), 75.8% and 78.3% (53"C), and 55.1% (49°C). C. jejuni was isolated from all chilling tank samples, 94.6% of feather picker water samples, and 60-100% of fecal samples. The results demonstrate that chilling water and feather picker water were the major sites for cross-contamination (52). A study of Cmzpylobacter contamination of turkey carcasses at various stages of processing showed reductions of contamination at scalding, but recontamination at defeathering. C.jejuni counts peaked during evisceration but declined after chilling (53). Harris et al. reported a case-control study in Seattle (King County), Washington, where almost 50% of poultry from the processing plant surveyed were positive for C. jejuni. The highest positive rate occurred during the period July-October (54). This finding

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is consistent with earlier studies in England, Belgium, the United States, and South Africa, which showed a summertime peak of Campylobacter infection (55,56).

C. OtherAnimalReservoirs Epidemiological studies of C. jejuni and other Cumpylobacter species in such pets as dogs and cats have led to several findings: (a) C. jejuni may be isolated from both healthy and diarrheal dogs, (b) isolation rates are higher in puppies and kittens than in adult dogs and cats, and (c) isolation rates are higher in kennel populations of both dogs and cats than in pets raised in households. C. jejuni and C. coli have been isolated from 45% of clinically normal cats-the high rate of isolation possibly being attributable to scavenging of contaminated food from a poultry-processing plant. Campylobacter species were isolated from 49% of 144 stray dogs, 39% of 38 clinically normal puppies, and 38% of 42 diarrhetic puppies seen in a veterinary practice (57). In one breeding kennel, C. jejuni was isolated from the feces of 4 of 47 healthy adult dogs, 7 of 71 healthy puppies, 10 of 31 puppies with blood-stained diarrhea, and from the intestines of a puppy that had died from hemorrhagic enteritis (58). Campylobacter counts in normal dogs and cats show a wide range; they are highest (49% dogs, 45% cats) in immature animals, particularly strays or those living in kennels, and lowest (1.6% of dogs and cats) in adult animals living in households (16). C. upsaliensis was isolated from the blood and fetoplacental material of a woman in Turkey who spontaneously aborted after an 18-weeks pregnancy. The woman had had contact with a household cat, which tests showed may have been the source of the infection (60). A 1996 study of dogs and cats in Sweden found a high proportion to be colonized with C. upsaliensis, also found in children. A significant proportion of cats were colonized with C. Iwlveticus, a diarrheal agent in humans (61). These and other studies have shown the risks of Campylobacter transmission to humans from pets, even asymptomatic pets, the need for more efficient procedures to detect campylobacters in pets, and the importance of taking preventive measures, such as hand-washing, after contact with pets (62,63).

D. HumanReservoirs Fecal-oral person-to-person infection has been reported for C. jejuni. As with other enteric pathogens, those in contact with excreta of infected people are at risk. Transmission from asymptomatic infected food handlers is uncommon (31). Interesting reports of spread by human-to-human contact appear in the published literature, implicating food handlers as sources of the organism. These findings support the exclusion of food handlers from the work environment when they are affected, as well as during the period of carriage of the organism (59). A temporary food handler was the most probable source of an outbreak of acute gastroenteritis due to Campylobacter jejuni that occurred at a military base in Israel. Stool cultures were taken from 17 clinically affected as well as 23 asymptomatic soldiers. In 6 of the 17 patients with enteritis (35%), Campylobacter jejuni serotype ii was isolated, while the stool cultures of all the asymptomatic soldiers were negative. The temporary food handler had suffered from acute gastroenteritis before the outbreak but had not reported the illness. He was found to harbor the same serotype as the affected patients (64).

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E.Environmental

Reservoirs

1. Water Outbreaks of waterborne campylobacteriosis in the United States have been traced to the drinking, during outdoor recreational activities, of contaminated spring water or other water not meant for drinking. A May 1983 outbreak in Florida consisting of 871 cases was found to have occurred after a community water system had been contaminated by wild birds (65). Domestic or wild animals and birds potentially are significant sources of Cnrnpylobacter organisms found in polluted water. Back country (i.e., thinly settled rural areas) surface waters can be an important source of C. jejzmi. Infection should be considered as a cause of diarrhea in those who recently have returned from wilderness areas (66). In nature, C. jejzlni has been isolated from stream and river water, from the effluent of poultry-processing plants, and even from seawater samples. Ccrnzpylobacter species have been isolated from 50.4% of 540 (riverine) water samples from the Southampton area in England (34). Survival studies in Norway of thermophilic Carnpylobncter species revealed that all organisms survived better at 4°C. Chlorination drastically reduced the survival of all three strains examined. All strains survived for 10-15 days in polluted river water at 4°C. Removal of interspecific competition by sterile filtration did not enhance survival significantly (67). In 1992, Norwegian researchers reported that thermotolerant campylobacters had been isolated from polluted surface waters most often in a temperature range of 2.18.0°C, least often at temperatures higher than 15"C, and more often in autumn than in other seasons (68). 2. Soil Under conditions favoring replication, fecal contamination of soil could be a source of infection. Isolations of Curnpylobacter species have been made from both mud and sewage sludge in the United States. A 1994 survey of livestock wastes in the United Kingdom found Cmrpylobucter in a high proportion of the wastes. Cnrnpylobacter spp. were isolated more frequently than other pathogens such as Sulmonelln. Though considering the relatively poorer survival of the Canlpylobncter to make them less of a water pollution hazard, the researchers acknowledged the need for a greater understanding of the survival and transport of Currzpylobncter. in soil (69). 111.

OCCURRENCE AND SURVIVAL IN FOODS OF ANIMAL ORIGIN

Transfer of C. jejzwi to carcasses and to other meat and poultry products has become a serious problem for the meat and poultry industries. The most frequently implicated meat is poultry, with a C. jejzmi recovery incidence of 50% in store-bought poultry meat. Red meat carcasses also have yielded this bacterium, although at lower incidence. Food surveillance results of samples collected from February 1982 to September 1983 by researchers of the Seattle-King County Department of Public Health showed C. jejuni cultures at the following levels from retail products: poultry, 192 of 862 samples (22.370);beef, 1 of 230 samples (0.4%); pork, 0 of 142 samples; and lamb, 0 of 37 samples (54,70).

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A Polish survey found Canzpylobncter species on 80.3% of chicken, 48.0% of duck, and 38.0% of goose carcasses but on only 3.0% of turkey, 2.9% of pork, and 0.9% of beef carcasses (71). Samples collected in a survey of Canadian abattoirs from 1983 to 1986 yielded isolates of thermophilic Ccmpylobacter from 16.9% of pork, 22.6% of beef, and 43.1% of veal carcasses. C. jejuni biotypes I and I1 were the most frequently isolated in beef and veal. Biotype I was the most frequently isolated in pork, but 41.1% of the isolates from pork were C. jejuni biotypes I and I1 (72). Of Campylobacter-positive Samples in a Trinidad study of pathogen prevalence in raw meats and seafoods, most were from chicken (73). C. jejuni was found in 78 of 94 (82.9%) chicken wing packages analyzed on the day of delivery to supermarkets and in 15.5% or 45 out of 290 packages obtained from the supermarket shelves a few days later (74). About 800 fresh and frozen meat and poultry samples were analyzed at different field service laboratories of the U.S. Department of Agriculture, Food Safety and Inspection Service. Isolation levels of C. jejuni from fresh tissues were more than five times as high (12.1%) as those from frozen tissues (2.3%) in the samples analyzed. The prevalence of C. jejuni in fresh tissue according to species was avian, 21.3%; bovine, 4.7%; porcine, 8.6%; and ovine, 20.0% (75). A total of 6169 meat samples was examined in 31 laboratories. C. jejurzi was isolated from 98 (1.6%) of the samples, showing a low level of red meat contamination (76). Surveys continue to show the frequent presence of C. jejuni on whole birds or parts obtained at retail outlets and the need for food handlers and homemakers to practice good hygiene when preparing meals to prevent cross-contamination (77). There is evidence that the levels of C. jejuni on broilers sold at retail are affected by seasonal factors and the carrier state of the broilers (78). Studies of the incidence of C. jejuni on retail red meats have shown its presence on these products to be considerably less than on poultry (33,101). Refrigerated storage appears to cause the number of Campylobncter-positive red meat carcasses to diminish, although the effect of chilling on the bacteria appears to depend on the age of the animals. An Australian study showing Cnmpylobacter to be more prevalent on carcasses of feedlot cattle than on those of range cattle also demonstrated that chilling adult cattle carcasses for 20-24 hours significantly reduces the incidence of Canlpylobacter. On the other hand, Canzpylobncter species were found on almost all calf carcasses examined after chilling (79). Most cases of milkborne Cnmpylobacter enteritis have been associated with the consumption of unpasteurized milk. Research workers in Wisconsin isolated C. jejurzi from 1 of 108 (0.9%) milk samples obtained from the bulk tanks of 9 Grade A dairy farms and from 50 of 78 (64%) cows producing Grade A milk (80). In the Netherlands, C. jejuni was isolated from 2 of 1200 milk samples from farm bulk tanks, but not from 600 samples milk cans or 750 samples from cows with clinical mastitis (81). C. jejuni is a rather unique enteric pathogen. It differs markedly from other food poisoning organisms in that it normally does not multiply in food in numbers sufficient to cause infection, as do Sabnorzella species or Staphylococcus aureus, which produce a toxin in food associated with disease. The factors affecting survival and growth of C. jejuni usually are strict microaerophilic conditions: an atmosphere of 5% oxygen, usually with about 8-10% carbon dioxide and 85% nitrogen; an optimum temperature of between 42 and 45°C; and a roughly neutral pH (6.5-7.5) (82). The organism survives better in foods at refrigeration temperature than at room temperatures.

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IV. MODES OF TRANSMISSION Transmission can be defined as the pathway by which a pathogen spreads from its source to the host. It may occur through one or more of four different routes: contact, common vehicle, airborne, and vectorborne (83). Cumpylobacter may be transmitted by direct contact with contaminated animals or animal carcasses, through the ingestion of contaminated food and water, person to person from excreters with active infections, and by perinatal and childhood transmission. The following aspects of transmission are discussed from the foodborne perspective. The majority of Cnmpylobacter infections appear to occur sporadically without a clear determination of the transmission mode. The vehicles incriminated as sources of infection include poultry intended for human consumption, uncooked or poorly cooked meat and poultry products, unpasteurized dairy products, and uncooked foods subjected to possible cross-contamination by meat and chicken. In a case-control study in an urban Swedish community, two diseased persons had blood specimens positive for C. jejuni. Of the diseased persons tested, 8 of 10 had immunoglobulin-M-specific indirect fluorescent antibody (FA) titers to C. jejurzi, a significant association of chicken consumption and Cumpylobucter enteritis (84). C.jejuni was found almost routinely in beef sold in food stores. The bacteria survived on the food at 4°C for one week and frozen at -20°C for 3 months (37). In a Lima, Peru, market, 80 chicken handlers were studied for C. jejuni. The same number of individuals from the same market who did not handle chicken were sampled as controls. Results showed 15 persons (19%) positive for C. jejuni among handlers and 1 person positive among the control group (85). Raw hamburger was implicated in an outbreak that occurred in the Netherlands (87). Raw clams, probably contaminated by sewage, were described as the vehicle in an outbreak in New Jersey (34). Milk is an important and frequent source in the transmission of CnmpyZobacter enteritis. A community outbreak of Carnpylobncter enteritis was associated with the consumption of untreated milk, apparently from two cows with Campylobncter mastitis (87). A New Zealand report implicated raw milk as the cause of gastrointestinal illness among children in two different camp sites. Cnmpylobncter jejuni was isolated in 50 of the 88 affected children (88). A drink prepared with raw milk was associated with an outbreak of C. jejuni enteritis involving more than 500 participants in a jogging rally in Switzerland, with an attack rate of over 75% (89). An outbreak of Cnmpvlobacter jejuni enteritis followed the consumption of unpasteurized milk at an attack rate of around 50%. There were cases in all age groups, with the highest number in the 1 to 10-year-old group (90). C. coli was isolated from a 9-year-old British boy with persistent diarrhea, whose family had consumed raw goat's milk from a local farm. C. jejuni and C. coZi were found in the feces of goats from the farm, and C. jejuni was identified in samples of bulk milk (91). A Swedish researcher has implicated C. jejuni in milk with mastitis, even though no cases of Cnrnpylobacter mastitis have been reported except those induced experimentally (92). In another study, some 2500 English schoolchildren between the ages of 2 and 7 became acutely ill after drinking free school raw milk (59). A case-control study conducted in Seattle and King County, Washington, revealed an association between infection by Campylobacter jejuni coli and the consumption of raw milk and, to a lesser extent, mushrooms. The subjects in the study were more likely to be infected by tetracycline-resistant than tetracycline-sensitive strains (54).

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It is important to remember that many zoonotic infections are acquired by contaminating food from diseased animal tissues or from infected animal feces. Under normal processing procedures, it is extremely difficult, if not impossible, to produce meat and poultry carcasses that are free of bacteria. The slaughter and processing environment has the potential to perpetuate contamination because of the close contact of the tissues in the slaughterhouse with animal and poultry feces. It is inevitable that some of these contaminating bacteria will be Campylobncter species (93). The same basic factors apply to raw or inadequately heat-treated milk contaminated by feces at the time of milking. Chronic consumption of raw milk contaminated with C. jejuni appears to stimulate the production of antibodies resistant to the organism in healthy individuals. Those without the immunity thus conferred are more likely to be susceptible to infection (94). Fortunately, most milk consumed in the United States is pasteurized and devoid of harmful pathogens. The cycle of actual or potential contamination does not always end at the processing establishments. Clean meat and poultry can be contaminated at retail outlets if hygiene at the facility is poor or if insufficient attention is paid to the sanitization and cleanliness of such operational equipment as knives, cutting blocks, and slicing machines.

V.

PATHOGENICITY-CLINICALMANIFESTATIONS

Campylobncfer can be found in almost every country and now is recognized as one of the most common causes of bacterial diarrhea. Acute enteritis is the most common presentation of C. jejurzi infection, with symptoms varying from one day to one week or longer. A prodromal phase with fever, headache, myalgia, and malaise usually occurs 12-24 hours (but may last for up to 48 hours) before the onset of intestinal symptoms. The most common symptoms with Campylobncter infection are diarrhea, malaise, fever, and abdominal pain. The diarrhetic pattern can vary from loose stools to profuse, bloody, slimy, and foulsmelling stools (3 1,95). In some patients, cramping abdominal pain, typically periumbilical, can be the predominant manifestation of illness (31). Vomiting may occur but is rarely a marked feature. The illness frequently is selflimiting to within 1-4 days and usually lasts no more than 10 days, although there may be occasional relapses (96). A recurrence of symptoms is experienced by 25% of patients, often characterized by abdominal pain and varying from a relatively mild gastroenteritis to an enterocolitis with bloody diarrhea and accompanying abdominal pain lasting for several weeks (95). As with other intestinal pathogens, the clinical picture of C. jejuni infection varies from symptomless excretion to severe disease. Although it is normally a self-limiting disease, such complications as cholecystitis, peritonitis, septicemia, and meningitis have been reported. The small intestine is thought to be the main site of infection, but the colon often is involved (97). In the mid- 1980s, two fatalities from C.jejuni infections were reported in the Denver metropolitan area. The first case was in a previously healthy 26-year-old woman who died following a 2-day diarrheal illness. The second case was in a 69-year-old diabetic woman who died 19 hours after developing a gastrointestinal tract illness one day following hospital discharge for an orthopedic procedure (98).

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Archer reviewed the role of foodborne enteric pathogens (including C. jejuni) in the development of three seronegative spondarthropathies jankylosing spondylitis, Reiter’s disease, and reactive arthritis) (99), following gastrointestinal infections with gram-negative enteric bacteria. A molecular explanation of the etiology of these complications has been explored ( 100). Another potential complication of Cmnpylobacter infection is acute inflammatory neuropathy. There is increasing recognition that recent C. jejuni infection is the most common precursor of Guillain-Barr6 syndrome (101). Clinical investigations of gastrointestinal cases during the last two decades have led to the identification and description of new types of Cnnzpylobcrcter and Cuwpylobacterlike organisms (CLO). Australian researchers have reported on the colonization by spiral bacteria of the stomach antrum of patients with peptic ulceration and gastritis. They were found in 100% of patients with duodenal ulcers, 80% of patients with gastric ulcers, and 95% of patients with “active chronic gastritis’’ (102). Another Australian study, of blood cultures at Alice Springs Hospital, showed that of 72 cases of bacteremia that were identified, C. jejuni was the causative agent (103). Fennel1 and coworkers from the University of Washington in Seattle described new CLOs associated with proctitis in homosexual men. The organisms were isolated from 28 of 181 symptomatic homosexual men compared with 7 of 77 asymptomatic homosexual men and 0 of 150 normal heterosexual men and women (104). A study of Campylobncter isolates collected over two decades from South African children with gastroenteritis found that, for those cultures that could be serotyped, the most common serotype (0:41) was one that included strains associated with Guillain-Barr6 syndrome cases. Others were obtained from kwashiorkor and severe, chronic diarrhea cases. It is suggested that this serotype may represent a particularly virulent strain (105).

VI.

ECONOMICIMPACTANDOTHERCOSTS

While the reporting of foodborne disease has increased, particularly in Europe, the effect of disease on national economies and health has not been documented thoroughly. Limited attempts to document such costs have been made, but extrapolations from one or two outbreaks to an entire nation have been unreliable (106). This is unfortunate, because the failure to document the true costs leads to underfunding of research in what is one of the most important and prevalent public health problems in the United States (107). The true incidence of foodborne diarrheal disease is nearly impossible to determine, and outbreaks may go unreported for a variety of reasons. First, because of the limited duration of many bacterially mediated foodborne infections, individuals are not inclined to report them. Second, affected persons who do seek a physician’s opinion because of the severity or duration of diarrhea normally are treated symptomatically and without diagnostic tests. Third, even when the episode is severe enough to require hospitalization, the appropriate diagnostic tests may not always be performed. Fourth, by the time the epidemiology is associated with a foodborne illness, the suspected food seldom is available for analysis. Fifth; the public considers diarrhea to be a self-limiting, unpleasant nuisance, but not life-threatening and with few negative long-term health consequences (108). Hauschild and Bryan estimated the number of cases of foodborne and waterborne illness in the United States to be 1.4-3.4 million per year (109). This estimate was based on data from follow-up surveys conducted by the CDC following disease outbreaks. The

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ratio of estimated cases to those reported initially was 25:l for all foodborne diseases. Todd estimated the number of foodborne disease cases in the United States to be 5 million per year (106). In a 1977 German study, Krug estimated the cost of salmonellosis among humans to be $266 for sickness and $30 million for deaths (1 13). Morbidity accounted for 30%, mortality lo%, and undetected cases 60% of the total costs in this assessment. Costs also were expressed as proportions of the social costs as follows: 42% loss of leisure 23% welfare losses 16% losses in consumption 12% treating costs 6% examination costs 1% other costs Since 1962, estimates have been made of losses in the food-service industry due to foodborne illness. Most of thedocumented illnesses have involved Salstzonelln contamination; total costs per case ranged from $315 (1962 dollars) to $4867 (198 1 dollars) (1 13). Todd some years ago estimated the cost of all foodborne disease in the United States to be $1-10 billion annually (1 13). Included in Todd’s estimate were such economic factors as direct medical costs, lost wages and productivity, and industry loss through embargo, voluntary destruction, and recall. Because Campylobacfer j e j m i is a recently recognized, important cause of foodborne disease in the United States and the rest of the world, its economic impact can be estimated by a comparison with Salmonella incidence and prevalence. The incidence of campylobacteriosis has been reported to be 2.5 times that of salmonellosis (54). Skirrow reported that the incidence of campylobacteriosis exceeds the combined total cases of salmonellosis plus those of shigellosis (16). Archer and Kvenberg reported on work done by Hauschild and Bryan, who devised a ratio for salmonellosis of 29.5: 1 cases to isolations, based on data from postoutbreak questionnaires (109). In 1983, it was estimated that 1,147,000 cases of salmonellosis, a reportable disease, occurred in the United States, An extrapolation for the prevalence of carnpylobacteriosis would approximate 2,867,000 cases for that year. Accurately assessing the incidence of foodborne disease has posed obvious problems. In the United States, there have been recent indications that the CDC estimate, dating from the 1980s, of approximately 9000 deaths annually, is an inflated number that has not been scientifically validated. A definitive determination of the true number will have to be made if real progress in assessing risk is to be made. In the United States, an acceptable reference baseline does not exist aside from the extrapolations made by CDC epidemiologists. The Clinton administration initiative to build a new early-warning system for foodborne disease outbreaks will yield better information on which to base future prevention strategy. Increasing the number of FoodNet surveillance sentinel sites and using DNA “fingerprinting” technology to identify the source of infectious agents will enhance the process (1 10). 1996 U.S. calculations of the economic impact of campylobacteriosis, based on an estimated total 2,500,000 cases, yielded a result of $1,169.8- 1,373.8 billion annually. This result is a function, among other variables, of an estimated 200-730 deaths. Assuming that the vehicle in 55-70% of human illness cases of campylobacteriosis in the United States is food, the number of foodborne deaths in the United States attributable to Campy-

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lobctcter ranges from 110 to 5 11 per year. The annual costs of foodborne campylobacteriosis are $0.6-1 .O billion (1 11). In the United States, estimating total diarrheal disease is a continuing challenge, especially with regard to such nonreportable conditions as Camnpylobncter enteritis. The question persists: How many people seek a physician's care for diarrhea or an associated symptom (e.g., vomiting, cramps, nausea)? The answer depends on many variables that will continue to elude the resources of professionals in the field of foodborne disease. The fact that no specific methodology exists for valuing economic loss of a given illness, and that there is also no way of comparing the seriousness of specific illness, adds to the complexity of making adequate resource allocations. Economic impacts and cost profiles of such specific foodborne diseases as campylobacteriosis and salmonellosis are needed to support policy decisions to attack specific diseases or study risk and co-risk factors. We must continue to maintain an interest in developing better methods of recognizing various forms and impacts of loss and suffering from foodborne illness, living with risk factors, and invasive treatments. In summary, we must be aware that a condition like campylobacteriosis is much more than an inconvenience. It is responsible for severe illness, complications, operations, and hospitalizations for previously well persons in all age groups. Casnpylobncter enteritis is also expensive, not only for those who are ill, but for society at large, because outbreaks affect the economy adversely. Because it is a preventable disease, the cost of public health programs for the prevention and control of campylobacteriosis must be measured against the unnecessary costs of medical treatment and productivity loss.

VII.PREVENTIONANDCONTROL Prevention and control of most zoonotic diseases is difficult at best but assumes greater importance and significance when the causative agent C. jejrnni is distributed so widely among animal reservoirs and the environment. The survival of Cnmpvlobncter on fruits and vegetables also has become a subject of increasing attention and concern (112). It should be recognized that even a comprehensive, conscientious approach might not result in complete elimination of the organism. Also, resources are not available to carry out an unlimited effort against a particular foodborne pathogen like Campylobacter without including a number of other such bacteria as Yersinia enterocolitica,Aeromonns, and Listeria monocytogenes.Thus, there can be no assurance that any raw meat and poultry will be completely free of Campylobacter. Control measures are facilitated by the fact that C. jejuni is not a hardy survivor outside the host and does not survive well in food because it is sensitive to (a) drying, (b) 21% oxygen, (c) storage at 25OC, (d) acidic conditions, and (e) heat (1 15). Preventive and control measures must include the use of existing resources to reduce the level of infection in animals and subsequently in humans. Achieving this goal will require the concerted effort of livestock producers, food processors, veterinarians, physicians, public health officials, regulatory agencies, food chains (distributors and retailers), consumers, and research scientists. The livestock farmer can (a) practice sound sanitation procedures around the farm, (b) maintain disease-free animals, (c) use only feeds tested and found free of Campylobacter, and (d) prevent contamination of animals or food before or during transfer from farm to market. Cleaning and disinfection and maintaining a sanitary environment are

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essential. Changes in rearing practices, including improvement of the quality of litter, can reduce the transmission of Cnmpylobncter through poultry flocks (1 16). Administration of defined competitive exclusion cultures to chicks, colonizing them with protective bacteria, has been demonstrated to reduce colonization with C. jejuni (1 17).The implementation of disease-control principles will cost money, but the benefits of healthier livestock and the potential for establishing Cnrnpylobacter-free farms has to be a meaningful long-term objective. The food processor and distributor can assure the wholesomeness of food products by making mandatory the use of the HACCP (hazard analysis critical control point) system in their food-protection programs. The HACCP system includes elements that deal with (22):

1. Animal production controls-the wholesomeness and safety of meat and poultry are based in part on the health of live animals, their feed, and the environment in which they are raised. 2. Sanitary slaughter and dressing-the sanitary conditions during transport, slaughtering, and dressing; the rate of carcass cooling; and the time-temperature conditions of storage and distribution all have an effect on the microbial load on carcasses. 3. Processed products-cross-contamination of raw meat is minimized by assuring the cleanliness of workers’ hands, cutting boards, knives, and saws and by maintaining the condition of other utensils and equipment. 4. Raw (eviscerated, ready-to-cook) poultry-use of microbiological guidelines periodically to evaluate equipment surfaces and processing practices by examination of freshly processed carcasses. Companies should educate employees to observe correct personal hygiene principles, establish effective rodent and insect controls, limit nonessential traffic in all foodprocessing areas, and employ technically competent managers and supervisors who are conscious of and committed to the principles of food hygiene. The consumer should be conscious of basic sanitation and health practices, especially hand-washing and the use of clean equipment and cutting boards; proper methods of storing, preparing, and serving food; and the potential microbial contaminants of raw meat and poultry. The veterinarian can (a) help farmers to maintain disease-free animals and clean environments in which to raise them, (b) seek laboratory confirmation of suspected cases of campylobacteriosis in animals and report positive cases, (c) conduct sanitation inspection of the slaughter environment and product inspection to ensure an acceptable level of compliance with established standards of hygiene, (d) assist in the education of food-processing management and employees of proper sanitation practices, and (e) assist in the education of the public regarding the basic principles of food hygiene and the attributes of personal cleanliness in the prevention and control of foodborne diseases. The physician can (a) seek laboratory confirmation of suspected cases of campylobacteriosis in humans, (b) report all infections of Cnmpylobncter species to the proper health authorities and assist in the epidemiological work, when appropriate, (c) inform patients of needed precautions to take against the spread of the organism, especially to family and close contacts, and (d) assist in the education of the general public on the prevention of foodborne disease. The public health official can (a) keep local physicians informed of suspected outbreaks of campylobacteriosis, (b) follow up reported cases and investigate outbreaks, (c) take special care to obtain proper specimens for laboratory examination, (d) impress upon food processors

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the need for proper sanitation control, (e) create public awareness of campylobacteriosis, and (Q promote campylobacteriosis prevention and control (1 18). The research scientist can (a) maintain a close liaison with clinicians and epidemiologists, (b) keep abreast of the latest information applicable to Ccmpylobncter species, (c) assist in the adoption of acceptable standardized methods for isolating CnmpyZobncter species, (d) keep current with new or improved techniques for their isolation, (e) handle all cultures of Cntnpylobcrcter species with the necessary precautions to prevent spread from the laboratory, and (f) serotype all isolations and share information with collaborators and other researchers. In addition to these cooperative efforts to control the organism, innovative technologies show promise in minimizing or eliminating the Cun?pylobucterproblem. One of these, food irradiation, has been studied intensively for its antimicrobial effects. A major study showed that a medium dose of 250 kilorads (krad), combined with a handling environment temperature of 1.6"C (34.9"F). resulted in a product essentially free of bacteria that could be kept safely under refrigeration for up to 20 days. Foods irradiated at doses not exceeding 100 krad have been shown to be wholesome and safe for human consumption, requiring no safety testing to market (1 19). While irradiation can be effective in enhancing the safety of fresh products, there are some disadvantages. Irradiation can affect metabolic food processes, making the food less resistant to spoilage by various fungal diseases. Also, changes in the flavor or texture of irradiated food may be unacceptable to some consumers ( 120). The preponderance of research indicates, however, that irradiated foods are safe, can extend a product's shelf life, and can reduce the number of food spoilage microorganisms (121). Significant research has been aimed at evaluating the use of acetic acid or lactic acid in chilled water in poultry-processing plants to help produce a Snlnzonelln- or Canzpylobacter-negativepoultry supply (R. W. Johnston, personal communication, 1985). A 0.5% lactic or acetic acid wash has been observed to reduce the number of indigenous Cnnzpylobucter 10-fold in chickens (122). Studies involving the use of lactic acid and other chemical solutions on chicken inoculated with various pathogens, including C.jejuni, have shown significant reductions in the populations of these bacteria (123,124). This response to acid media in the water presumably was due to the acid sensitivity of C. jejuni, the preponderant strain in poultry (125). Other studies have found that 1.5 and 2% sprays of lactic acid or other organic acids reduce the numbers of Cnnzpylobacter and other organisms that could be isolated from pork carcasses (126,127). Recent research also has focused on the effects of trisodium phosphate treatments on poultry carcasses, with promising results ( 128,129). There are both immediate and long-term solutions to the problem of Cn~npylobcrcter. Immediate solutions involve pasteurization or cooking of animal-derived foods before consuming them and preventing cross-contamination of cooked food by raw food or surfaces and other utensils that contact raw food (130). The control of milkborne campylobacteriosis is simple because C. jejuni cannot survive pasteurization or such other forms of heat treatment as ultra-heat treatment. Freezing foods will reduce substantially the initial Ccmpylobncter population, but some organisms will survive and may remain viable for months. Although C. jejuni does not survive well in foods,refrigeration tends to prolong survival. Long-term solutions include reducing the organism's presence in food animals, developing systems to inactivate the organism in food before it reaches the retail level, and making the general public aware of the importance of food hygiene (e.g., through educational programs in the public schools).

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VIII. SUMMARYANDCONCLUSIONS Cninpylobacter jejmi was isolated first from human diarrheal stools in 1971. Since its identification as a human enteric pathogen, the organism has been recognized increasingly as one of the mostimportant causes of diarrheal disease in the UnitedStates and throughout the world. The study cited at the beginning of this chapter of Campylobncter infection on 303 U S . college campuses ( 2 ) illustrates the public health significance of the organism and highlights the need for further research. The study showed an incidence of Cnrnpylobncteer- in stool cultures of 13.2%, which exceeds the normal expected rate. C. jejmi enteritis is primarily a zoonotic disease with apparently different modes of transmission in the industrialized and developing countries. In the developed countries, the organism is spread primarily through foods of animal origin: on the other hand, evidence for the less developed countries is that fecal contamination of food and water and contact with sick people or animals predominate as the modes of transmission. These contrasting patterns of transmission are explained by the different levels of environmental hygiene in the developed and less developed countries, which have corresponding implications for international control of the organism. Transmission of the organism is minimized when the microbial environment is inhospitable to it. C. jejuni dies off rapidly at ambient temperatures and atmospheres and grows poorly in food. Food that has been mishandled, of course, can become a medium. Although milk has been cited tnost frequently as a vehicle for Carrzpylobncter worldwide, increasing attention is being paid to meat and poultry products as major reservoirs and vehicles. The principles of animal husbandry will play a significant role in controlling this ubiquitous organism. Using Canzyylobacter-free parent animals and raising Canpylobncter-free animals for slaughter will help immeasurably to protect the consumer from foodborne infection. The strictest standards of livestock hygiene and management will be required. Hygienic slaughter and processing procedures will preclude cross-contamination, while adequate cooling and aeration will cause a decrease in the microbial load. In addition, thorough cooking of meat and poultry products followed by proper storage should help in maintaining food integrity with less contamination. Campylobacteriosis is a universal problem and a challenge to all who work in the food safety arena. The solutions for control and prevention are not simple. More research is needed and close national and international cooperation between the veterinary and medical professions is imperative if progress is to be achieved in the long-term control of Cmlyylobacteer.

REFERENCES Tauxe, R. V., Deming. M. S., and Blake, P. A. (1985). Cnmpylobacter jejuni infections on college campuses: A national survey. AJPH, 75:659-660. 2. Tauxe, R. V. (1 992). Foreword: Transmission of human bacterial pathogens through poultry. In Colonization Control of Human Bacterial Enteropthogens in Poult91 (L. C. Blankenship. ed.), Academic Press, New York. Pp. xv-xxxiii. 3. Butzler, J. P., and Skirrow, M. B. (1979). Curzzpylobacter enteritis. Clin. Gastroenterol., 8: 737. l.

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29. Kaplan, R. L. (1980). Campylobacter. In Mrrnunl of Clinical Microbiology, 3rd ed. (E. Lennette, A. Balows, W. J. Hausler, Jr., et al., eds.), American Society for Microbiology. Washington, DC, p. 235. 30. Hebert, G. A., Hollis, D. G., Weaver, R. E., et al. (1982). 30 years of campylobacters: Biochemical characteristics and a biotyping proposal for Cumpylobacter jejuni. J. Clin. Microbiol., 15:1065. 31. Blaser, M. J. (1985). Campylobacter species. In Principles and Practice of Znfectious Diseases. 2nd ed. (G. L. Mandell, R. G. Douglas, Jr., and J. E. Bennett, eds.). John Wiley and Sons, New York, pp. 1221-1222. 32. Vandamme, P., Falsen, E., Rossau, R., Hoste B., Segers, P.. Tytgat, R., Ley, J. de. (1991). Revision of Campylobacter,Helicobacter, and Wolirzella taxonomy: Emendation of generic descriptions and proposal of Arcobacter gen. nov. Znt. J. Syst. Bacteriol., 41(1):88103. 33. Slurrow, M. B. (1994). Diseases due to Campylobacter, Helicobacter, and related bacteria. J. Cony. Path., 111:113-149. 34. Blaser, M. J., Taylor, D. N., and Feldman, R. A. (1984). Epidemiology of Campylobacter infections. In Campylobacter Infection in Man and Animals (J. P. Butzler, ed.), CRC Press, Boca Raton, FL, pp. 143-161. 35. Garcia, M. M., Lior, H., Stewart, R. B., Ruckerbauer, G. M., and Skijarevski, A. (1 985). Isolation, characterisation, and serotyping of Canzpylobacterjejltni and Carnpylobacter coli from slaughter cattle. Appl. Environ. Microbiol. 43:977-980. 36. Hanninen, M. L., and Raevuori, M. (1981). Occurrence of Campylobacterfetus subsp. jejuni and Yersinia enterocolitica in swine carcasses and the slaughterhouse environment. J. Food Prot., 33:441-445. 37. Svedhem, A., Kaijser, B., and Sjogren, E. (1981). The occurrence of Campylobacter jejzrni in fresh food and survival under different conditions. J. Hyg., 87:421-425. 38. Vogt, R. L., Little, A. A., Patton, C. M., Barrett, T. J., and Orciari, L. A. (1984). Serotyping and serology studies of campylobacteriosis associated with consumption of raw milk. J. Clin. Microbiol., 20:998-1000. 39. Mafu, A. A., Higgins, R., Nadeau, M., and Couisineau, G. (1989). The incidence of Salmonella, Car.~~pylobacter, and Yersinia enterocoliticnin swine carcasses and the slaughterhouse environment. J. Food Prot. 52:642-645. 40. Van D a m e , L. R. (1983). Isolation of Campylobacter jejuni from animals in Zaire. In Canzpylobacter ZZ (A. D. Pearson et al., eds.), PHLS, London. 41. Borch, E., Nesbakken, T., and Christensen, H. (1996). Hazard identification in swine slaughter with respect to foodborne bacteria. Znt. J. Food Microbiol., 30(1/2):9-25. 42. Oosterom, J., Beckers, H. J., van Noorie Jansen, L. M., and van Schothorst, M. (1980). An outbreak of Calnpylobacter infection in a barrack, probably caused by raw hamburger. Ned. Tijdsclzr. Diergeneeskd., 124:1631-1634 (in Dutch). 43. Monroe, D. L., Prescott, J. F., and Penner, J. L. (.1983). Canzpylobacterjejuni and Campylobacter coli serotypes isolated from chickens, cattle, and pigs. J. Clin. Microbiol., 18377878. 44. Oosterom, J. (1983). Carnpylobacter jejuni in poultry processing and pig slaughtering. In Campylobacter ZZ (A. D. Pearson et al., eds.), PHLS, London. 45. Rosef, D., Gondrosen, B., Kapperud, G., Underdal, B. (1983). Isolation and characterization of Campylobacter jejuni and Cmnpylobacter coli from domestic and wild mammals in Norway. Appl. Environ. Microbiol., 46:855-859. 46. Gumbrell, R. C. (1980). Preliminary Report on the Results of the First Year of a Five-Year Survey on Lamb Perinatal Mortality. Animal Health Laboratory, Lincoln, New Zealand, pp. 94-99. 47. Doyle, M. P. (1984). Association of Campylobacter jejuni with laying hen and eggs. Appl. Environ. Microbiol., 47533-536.

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48. Genigeorgis, C.. Hassuneh, M., and Collins, P. (1986). Cnnrpylobncterj e j m i infection in poultry farms and its effect on poultry meat contamination during slaughtering. J. Food Prot., 49:895, 899-903. 49. Hartog. B. J., DeWild,G. J. A., and DeBoer, E. (1983). Poultry asa source of Crtnzpylobncter 34: 116-122. (in German). jujurzi. Arch. Lebelzsmittellr?~g., 50. Casanovas, L., de Simbn, M., Ferrer, M. D., ArquCs, J., and Monzbn, G. (1 995.) Intestinal carriage of campylobacters, salmonellas, yersinias, and listerias in pigeons in the city of Barcelona. J. Appl. Bmteriol., 78:ll-13. 51. Jocobs-Reitsman, W. F., Bolder, N. M., and Mulder, R. W. A. W. (1994.) Cecal carriage of Cantpylobncter and Salmonelllr in Dutch broiler flocksat slaughter: a one-year study. Poultry Sci.. 73(8):1260-1266. 52. Wempe, J. M.. Genigeorgis, C. A., Farver, T. B., and Yusufu, H. I. (1 983). Prevalence of Cnnzp$obacter jejuni in two California chicken processing plants. Appl. Environ. Microbiol., 45:355-359. 53. Acuff, G. R., Vanderzant. C.. Hanna, M. 0..Ehlers, J. G., Golan, F. A., and Gardner, F. A. (1986). Prevalence of Canzpylobncter j e j m i in turkey carcass processing and further processing of turkey products. J. Food Prot., 49:712-717. 54. Seattle-King County Department of Health. (1984). Surveillance of the flow of Salmollella and Cnnzpylobacter in a community. Communicable Disease Control Section, Seattle-King County Department of Public Health. Contract #223817041. Report to U.S. Food and Drug Administration. Seattle-King County Department of Public Health, Seattle, WA. 55. Blaser, M. J., Wells, J. G., Feldman, R. A., Pollard, R. A., and Allen, J. R. (1983). Cnrnpylobircter enteritis in the United States. A multicenter study. Ann. Zrrt. Med., 98:360. 56. Mauff, A. C.. and Chapman, S. R. (1981). Campylobacteriosis in Johannesburg. S. Afi. Med. J., 59:217. 57. Burce, D.. Zochowski, W., and Fleming, G. A. (1980). Cunzpylobucter infections in cats and dogs. Vet. Rec. 107:200-201. 58. Crotti, D., andBoscato, U. (1983).Enteritisinthedog:Possibleetiopathogenicrole of Cnrtzpvlobncter. Obiettivi Doc. Vet., 5:51-54 (in Italian). 59. Jones, A., and Harrop, C. (1981). A study of Cnwrp?lobncter enteritis. J. Znt. Med. Res.. 9: 40-43. 60. Gurgan, T., and Diker. K. S. (1994). Abortion associated with Crrnrpylobacter rpaliensis. J. Clin. Microbiol. 32( 12):3093-3094. 61. Sjogren, E., Falsen, E., Kaijser, B., and Lindblom, G.-B. (1996). Carnpylobcrcter species in faeces from healthy pets in Sweden isolated by filter technique. In Cnnrpylobacters, Helicobacters, o?rdRelated Organism (D. G. Newell. J. Ketley, and R. A. Feldman, eds.), Plenum Press, New York. Pp. 471-473. 62. Moreno, G. S., Griffiths, P. L., Connerton, I. F., and Park, R.W. A. (1993.) The occurrence of campylobacters in small domestic and laboratory animals. J. Appl. Bacteriol., 75:49-54. 63. Altekruse, S. F., Hunt, J. M.. Tollefson, L. K., and Madden, J. M. (1994). Food and animal sources of human Carnpylobcrcter. jejzcniinfection. J. Am. Vet. Med. Assn., 20457-61. 64. Cohen, D. I., Rouach. T. M., and Rojol, M. (1984). A Cmnpylobacter enteritis outbreak in a military base i n Israel. Zsr. J. Med. Sci., 20:216-218. 65. U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control. (1983). Water-Reluted Disease Outbreaks. Arlrmnl Summni?:. CDC, Atlanta. GA. 66. Taylor, D. N., McDermott, K.T., Little, J. R., Wells, J. G.,and Blaser, M. J. (1 983). Cnnzyylobrrcter enteritis from untreated water in the Rocky Mountains. Ann. Znt. hled.. 99:38-40. 67. Gondrosen, B. (1983). Survival of thernlophilic canlpylobacters in water. In Canlpylobncter ZZ (A. D. Pearson, et al., eds.), PHLS, London. 68. Brennhovd. O., Kapperud, G., and Langeland, G.(1992). Survey of thennotolerant Cnnzpylobcrcter spp. and Yersirtia spp. in three surface water sources in Norway. Znt. J. Food Microbiol., 15(3/4):327-338.

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69. Easton, J. (1 996). Fats and transportof campylobacters in soil arising from farming practices. In Camnpylobacters, Helicobncters, and Related Orgrrnisrm (D. G. Newell, J. Ketley, and R. A. Feldman. eds.), Plenum Press, New York. Pp. 461-465. 70. Kotula. A. W., and Stern, N.J. (1984). The importance of Cantpylobncterjejuni to the meat industry: A review. J. Anint. Sci., 58:1561-1566. 71. Kwatiek, K., Wojton, B., and Stern, N. J. (1990). Prevalence and distribution of Cnnzpylobacter spp. on poultry and selected red meat carcasses in Poland. J. Food Prot., 53:127130. 72. Larmnerding, A. M., Garcia, M. M.. Manu, E. D., Robinson, Y . ,Dorward, W. J., Truscott. R. B., and Tittiger, F. (1988). Prevalence of Salnzonellu and thermophilic Cmzpylobacter in fresh pork, beef, veal, and poultry in Canada. J. Food Prot., 51:47, 51. 73. Adesiuyn. A. A. (1993). Prevalence of Listeria spp., Cnrnpylobrrcter spp., Salrnortelln spp., Yersinia spp., and toxigenic Esclzerichin coli on meat and seafoods in Trinidad.Food Microbiol., 10(5):395-403. 73. Kinde, A., Genigeorgis. C. A., and Pappaioanou, M. (1 983). Prevalence of Cnnzpylobncter jejuni in chicken wings. Appl. Emiron. A/licrobiol., 45: 11 16-1118. 75. Stern, N. J.. Green, S. S., Thaker, N., Krout. D.J., and Chiu, J. (1 984). Recovery of Cnnzpylobacter jqjrrni from fresh and frozen meat and poultry collected at slaughter. J. Food Prof., 47:37"373. 76. Turnbull, P. C. B., and Rose, P. (1982). Canzpylobacter jejuni and Salmonella in raw red meats. A Public Health Laboratory Service Survey. J. Hyg., 8829-37. 77. Flynn, 0. M. J., Blair, I. S., and McDowell, D. A. (1994.) Prevalence of Cunlpylobrrcter species on fresh retail chicken wings in Northern Ireland. .I. Food Prot., 57(4):334-336. 78. Willis, W. L. and Murray, C. (1997.) Cnnyylobcrcter jejzjlani seasonal recovery observations of retail market broilers. Pozrltly Sci. 76(2):314-317. 79. Grau, F. H. (l 98s).Ccmpylobncter jejzrrzi and Cmtpylobacter hyointesti~rnlisin the intestinal tract and on the carcasses of calves and cattle. J. Food Prot., 5 1957-861. 80. Doyle, M. P., and Roman. D. J. (1982). Prevalence and survival of Carnpylobacter jqjuni in unpasteurized milk. Appl. EmVror1. Micr-obiol., 44: 1154-1 158. 81. de Boer, E.. Hartog, B. J., and Borst, G. H. A. (1984). Milk as a source of Canzpylobucter jejuni. Netlt. Milk Dairy J., 38:183-194. 82. World Health Organization (WHO). (1984). Report of the WHO consultation on veterinary public health aspects of prevention and control of Cniizpylobncter infections in Moscow. WHO, Geneva. 83. Emory University School of Medicine, Department of Community Health. (1985). Course notes: Control of enteric infections in Third World countries. Emory University, Atlanta, Georgia. 84. Norkrans, G., and Svedhem, A. (1982). Epidemiological aspects of Cnnrpylobacter jejuni enteritis. J. Hyg.. 89:163-170. 85. Grados, O., Bravo, N., Butzler, J. P., and Ventura, G. (1983). Cnntpvlobncter infection: An occupational disease risk in chicken handlers. In Cnrlzpylobacter ZZ (A. D. Pearson et al., eds.), PHLS, London. Cnmpylobacter fetus subspecies jejurli innormal 86. Oosterom, J. (1980).Thepresenceof slaughtered pigs. Ned. Tijdscht-. Diergeneeskd., 105:49-50 (in Dutch). 87. Hutchinson, D. N., Bolton, F. J., Hinchliffe, P. M., Dawkins, H., Horsley, S. D., Jessop, E. G., Robertshaw, P. A., and Counter, D. E. (1985). Evidence of udder excretion of Campylobncterjejzmi asthecause of milkborne Canzpylobacter outbreak. J. Hyg., 94205215. 88. Brieseman, M. A. Raw milk consumption as a probable cause of two outbreaks of Canzpylobacter infection. N. 2. Med. J., 97:411-413. 89. Stalder, H., Tsler, R., Stutz, W., Salfinger,M., Lauwers, S., and Vischer, W.(1983). Contribution to the epidemiology of Canzpylobucter jejrcni. From asymptomatic excretion by a cow

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and

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in the cowshed to overt diseasein over 500 persons. Sclzwei:. Med. Wochenschr., 113245249 (in German). Porter, I. A., and Reid, T. M. (1980). A milkborne outbreakof Campylobacter infection. J. Hyg., 841415-419. Hutchinson. D.N., Bolton, F. J., Jelley, W. C.N., Matthews, W. G., Telford,D. R., Couitner. D. E., Jesop, E. G., and Horsley, S. D. (1985). Campvlobacter enteritis associated with consumption of raw goat's milk. Lancet, 1, 8436:1037-1038. Sandstedt, K. (1982). Carrzpylobcrcter transmission via food, especially milk. Statens Vet. Anstalt, Uppsala, Sweden, pp. 225-226 (in Swedish). Andrewes. C. H., and Walton, J. R. (1977). Viral and Bacterial Zoonoses. Balliere Tindall, London, pp. 18-19. Blaser, M. J., Sazie, E., and Williams, L. P. (1987). Theinfluence of immunity on raw-milkassociated Carnpylobncter infection. JAMA, 257:43-46. Mandel, B. K., De Mol. P., and Butzler, J. P. (1984). Clinical aspects of Canzpylobacter infections in humans. In Campylobacter Iizfection in M m and Animals (J. P. Butzler, ed.), CRC Press, Boca Raton, FL. Pp. 22-30. Benenson. A. S. (1980). Control of Cor)tnz~nicableDiseases in Man, 13th ed. American Public Health Association, Washington, DC, pp. 112-113. Butzler, J. P. (1982). Campylobacter enteritis. Itlfection 10 (supp1.2):567-569 (in German). Smith, C.S., and Blaser, M. J. (1985). Fatalities associated with CampyZobacterjejzmi infections. JAMA. 253:2873-2875. Archer, D. L. (1985). Enteric microorganisms in rheumatoid diseases: Causative agents and possible mechanisms. J. Food Prot., 48:538-545. Smith, J. L., Palumbo, S. A., and Walls, I. (1993). Relationshipbetween foodborne bacterial pathogens and the reactive arthritides. J. Food Sci., 13 (3):209-236. Wesley, I. (1997). Cnmpylobacter and related microorganisms incattle. In Beef Safery Synzp o s i m Proceedings, December 3-4, 1997. American Meat Science Association/National Cattlemen's Association, Chicago. Pp. 30-41. Marshall, B. J., and Warren, J. R. (1984). Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet, l , 8390: 13l l - 1315. Morey, F. (1996). Five years of Canzpylobacter bacteremia in Central Australia. In Cnq$obacters, Helicobacters. and Related Organisms (Newell et al., eds.), Plenum Press, New York. Pp. 491-494. Fennell, C. L., et al. (1984). Characterizationof Campylobncter-like organisms isolated from homosexual men. J. Irzfect. Dis. 149(1):58-66. Lastovica, A. J. (1996). Serotype distributionof isolates of C.jejzsni subsp. jejuni, C. jejuni subsp. doylei, C. coli, and C. upsaliensis from paediatric enteritis patients. In Campylobacters, Helicobacters, alld Related Organism (D. G. Newell et al., eds.), Plenum Press. New York. Pp. 481-485. Todd, E. C. D. (1985). Economic loss from food borne disease outbreaks associated with foodservice establishments. J. Food Prot., 47:322-328. Levy, B. S., and McIntire, W. (1974). The economic impact of a foodborne salmonellosis outbreak. JAMA, 230:1281-1282. Archer, D. L. (1984). Diarrheal episodes and diarrheal disease: Acute disease with chronic implications. J. Food Prot., 47322-328. Archer, D. L., and Kvenberg, J. E. (1985). Incidenceand cost of foodborne diarrheal disease in the United States. J. Food Prot., 48:887-894. U.S. Food and Drug Administration, U S . Department of Agriculture, U.S. Environmental Protection Agency, Centers for Disease Control and Prevention. (1997). Food safety from farm to table: A national food safety initiative. Report to the President, May 1997. Buzby, J. C., Roberts, T., Lin, C.-T. J.. and MacDonald, J. (1996). Bacterial Foodborne

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Clostridium botulinum John W. Austin and Karen L. Dodds Health Canada, Ottawa, Ontario. Cunncla

I. Introduction

108

11. The Organism: Classification and Characteristics

111. Epidemiology

IV.

V. VI.

VII.

108

109

A. Occurrence of spores in the environment 109 B. Occurrence of spores in foods 111 C. Incidence of botulism in North America 113 D. Incidence of botulism in Europe 115 E. Incidence of botulism in Asia 115 F. Incidence of botulism in other areas 116 G. Infant botulism 117 Clinical Aspects 118 118 A. Symptoms 119 B. Treatment C. Diagnosis 119 120 D. Case history 120 E. Prevention Detection 121 Neurotoxin 121 A. Structure 122 B. Mode of action 122 C. Therapeutic uses 123 Control in Foods 123 A. Factors affecting growth and toxin production 123 B. Thermal Inactivation 126 C. Inactivation by irradiation 126 References 127

108

I.

Austin and Dodds

INTRODUCTION

The species Clostridiun~botsrlinzun comprises gram-positive, anaerobic, rod-shaped, spore-forming bacteria. They are distinguished from other bacteria by the production of the most potent biological toxin known, botulinum neurotoxin (BoNT). The lethal dose for humans is estimated to be in the range of 0.1-1 ng/kg (l). Strains of C. botulim~rn are classified into seven types, A through G, depending on the serological specificity of the neurotoxin produced. Foodborne botulism, the most common form of botulism worldwide, is caused by ingestion of food contaminated with preformed BoNT, usually type A, B, or E. Because of the severity and life-threatening nature of this disease, C. botulimm has been extensively studied. Indeed, botulism has been described as “an archetype of catastrophic illness” (2). Originally, botulism was associated with sausages in central Europe (3). Presently, a wide variety of foods have been implicated in botulism, including such unusual products as hazelnut-flavored yogurt (4), garlic in oil (5), commercial cheese products (6,7), vacuum-packaged clam chowder, and commercial bean dip (8). However, the largest problem worldwide remains foods preserved in the home, whether from home canning, home curing, or home fermentation. Human botulism is currently classified into four categories. Foodborne botulism is caused by ingestion of food contaminated with preformed BoNT. Wound botulism is very rare and is due to infection of a wound with spores of C. botulirzurn, which grow and produce toxin in situ (9,lO). Infant botulism, first recognized in 1976 (11,12) and now the most common form of botulism in the United States, is caused by ingestion of viable spores that germinate and colonize the intestinal tract of infants under one year of age and are responsible for the local production of toxin. The fourth category, unclassified, includes cases of unknown origin and adult cases which resemble infant botulism (13). Animal botulism, which will not be discussed further, is typically caused by types C and D.

II. THEORGANISM:CLASSIFICATIONAND CHARACTERISTICS C. botulinum was defined in 1953 as the species designation for all organisms known to produce BoNT (14), although recent studies have identified strains of Clostridiunz baratii ( 1S ) and Clostridium butyricum(1 6- 18) as capable of producing neurotoxin (19). Strains of C. botulirzum are separated into seven types, A through G, based on the serological specificity of the toxin produced. Although serologically different, the toxins are structurally similar and have the same biological effect. Based on physiological differences, the species is also divided into four groups (designated Groups I to IV) (19,20). The classification of strains of C. botulin~rminto four physiological groups also agrees with results of nucleic acid hybridization and 16s ribosomal RNA sequencing studies (21-23). Group I strains are proteolytic, produce spores of high heat resistance, and have a minimum growth temperature of 10°C. This group includes all type A strains, which are all proteolytic, and proteolytic strains of types B and F (both of these types may be proteolytic or nonproteolytic). Strains in Group I1 are nonproteolytic, produce spores of a low heat resistance, and are capable of growing at refrigeration temperatures. Included in Group I1 are all type E strains, which are all nonpro-

Clostridium botulinum

109

teolytic, and nonproteolytic strains of types B and F. Group I11 includes type C and D strains. This group is generally nonproteolytic and, since it is not involved in human botulism, little is known regarding its heat resistance. Group IV includes type G strains and is classified as a distinct species, C. nrgerztinerzse (24). The emphasis in this chapter will be on Groups I and I1 since they are involved in human illness.

111.

EPIDEMIOLOGY

A.

Occurrence of Spores in the Environment

Fundamental to a knowledge of the potential botulism hazard from any food is a knowledge of thepossibility of contarnination of the food with C. botulinum. This contamination depends largely on the distribution and incidence of C. botulirzum in the environment (25). The serious nature of botulism has led many investigators worldwide to conduct surveys to establish the presence or absence of C. botulimrn in different environments. Summaries of these surveys are presented here, but readers should refer to the original publications or other reviews (25,26) for details regarding sampling. Spores of C. botulinum are commonly present in soils and sediments, but their numbers and types vary, depending on the location (Table 1). C. botulinum is often described as a soil organism, ubiquitous in nature, but this does not reflect veryaccurately its distribution in the environment. As the survey data show, spores of C. botuZinurn are spread widely in the environment, but the factors governing the geographical distribution of spores and the different types are still not understood well. C. botulinum spores are distributed widely in North America, but the spore load varies considerably, as does the predominating type (Table 1) (25). Soils in eastern and central United States usually contain type B spores, usually proteolytic, while type A spores predominate in the west (27). Type E is found infrequently, and only in damp to wet locations. However, in the region around the Great Lakes, and particularly around Green Bay of Lake Michigan, high numbers of type E are found in shoreline and sediment samples (28). Type E is also found in the coastal areas of Washington and Alaska (29,30). Type B predominates in the terrestrial environments of Britain, Ireland, Iceland, Denmark, and Switzerland (Table 1) (25). It is associated with most botulistn outbreaks in Spain, Portugal, Italy, France, Belgium, Germany, Poland, Hungary, and the former Czechoslovakia and Yugoslavia (3l), indicating wide distribution of this type in the European environment. Type B also predominates in the aquatic environtnents of the United Kingdom. Most European type B strains are nonproteolytic. The predominant serotype from all other aquatic environments studied in Europe is E. High numbers of type E spores are found in Scandinavian waters, particularly in the Sound and Kattegat, between Denmark and Sweden. C. botulirzurn type E is also prevalent in samples from the Baltic sea (32,33). The highest overall spore concentrations detected in Europe were reported in the Netherlands, where a mixture of types was found. Type E spores also predominate in most parts of the former U.S.S.R., except for the central portion, where type B spores are found most frequently (Table 1) (34). In general, surveys of Asia report lower numbers, with the exceptions of a high incidence of type E spores around the Caspian Sea (35), and a very high incidence of types A and B in the Xinjiang district of China (36,37). Types A, B, E, and F are found in areas of China north of 30" latitude, while types C and D are more common in parts of China south of 30" latitude (36). The incidence of type E spores is also high in much of northern

Austin and Dodds

I10 Table 1 Soiland Sediment Surveys for C. botztlinzrnz

Location

9% of Sample samples MPN size (g) positive per

North America Eastern U.S., soil 10 Western U.S.. soil 10 1 Great Lakes, soil S Green Bay, sediment 5 Washington coast, sediment l Alaska coast, soil Europe 50 Britain, soil 2 Britain coast, sediment Ireland, sediment 50 10 Iceland, soil 10 Faroe Islands coast, sediment Greenland coast, sediment 10 Scandinavian coast, Skagenak, 6 Kattegat. sediment 5 Norway coast. soil Sweden coast, soil 3-6 Denmark coast. sediment 10 10 Denmark, soil 0.5 Netherlands, soil Poland. Baltic coast, soil 5 Finland freshwater 300 300 Baltic coast Baltic offshore 500 Czechoslovakia, sediment 12 Switzerland. soil Italy. Rome, soil 7.5 Asia Former United Soviet Socialist Republic, soil Northeastern Southeastern Central Northwestern Far East 2 Iran, Caspian Sea. sediment China, Sinkiang. soil 10 Taiwan, soil 10 Japan. Hokkaido, soil 5-10 Japan, Ishikawa, soil 40-50 Japan. Honshu. sediment 5 Southem Hemisphere Brazil, cultivated soil 5 Paraguay, soil 5 Argentina, soil

19 29 2 77 71 41

Type kga A

B

(%'C)h

C/D

Ref. 27 27 28 28 29 30

64 16 0 0 3 0

12 14 0 0 0 0

12 8 100 100 96 100

0 0 0 0 0 0

100 100 100 100 0 0

0 0 0 0 0 0

0 0 0 0 100 100

0 158 0 159 0 160 0 41 0 41 0 41

0

100 100 99 100 0 32 97

0 32 0 32 0 32 0 41 0 41 0 161 0 162

0

100 100 100 0 0 0

0 33 0 33 0 33 0 163 27 164 0 165

0 86 0 73 6 0 86 0 5 87 92 0 19 2 3 20 0 100 100 0 9 33-82

0 34 0 34 0 34 0 34 0 34 0 35 0 37 5 166 0 167 0 40 0 168

4 3 l 46

0 0 0 0 0 0

100 7 84 67 13 94 32

>780 7 410 110 15 2500 76

0 0 0 0 0 0 3

0 0 93 22 0

0 0 1 0 7 46 0

61 58 88 32 34 1

372 500 1020

0 0 0 0 48 28 2 86

0 0 0 0 83 l4

0 0 100 6 0

0 8 16 14 0 0

14 17 77 0 8 8

0 0 0

0 0 7

86 57 10 14 67

7 0 20

35 24 34

F

21 12 33 62 24 0 1280 0 250 1 660 0

6 4 18 3 1 37

2 13 14 4 14 S7 70 78 4 56

E

2 18

93 25000 3247 373430 4 16

0

29 14 0

0 0 0

7 71 5

43 45 44

Clostridium botulinum

111

Table 1 Continued

Location Kenya. soil South Africa, soil Bangladesh, sediment Thailand. Hua-Hin, sediment Indonesia, sediment Java coast, sediment New Zealand, sediment

5% of Sample samples MPN size (g) positive per kg' A 5 30 10 10 50 10 20

25 3 37 3 21 13 55

Type (%)" B

0 33 S9 1 0 100 0 19 0 0 3 0 0 5 0 14 0 33 0 40 0

MPN calculated using the Halvorson-Ziegler equation In(n/q).where n and q = the number o i nontoxic samples. h As percentage of the types identified. Source: Adapted from Ref. 26.

=

C/D 11 0 100 83 100 67 100

E

F Ref.

0 0 0 17 0 0 0

0 46 0 47 0 41 0 43, 0 169 0 170 0 171

the total numberof samples analyzed

Japan (38,39). In other areas of Japan, the distribution of types is different, and type C predominates (40). In the tropical regions of Asia, types C and D replace type E as the predominant type in aquatic environments (41,42). Fewer surveys have been carried out in the Southern Hemisphere (see Table 1). Type A spores predominate in Brazilian and Argentinian soils (43.44). In Paraguay, the prevalent spore type was F, followed by A and C (45). In Africa, few surveys have been done, but one following an outbreak in Kenya found type A and C spores in soil (46), whereas in South Africa only type B spores were found (47). In summary, type A spores predominate in soils in the western United States, China, Brazil and Argentina, and type B spores in the eastern United States, the United Kingdom, and much of continental Europe. However, most American type B strains are proteolytic, while most European strains are nonproteolytic. Type E is the predominant type in northern regions, and in most temperate aquatic regions and their surroundings. Types C and D are found more frequently in warmer environments. The reasons for this distribution pattern are not understood well. Type A appears to be favored by neutral to alkaline soil with low organic content: this is consistent with its virtual absence in the highly cultivated soils of the eastern United States and Europe. Type E is psychrotolerant, which undoubtedly plays a role in its prevalence in the North and in many aquatic environments. Sirnilarly, the higher optimum growth temperatures for types C and D are consistent with their presence in warmer environments. B. Occurrence of Sporesin Foods The sequence of events resulting in foodborne botulism begins when C. botulitlrm contaminates a food. This often occurs during growth or harvesting, especially if a product originates in an environment with a high incidence of spores. However, contamination can also occur during or after processing. The presence of C. botulir~zm,mostly type E, in fish is demonstrated readily (see Table 2), although the incidence is lower than in environmental surveys (48). The level of contamination of prepared fish in the United States appears to be higher than in Europe. Numbers vary in Asia, with a particularly high result noted in fish from the Caspian Sea (35).

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Table 2 Incidence of C. botulinnm Spores in Prepared Fish Product Whitefish chubs eviscerated fresh smoked Frozen flounder, vacuumpacked Frozen, packaged fish Filleted fish Smoked fish Dressed rockfish Fish and seafood Salmon Vacuum-packed fish Vacuum-packed fish Smoked herring Smoked salmon Smoked eel Salted carp Smoked carp Rainbow trout eviscerated intestines Fish Fish and seafood

Origin

Sample 7o positive MPN size (g) samples per kg

Types identified

Ref.

Great Lakes

Atlantic Canada New York City Pacific NW California California Alaska England Viking Bank Sweden Denmark Baltic Sea Caspian Sea Caspian Sea Finland Finland Indonesia Osaka

10 10 1.5

100 5-10 10 70 24-36

20 20 2 2

1-5 5 30

13 1 10
20 63 5 0 0 3

8

128 128 172

14 1 70


173 130 174 175 176 131 177 177 178 179 180 35 35

0 0 6 3

181 181 182 183


A, B, C, D, F C, D

MPN determined by authors. All lots examined were contaminated. Soz4rce: Ref. 48. h

The level of contamination of meats is generally low (see Table 3) (48). These products are less likely than fish to be contaminated with spores prior to slaughter of the animals since the farm environment is generally less contaminated than the aquatic environment. Studies indicate a very low incidence of C. botrrlirzurn spores in meats in North America, where the average most probable number (MPN) is approximately equal to 0.1 spore per kg, while the incidence in Europe, where the average MPN is approximately equal to 2.5 spores per kg, is somewhat higher. The types most often associated with meats are A or B. C. botulinum often is present on fruits and vegetables, particularly those in close contact with the soil (49). Types A or B usually are identified. Products in which contamination has been detected include asparagus, beans, cabbage, carrots, celery, corn, onions, potatoes, turnips, olives, apricots, cherries, peaches, and tomatoes (48). A product of particular concern due to the high number of spores found is cultivated mushrooms, in which up to 2.1 X 10’ type B spores per kg have been detected (50). Spores in honey and other infant foods pose a unique hazard because, in some infants, the spores are able to colonize the intestines, produce toxin, and cause infant botulism. Honey is the only food implicated in infant botulism. Surveys for the presence of C. botulinum spores in honey suggest that the botulinum spore level in random samples

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Table 3 Incidence of C. botulinunz Spores in Meat and Meat Products Product Raw meat Cured meat Vacuum-packed bacon Cured meat Liver sausage Raw pork Vacuum-packed bacon

Sample size (g)

% positive

Origin North America North America Canada Canada Canada United Kingdom United Kingdom

3 30 75 75 75 30 25-175’’


samples

2 0-14b 4-73’

MPN per kg 0.1 0.5 0.1 0.2 0.2 <0.1-5” 1.0-7”

Types identified Ref.

c A, B A or Ba A A A, B, C A, B

184 185 186 186 187 188 189

Not specifically typed. Variation found on different sampling occasions. Sowce: Ref. 48. a

of honey is in the order of 1-10 per kg (48). However, in honey samples associated with infant botulism, the level is approximately lo4spores per kg (51-53). While C. botulinum has been detected in such other infant foods as corn syrup (54) and rice cereal (52), exposure of infants to botulinum spores via these foods seems to be minimal because the levels are low and unlikely to increase during manufacture and storage. Other foods exanlined, including dairy products (55), vacuum-packed products (56), fresh-cut packaged salads (57), and convenience foods (58), show a very low incidence of C. botulinum spores.

C. Incidence of Botulism in NorthAmerica Unlike other foodborne diseases, botulism outbreaks are defined as incidents involving one or more botulism cases. Table 4 shows the data for countries that have reported relatively frequent outbreaks. The accuracy of these data depend uponhow thorough the reporting system records incidents of botulism. As with other foodborne diseases, unrecognized and misdiagnosed cases of botulism occur, as evidenced by a 1985 outbreak in Vancouver, Canada, in which the initial diagnoses for 28 patients included psychiatric illness, viral syndrome, laryngeal trauma, overexertion, and a variety of other maladies (5). Similarly, initial diagnoses for patients in a 1993 outbreak in Georgia included labyrinthitis, transient ischemic attack, allergic reaction, mild glaucoma, and astigmatism (6). As well, in large areas of the world, particularly where botulism occurs infrequently, epidemiological data are scarce. In Canada, most botulism outbreaks have occurred in northern native communities (3 1). The foods involved were mainly such traditional native dishes as raw and parboiled meats from sea mammals, such fermented meats as muktuk (meat, blubber and skin of the beluga whale), and fermented salmon eggs. Type E was implicated in the majority of incidences. Toxin production in these outbreaks was attributed to keeping meats at ambient temperature for some time, often to attain a desired decomposition (59). The problem with the so-called fermented products urraq (seal flippers in oil), muktuk (meat of the beluga with blubber), nlicerak (mostly seal or whale fat), and ogsuk (mostly seal oil) is that the level of fermentable carbohydrates is too low to ensure a sufficiently rapid pH reduction to prevent growth of C. botulinum. Commercial products have been implicated in only four incidents in Canada since 1971 (31,53) involving bottled marinated mushrooms im-

Dodds 114 Table 4

and

Austin

Recorded Outbreaks of Foodborne Botulism

Countly Poland China United Statesd Italy France Japan U.S.S.R. Iran Canada Spain West Germany Alaska East Germany Hungary Portugal Norway Czechoslovakia Argentina Yugoslavia Belgium Denmarkg

Period 1984-87 1958-83 197 1-90 1979-87 1978-89 1951-87 1958-64 1972-74 1971-91 1969-88 1983-88 1971-88 1984-89 1985-89 1970-89 1961-90 1979-84 1980-89 1984-89 1982-89 1984-89

No. of outbreaks

No. of casesa

Predominant typeh

1301 986 286

1791 (3) 4377 (13) 619 (15) 310 304 (2) 479 (23) 328 (29) 314 (11) 213 (15) 198 (6) 154 (4) 117 (6) 52. (8) 57 (2) 80 (0) 42 (7) 20 (0) 36 (36) 51 25 (4) 16 (12)

B

-e

175 97 95 -e

87 63 63 48 33 31 24 19 17 16 12 11 11

A

A B B E B E E B B E B B B B, E B A

B E

Predominant food type' Meats Vegetables Vegetables Vegetables Meats Fish Fish Fish Meatst Vegetables Meats Fish Meats Meats Meats Fish Meats Vegetables Meats Meats Meats

Fatality rate shown in parentheses. Of outbreaks with type identified. c Of outbreaks with the food vehicle identified (vegetables includes fruits). Includes Alaskan data (mostly traditional lnuit meat dishes). e Number of outbreaks unknown. ' Mostly traditional lnuit meat dishes. e Greenland data included. Sorrrce: Ref. 31.

ported from the United States, bottled garlic in oil, also imported from the United States but temperature-abused locally, in-house bottled wild mushrooms, and commercial pati. Bottled garlic in oil also caused an outbreak in 1989 in the United States (60) and, as a result of these two outbreaks, garlic in oil can now be sold in North America only if a second barrier (e.g., acidification) in addition to refrigeration is present. In Canada, fatality rates have decreased from 58% during the 1950s to 10% in the 1990s. In Alaska, the situation is similar to that in northern Canada (61). All of the Alaskan outbreaks involved native Alaskans; they had ingested raw or fermented native foods, and type E was usually implicated. In the continental United States, the situation is quite different (31). Home-preserved vegetables are the foods most often implicated, and most outbreaks are caused by type A. Type E accounts for less than 5% of the outbreaks in the continental United States. Commercial foods seldom have been involved, but five outbreaks were associated with food service establishments, involving 130 cases from 1977 to 1989 (62). Temperature abuse of either food ingredients or the final product was often the problem. For example, one outbreak was caused by preparing a potato salad using leftover baked potatoes that

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had been held for up to 5 days at ambient temperature wrapped in aluminum foil (63). Temperature abuse of a commercial, canned cheese sauce resulted in a botulism outbreak in Georgia in 1993, involving eight cases and one fatality (6). Fatality rates in the United States have shown a steady decline, from 34% during the 1950s to 7% inthe 1980s. Fatalities due to type E showed the greatest decline, primarily due to the general distribution of type E antiserum, which first became available commercially in 1964.

D. Incidence of Botulism in Europe By far the highest number of cases of any country in the world reported on an annual basis is in Poland (see Table 4) (31). This probably reflects both a high local incidence of botulism and a very thorough surveillance system. In Poland and several other European countries, including France, Germany, Hungary, Portugal, the former Czechoslovakia, and Belgium, the predominant type involved was B and the foods most often implicated were such home-preserved meats as ham, fermented sausages, and canned products. A significant number of implicated foods were of commercial origin in Poland (25%), the former East Germany (27%), and Belgium (38%). In France, where 12% of implicated foods were of commercial origin, the manufacturers were generally small, local establishments. In Italy and Spain, type B also predominates, but the most commonly implicated foods were home-preserved vegetables. In Italy, most of the vegetables had been preserved in oil. A notable exception to botulism caused by home-preserved vegetables in Italy is a recent outbreak involving eight people in southern Italy who had consumed commercial acidified dairy cream (mascarpone cheese) (7,64). All patients required artificial ventilation, and one died. In Spain, all of the incriminated vegetables had been canned. Scandinavian countries recorded fewer outbreaks, and these mainly were associated with fish and type E. Botulism outbreaks have been rare in the United Kingdom; however, a large outbreak in 1989 involved 27 cases with 1 death (4). Type B toxin had been produced in an underprocessed hazelnut puree, which was then used to flavor a yogurt produced by a local dairy. Other European countries, including Austria, Switzerland, Bulgaria, and Greece, either reported few or no botulism outbreaks (3 1). There is no recent summary on botulism outbreaks in the former U.S.S.R. because epidemiological workon botulism there was discontinued (31). During the period of 1958-64, types A, B, and E were implicated in approximately equal numbers of outbreaks, with a slight predominance of type B (see Table 4). The type B outbreaks occurred mostly in the western region of the former U.S.S.R. and usually involved meats, nearly always home-cured ham, very similar to the situation in central Europe. Incidents associated with fish occurred mainly in regions around the Black Sea, the Sea of Azov, the Caspian Sea, and Lake Baikal. Surprisingly, type A was involved in as many outbreaks from fish as type E. The news agency TASS recently reported an outbreak of type E botulism in Irkutsk involving 12 cases and 1 death that was caused by smoked and salted omul fish.

E. Incidence of Botulism in Asia Foodborne botulism has been reported from only a few countries in Asia (see Table 4) (31). Israel reported an outbreak in 1987 that affected six people with one fatality (65). The incriminated food was kapchunka (salted, uneviscerated whitefish) contaminated with type E toxin. It had been bought in New York City and transported to Israel unrefrigerated. Iran reported a high incidence of botulism. The majority (97%) of investigated outbreaks

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was associated with type E and with fish or fish products. However, fish fleshwas responsible for only 10% of the outbreaks; the other 90% were caused by fish eggs. These eggs (ashbal) are salt-cured for several months and then eaten without further treatment. China recorded almost 745 outbreaks, involving 2861 cases and 421 fatalities, from 1958 to 1989 (36). Most were associated with type A, followed by types B and E. The northwestern province of Xinjiang recorded the majority of outbreaks (-80%), which were usually type A. Typically, vegetable products were incriminated, particularly fermented bean curd (74% of outbreaks) and fermented bean sauce (12%). The few incidents reported from the northeastern region were all associated with type E, but fish were not identified as the causative food. The only outbreak of foodborne botulism caused by neurotoxigenic C. butyric~rnoccurred in Guanyun, Jiangsu province in China. An isolate producing type E BoNT and giving the lipase- and lecithinase-negative colonies typical of C. bu~ricurnwas isolated from the salted and fermented paste of soybeans and wax gourds implicated in the outbreak (18). The majority of Japanese outbreaks occurred in northern areas and were associated with type E and fish or fish products. Izushi was the food most often implicated. To prepare izushi, fleshy pieces of fish are soaked in water for a few days, and then packed tightly into a tub with cooked rice, vegetables, salt, vinegar and spices and left to ferment, often for 3 weeks or longer. Izushi is eaten without further cooking. Two outbreaks in Japan were associated with commercial food. In one, vacuumpackaged, stuffed lotus rhizome caused a type A outbreak and involved 36 cases with 11 deaths. The second such outbreak was caused by imported bottled caviar with 21 cases and 3 deaths and was due to type B toxin. Taiwan also has reported a few botulism outbreaks, including an unusual one involving commercially canned peanuts (66).

F. Incidence of Botulism in Other Areas Argentina is the only country in the Southern Hemisphere to report a substantial number of botulism outbreaks (Table 4) (3 1). These occurred mainly in the provinces of Mendoza and Buenos Aires and between 30" and 40" south latitude. Most outbreaks were associated with type A, and the implicated foods were usually vegetables. For the period 1980-1989, the reported mortality rate was high, 36%, likely due to the frequent involvement of type A (67). Mexico, Guatemala, Venezuela, Peru, Brazil, and Chile are the other Latin American countries that have reported outbreaks. Chad, Egypt, Kenya, Madagascar, and Rhodesia are the only African countries with reported outbreaks (3 1). The first cases of type E botulism in Egypt were reported in 1991 (68). The outbreak, which involved 91 hospitalized patients and 18 deaths, was associated with ungutted, salted, uncooked fish called faseikh. In Kenya, two outbreaks were caused by native foods, one by sour milk prepared in a gourd and one by consumption of raw termites, considered a delicacy by several tribes. The termites were consumed, either raw or fried, after being kept for 3 days in a closed polyethylene bag (69). One of the two outbreaks reported from Madagascar was unusual because type E toxin was associated with a meat product (70). About 60 people were involved with 30 deaths, and locally manufactured bologna was the vehicle. Since 1942, Australia has recorded only six outbreaks of foodborne botulism, five due to vegetables and one due to canned tuna; the last reported outbreak was in 1992 (71). One outbreak of type A botulism was recorded in New Zealand, caused by homebottled fermented mussels and watercress, a traditional Maori dish (72).

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G. InfantBotulism Since the first two cases of infant botulism were diagnosed in the United States (1 l), over 1000 cases of infant botulism have been reported around the world, predominantly (more than 90%) from the United States (see Table 5) (73), where the annual number of infant botulism cases now exceeds the number of foodborne and wound botulism cases combined (74). California, Utah, and Pennsylvania report more than half of the cases (74). This distinct geographic disparity probably reflects both regional variation in physician awareness and the worldwide distribution of C. botulinum spore types. The disease, which affects otherwise healthy children usually less than 1 year old, is characterized by constipation, generalized weakness, and various neurological disorders (75,76). While most patients require hospitalization, fatal cases are rare. After its recognition asa disease, it quickly became evident that infant botulism presented a new pathogenic role for C.botulinum. The nature of the foods most often associated with foodborne botulism usually preclude the involvement of infants. The etiology of infant botulism involves ingestion of clostridial spores, apparently most often from the general environment, and germination, multiplication, and toxigenesis in the infant’s intestines. The only food implicated thus far is honey, which, as discussed in the section on the occurrence of spores in food, occasionally carries C. botuZinum spores in large numbers. Originally limited to C. botulirzum types A and B, the toxin types and organisms associated with the disease now include types E and F and toxigenic C. b u ~ r i c u mand C. bnrutii (16,17,77,78). There are several lines of evidence suggesting that the greatest exposure of infants to spores is from the environment. Since C. botulinum spores are widely distributed in soil and dust, people are always at some risk of ingesting them. Spores adhering to the surface of fresh fruit and vegetables may be ingested and, in the case of infants, spores in dust or dirt adhering to such toys as rattles that infants commonly put in their mouths

Table 5 Worldwide ReportsofInfantBotulism of

Country Argentina Australia Canada Chile Czechoslovakia Denmark France Hungary Italy Japan Spain Switzerland Sweden Taiwan United Kingdom United States

Number of cases infant botulism

Number of fatalities

>50

6 0 0 0 0 0 0 0 1 0 0 0 0 0 0

6 6 1 1 1 1 1

6 15 2 1 1 1 5 876a

Number of cases from 1986 to 1996.

?

Toxin type

Ref. 190, 191 81, 192-194 53, 195-197 198 199 200 20 1 202 16, 203-205 206-2 13 214, 215 216 217 218 2 19-222 76, 223

I18

Dodds

Austin and

maybe ingested. However, it appears that healthy children and adults are completely resistant to this form of exposure to C. botr~lirlrm,whereas many infants are not (79). A strong association between worldwide distribution of spores types and the incidence and type associated with infant botulism is best demonstrated in the United States. Type A cases predominate in the West while type B cases predominate in the East, reflecting the known distribution of spore types in the soil. This also is evident in Argentina, where all cases have been type A, the prevalent type in the soil. As well, more direct studies have implicated dust and dirt in the environment as the source of spores causing infant botulism. In every instance where C. botulirwrl was isolated from a case in California, the environmental isolate had the same toxin type as the clinical isolate (79,80). In Australia, C. botdirzm was isolated from environmental samples associated with three of four cases, and in each case the toxin type was the same as that isolated from the patient (81). Since one of the earliest studies of infant botulism implicated honey as the source of C. bofulirturrz spores (12), honey continues tobethe only food implicated in infant botulism (76). Spores of C. botrdimm have been isolated repeatedly from leftover honey fed to afflicted infants, and the spores' toxin types consistently matched that of the isolates from the infants' stools ( 5 3 3 ) . The California Department of Health Services now distributes a pamphlet advising against giving babies honey during their first year. This is the position of Health Canada, the U.S. Centers for Disease Control, the American Academy of Pediatrics, and the Sioux Honey Association of the United States, the world's largest honey producer.

W . CLINICALASPECTS A. Symptoms Foodborne botulism may vary from a mild illness, which may be disregarded or misdiagnosed, to a serious disease, which may be fatal within 24 hours (20). The onset of symptoms typically occurs 12-36 hours after ingestion of toxin, with a range from a few hours to 14 days. In general, the earlier symptoms appear, the more serious the disease. The first symptoms are generally nausea and vomiting, followed by neurological signs and symptoms, including visual impairments (blurred or double vision, ptosis, fixed and dilated pupils), loss of normal mouth and throat functions (difficulty in speaking and swallowing: dry mouth, throat, and tongue: sore throat), general fatigue and lack of muscle coordination, and respiratory impairment. Other gastrointestinal symptoms may include abdominal pain, diarrhea, or constipation. Diarrhea occurs early in the course of the disease, whereas constipation persists in the advanced stages. Nausea and vomiting appear more often in cases associated with type B and E than with type A. Dysphagia and muscle weakness are more common in outbreaks of types A and B than of type E. Dry mouth, tongue, and throat are observed most frequently in type B cases. Type E botulism tends to have the most rapid onset of symptoms, while type A botulism tends to be the most severe, as measured by intubation rates of patients (82). Respiratory failure and airway obstruction are the main causes of death. Fatality rates in the first half of the century were about 50% or higher, but with the availability today of antisera and modern respiratory support systems, they have decreased to about 10%. Botulism is often confused with other illnesses, including other forms of foodborne poisoning, myasthenia gravis, and carbon monoxide poisoning, but most commonly with

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119

Guillain-Barr6 syndrome. The neurological signs of botulism appear first in the cranial nerve area (eyes, mouth, and throat) and then descend; in Guillain-Barr&,the syndrome progresses in an ascending fashion, beginning in the extremities. The initial symptoms of infant botulism are less clear-cut. The most common, and usually the earliest, symptom is constipation (73). Medical attention is generally requested several days to a week later. The infants usually show a generalized weakness and a weak cry. Other symptonls may include feeding difficulty and poor sucking, lethargy, lack of facial expression, irritability, and progressive “floppiness.” Respiratory arrests occur frequently but seldom are fatal.

B. Treatment Initially, treatment of foodborne botulism is directed toward removing or inactivating the toxin by (a) neutralizing circulating toxin with antiserum, (b) enema or treatment with cathartics to remove residual toxin from the bowel, and (c) in the absence of vomiting, gastric lavage or treatment with emetics (26). Treatment with antiserum is most effective in the early stages of the illness. The impact of antiserum is obvious from the Chinese data; prior to the availability of antisera in 1960, the death rate in China was approximately 50%, but it was only 8% in the nearly 4000 patients who received antitoxin. Subsequent treatment is mainly to counteract the paralysis of the respiratory muscles by artificial ventilation. Optimal treatment for infant botulism consists primarily of high-quality supportive care (73). Approximately 25% of all affected infants require mechanical ventilation, and many require gavage feeding. The use of equine antitoxin and antibiotics are not recommended and do not appear to affect the course or outcome of infant botulism. A trial study using human botulism immune globulin to treat infant botulism began in 1992 and was completed in 1997 (83,234). Treatment with botulism immune globulin reduced hospital stay, intensive care unit stay, ventilator time, and tube feeding time.

C.

Diagnosis

The initial diagnosis of foodborne botulism is based on the patient’s signs and symptoms and, perhaps, food history. Outbreaks in which more than one patient presents with typical symptoms and have a common food history are recognized quickly, even in areas where botulism is not frequent, such as the massive outbreak in Egypt (68). However, the difficulties in diagnosing botulism were highlighted by the 1985 outbreak in Vancouver, Canada, in which the initial diagnoses for 28 patients included psychiatric illness, viral syndrome, laryngeal trauma, overexertion, and a variety of other maladies (5). This outbreak was particularly difficult to diagnose due to the slow onset of rather mild symptoms and the geographic distances between patients. The clinical diagnosis should be confirmed by detecting toxin or viable C. botulimnz in a suspect food or clinical sample or by epidemiological association with a laboratory-confirmed case (36). Serum, feces, enema fluid, stomach contents, and autopsy sections of the small and large intestines and of the liver are suitable specimens for toxin detection. Except for serum, these specimens are also suitable for detecting viable C. botulinzm. Diagnosis of infant botulism is difficult due to the lack of specific symptoms and variation in severity (73,76). It also requires identification of C. b o r ~ d i r rtoxin ~ ~ ~in sel’un1 or toxin and/or spores in the patient’s stools. Patients’ stools usually contain moderate to

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high toxin levels (up to 10' mouse lethal doses per g) and up to lo* viable spores per g, while toxin is seldom detected in theserum (77). Lack of physician recognition and limited diagnostic abilities in some areas are thought to contribute to the low numbers of cases reported from some areas.

D. CaseHistory The following case history is presented as one of the most severe, but nonfatal, cases of botulism documented (85). Within approximately 10 hours of eating only two mouthfuls of the suspect food, a patient in the United Kingdom had blurred and double vision followed by nausea and vomiting, difficulty in swallowing and talking, dryness of the mouth, and arm weakness. He suffered a respiratory arrest on arrival at the hospital and was intubated. A progressive flaccid paralysis ensued. He was given polyvalent antitoxin, a tracheostomy was done, and total parenteral nutrition (TPN) was started. While the patient was conscious, he essentially was paralyzed totally and blind due to complete ptosis. He could respond to questions by clenching his fists. There was little change in his condition for 100 days. His ptosis did not resolve until 46 days after onset of symptoms. TPN was stopped on day 158, and respiratory support was withdrawn after 173 days. He was discharged after 237 days, able to walk with the aid of crutches.

E. Prevention The only effective means of preventing foodborne botulism is by preventing toxin production in foods. Immunization of high-risk populations with botulinal toxoids has been considered; it is not considered effective and, at present, only laboratory workers at risk normally are immunized. In most cases, the preservation of high-moisture foods is geared toward control of C. botzrlirzum,which usually involves inhibition rather than destruction. Such control generally also ensures control of other foodborne pathogens and of many spoilage microorganisms. The effects of different factors on the growth of C. botulinrm~ in foods are described in a separate section below. Control of C. botulinum in foods is generally achieved by one of the following methods. 1. Low-acid, shelf-stable canned foods are preserved by a full thermoprocess. 2. Shelf-stable, canned, cured meats are preserved by a combination of thermoprocessing and addition of salt and nitrite. 3. Canned acid foods are preserved by a pasteurizing thermoprocess and acidity. 4. Products such as dry-fermented sausages are preserved by reduced water activity (a,J and pH and added nitrite. 5. Packaged raw or cooked meats or fish and shellfish are preserved by refrigeration alone. 6. Many meat and fish products are preserved by a combination of added salt and refrigeration. 7. A nutnber of cured meat products are preserved by a combination of added salt and nitrite and refrigeration. 8. Vacuum-packaged smoked fish are preserved by a combination of thermoprocessing, added salt, smoking, and refrigeration. 9. A few perishable products such as processed cheese, caviar, pickled fish, and acidified meats are preserved by decreased a, and pH and refrigeration.

Clostridium botulinum

V.

121

DETECTION

It is not necessary to isolate C. botulinum in pure culture from foods or a clinical sample in order to demonstrate its presence. Usually, the sample is inoculated into a nonselective enrichment medium. If the neurotoxin is present in the culture after incubation, the toxinproducing organism must have been present originally. BoNTs continue to be recognized by their lethal action in mice and neutralization with specific antisera. The mouse bioassay is generally more sensitive and more specific than several in vitro tests that have been developed. The sample, or an extract prepared by homogenizing it in a slightly acidic buffer, is clarified by centrifugation and normally filter-sterilized. Trypsin treatment may be required to activate low levels of toxin from nonproteolytic strains. The prepared sample is injected intraperitoneally into mice with and without neutralization with specific antitoxin. Typical signs of botulism are ruffled fur, pinched waist, labored breathing, limb paresis, and general paralysis before death. Definitive results are obtained if mice injected with untreated sample die within 72 hours, whereas mice injected with neutralized sample survive. Several investigators have developed ELISA (enzyme-linked immunosorbent assay) protocols for detecting the neurotoxin that approach or meet the sensitivity of the mouse bioassay (86). A new procedure based on detection of the endoproteinase activity of BoNTs has recently been developed (87,88). Common enrichment media for detecting viable C. botulinum are cooked meat medium (CMM), CMM glucose, chopped meat glucose starch (CMGS) medium, and trypticase-peptone-glucose-yeast (TPGY) extract broth, to which trypsin maybe added (TPGYT). Trypsin is necessary to activate the toxin produced by Group I1 organisms, and also may inactivate such potential inhibitors of C. botulinurn as boticins in mixed cultures. While foods may beinoculated directly, the sediments of centrifuged samples are preferred because potential growth inhibitors are removed. At least two tubes of media are inoculated. To select for spores, one tube is heated at 75-80°C or 60"C, respectively, depending on whether the suspected type belongs to Group I or 11. Spores of group I1 also may be selected by holding samples in 50% alcohol for 1 hour before inoculation. The other tube is incubated without any treatment to allow development of vegetative C. botulinunl cells in case few or no spores are present. Adding lysozyme to the medium may increase recovery of heat-injured spores. C. botulinum is identified after incubation of the enrichment medium by toxin analysis of the supernatant fluid using the mouse bioassay as outlined above.

VI.

NEUROTOXIN

As stated in the introduction to this chapter, seven serologically different neurotoxins are produced by various strains of C. botulinun~belonging to the four groups (19,89). They are produced during growth and generally accumulate in the culture fluids toward the end of the growth phase, largely through cell lysis, but toxin also is released during the logarithmic phase before any significant lysis occurs (90). In general, the designation of the strain is that of the toxin it produces. Group I strains produce type A, B, and F neurotoxins or, rarely, combinations of subtypes AB,AF, BA,and BF, where the subscripted type is produced in smaller amounts (19,23). Group I1 strains produce types B, E, and F neurotoxins. Group I11 strains produce C l and D neurotoxins and C? toxin and exoenzyme C3. C2toxin and exoenzyme C3 are not neurotoxins, but are ADP-ribosylating toxins, which have actin

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or the small GTP-binding proteins of the Rho family as substrates (91). Type G neurotoxin is produced by Group IV strains, which are now classified as C. nrgenfirzense (24).

A.

Structure

The neurotoxins are all very similar proteins with a molecular weight of approximately 150 kDa (92). They are synthesized as a single-chain protein with relatively low toxicity, which is activated when it is ‘nicked” by a number of proteases, including proteases of Group I, into a dichain molecule held together by a disulfide bond (93). The two components of the nicked toxin, a light (L) and heavy (H) chain, have molecular weights of approximately 50 and 100 kDa, respectively. Individually, the two components are not toxic, but toxicity can be restored by reestablishing the disulfide bond. The neurotoxins are released by bacterial autolysis and exist as complexes with other, nontoxic, proteins; these complexes are referred to as “progenitor toxins.” Three forms of progenitor toxins have been identified: 12S,16S, and 19S, respectively designated M (medium), L (large), and LL (extra large) toxins (1,201. The molecular weights of the progenitor toxins range from 500 to 900 kDa. The M form is the most common natural form, found in foods and cultures, along with the L form, which is a complex of the 150 kDa neurotoxin with an atoxic component. In the L form, a hemagglutinin is also part of the complex. M and L forms dissociate under mild alkaline conditions into the S, or derivative toxin, which is the single-chain protein. The LL form is only known for type A and was the first form of the botulinum toxin to be purified and crystallized. It has not been found in cultures and is likely an artificial aggregate. L

B. Mode of Action The detailed mechanism of action of the various BoNTs has recently been fully elucidated (94-96). The neurotoxins cause flaccid paralysis by blocking the release of the neurotransmitter acetylcholine at neuromuscular junctions (97). The mode of action can be divided into three steps: binding. internalization, and intracellular action (98). The C-terminal half of the H chains are responsible for binding, while the N-terminal half contains the domain required for internalization. The Lchains possess catalytic activity, which results in inhibition of exocytosis when they are released into the cytosol. The neurotoxin binds to a type-specific receptor on the presynaptic membrane, mediated by the carboxy terminus of the H chain (99). It appears that the different BoNTs bind to different receptors (1 00). While BoNT/B was recently shown to bind to synaptogamin I1 complexed with gangliosides (101-104), the receptor(s) for the other BoNTs have not been identified. The neurotoxin is internalized into the neuron by an initial receptor-mediated endocytosis (99), followed by pH-dependent membrane penetration. The amino terminus of the H chain then creates channels in the membrane, which permit the 50 kDa L chain to enter the cytosol (105,106). After this step, the toxin can no longer be neutralized. Finally, the internalized L chain acts as a zinc-dependent endopeptidase, which specifically cleaves proteins involved in vesicle docking and membrane fusion. BoNT types B, D, F, and G selectively cleave vesicle-associated membrane protein (VAMP) (also referred to as synaptobrevin) (107-109), while BoNT types A and E cleave the synaptosome associated protein SNAP-25 (110- 112). BoNT type C l degrades syntaxin/HPC-l ( 1 13,114). The

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cleavage of theproteins involved in vesicle docking and membrane fusion results in inhibition of acetylcholine release from the neuron and paralysis.

C. TherapeuticUses The use of BoNT to treat patients with disorders of muscle function originated with the use by Dr. Alan B. Scott of the Smith-Kettlewell Eye Research Institute (San Francisco, CA), in the 1970s of small doses of toxin A in the treatment of strabismus (crossed eyes caused by an imbalance of eye muscles) (115). Toxin injected into the problematic muscles, usually muscles that are contracting too much, causes temporary local paralysis and often results in long-lasting muscle realignments. Results of the first clinical trial in 1979 and many thousands of cases since have demonstrated its effectiveness and safety, with a few limitations (1 16). Since then, many other muscle disorders have been treated successfully including blepharospasm (an involuntary, spasmodic, repetitive eyelid closure) (1 17), torticollis (an involuntary contraction of neck muscles producing abnormal turning of the head) (1 18), spasmodic dysphonia (a contraction of the vocal chords causing a strained, strangled voice) (1 1g), hemifacial spasm (involuntary spasms on one side of the face) (119j, and various dystonias (involuntary muscle contractions in diverse areas of the body) including cervical dystonia (120), oromandibular dystonia (121), and focal dystonias of the hand (119). More recently, BoNTs have been used to treat common nondystonic conditions including hyperfunctional facial lines (122j and chronic anal fissure (123).

VII. A.

CONTROL IN FOODS Factors Affecting Growth and Toxin Production

The main factors controlling growth of C. botulinum in foods are temperature, pH, water activity (a%),redox potential, added preservatives, and other microorganisms. The growth of types A, B, and E, of Groups I and 11, has been intensively studied due to their involvement in foodborne botulism. Traditionally, food microbiologists have established maximum and/or minimum limits for these parameters which would permit growth of C. botulin z m (Table 6). and these limits have often been used in the control of C. botulinum. However, these factors seldom function independently; usually they act in concert, often having synergistic or additive effects.

Table 6 Properties of Groups I and I1 C. botzdinztm Property Toxin types Minimum temperature for growth Optimum temperature for growth Minimum pH for growth Inhibitory [NaCI] Minimum a, for growth D,,,IyCof spores D,?,'c of spores Source: Ref. 26.

Group I A, B, F 10°C 3540°C

4.6 10% 0.94 25 min 0.1-0.2 min

Group TI

B. E, F 3.3"C 18-25°C 5.0 5% 0.97 <0.1 min <0.001 min

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1. Temperature Due to the importance of refrigeration as a barrier to growth, studies have focused on determining the minimum temperatures permitting growth. The established lower limits are 10°C for Group I (20) and 3.0"C for Group I1 (124). However, these limits apply to relatively few strains and depend on otherwise optimum growth conditions. Irrespective of the actual minimum growth temperature, production of toxin generally requires several weeks at the lower temperature limits. The optimum growth temperature is in the range of 35-40°C for Group I organisms and in the range of 25-30°C for Group 11. The upper temperature limits, respectively, for Group I and Group I1 organisms are approximately 45-50 and 40-45°C.

2. pH It is generally accepted that the minimum pH permitting growth of C. botulinum Group I is 4.6, and many regulations worldwide use this limit. For Group 11, the limit is about pH 5.0. Thus, many fruits and vegetables are sufficiently acidic to inhibit C. botulinum by their pH alone, while other products, such as marinated mushrooms, are preserved by added acidulants. Several factors influence the acid tolerance of C. botulinum, including strain, substrate, temperature, nature of the acidulant, the presence of preservatives, a,, and redox potential (125). The growth of such acid-tolerant microorganisms as yeasts and molds may raise the pH in their immediate vicinity to a level that permits growth of C. botulinum (126). C. botulinunz also can grow in some acidified foods if excessively slow pH equilibration occurs. While high concentrations of proteins in laboratory media appear to protect C.botuZinum and permit growth at pH levels below 4.6, this does not occur in foods preserved by acidity (26). Current regulations stipulating a minimal acidity of pH 4.6 for the control of C. botulinum therefore are valid. The upper pH limits for growth are in the range of pH 8-9 but are of no practical consequence. 3. Salt and Water Activity Salt (NaC1) is one of the most important factors controlling C. botulinum in foods. Its inhibitory effect is primarily due to the depression of a, and consequently to its concentrationin the aqueous phase, also called the brine concentration (% brine = % NaCl X loo/% H20 + % NaC1). Under otherwise optimal conditions, the growth-limiting brine concentrations are about 10% for strains of Group I and 5% for strains of Group I1 (125). These concentrations correspond well to the limiting a, of 0.94 for Group I and 0.97 for Group I1 in foods in which NaCl is the main a , depressant. The type of solute used to control a, may influence these limits. Generally, NaC1,KC1, glucose, and sucrose show similar patterns, while glycerol permits growth at lower a, by up to 0.03 units (127). Full or partial replacement of NaCl with other salts has been evaluated in many products, with a slight reduction in control if a mixture of salts was used (125). The limiting a, may be raised significantly by such other factors as increased acidity or preservatives. 4. Atmosphere, Redox Potential Modified-atmosphere packaging (MAP) is being increasingly used to extend the shelf life and improve the quality of foods. However, MAP of perishable foods has been a concern because the extension of shelf life and inhibition of spoilage microorganisms might promote growth of C. botulinurn. The safety of MAP of fish has been studied extensively due to the high incidence of type E spores in fish (26,30,128,129). Furthermore, various researchers have shown that C. botuliizum can grow and produce toxin in MAP fish and,

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depending on conditions, toxin may be present before the fish is considered spoiled (130134). While there is a common assumption that C. botulinurn cannot grow in foods that are exposed to 02,the Eh (oxidation reduction potential) of most such foods is usually low enough to permit its growth (125). For example, initial atmospheres containing 20% O2 did not delay toxin production by C. botulinum in inoculated pork as compared to samples packaged with 100% N2, and some toxic samples contained 15% residual O2 ( l 35). Most MAP products rely on CO2to inhibit spoilage and pathogenic microorganisms (136), but CO1 may actually stimulate C. botulinum (137). Initial levels of 15-30% CO? did not inhibit C. botulinum in inoculated pork; only 75% CO2 showed significant inhibition (138). The safety of different atmospheres with respect to C. botulinum should be carefully investigated before use. C. botulinum grows optimally at an Eh of -350 millivolts (mV), but growth initiation may occur in the Eh range of 30-250 mV. The presence of other inhibitory factors lowers this upper limit. Once growth is initiated, the Eh declines rapidly (125).

5. Preservatives While effects on color and flavor are important organoleptic considerations in adding nitrite and nitrate to cured products, the most important role of nitrite in cured foods is the inhibition of C. botulinum. Nitrite is the functional constituent. While nitrate has little or no antibacterial activity, it is partially converted to nitrite by endogenous or added microorganisms. The exact mechanism of botulinal inhibition by nitrite is not known yet. Its effectiveness is dependent upon complex interactions among pH, salt, heat treatment, time and temperature of storage, and composition of food (125). Nitrite is rapidly converted to forms undetectable as nitrite during processing and continues to be converted with time until a constant low level is reached. Losses of 48-93% during processing alone have been reported (139). The effectiveness of nitrite and the duration of its effect may depend on a balance between the rate of nitrite depletion and the death rate of spores. The rate of nitrite depletion is dependent on product formulation, pH, and time and temperature during processing and storage. While nitrite is more effective at low pH, it is also depleted faster at low pH. However, a significant contribution of nitrite to the inhibition of C. botulirzurn continues even when nitrite is no longer detectable (26). Nitrite reacts with many cellular constituents and appears to inhibit C. botulinum by more than one mechanism, one of which is probably its reaction with essential ironsulfur proteins to inhibit the phosphoroclastic system that supplies the cell with energy (140,141). The reactions of nitrite, or nitric oxide, with secondary amines in meats to produce nitrosamines, some of which are carcinogenic, has led to regulations limiting the amount of nitrite used. Other compounds that are active against C. botulinum include sorbates, parabens, nisin, phenolic antioxidants, polyphosphates, ascorbates, EDTA, metabisulfite, n-monoalkyl maleates and fumarates, and lactate salts (125). The use of natural or liquid smoke has a significant inhibitory effect against C. botulinuln in fish, but appears to be insignificant in meats. 6. Other Microorganisms Other microorganisms have a very significant role in the control of C. botulinum in foods (26,125). Acid-tolerant yeasts and molds may make the environment more favorable for

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growth of C. botulirzurtz (126,143). Other microorganisms may inhibit C. botzdirzzrrn either by changing the environment or by producing specific inhibitory substances, or both. Lactic acid bacteria, including Lactobacillus, Pediococcus, and Streptococcus, can inhibit growth of C. botzrlirzzrrn in meat products, largely by reducing the pH, but perhaps also by the production of bacteriocins (143-146). The use of lactic acid bacteria and a fermentable carbohydrate, the “Wisconsin process,” has been permitted for producing bacon with a decreased level of nitrite in the United States (147,148). The growth of other microorganisms also may protect the consumer by causing spoilage that would make a toxic product less likely to be consumed.

B. ThermalInactivation Thermal inactivation is the most common method of sterilizing foods. C. botzrlir~urnspores of Group I, which are very heat resistant, are the target organisms for thermal processes. D-values (the time required to inactivate 90% of the population at a given temperature) vary considerably among C. botzrlirmnz strains and depend on how the spores are produced and treated, the heating environment, and the recovery system (149-153). Spores of types A and B are the most heat resistant, having D,2IrC values in the range of 0.1-02 minute. These spores are of particular concern in the sterilization of canned low-acid foods, and the canning industry has adopted a D-value of 0.2 minute at 121°C as a standard for calculating thermal processes. For the most resistant strains, z-values (the temperature change necessary to bring about a 10-fold change in the D-value) are approximately 10°C, which has also been adopted as a standard. Actual z-values may vary by several degrees. Despite the variations in D- and z-values, the adoption of a 12-D process as the minimum thermoprocess applied to commercial canned low-acid foods by the canning industry has ensured the production of safe products (26). Although strains of Group I1 are considerably less heat resistant (DInusC < 0.1 min) than those of Group I, their survival in pasteurized, refrigerated products is of concern because of their ability to grow at refrigeration temperatures (154-156). D*. c values of type E in neutral phosphate buffer are generally in the range of 0.2- 1.0 minute. While the D-values often are higher in foods, the z-values are essentially the same. The pasteurization of such products as crabmeat and processed fish should achieve a 10-log reduction of type E strains. Various regulations and guidelines for the safe production, distribution, and sale of refrigerated foods of extended durability have been published. The Advisory Committee on the Microbiological Safety of Food Working Group on Vacuum Packing and Associated Processes has produced recommendations for vacuum packed and sous vide foods to control growth of C. botuliwnz in foods (157). These recommendations include maintenance of chill temperatures and one or more of the following: a heat treatment of 90°C for 10 minutes or equivalent lethality, a pH of 5 or less, a minimum salt level of 3.5% in the aqueous phase, an a,of 0.97 or less, or a combination of heat and preservative factors that can be shown to prevent growth and toxin production by psychrotrophic C. botulinum. American recommendations from the National Advisory Committee on Microbiological Criteria for Foods include inoculated pack studies with C. botulimrm to determine shelf life.

C.

Inactivation by Irradiation

C. botzrlinzrr~spores are probably the most radiation-resistant spores of public health concern. D-values (irradiation dose required to inactivate 90% of the population) of Group

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I strains at -50 to - 10°C are in the range of 2.0-4.5 kGy in neutral buffers and in foods (125). Spores of type E are only marginally more sensitive, having D-values in the range of1-2 kGy. The goal of radappertization is to reduce the number of viable spores of the most radiation-resistant C. botulimlrn by 12 log cycles. D-values are affected by any pretreatment of spores, the presence of 02,irradiation temperature, and irradiation and recovery environments. In general, spores are more sensitive in the presence of 0, or preservatives and at temperatures above 20°C.

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173. Thatcher, F. S., Erdman. I. E., and Pontefract, R. D. (.1967). Some laboratory and regulatory aspects of the control of Clostridium botulinum in processed foods. In Botulism 1966 (M. Ingram and T. A. Roberts, eds.), Chapman and Hall Ltd., London, pp. 511-521. 174. Hayes, S., Craig, J. M., and Pilcher, K. S. (1970). The detection of Clostridium botulinur~r type E in smoked fish products in the Pacific Northwest. Can. J. Microbiol., 16:207209. 175. Lindroth, S. E., and Genigeorgis, C. A. (1986). Probability of growth and toxin production by nonproteolytic Clostridiuw botulinun? in rockfish stored under modified atmospheres. Znt. J. Food Microbiol., 3:167-181. 176. Baker, D. A., Genigeorgis, C., and Garcia, G. (1990). Prevalence of Clostridium borulinurn in seafood and significance of multiple incubation temperatures for determination of its presence and type in fresh retail fish. J. Food Prot., 53:668-673. 177. Cann, D. C., Wilson, B. B., Shewan, J. M., and Hobbs, G. (1966). Incidence of Clostridiuln botulinum type E in fish products in the United Kingdom. Nature, 211:205-206. 178. Johanssen, A. (.1965). Clostridiunz botulinum type E in foods and the environment generally. J. Appl. Bacteriol., 28:90-94. 179. Nielsen, S. F., and Pedersen, H. 0. (1967). Studies on the occurrence and germination of Cl. botzdinzrm in smoked salmon. In Botulism 1966 (M. Ingram and T. A. Roberts, eds.), Chapman & Hall, London, pp. 66-72. 180. Abrahamsson, K. (1967). Occurrence of type E Cl. botulinum in smoked eel. In Botulism 1966 (M. Ingram and T. A. Roberts, eds.), Chapman and Hall, London, pp. 73-75. 181. Hielm, S., Hyytia, E., Ridell, J., and Korkeala, H. (1996). Detection of Clostridium botulinum in fish and environmental samples using polymerase chain reaction. Znt. J. Food Microbiol. 31:357-365. 182. Suhadi, F., Thayib, S. S., and Sumarsono, N. (1981). Distribution of Clostridizrm botulinum around fishing areas of the western part of Indonesian waters. Appl. Environ. Microbiol., 41:1468-1471. 183. Haq, I., and Sakaguchi, G. (1980). Prevalence of Clostridizrnz botulinum in fishes from markets in Os&a. Jpn. J. Med. Sci. Biol., 33:l-6. 184. Greenberg, R. A., Tompkin, R. B., Bladel. B. O., Kittaka, R. S., and Anellis, A. (1966). Incidence of mesophilic Clostridium spores in raw pork, beef, and chicken in processing plants in the United States and Canada. Appl. Microbiol., 14:789-793. 185. Abrahamsson, K., and Riemann, H. (197 1). Prevalence of Clostridium botzrlinwn in semipreserved meat products. Appl. Microbiol., 21543-544. 186. Hauschild, A. H. W., and Hilsheimer, R. (1980). Incidence of Clostridiunl botulinum in commercial bacon. J. Food Prot., 43564-565. 187. Hauschild, A. H. W., and Hilsheimer, R. (1983). Prevalence of Clostridiurn botulinum in commercial liver sausage. J. Food Prot., 46:242-244. 188. Roberts, T. A., and Smart, J. L. (1976). The occurrence and growth of Clostridium spp. in vacuum-packed bacon with particular reference to Cl. perj?ignens (welchii) and Cl. botulinum. J. Food Technol., 11:229-244. 189. Roberts, T. A.. and Smart, J. L. (1977). The occurrence of clostridia, particularly Clostridium botulinum, in bacon and pork. In Spore Research 1976 (A. N. Barker, J. Wolf, D. J. Ellar, G. J. Dring, and G. W. Gould, eds.), Vol. 2, Academic Press, New York, p. 91 1. 190. Lentini, E., Fernandez, R., Ciccarelli, A. S., and Gimenez, D. F. (1984). Botulismo en el lactante, una neuva enfernledad? Arch. Arg. Pediatr., 82:197-198. 191. de Centorbi, 0. P., Centorbi, H. J., Demo, N., Pujales, G., and Fernandez, R. (1998). Infant botulism during a one year period in San Luis, Argentina. Zent. Bl. Bakteriol., 287:61-66. 192. Shield, L. K., Wilkinson, R. G., and Ritchie, M. (1981). Infant botulism in Australia-a case report. Aust. Paediatr. J., 1759. 193. Ryan, P. J. (1987). Infant botulism-the first reported case from Queensland. Med. J. Arrst., 1461105-106.

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194. Thomas, D. G. (1993). Infant botulism: Areview in South Australia(1980-89). J. Paediatr. Child Health, 29:24-26. 195. Austin. J., Blanchfield, B., Proulx, J.-F., and Ashton, E. (1997). Botulism inCanada-summary for 1996. Caw Commun. Dis. Rept., 23:132. 196. Hauschild, A.. Gauvreau, L., and Black, W. A. (1987). Botulism in Canada-summary for 1986. Can. Dis. Week. Rept., 13:47. 197. McCurdy. D. M., Krishnan, C., and Hauschild, A. H. (1981). Infant botulism in Canada. Can. Med. Assoc. J., 125:741-743. 198. Erazo, R., Marambio, E., Cordero, J., Fielbaum, O., and Trivino, X. (1987). Botulismo infantil: comunicaion de un caso. Rev. Med. Chi., 115:344-348. 199. Neubauer, M., and Milacek, V. (1981). Infant botulismtype B in central Europe. Abl. Bakt. Hyg., I. Abt. Orig. A., 250:540-547. 200. Balslev, T., Ostergaard, E., Madsen, I. K., and Wandall, D. A. (1997). Infant botulism. The first culture-confirmed Danish case.Neuropediatrics, 28:287-288. 201. Paty, E., Valdes, L., Harpey, J. P., Dore, F., Hubert, P., Roy, C., and Caille, B. (1987). Un cas de botulisme chez un nourrisson de 11 mois. Arch. Fr. Pediatr., 44:129-130. 202. Trethon, A., Budai,J., Herendi, A., Szabo, V.,and Geczy, M. (1995). Csecsemokori botuliznlus. Owosi Hetilap, 136:1497-1499. 203. Aureli, P., Fenicia, L., Creti, R., Bertini, E., Vigevano, F., DiCapua, M., and Pirozzi, N. (1989). Botulismoinfantile in Italia Aspetti clinici e microbiologicidi 5 casi. Riv. Ital. Pediatr., 15:442-447. 204. Fenicia, L., Ferrini, A. M., Aureli, P., and Pocecco, M. (1993). A case of infant botulism associated with honey feeding in Italy. Eur. J. Epidemiol., 9:671-673. 205. Moli, 0.L., Quarto, M., Larosa, C. G., Germinario,C., Zimbalatti, F., and Barbuti, S. (1996). A case of infant botulism: Clinical and laboratory observations. Ital. J. Pedintr., 22:234237. 206. Noda, H., Sugita, K., Koike, A., Nasu, T., Takahashi, M., Shimizu, T., Ooi, K., and Sakaguchi, G. (1988). Infant botulism in Asia. ANI.J. Dis. Child., 142:125-126. 207. Oguma, K., Yokota, K., Hayashi, S., Takeshi, K., Kumagai, M., Itoh, N., Tachi, N., and Chiba, S. (1990). Infant botulism due to Clostridizm botulinurzl type C toxin. Lancet, 336: 1449-1450. 208. Nabeya, T., Yano, R., Saito, T., Inoue, H., Shinohara, N.,Yokoyama, T., Nagai, S., Nishibayashi. Y., and Sakaguchi. G. (1989). Infant botulism was confirmed in Ehime Prefecture. Karlsenshogaku Zasshi-J. Jpw. Assoc. Infect. Dis., 63:268-272. 309. Tabita, K., Sakaguchi, S., Kozaki, S., and Sakaguchi, G. (1991). Distinction between Clostridium botulinum type A strains associated with food-borne botulismand those with infant botulism in Japan in intraintestinal toxin production in infant miceand some other properties. FEMS Microbiol. Lett., 79:251-256. 210. Morikawa, Y., Shishida, N., Toshima, M., Yoshioka, Y., and Nukina, M. (1994). A case report of infant botulism without a history of honey ingestion. Karlsenshogaku Zusshi, 68: 259-262. and Nakamura, S. (1996). The 21 1. Kakinuma, H., Maruyama, H., Takahashi, H., Yamakawa, K., first case of type B infant botulism in Japan. Acta Paediatr. Jpn., 38:541-543. 212. Toyoguchi, S., Tsugu, H., Nariai, A.. Kaburagi, Y., Asahina, Y., Ambo, K., and Katou, K. (1991). Infant botulism with Down syndrome. Acta Paediatr. Jpn., 33:394-397. H., and Nakamura, 213. Yamakawa, K., Karasawa, T., Kakinuma, H., Maruyama, H., Takahashi, S. (1997). Emergence of Clostridiurn botulinum type B-like nontoxigenic organisms in a patient with type B infant botulism. J. Clin. Microbiol., 35:2163-2164. 214. Torres Tortosa, P., Martinez Villalta, E., Rodriguez Caamano, J.. Lorca Cano, C., Puche Mira, A., and Borrajo, E. (1986). Botulismo dellactante. Presentacion de un caso. An. Esp. Pediatr., 24:193-196. 21 5. Lizarraga Azparren, M.A., Lopez Fernandez,Y., Pilar Orive,J., Latorre Garcia,J., Hermana

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216. 217. 218. 219. 220. 221. 222. 223.

Austin and Dodds Tezanos, M. T., and Rivera Aranda, A. (1996). Botulismo infantil: a proposito de un caso. An. Esp. Pedintr., 44:399-401. Gautier, E., Gallusser, A., and Despland, P. A. (1989). Botulisme infantile. HeIv. Pnediat. Acta, 43:521-530. Jansson, L., Bjerre, I., Schalen, C., and Mardh. P. A. (1985). Forstafallet av infantil botulism i Sverige? Lnkartidningen, 82:955-956. Wang, C. C., Chu, M. L., Liou, W. Y., Twu, P. H., Lin, C. H., and Lee, C. L. (~1988). Infant botulism: Report of a case. J. Formosan Med. Assoc., 87:919-922. Turner, H. D., Brett, E. M., Gilbert, R. J., Ghosh, A. C., and Liebeschuetz, H. J. (1978). Infant botulism in England. Lancet, i: 1277-1278. Smith, G. E., Hinde, F., Westmoreland, D., Berry, P. R., and Gilbert, R. J. (1989). Infantile botulism. Arch. Dis. Child., 64:871-872. Gilbert, R., Brett, M., and Kramer, J. (1993). A case of infant botulism. CDR Weekly, 3: 129. Cochran. D. P., and Appleton, R.E. (1995). Infantbotulism-Is it that rare? Dev. Med. Child Neurol., 37:274-278. Breuer, T., and Maslanka, S. E. (1997). Laboratory-confirmed botulismin the United States. January 1,1996-December 3 1, 1996. In 1997 Meeting of the InteragencvBotulism Research Coordintcting Committee, Bethesda, MD., 12-14 November, 1997.

8 Clostridium perfringens Dorothy M. Wrigley Minnesota State UniversiQ, Mankcrto, Mirtnesotu

I. Introduction 140 11. General Characteristics

140

Morphology A. 140 Toxins B. 141 C. Growthandsurvival of vegetative cells D. Sporulation and enterotoxin production 144 E. Germination 145 F. Genetics 146 G. Antibiotic sensitivities 147

142

111. Diseases 147 Gastroenteritis A. B. Relateddiseases

137 148

IV. Epidemiology 148 Human-to-human A. spread 148 B. Animal-to-human spread 149 V. Isolation and Identification 149 Plating A.techniques 149 B. Biochemical/Physiological tests C. Other tests 152 153 Transport D. of specimens VI. Enterotoxin Pathogenicity

152

153

Feeding A. studies 153 B. Biochemistry and mode of action C. Production 154 Purification D. 154 Enterotoxin E. assays 155 F. DNAprobes of theenterotoxingene156

153

VII. Prevention 156 References 157

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1.

INTRODUCTION

Clostridiun~ yegringens type A ranks among the most common agents of foodborne gastroenteritis (1-5). Some estimations place it as the leading cause because most cases are unreported or undiagnosed due to the mild nature of the symptoms. C. pegrilzgens gastroenteritis was described first in the early 1900s but received little attention until 1953 when Hobbs et al. (6) described the disease and began classification of gastroenteritis-associated strains (7). The organism is considered by many to be ubiquitous and has been isolated from soil, plants, and a variety of animals that includes cattle, poultry, and humans. All these sites may serve as reservoirs for infection, although animal and human sources appear to bethemost common. Its nutritional requirements and temperature ranges for growth are such that it may only replicate in mammals or other warm, nutrient-rich sites. The foodborne gastroenteritis follows ingestion of high numbers of vegetative cells. The cells sporulate in the intestines. It is during sporulation that the enterotoxin is produced. This protein, which appears to be a spore-coat protein, is released into the lumen of the gut and triggers the onset of diarrhea. Resolution of the diarrhea usually requires no treatment and is complete in several hours (7,s).Confirmation of C. perfringens as the etiological agent often is difficult due to the short duration of the spores in fecal material and the sensitivity of the organism to cold storage. Culture of C. perfringens from fecal material needs to be supplemented with confirmation of enterotoxin production (9,10). More serious diseases are also associated with C. perfringeus. Enterotoxigenic C. perfringem type A has been linked with sudden infant death (SID) more than any other bacterial species (1 1- 13). C. peI$”ngens type A also causes wound infections and is one of several agents associated with gas gangrene and myonecrosis. These infections usually are not caused by foodborne bacteria. While C. pe~fr-irzgenstype A is the most common of the foodborne clostridia, type C also is afoodborne pathogen. The first recorded outbreak was described as a necrotizing gastroenteritis and followed consumption of contaminated rations in Germany (14,15). More recent outbreaks have been seen in Papua New Guinea, where the disease is known as pig-bel (16).

II. GENERALCHARACTERISTICS

A.

Morphology

C. perfiilzgerzs is a nonmotile, gram-positive, anaerobic bacillus that has the capability to form an endospore. Strains associated with foodborne gastroenteritis more readily form spores than those associated with myonecrosis and gas gangrene. Spores are oval and subterminal. The length of the bacillus varies greatly. In glucose-rich media, the rods are short; in starch-based media, the rods are long. Capsules may be present, especially on bacilli from tissue specimens (l 7). Colonies of C. perfringens are generally white. When cultured on blood agar, colonies are typically surrounded by double zones of hemolysis due to the hemolytic activity of the alpha (a)-and theta (0)-toxins. On egg yolk agar, a zone of opalescence due to atoxin activity surrounds each colony. On sulfite-containing agars, black colonies form due to sulfite reduction.

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Table 1 Toxins of Clostridim perfringens Toxin (C. pe?jhkgens Activity type) a-Toxin (A,B,C,D,E)a P-Toxin (B,C) y-Toxin (B,C) &Toxin (B,C) €-Toxin (B.D) q-Toxin (A) 0-Toxin (A,B,C,D,E) L-Toxin (E) K-Toxin (A,C,D,E) h-Toxin (B,D,E) p-Toxin (A,B,D) v-Toxin (A,B.C,D,E) Enterotoxin (A)h

Phospholipase C, lethal, necrotizing Lethal, necrotizing Lethal Lethal, hemolytic Lethal, necrotizing Lethal Hemolytic, lethal ADP-ribosyltransferase, necrotizing, lethal Collagenase Proteinase Hyaluronidase Deoxyribonuclease Complex with plasma membrane

'C. pelfringens types shown to secrete the toxin. Types C and D secrete an enterotoxin distinct from type A. Source: Adapted from Ref. 17.

B. Toxins As a species, C. ye~fringensproduces a wide variety of toxins, which are used for typing the species. The activities are listed in Table 1. Two of the toxins, epsilon (E) and iota (I), are activated by trypsin. Theta-toxin is oxygen labile. The proteinase, lambda (1)toxin, degrades casein but not collagen (18). The types of C. perfringens (A, B, C, D, and E) are based onthe secretion of a-,beta (p)-, gamma (y)-, E-, and t-toxins. The characteristics of these five major toxins are given in Table 2. Numerous other toxins and enzymes are produced. 1. Toxins of C.perfringens Type A The toxins of C. ye$ringens type A have been studied extensively. The enterotoxin associated with gastroenteritis is discussed in Section VI. Alpha-toxin (phospholipase C) has received the most attention and the gene has been cloned and sequenced (19-21). The recombinant phospholipase C sequence has a promoter similar to the sigrng5 of Esclw richict coli. The recombinant protein at 42,528 daltons is identical to native protein. The N-tenninus has a 77-amino-acid sequence similar to the phospholipase C of Bacillus cereus and a 64% homology with thephospholipase C of C. bifermmztnns (21). The recombi-

Table 2 Conlparison of Major Toxins of Clostridium perfrirlgens Toxin

Molecular weight (daltons) producing Types

PI

Ref.

~~~

a-Toxin P-Toxin €-Toxin t-Toxin (a binary toxin)

43,000 28,000 34,250 47,500 7 1,500

5.4 5.4-5.5 5.6 5.2 4.2

A,B,C,D,E B,C B ,D E

19-21 33.138 34.35 39-41

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nant a-toxin has activity distinct from that of 0-toxin and no hemolytic activity against horse. sheep, rat, or rabbit erythrocytes. Alpha-toxin preferentially attacks the phosphorylcholine and sphingomyelin in tnenlbranes (22j. Alpha-toxin has no apparent activity in human gastroenteritis. Gastroenteritis associated strains usually produce less a-toxin than wound-associated strains (23). Whether a toxin has any true pathogenic effect in other intestinal diseases has not been determined conclusively. The toxin can kill intestinal villi cells (34). Alpha-toxin plays an important role in C. pelfriizgens gas gangrene and myonecrosis (25,26). Since these infections are complex and may involve more than one bacterial species, it is difficult to associate in vivo effects with a single agent or toxin. The epitopes of a-toxin have been mapped (27). Theta-toxin, perfringolysin 0, may have a significant rolein wound infections caused by C. pelfiiugerzs type A. It is thio-activated, binds cholesterol. and fornls pores in susceptible cells (28). This cytolysitdhemolysin kills polymorphonuclear cells and inhibits chemotaxis (29,30). It may act by a colloid-osmotic mechanism (3 1) that separates it from other hemolysins of similar function but different mechanism (e.g., streptolysin 0 )(32j. The gene for 0 toxin codes for a 54,000 dalton protein (29,30). 2. Major Toxins from Other C. perfringens Types The damage caused by p-toxin may be a major component of the disease pig-bel, a necrotic enteritis of humans. Beta-toxin is elaborated during vegetative growth. Production is optimal if the pH of the medium is maintained at pH 7.5 (33) The toxin is inactivated by trypsin. A recombinant p-toxin was not toxic in mice and induced neutralizing antibodies against C. p e ~ f i i ~ g e ltype z s C-produced p-toxin (34). Epsilon- and delta-toxins are associated with gastrointestinal disease in livestock. Epsilon-toxin is the principal toxin of C. per$-irzgens type D, which is associated with enterotoxemias in ruminants. The mode of action of €-toxin is not known completely, but it increases intestinal permeability and enters the blood stream: from there, it binds to brain endothelial cells and increases blood pressure. It also binds to Henle's loops and convoluted tubules in the kidney (35). Epsilon-toxin is synthesized as a prototoxin and then cleaved (36); the h-toxin may be responsible for the cleavage (37). Delta (@-toxin, a lytic toxin of 42,000 daltons, is amphiphilic. Its lytic activity is inhibited by Gm2 gangliosides (38). As one of the major toxins produced by C. perfringens types B and C, it is associated with dysentery in lambs and enterotoxelnias for sheep, goats, calves, and pigs (17). Iota-toxin is produced only by C. perfringem type E. It shares homologies with other binary toxins with ADP-ribosylating activity similar to the binary toxin C2 produced by C. botrdirzurn (39-41). To date, no disease state has been linked with C. peIfiingem type E.

C. Growth and Survival

of Vegetative Cells

C. perj?ingens has been studied extensively for factors that affect growth, enzyme/toxin production, sporulation, and spore germination and has been the subject of a number of reviews (17.43). The following sections focus on those factors associated with growth and recovery of C. perfringem in foods. As with other foodborne pathogens, many factors interrelate to stimulate optimal growth. In addition, strain characteristics differ, especially as relates to sporulation. At optimal temperature, pH, and nutritional requirements, generation times usually are below 15 minutes. One of the shortest reported is for strain NCTC

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8328, which can double every 7.1 minutes at 41°C (43). Theshort generation times permit the bacteria to rapidly reach infective doses necessary for disease. 1. Nutritional Requirements A defined medium for C. per-ingem developed by Boyd et al. (44) contains 19 amino acids. Of these, 14 are essential for growth. The lysine requirement can be eliminated by the addition of high concentrations of pyridoxamine or pyridoxal. Addition of uracil and adenine accelerates growth. With these growth requirements, C. perfiingens does well in many foods, especially meat and poultry. Sterilized ground beef is an excellent growth medium and supports a-toxin production (45,46).

2. Temperature Reported optimal temperatures for C. yeqfiringeru replication range from 41 to 45°C. Most reported optima are between 43 and 45°C. Maximum temperatures for growth are reported at 50-51°C (17). Above 50°C, the death rate increases. Culture at high temperatures increases thermal resistance and decreases the death rate (43,47). Minimum temperatures for growth range from 14 to 18.5"C. Incubation of cells between 0 and 10°C results in rapid cell death (17,48). The rapid loss of viable cells at 1-4°C accounts for the difficulty in isolating organisms or obtaining accurate estimates of the number of organisms from many refrigerated foods. Cold shock, the transfer of cells from 37 to 4 ° C on logarithnlically growing cells kills 96% within 10 minutes. Cultures become less sensitive to cold shock as the population approaches stationary phase growth (48). Maximum and minimum temperatures and survival in cold and heat vary with pH and growth medium. Cultures in cooked meat medium survive longer at 4°C than cultures in thioglycollate medium (1 7). 3. Oxidation Reduction PotentiallOxygen Requirements C. yer:fringens cells do not initiate binary fission in an oxygenated environment. Culture media are routinely steamed prior to use to drive off dissolved oxygen. To maintain anaerobiasis during culture, the reducing agents thioglycollate, cysteine, and OxyraseR(Oxyrase Inc., Mansfield, OH) are often added to media. Low concentrations of agar in a liquid medium retard oxygen diffusion into the medium. Overlays of agar or paraffin mixtures also slow or prevent reoxygenation. Anaerobic chambers perfused with a variety of gas combinations support C. pe$-ingens growth. In spite of its sensitivity to oxygen, C. perfringens will grow over a wide range of oxidation reduction potentials (Eh). Growth between - 125 and +350 mV (millivolts) has been reported. Walden and Hentges examined growth of C. yetfringens from fecal isolates and determined that elimination of oxygen is more important than Eh (49). The organism lacks an electron transport system and is, therefore, insensitive to electron inhibitors such as sodium azide, At least one enzyme essential for growth is oxygen sensitive (50). 4. Water Activity Minimum water activity (alv)ranges from 0.97 to 0.93 depending on the solute used. When glucose is used to adjust a , ,the minimum a, for growth is 0.96 (51). Extremes in temperature and pH raise the tolerated a, (40). The rate of growth is also affected by the solute in the medium (52).

5. pH Optimal pH for growth is between 6.0 and 7.0. Below pH 5.5 growth is inhibited. Below pH 5 , the bacterial cells die in several days. Metabolizing cells dramatically alter the pH

144

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in culture. In poorly buffered, glucose-supplemented media, the bacteria produce acids and drop the pH from an initial pH 7 to below pH 5 in several hours. The low pH affects survival and diminishes toxin production (53,54). 6. Curing Salts Sodium nitrite and sodium chloride act together synergistically to inhibit C.yelfiingens growth. The sensitivity of C. pelfringens to this combination of curing salts helps to make cured meats a rare vehicle for C'. yegringem gastroenteritis (one case from 1978- 1982 in the United States) (17). Individually, 7 4 % sodium chloride and 300 pg/mL sodium nitrite inhibit growth. Sodium nitrite inhibitory concentrations vary depending on bacterial strain, preparation of the salt solution, pH, and number of bacteria (55). D. SporulationandEnterotoxinProduction 1. Sporulation and Its Relation to Gastroenteritis Only endospore-forming strains cause gastroenteritis. Endospores form during the first 6 hours of culture in a sporulation medium. Depending on nutrient supplements in the sporulation medium, vegetative growth may continue, but the replicating cells do not contribute to the number of spores (53). Free spores are released between 7 and 8 hours after culture initiation (56,571. Under anaerobic conditions, the maximum number of spores in DuncanStrong sporulation broth (58) or ground beef (59) is reached by 10-12 hours. Spore also will form under aerobic conditions, but the maximum number is reached in 24 hours (60). Although there is evidence that some vegetative cells produce low levels of enterotoxin (61-66) and that some strains sporulate well without enterotoxin production (67), the connection between spore formation and enterotoxin production is well established. Enterotoxin-negative spore-fonning strains are not associated with gastroenteritis. The enterotoxin appears to be a spore coat protein (68) and is detectable in sporulation cultures after 2 hours of culture (69). One spore-forming strain considered negative by biological assays was shown to produce about 1 ng/mL enterotoxin by a more sensitive enzyme immunoassay. One ng/mL may be below the concentration needed to cause gastroenteritis (64). The sporeformers associated with C. yelfiingens gastroenteritis are divided into heat-sensitive and heat-resistant strains. Most heat-resistant strains that sporulate well in Duncan-Strong medium produce high concentrations of enterotoxin. Heat-sensitive spore-fommers do not sporulate as well. Therefore, enterotoxin is not always detectable by reverse-phase passive latex agglutination assays (70). 2. Nutrient Requirements for Sporulation Detection of spore formation by C.ye?fringens is important for the diagnosis of C. perfiirzgens gastroenteritis. Many strains do not form spores well in culture. Several media have been developed that provide good sporulation from a number of strains (58,7174). No one medium supports spore formation in all strains. Duncan-Strong medium or modification of Duncan-Strong medium are the most widely used. The components for 100 mL of Duncan-Strong mediutn are protease peptone, 1.5 g; yeast extract, 0.4 g; soluble starch, 0.4 g; sodium thioglycollate, 0.1 g; and Na2HP04-7H20,1.O g (58). Ting and Fung (75) and Sacks and Thompson (76) have developed defined media for sporulation studies. One study indicated that mutants of C. perfkingens type A (NCTC 8798) sporulate well in the defined medium of Sacks and Thompson but not in Duncan-Strong broth (77).

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While many components affect sporulation, carbohydrate composition is one of the most important. Starch, dextrin, and raffinose are used as carbodenergy sources. Glucose or lactose added to Duncan-Strong medium inhibit sporogenesis (78). However, Decaudin and Tholozan (77) demonstrated the 11 tnMof glucose in D medium resulted in anincrease in the percentage of spore formed for two mutants of C. pelfringem NCTC 8798. Depression of thepH of themedium due to fermentation of sugars appears to cause the inhibition. Sacks et al.demonstrated that the methylxanthines caffeine and theophylline enhance sporulation (79). In addition, they suppress the growth rate of C. yerjkingens. Guanosine also enhances spore fortnation in some strains (54). Several foods support good sporulation of C.pelfringens. Kim et al. cotnpared sporulation in a sporulation broth, Noyes veal broth, pea puree, and tuna (80). The best sporulation occurred in the sporulation medium and veal broth, but the pea and tuna also supported sporulation. In turkey, chicken, and beef, sporulation occurs in 14-18 hours (60,81,82). 3. Other Environmental Requirements The optimal temperature for sporulation is 35-40°C. Kim et al. examined five strains of C. yerjkkgerzs for temperature effects (80). Two strains produced a few spores (2-37 spores/mL) at 5°C; all five strains produced spores (1-63 sporednll) at 22°C. At 37"C, the number of spores in the culture ranged from 5 1 to 12 X 10' spores/mL. Provision of exogenous, heat-resistant amylase permits sporulation at higher temperatures (83,84). The role of amylase in sporulation is still being studied. Although vegetative cells of C. yerfiingems produce several amylases, a significant increase in one amylase occurs during sporulation (85). Extracellular amylase produced by C. ye@ingens during sporulation is rapidly inactivated above 43°C (83). Acid conditions in sporulation media inhibit spore formation. The pH range for spore formation is pH 6.0-8.0 (17,56). Spore formation decreases as vegetative cells in culture ferment sugars to acids. However, a brief exposure of the cells to acid prior to inoculation into sporulation media may enhance sporulation (86). Sporulation requires a higher a, than vegetative cell growth. Kang et al. induced sporulation in Ellner's medium at different a,v adjusted with NaC1, glycerol, or sucrose (51). Spores were found in the glycerol adjusted medium at an a,v of 0.98. Few studies have examined the effect of oxidation/reduction potential and oxygen on sporulation. The Eh of a medium at -450 mV changed to -400 mV during sporulation. In ground beef, spores formed more rapidly under anaerobic conditions than aerobic conditions (60,82). 4. Regulation of Sporulation The sporulation gene map appears to be similar to mapped positions for sporulation genes of Bncillus subtilis (87). Like B. subtilis, sporulation gene expression appears regulated through the activity of sigma factors. Three promoters with similarity to sigK- and sigEdependent promoters have been described (88). Some antibiotics interfere with sporulation at distinct stages. The antibiotics netropsin and distamycin allow sporulation to initiate but block that process at stage 111 (89).

E. Germination Spores can be classified as either heat resistant or heat sensitive based on their ability to germinate following heating. Heat generally enhances the recovery of cells from spore

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preparations. Heat-resistant strains require heat activation at 75°C for 20 minutes before they germinate well. It should be noted that under those conditions heat-sensitive spores will not germinate (70). Heat (90), alcohol (91), and the chaotropic salt CaCI, (92) enhance gemination. The disruption of hydrophobic interactions in spore proteins is essential for spore activation and suggests that spore activation involves conformational changes in spore proteins. Addition of lysozyme to plating medium helps in recovely of heat-injured spores (93-95). During germination, two acid-soluble proteins in the spore rapidly degrade. The two proteins are of the a/b type. One has 59 anlino acids and the other 60 (96). Another feature of C. perfringens spore proteins is a high degree of glycosylation (97). Two spore lytic enzymes, an amidase and a muramidase, are located on the external coat of the spore (98100). The deduced amino acid sequence for the muramidase indicates that it is mature as formed. The amidase is synthesized as a prepro-enzyme. The proform is thought to be noncovalently attached to the coat and is activated during germination (98,101). Spore activation is affected by components in the medium. Heat-resistant spores germinate well in medium with KC1 and Tris buffer. Heat-sensitive spores germinate better in medium supplemented with alanine, inosine, CaCL, and Tris buffer (70). Some spores require lysozyme for activation. Spores may contaminate food and germinate in the food. One study examined the sporulation and outgrowth of C. pei$-ilzgens in ground beef. Spores germinated well. The resulting vegetative cells were readily killed by 60°C (102). F. Genetics 1. Genetics of C.perfringens Type A Toxins The enterotoxin produced by C. yerfr.ingens type A has only one serological type (103); cloning and hybridization studies have confirmed one, highly conserved gene (104-106). Van Damme-Jongsten et al. constructed four probes from the cloned gene (10). The probes detect the enterotoxin gene in many spore-forming isolates from food, fecal material, and soil. Probe hybridization with strains of known enterotoxin activity corresponded to enterotoxin production. Several isolates from food and fecal material associated with C. perfriizgens gastroenteritis did not hybridize with any of the probes, which implies that those isolates might not be responsible for the outbreaks. Expression is associated with sporulation. Enterotoxin-negative strains, transformed withthe enterotoxin gene, express the gene on sporulation (107). Three promoters lie upstream from the enterotoxin gene. P1 is similar to the sigK-dependent promoters, and P2 and P3 are similar to sigE-dependent pronloters active in the mother cell of sporulating B. subtilis (88). The phospholipase C or a-toxin gene (plc) has been cloned (19-21). The gene is expressed from a single promoter (22). Mutagenesis of the histidine-68 or -148 results in loss of activity (108). Gene expression is partially regulated through the virR/virS locus (109). Homologies with other bacterial phospholipase C genes are limited to the genus Bacillrrs. The gene for @toxin (PfoA) has also been cloned into E. coZi (29,30). Like the phospholipase C gene, &toxin expression is positively regulated through the virR/virS locus (1 09). Expression is also stimulated by the product of yfoR, which codes for aDNAbinding protein (1 10). A small (<2000 daltons) secreted product from %positive strains derepresses the 8 gene and allows some ‘%-negative” stains to produce the toxin (111).

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2.

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Plasmids

C. pelflingens has plasmids that code for bacteriocins (1 12.1 13) and antibiotic resistance. C. yer.fringens has attracted some attention as a potential host for cloned genes (1 14,115).

Recovery of lysozyme-treated cells is good. Of the cells converted to protoplasts by lysozyme treatment in an osmotic stabilizing medium, 5% are recoverable on regeneration medium supplemented with bovine serum albumin (116). Transformation of L-forms of C. pelfiirzgens has been moderately successful (1 15,117). Stable L-forms are produced in brain-heart infusion with 10% sucrose and 2 units of penicillin G per ml. Stable L-forms also accept shuttle plasmids designed to transform both E. coli and C. pelfiingerzs (118).

G.AntibioticSensitivities There is little interest in determining antibiotic sensitivity of food isolates or fecal isolates from cases of C. pe@ingens type A gastroenteritis due to the short-term nature, role of sporulation, enterotoxin release, and mildness of the disease. Antibiotic sensitivity of isolates from chickens and swine indicate an increase in resistance to antibiotics, particularly tetracycline and macrolides, which are common additives to animal feeds (1 19-121). Strains associated with gas gangrene and other wound infections are susceptible uniformly to benzylpenicillin, metronidazole, and rifampin (122,123). Susceptibility to other antibiotics varies. Traub cautioned that minimum inhibitory concentration (MIC) data is not an adequate predictor for good prognosis (124) due to inadequate drug accumulation in the wound. Protein inhibitors affect phospholipase C production, but benzylpenicillin does not (125). Resistance genes against tetracycline (126,127), erythromycin (128), and chloramphenicol (129,130) have been determined. Chloramphenicol resistance is conferred by two separate genes. One resistance gene, cutP, is located on a large plasmid and is carried on transposons (129). The second resistance gene, cntQ, appears to be located on the chromosome (1 30). The e m P and e m B genes for macrolide resistance have been cloned (128). Other resistance genes for the macrolides probably exist because only 5 of 40 resistant strains hybridized with e m P and e m B probes (128). Only one tetracycline resistance gene (tetp) has been identified (126,127).

111.

DISEASES

A.

Gastroenteritis

Hobbs et al. were the first to confirm C. perfringens as a foodborne pathogen by finding bacteria of the same serotype in both the contaminated food and fecal specimens from an outbreak of gastroenteritis (6). The symptoms associated with C. yetfiingerzs gastroenteritis are abdominal pain and diarrhea. Nausea is rare (6,8). Onset is usually within 18 hours of ingestion of large numbers of C. pelfringerzs type A vegetative cells. Recovery is usually within 12 hours of onset. The contaminated food is most often meat, poultry, and such associated material as gravy or stock ( 1,2). The food often is precooked the day before serving and rewarmed. During the time between cooking and serving, surviving spores or newly introduced organisms replicate to high numbers. In cases in which the suspect food is available for analysis, the major species isolated is C.yelfiilzgerzs.

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The actual incidence of C. perfringens foodborne gastroenteritis is not known. Todd estimated that most cases are not diagnosed and thus are not reported due to the mild nature of the disease (5). In the United States, outbreaks are common and most often are associated with mass catering (13 1- 133). The disease is not limited by age, sex, or other criteria. C. pelfii1?gens has also been associated with a chronic gastroenteritis in patients on extended antibiotic therapy (134,135). In this nonfoodborne gastroenteritis, the organism carries the ent gene on an episome rather than on the chromosome as found in the traditional foodborne cases (136).

B. RelatedDiseases C. pe$ringer?s type C is the etiological agent in a foodborne, hemorrhagic enteritis first described in Germany (14,15) and later in Papua New Guinea, where it is called pig-bel (16,137). The enteritis is accompanied by diarrhea and abdominal pain. The stools are bloody with mucus. Death due to the enteritis has been reported. The disease appears to be due to the activity of the p-toxin, which causes necrosis of the intestine. Enterotoxin has no noticeable role. Delta- and theta-toxins may also contribute to the pathology (138). The disease is sporadic and occurs in epidemics. A predisposing factor appears to be a low-protein diet (16). Epidemics of pig-bel occur when youths who normally consume low-protein foods partake in a feast of roast pig. The contaminated pig is the vehicle for the organism (16,137). C. perfrilzgens type A is isolated from a majority of anaerobic infections (139). Infections include cellulitis, myonecrosis, and gas gangrene. The source of the infecting organism usually cannot be determined, and introduction into the body usually follows trauma. However, non-trauma-associated C. pelfiingens infections also are seen in patients with malignancies (140). Several of the toxins, but not the enterotoxin, are responsible for bacterial spread and tissue destruction (25,141). Alpha- and theta-toxin cause the most damage. Bacterial toxins have been implicated in sudden infant death syndrome (SID). C. per-ingens was found in 45.5% of affected infants but only in 19.6% of healthy babies. Furthermore, the enterotoxin was detected in 34.4% of fecal extracts from SID cases but not from healthy babies. Alpha-toxin was also present in SID fecal extracts (17.5%); none was found in the control group. Although other bacterial toxins, from E. coli, Staphylococcus uureus, and Clostridium diifJicile, were found, C. pe$ringelzs had the highest incidence (1 1). In another study, greater than 80% of SID fecal specimens had C. perfEngerls type A. Less than 3% of control non-SID cases were positive (142). IV. EPIDEMIOLOGY

A.

Human-to-HumanSpread

Humans appear to be a common source for many C. perfringens outbreaks. The contaminant often is introduced into a food during preparation (2). Adult levels of C. perfringens in fecal material are established by 6 months of age (17). Of populations studied, 1-52% of the individuals possessed heat-resistant spores in their feces (17,143,144). Heat-resistant spores commonly are associated with diarrheal disease, especially in Great Britain (7). In the United States, outbreaks have been associated with both heat-resistant and heat-sensi-

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tive spores of C. perfringens. When heat-sensitive spores are included in incidence studies from human fecal material, close to 100% of adults humans are positive for C. perfringens type A (17,145). However, at least one report indicates that vegetarians may lack C. perfringens (146j. The high incidence of C. perfringens in normal flora compounds the problems associated with outbreak confirmation in that positive cultures will not necessarily indicate the cause of the diarrhea. Detection of enterotoxin from isolates is necessary for confirmation. Spore counts of greater than 10" per g of fecal material are also needed for disease confirmation (147- 149).

B. Animal-to-HumanSpread C. perfringens type A is a ubiquitous organism and has been isolated from soil, animals, spices and herbs, dehydrated foods, and human fecal material ( l 19,120,150-153). Organisms associated with soil and dried foods tend to be in spore form. Whether isolates from all potential sources of C. yerfriMgerzs cause diarrhea in humans is not known. Fish harbor bacterial flora containing C. p e $ - i n g e n stype A but have not been associated with C, per$-ingens gastroenteritis. The prevalence of C.per$-ingens on pork, beef, and poultry and the number of outbreaks associated with these foods indicate that animals are a potential source of enterotoxin-producing strains. Pets may harbor the organisms (1 54). Examination of pigs with diarrhea revealed that the enterotoxin gene was episomal rather than chromosomal as typical of foodborne outbreaks (155). Meats that require extra handling in preparation are more likely to be contaminated with C. pelfringerzs than foods that require little handling (59,156), which implies that humans are the most common reservoir for human disease. The foods most likely to be contaminated with C. perfringens are beef, pork, lamb, chicken, and turkey (150153,157). Stews and other casserole-style foods made with meat products also provide an excellent growth environment for C. perfringem. Meat products in the United States are more likely to be contaminated with heat-sensitive spores than meat products in Great Britain (59). Cured meats, though, are poor vehicles for C. pelfiingerzs. In many outbreaks suspected of being caused by C. perfringens, the food is not available for analysis or has been stored improperly for assessment of C. pe$rirzgens (17). One hospital outbreak in Japan implicated a crab salad as the vehicle (l%), but the origin of the contamination was not determined. The bacterial source for domestic animals may be their feeds. Many chicken feeds and soy products are contaminated with C. perfringem (159,160). Soy meals also support bacterial growth, and the addition of soy meals to meat products may aid bacterial replication (45,46,16lj.

V.

ISOLATIONANDIDENTIFICATION

A.

PlatingTechniques

1. Isolation Isolation of C. perfringens from mixed cultures is relatively easy based on several characteristics: the ability to grow at 46"C, resistance to specific antibiotics, the ability to reduce sulfite to sulfide, and anaerobic metabolism. The difficulty in isolation lies in the number of

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other bacteria that contaminate the specimens, especially fecal specimens. Media selection depends of whether the specimen is fecal or food. C. perjhkgens requires an anaerobic environment for growth. C. perj?ingens may be isolated if oxygen concentrations are low and sufficient bacterial load is present to reduce the oxygen and permit the anaerobe to grow. However, if the number of C. perfiingens is low, then it is necessary to establish anaerobiasis in order to enrich for the organism. If sulfite reduction is to be used to enhance colony visualization, anaerobiasis also must be established (149). A variety of gases can be used to establish an anaerobic environment. Nitrogen, nitrogen-hydrogen, or nitrogen-hydrogen-carbon dioxide mixtures all permit growth. Hydrogen-carbon dioxide-generating systems that deplete oxygen (e.g., the GasPak anaerobic system [BBL, Becton Dickinson Microbiology Systems, Cockeysville, MD]) work well (17). Alternatively, oxygen scavengers, such as OxyeraseTM,can be added tothe media. A typical isolation medium for suspected C. perjl-ingem in meat and poultry products contains, tryptone, sulfite, and cycloserine (TSC) (see Table 3) and is supplemented with egg yolk to permit detection of a-toxin-producing strains (149,162). A variation of this medium is made without egg yolk (163) and is preferred at times when weak a-toxin producers are suspected. While most commercial media support the growth of pure cultures of C. per-ingem, they do not necessarily do as well for the organism in mixed cultures. Isolation of C. pe1;fiingelzsfrom chicken intestines on four different media demonstrated that sulfite-polymyxin-sulfadiazine nledium (164), tryptose-sulfite-neotnycin medium, Clostrisel (BBL), and lecithin-lactose agar were inadequate for good isolation (165). Another medium that contains oleandomycin, polymyxin, and sulfadiazine (OPSO) (see Table 3) is good for the selection of C. perj?ingens, but OPSO also supports fecal enterococcal growth (149). McClung-Toabe agar with added egg yolk also has been used for isolation of C. perfringens from foods (Difco, Ann Arbor, MI). Antibiotic addition to media is helpful in selecting for C. yeqringens. Cycloserine is added at 400 pg/mL final concentration. In OSPS, oleandomycin phosphate (Pfizer) and polymyxin B sulfate are added to give final concentration of 0.5 yg/mL oleandomycin phosphate and 10 IU/mL polymyxin B sulfate. Neomycin can be used with blood agar by spreading 0.06 mL of a 1% (w/v) sulfate over the plate (149). Due to C. perfi*ingens’optimal growth temperatures, the effectiveness of isolation can be improved by culture at 46°C rather than 35-37°C. However, media composition

Table 3 Media for Isolation _____

Tryptose (Difco) Soytone (Difco) Yeast extract Sodium metabisulfite Ferric ammonium citrate Ag‘W Source: Ref. 139.

15 15 5 1 1 20

For TSC: Cycloserine Egg yolk (optional)

400.5 pg/mL 0.08 mL/mL

For OSPS: Oleandomycin phosphate Polymixin B sulfate

0.05 Ing/InL 10 IU/mL

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affects culture growth at high temperatures. Marshall et al. demonstrated that C. perfiingerzs has difficulty growing at 46°C when cultured on tryptone-sulfite-neomycin agar (166j. Fecal specimens with spores may needto be heated to activate the spores. To activate spores, the fecal specimen typically is heated for 10 minutes at 80"C, which activates heat-resistant spores. Heat-resistant spores can survive heating at 100°C for 60 minutes (17). Heat-sensitive spores do not require heat for germination and may be killed during the 80°C incubation used to activate heat-resistant spores (167). Heating the specimen is also useful for decreasing the number of other species in the specimen.

2. Enumeration Enumeration of vegetative cells and spores aids in confirmation of C. pel-j'ritzgensas the cause of an outbreak. In suspect foods, high numbers (> 10"g food) of C. petfrirzgem suggest it as the cause of gastroenteritis (168,169). High numbers of spores also should be found in fecal specimens collected within 24 hours of symptom onset. Colony enumeration is performed by either tube methods or plate methods. Tube methods have the advantage of not requiring special anaerobic incubation chambers. A disadvantage of tube methods is that egg yolk cannot be incorporated into the agar for detection of a-toxin production. Typically, 150 X 19 mm tubes are filled with a selective and differential medium (e.g., TSC) and inoculated with dilutions of the fecal specimen or suspect food. To preserve anaerobiasis, the inoculated medium is topped with sterile medium or paraffin (149). An opaque white bar inserted in the tube at the time of inoculation enhances visibility of the colonies (170). Ali and Fung developed a double-tube assay for C. per-j'rirzgerzsenumeration (171). This entails insertion of an empty, thin, sterile test tube into a larger screw cap tube that contains the melted, inoculated TSC agar. The advantage of this method is that isolated colonies can be selected from the agar medium after removal of the thin inner tube. For plate counts, sealed pour plates or anaerobic culture chambers are necessary. With sealed pour plates, 14 mL of medium are inoculated with the sample and poured into a deep petri dish. This is covered by 10 mL of freshly molten medium. Reducing agents in the medium help to maintain anaerobiasis (149). Traditional spread plates on the appropriate medium will support growth when placed in anaerobic chambers such as the GasPak system or anaerobic incubators with the appropriate gas mixture. Alternatively, addition of the supplement Oxyrase permits incubation without specialized equipment. Although C. perjringens is relatively aerotolerant for an anaerobe, some organisms may be lost during plating and during the time needed to establish anaerobic conditions. Comparison of five media for recovery of heat-resistant spores indicates that TSC agar or Shahidi-Ferguson perfringens agar are better than tryptone-sulfite agar, sulfitepolymyxin-sulfadiazine agar, and tryptone-sulfite-neomycin agar (172). Plating on an antibiotic-containing blood agar has been reported as helpful in the isolation of heat-sensitive, spore-forming C. pe$&gens. Sutton and Hobbs demonstrated a correlation between hemolytic activity and heat sensitivity (167). Low numbers of C. per-j'rirzgem in foods are difficult to detect. Enrichment cultures coupled to most probable number (MPN) determinations can be used but are considered inaccurate and may represent the activity of nonclostridial species (173,174). Selective agents in themedium and incubation at 46°C improve recovery and aidpresumptive identification (148). Erickson and Deibel developed a rapid perfringens medium (RPM) that contains litmus plus supplement nutrients, polymyxin B, and neomycin sulfate (175). Iron-

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milk medium, a mix of whole milk and iron powder, also has been used for MPN enumeration of presumptive C. pel@ingerzs contamination of foods (176,177). When meat samples have low numbers of C. perfiiqerw, the organism has been enriched by placing the meat in vacuum packaging and incubating at 43-45°C. The gas produced by the organisms swells the bag, and drippings from the bag can be cultured for isolation (149). All methods used to detect C. perfringens need to be supplemented with additional tests for species confirmation and for enterotoxin production. B. BiochemicaVPhysiological Tests TSC and other selective media permit the growth of other clostridial species; therefore, species identification is necessary. C. perfringem has the following characteristics for identification: nonmotile, reduces nitrate, liquefies gelatin, and ferments glucose. Presumptive identification can be made based on “stormy fermentation” in iron-milk medium. Another characteristic is the ability to ferment raffinose but not salicin. C. perjhhgerzs has all the enzymes of the Embden-Meyerhof pathway for glucose degradation. It lacks glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (50). Alpha-toxin production can aid in identification. Bacteria growing on egg yolk agar and producing a-toxin will be surrounded by a zone of opalescence (149). Addition of anti-a-toxin to the medium will prevent the opalescence. Some strains associated with foodborne illness are weak producers of a-toxin. An enzyme immunoassay to detect a toxin is sensitive to 16 ng/mL (178). Rapid tests and multiple test strips with a number of biochemical tests in one system have become popular in laboratories for anaerobic, wound isolate identifications (179182). With these systems, C. perfrilzgens is identified with great reliability. The Vitek ANPMcard (Vitek Systems, bioMerieux, Hazelwood, MO) was used to correctly identify eight of eight strains of C. perfringens. The Patho-Tec (General Diagnostics, Cincinnati, OH), API ZYMTM(API Systems SA, La Balme les Grottes, Montalieu, Vericieu, France), and RapID ANA I1 (Innovative Diagnostic Systems, Atlanta, GA) correctly identified seven of seven. An-Ident (Analytab Products, Plainview, NY) also correctly identified known strains of C. perfkingens (179).

C. Other Tests Polymerase chain reactions (PCRs) using primers specific for C. per-ingens permit sensitive detection of the organism in a variety of specimens and give typing results similar to more classical methods (183,184). When coupled with electrophoresis, PCRs with a combination of primers can be used to look for the a-toxin, P-toxin, etoxin, and enterotoxin simultaneously. Alpha-toxin genes are detectable from direct enrichments of intestinal contents (185). One PCR reaction had a lower detection limit of 5 X 10’ bacteria from fecal specimens (186). Bacteriocin typing also may behelpful in identification of bacterial isolates responsible for outbreaks. Watson et al. typed 31 1 strains by bacteriocin patterns (187). Of the enterotoxin-producing strains, 92% were typeable. Serological typing initially was used to demonstrate that C. perfiirzgens was the cause of foodborne diarrhea (168,188). It still provides one of the best ways to determine if the organism found in the suspected food is the same as that isolated from fecal material and thus the agent of disease. Unfortunately, a significant number of isolates are still

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untypeable. One study found 24% of the isolates could not be typed by a panel of 75 antisera (188). Phage typing has not been adapted for use with C. perj%?gerzs. Yan used hostmodified phage to develop a typing scheme for C. peffringens isolated from human fecal material and other clinical specimens (189). Nine phage groups were used to define 10 typeable groups of C. pe@-ingens. Of the isolated organisms, 39% were not typeable by this method. Food isolates were not tested, and no correlation with enterotoxin production was made. Gas chromatography profiles of the fatty acid products of anaerobes are helpful in identifying some Clostridium species, but not C. yerjringens, which produces both acetic and butyric acid. Since C. pelfringens dies rapidly in cold storage, food samples may not yield high enough numbers to implicate it in a foodborne outbreak. Furthermore, the presence of carbohydrates in food may inhibit toxin production (46,54,76). Thus, direct toxin detection from foods may be unreliable.

D. Transport of Specimens Culture of C. yeifringens should be performed as rapidly as possible. Storage of specimens at 4°C can result in a loss of organisms as some die rapidly at that temperature. If plating cannot be immediate, then the food sample can be mixed 1:l (wthol)with 20% glycerol and stored on dry ice or at - 60°C (163).

VI.

ENTEROTOXINPATHOGENICITY

A.

FeedingStudies

The association of C. pelfkiizgens with gastroenteritis was not confirmed until 1953 when it was observed that both the associated food and the patients stools had high number of C. pelfiirtger~s.Of human volunteers fed 5.1 X lo*to 3 X 1O9 viable cells, 16 or l 8 became sick within 48 hours (8). Fecal specimens from the volunteers had high concentrations of spores. There is good correlation between the ability to cause gastroenteritis and positive rabbit ileal loop tests for enterotoxin (190). Ingestion of enterotoxin does not appear to play a major role in the disease. Eight to 10 mg of ingested toxin causes diarrhea in healthy adults (191j. Effectiveness of the toxin alone to cause disease is enhanced if gastric acids are buffered. Monkeys fed sodium bicarbonate developed gastroenteritis when fed 5 mg (192). Enterotoxin injected into rats or mice accumulates in the liver and kidneys, preferentially binding and killing parenchymal cells (193,194).

B. BiochemistryandMode of Action The enterotoxin of C. peffringerzs appears to be unique among the many bacterial enterotoxins. The single, 35,317 dalton polypeptide with 3 19 amino acids is produced in large quantities during sporulation and is released as free enterotoxin into culture medium concurrent with the release of spores from vegetative cells. The enterotoxin’s function in sporulation is not yet known. Hybridization studies using gene probes for the enterotoxin and numerous isolates of C. pelfringens reveal that only a small percentage of isolates

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have the enterotoxin gene (10) and that the protein is probably not essential to sporulation because there are spore-positive, enterotoxin-negative strains. The native protein is active. However, trypsinization increases activity threefold (195). The toxin binds to a 50,000 dalton protein in brush border membranes but is not enzymatic in activity. It forms a 160,000 dalton complex in the membrane with two membrane proteins of 50,000 and 7,000 daltons (196). Binding and cytotoxic activity are on different fragments of the toxin. Horiguchi et al. isolated a fragment that inhibits enterotoxin binding but has no cytotoxic activity (197). Binding of the toxin can be inhibited by monoclonal antibodies (198,199). Once bound, the toxin inserts into the membrane and remains associated with the membrane (200,301). A portion of the toxin remains on the external surface of the membrane (202). The result of the toxin’s presence is an increased permeability of the cell membrane to small molecules (203,204). The toxin creates ion-permeable “gates” in asolectin bilayers. The gates which are dynamic and bi-directional permit passage of molecules under 200 daltons (205j. Nucleotides and amino acids move through the gates and lead to cell death (206210). Addition of Ca+: may stabilize and enlarge the hole to permit small polypeptide movement (211,212). Calcium ions may not be necessary for cytotoxicity, McClane and Wnek examined both binding and permeability changes and concluded that there were two phases of enterotoxin activity (209). The first phase occurs during the first 7.5-8 minutes, is Ca+’ independent, and includes binding, insertion, complex formation, ion permeability, and inhibition of macromolecular synthesis. The second phase is Ca+?dependent and is accompanied by morphological damage to the cells. Permeability continues to increase, and the injured cells eventually lyse. In the intestines, cell death leads to desquamation of the villi, loss of fluid, and diarrhea.

C. Production Enterotoxin production is associated with sporulation frequency. Strains with a low frequency of sporulation in vitro tend to produce low concentrations of enterotoxin. Carbohydrates have a profound effect on sporulation. Many monosaccharides inhibit sporulation, while starch enhances it (54). A number of xanthines, such as caffeine, also enhance sporulation (79). Enterotoxin has a positive effect on its own production and enhances sporulation. Enterotoxin added to cultures decreased the lag phase, enhanced sporulation, and stimulated macromolecular synthesis in preexponential phase cells (213). Many sporulation media have been developed to enhance sporulation (61,69,7 173,75). Duncan-Strong medium continues to be the most widely used (61). Sporulation frequency in Duncan-Strong ranges from less than 0.1% to 90% (214). D medium is a defined sporulation and is used extensively in studies on enterotoxin production (76). All sporulation media need a complex carbohydrate such as starch or dextrin or the complex sugar raffinose. D. Purification The purification protocol of McDonel and McClane is used widely (214j. Preparation of the enterotoxin begins with an inoculum of a 9-hour culture of C. yerfi-ingem grown in fluid thioglycollate medium transferred to 20 L of fresh Duncan-Strong medium cooled

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155

to 37°C. For best production, the Duncan-Strong culture should be incubated until 80% of the vegetative cells form endospores. The bacterial cells are collected in a cold potassium phosphate buffer (pH 6.8) and sonicated to release the enterotoxin. Supernatant is collected by repeated centrifugation at 4°C. Enterotoxin in the supernatant precipitates on adjustment to 40% saturated ammonium sulfate at pH 7. Purification is enhanced by a second precipitation at 15% saturation with ammonium sulfate. The second precipitate then is resuspended in a sucrose buffer, and final purification is achieved on a Sephacryl 200TM column (Pharmacia LKB Biotechnology, Uppsala, Sweden). The fractions eluted off the column should be assessed for enterotoxin activity. The enterotoxin routinely is found in the second protein peak (214). A similar method is reported by Heredia et al. (215), with Duncan-Strong medium supplemented with raffinose. Other purification methods were described by Granum and Whitaker (69), Reynolds et al. (216,217), Dasgupta and Pariza (218), and Sakaguchi et al. (219). Purity is assessed by polyacrylamide gel electrophoresis. Satellite bands occasionally are seen and appear to be due to an endogenous protease (220).

E. Enterotoxin Assays Three basic groups of assays are used to identify or quantify C. yerjYingens enterotoxin: biological, serological, and tissue culture. DNA probes can be used to detect the presence of the enterotoxin gene (10). 1. Biological Assays Biological assays are some of the least sensitive assays to detect enterotoxin but have been useful in studies on mechanisms. The earliest assays were ileal loop assays. Rabbit, chicken, sheep, monkey, mouse, and calf intestinal loops have been used (17,190). LDS,,, mouse diarrheal, and rabbit and guinea pig erythemic reactions also have been used. Excised brush border membranes from the small intestine of rabbits have been used to examine the binding of radiolabeled enterotoxin (196,209).

2. Serological Assays Serological assays to detect enterotoxin have a long history (208,210,221-225). The most traditional serological tests are immunoprecipitation reactions in gels. In these assays, antibodies against the enterotoxin bind to the enterotoxin, forming a complex that precipitates. The most sensitive precipitations are counterimmunoelectroprecipitations (CIEPs), in which antiserum and enterotoxin are electrophoresed in a gel, and electroimmunoprecipitation (also known as rocket immunoelectrophoresis), in which the antiserum is in a gel and the enterotoxin is electrophoresed (223,226). The advantage of rocket immunoprecipitations is that the enterotoxin can be quantified based on the rocket height, which is proportional to the concentration of the enterotoxin. Both assays require a thin gel of buffered agar and predetermined optimal concentration of anti-enterotoxin antiserum (210). Enzyme immunoassays (EIAs), originally called enzyme-linked immunosorbent assays, have been modified both to detect and to quantify enterotoxin (208,224). Detection of 5 ng/g fecal material is possible. The absorbance for the samples can be compared to absorbance for known enterotoxin standards for quantitation (227). Enterotoxin inoculated into fecal samples has been detected at 1 ng/mL using an EIA (228,229).

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A variation on the liquid EIA is the DOT-EIA performed on nitrocellulose paper instead of microtiter plates. Mehta et al. diluted enterotoxin containing fecal samples and transferred 2 pL to nitrocellulose (230). The blots were visualized by treating with rabbit anti-enterotoxin, then anti-rabbit Ig conjugated with horseradish peroxidase, followed by the chromogen 3,3'-diaminobenzedine tetrahydrochloride. Reverse passive hemagglutination reaction with anti-enterotoxin antibodies fixed to human erythrocytes also detects enterotoxin in fecal extract (9). Reverse passive latex agglutination has also been reported (222). Unipath (Hampshire, England) markets a kit for perfringens enterotoxin reverse-phase latex agglutination (OxoidTM PET-RPLA). Sensitivity is approximately 2 ng/mL enterotoxin, and it is comparable to an EIA test for enterotoxin in fecal specimens (231). Other serological reactions have proved useful: single and double diffusion in gels, radioimmunoassay, and fluorescent antibody staining. EIA, radioimmunoassay, and reverse-phase assays are the most sensitive. The assays are less sensitive when the enterotoxin is in a biological specimen (e.g., fecal material) (208). 3. Tissue Culture Assays Tissue culture assays have used a variety of cell types. One of the more sensitive cell types is the Vero cell line. McClane and McDonel monitored enterotoxin by Vero cell assays based on changes in cellular morphology, cell detachment, decreased viability, plating efficiency, and alteration in cellular biochemical activity (207). These assays rely on microscopic observation; other studies have used "Rb efflux or neutral dye uptake to monitor enterotoxin damage to Vero cells (209,210,232).

F. DNA Probes of the Enterotoxin Gene Confirmation of a C. perj?ingerzs isolate as the cause of a gastroenteritis is enhanced if the isolate produces enterotoxin. Confirmation has been difficult since many strains do not sporulate well in vitro and therefore do not produce detectable enterotoxin. The use of DNA probes to detect the enterotoxin gene appears to be a useful alternative. They are sensitive and avoid some of the problems associated with enterotoxin detection in biologic a1 materials. Van Damme-Jongsten et al. cloned and sequenced the enterotoxin gene (erzt) (104) and constructed j'P-labeled probes. The probes hybridized with DNA from C. perfi-irzgens isolated from food and fecal material collected during confirmed outbreaks. The probe also detected the gene in one outbreak-associated strain that did not produce enterotoxin in culture. Comparisons of Western immunoblotting for the enterotoxin and PCRs to detect the gene further indicate the inability of some erzt gene-positive isolates to sporulate and produce the enterotoxin in culture (233). PCR reactions on beef samples could detect less than 10 colony-forming units per gram (234). The enterotoxin probes have indicated that the enterotoxin may have a major importance only in human disease. Isolates from sheep with C. pe$+lgens-induced diarrhea did not have the enterotoxin gene (10,235).

VII.

PREVENTION

Prevention of C. yegringens gastroenteritis depends on the elimination of spores from food or halting contamination during food processing and handling. Proper cooling of prepared foods and sufficient reheating to kill vegetative cells also will aid in prevention.

Clostridium perfringens

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Heat killing of spores can be difficult (93). Heat-resistant spores can survive heating at 80°C for over 1 hour. Heat-resistant spores have Dgs values ranging from 17.6 to 64 minutes (70). Viable spores of C. ye~fringensNCTC 8798 were recovered after 10 minutes at 105°C. At 120"C, 0.01% of this strain survived for 20 seconds (172). Heat-sensitive spores have Dgsvalues between 1.3 and 2.8 minutes (70). Thus, elimination of contamination by good hygiene and high-quality food sources becomes the best means to prevent infection.

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222. Harmon, S. M., and Kautter, D. A. (1986). Evaluation of a reversed passive latex agglutination test kit for Clostridium pe$ringens enterotoxin. J. Food Prot.. 49523-536. 223. Naik, S.. and Duncan, C. L. (1997). Rapid detection and quantitation of Clostridium perfr-ingeru enterotoxin by counterimmunoelectrophoresis. Appl. Empiron.Microbiol., 34: 125128. 224. Narayan, K. G.. Genigeorgis, C., and Behymer, D. (1984). Use of enzyme-linked immunosorbent assay (ELISA) in the quantitation of Clostridizlnz pe$ringens type A enterotoxin and antienterotoxin antibodies. Int. J. Zoorz., 10:105-1 10. 225. Uenlura, T., Sakaguchi, G., and Riemann, H. P. (1973). In vitro production of Clostridizrnz petfiingerzs enterotoxin by reversed passive hemagglutination. Appl.Microbiol., 26:381385. 226. Holliday. M. G. (1985). Rapid identification of Clostridium perfringeitsby counter-immunoelectrophoresis. Med. Lab. Sci., 42:322-325. 227. Bartholomew. B. A., Stringer, M. F., Watson, G. N., and Gilbert, R. J. (1985). Development and application of an enzyme linked immunosorbent assay for Clostr-idiunz per-ji-irzgens type A enterotoxin. J. Clin. Pnthol., 38:222-228. 328. Olsvik, O., Granum, P. E., and Berdal, B. P. (1982). Detection of Clostridim pe$ringens type A enterotoxin by ELISA. Acta Pcuhol. Microbiol. Immmol. Scnrzd., Sect. B, 90:445447. 229. Jackson, S. G., Yip-Chuck, D. A., and Brodsky. M. H. (1986). Evaluation of the diagnostic application of an enzyme immunoassay for Clostridium pe$r-ingens type A enterotoxin. Appl. Environ. Microbiol., 52969-970. 230. Mehta. R., Narayan, K. G., and Notermans, S. (1989). DOT-enzyme linked immunosorbent assay for detection of Clostricliurnperfringens type A enterotoxin. Int. J. Food Microbiol., 9:45-50. 231. Berry, P. R., Stringer, M. F., and Uemura, T. (1986). Comparison of latex agglutination and ELISA for the detection of Clostridium perfrirlgens type A enterotoxin in faeces. Lett. Appl. Microbiol., 2: 101-102. 232. Mahony, D. E., Gilliatt, E.. Dawson, S., Stockdale, E., and Lee, S. H. S. (1989). Vero cell assay for rapid detection of Clostridium ye@-ingewsenterotoxin. Appl. Erwiron. Microbiol., 552141-2143. 233. Kokai-Kun, J. F., Songer, J. G., Czeczulin, J. R., Chen, F., and McClane, B. A. (1993). Comparison of Western immunoblots and gene detection assays for identification of potentially enterotoxigenic isolates of Clostridium perfringens.J . dirt. Microbiol., 322533-3539. 234. Baez, L. A., and Juneja. V. K. (1995). Detection of enterotoxigenic Clostridium pe$ringens in raw beef by polymerase chain reaction. J. Food Prot., 58:154-159. 235. Van Damme-Jongsten, M., Haagsma, J., and Notemans, S. (1990). Testing strains of Clostridiiun? pe$ringerzs type A isolated from diarrhoeic piglets for the presence of the enterotoxin gene. Vet. Rec., 126:19 l - 192.

9 Escherichia coli Marguerite A. Neil1 Brown Universiq School of Medicine. Providence, Rhode Islarld

Phillip 1. Tarr University of Washingtoil and Children's Hospital ami Medical Center, Seattle. Washingtoll

David N. Taylor and Marcia Wolf Walter Reed Army Institute of Research, Washington, D.C.

I. Introduction

170

11. Enteropathogenic E. coli

170

A. Historical aspects 170 B. Characteristics of EPEC 171 Clinical C. manifestations 172 D. Epidemiology 172 E. Isolation and identification 173 F. Pathogenicity 173

111. Enterotoxigenic E. coli

175

Historical aspects 175 B. Characteristics of the ETEC 176 C. Characteristics of disease D. Epidemiology 177 E. Pathogenicity 178 IV. Enteroinvasive E. coli 180 A.

A. B. C. D. E.

Historical aspects 180 Characteristics of disease Epidemiology 181 Identification 181 Pathogenicity 182

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180

V. E. coli 0157:H7 and Enterohemorrhagic E. coli

182

A. Historical aspects 182 B. Characteristics of E. coli 0157:H7 and EHEC C. Characteristics of disease 184 D. Epidemiology 186 E. Isolation and identification 186 F. Pathogenicity 187

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7 70 VI. Enteroaggregative

E. coli

A. Historical aspects 189 B. Characteristics of the EAEC C. Clinical manifestations 191 Epidemiology D. E. Pathogenicity 19 1

189 190 190

VIT. Role of Food andWaterinTransmissionofDiarrheagenic VIII. Isolation

from Foods

E. coli

191

194

References 196

1.

INTRODUCTION

Over 100 years have elapsed since the original description of Bacterium coli cornnrzme by a German pediatrician, Dr. Theodore Escherich, in 1885 (l). His useofthesuffix “commune” recognized that this gram-negative organism, today known as Escherichia coli, was widely distributed in the intestinal flora of humans and warm-blooded animals. E. coli is the predominant facultative anaerobe in the human bowel and helps to maintain normal physiological function of the intestine (2). It was precisely this widespread and apparent commensal relationship between bacterium and host that obscured for many years the role of E. coli in causing enteric disease. When serological characterization was able to incriminate certain strains in the etiology of diarrheal illness, the species was finally recognized to be heterogeneous, containing both pathogenic strains and harmless inhabitants of the gastrointestinal tract. Previously, the diamheagenic E. coli were divided into four major categories: (a) the enteropathogenic E. coli (EPEC), (bjthe enterotoxigenic E. coli (ETEC), (cj the enteroinvasive E. coli (EIEC), and (d) the enterohemorrhagic E. coli (EHEC). Within each category, strains share distinct, category-specific virulence traits that are often plasmid encoded, common epidemiological and clinical features, and a similar pathophysiological mechanism of action. A fifth category is constituted by the enteroaggregative E. coli (EAEC), a recently recognized diarrheagenic E. coli. This group is composed of strains that have been epidemiologically linked to diarrheal disease but lack properties typical of the first four groups and possess a particular adherence phenotype. Another category of potentially diarrheagenic E. coli is the diffusely adherent E. coli (DAEC); because epidemiological and pathogenic features of the DAEC are less well understood, this group will not be discussed here. The five major categories of the diarrheagenic E. coli are the subject of this review.

II. ENTEROPATHOGENIC E. COLI

A.

Historical Aspects

The power of epidemiology to lay the groundwork for the discovery of an etiological agent is elegantly illustrated in the story of how EPEC came to be recognized as causes of infantile diarrhea (3,4). Pediatric diarrhea in the late 1700s was characterized by Dr.

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Benjamin Rush as a severe diarrheal illness occurring mostly in urban areas during the summer months (5), and from these observations came the terms “cholera infantum” and ‘‘summer diarrhea.’ ’ Even as epidemiological studies developed the corollary observations that disease was most severe in those less than 2 years old and those not breast-fed, mortality from childhood diarrhea fell dramatically in the United States and Great Britain. Explosive outbreaks in hospital nurseries during the 1930s were accompanied by high mortality rates and focused attention on the outbreaks’ likely infectious, but as yet undiscovered, causative agent. The association linking the bacterium E. coli with infantile diarrhea was made by Dr. John Bray, a British physician, who used his nose as well as his intellect in his investigations. Dr. Bray recognized that certain strains of E. coli from the stool cultures of children with diarrhea had a peculiar seminal odor, and that this same odor had been ascribed to the patients themselves by a pediatrician, Dr. Thomas E. D. Beavan, at the Hillingdon Hospital in Uxbridge, England. The antiserum prepared by Bray using these strains as an immunogen in a pet rabbit named Snowy agglutinated strains of E. coli in the stools of 95% of 50 infants with diarrhea and almost none (4%) of control E. coli from healthy children (6). The subsequent development of the Kauffman-White serotyping system, based on bacterial somatic (0),flagellar (H), and capsular (K) antigens (7j, was instrumental in proving that the hospital nursery outbreaks were related to specific serotypes. Studies of experimental infection helped to strengthen the epidemiological and serological association of these E. coli strains with diarrhea and convince the remaining skeptics that at least some serotypes of this normal inhabitant of the gastrointestinal tract could cause diarrhea, albeit mechanism unknown. In 1955, Neter (8) captured the diarrheal potential of these strains in his proposal of the term “enteropathogenic” E. coli (EPEC) for those serotypes with definite epidemiological association with childhood diarrhea. The recognition and definitionof the enterotoxigenic and enteroinvasive E. coli led to a renewal of skepticism that EPEC strains were pathogens, because the EPEC strains lacked the diarrheal mechanisms of the ETEC and EIEC and had no alternate explanation for their capability to cause diarrhea. Studies in volunteers by Levine et al. (9) demonstrated that EPEC strains lacking toxin production or invasiveness could cause diarrhea in humans. EPEC are now accepted as a distinct group of diarrheagenic E. coli, and considerable advances have been made in our understanding of the molecular pathogenesis of diarrhea from these strains.

B.Characteristics

of EPEC

1. Nomenclature The definition of EPEC strains has evolved over the past 40 years. Initially EPEC were defined solely on the basis of possession of certain somatic 0 antigens. This serogroupbased definition changed to a serotype-based categorization when EPEC were associated with particular O:H types. EPEC were subsequently shown to produce a characteristic histopathological finding, the attaching-effacing (A/E) lesion, which is now recognized as a hallmark of both their identification and their diarrheagenic properties. Other strains of E. coli, particularly those that produce Shiga toxin (Stx), may also produce A/E lesions. The current working definition formulated at the Second International Symposium on EPEC is that EPEC strains produce A/E lesions and lack the ability to produce Stx (10). Most EPEC by this definition cluster within certain O:H serotypes, but serotype alone is no longer the basis for definition of EPEC.

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Occasionally the tern1 “enteropathogenic” E. coli is used in a more broad fashion to denote any strain of E. coli that causes diarrhea. Such usage, while perhaps philosophically appealing, dilutes rather than enhances our conceptual understanding of the diarrheagenic E. coli and should be discouraged.

2. Serogroups Fifteen 0 serogroups had been described previously as including members of the EPEC: 018ab, 018ac, 026, 044, 055, 086, 0111, 0 1 14, 0119, 0125, 0126. 0127, 0128ab, 0142, and 0158 (1 1-14). Serotyping using the H antigen has shown that strains of different serotypes within the same 0 serogroup do not have equal pathogenic potential or occurrence in disease. Some serogroups, such as 055, are so rarely found in healthy persons that detection of this serogroup in a child with diarrhea may make further serotyping unnecessary as the strain is highly likely to be pathogenic. Other serogroups, such as 086, can occur commonly in normal bowel flora so detection of an 0 8 6 strain in a child with diarrhea necessitates further serotyping to determine if it falls into the one serotype (086: H34) associated with diarrheal disease anlong the 21 known serotypes in serogroup 086. Serological differentiation alone is insufficient to fully characterize the pathogenicity of an individual isolate and additional testing is required for classification into one of the groups of the diarrheagenic E. coli. Based on the current definition of EPEC and including genotypic characterization, those serotypes that characteristically contain EPEC include 055:H6,05S:NM, 086:H34, 0 1 11:H2. 011 1:H12, 0 1 11:NM, 0 1 19:H6, 0 1 19:NM, 0125ac:H21, 0126:H27, 0126: NM, 0127:H6, 0127:NM. 0128:H2, 0128:H12, and 0142:H6 (15). C. Clinical Manifestations There are no major distinguishing features of the diarrhea due to EPEC infection that can be considered pathognomonic. Typically the illness is an acute watery diarrhea with lowgrade fever and vomiting occurring in young infants (9,16). Severe disease resulting in death or prolonged diarrhea ( B 14 days) is not uncommon in young children, especially those less than 6 months of age (16- 19). In a study of community-acquired gastroenteritis, hospitalized children with illness attributed to EPEC had diarrhea for an average of 4.6 days compared to 2.4 days for children with nonbacterial gastroenteritis (20). These findings on duration and severity support the observation that the diarrhea due to EPEC infection may be more severe than that due to the other common causes of pediatric diarrhea such as rotavirus. The lack of symptomatic infection in older children and adults coupled with the not infrequent finding of such persons asymptomatically excreting EPEC strains (21,22) has led to the concept that imnlunity is acquired. However, EPEC are not common causes of traveler’s diarrhea even in geographic areas with a high incidence of EPEC, which may suggest that immunity is developmentally regulated. D. Epidemiology EPEC have been reported as a cause of childhood diarrhea from around the world (17,20,22,23), predominantly in children less than 2 years of age. Case-control studies comparing fecal isolation of EPEC with disease status have shown the most significant correlation of EPEC isolation and diarrheal illness in infants younger than 6 months (19). Both colostrum and breast milk have been shown to have both specific and nonspecific

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factors that inhibit EPEC adherence to HEp-2 cells (24) and breast feeding is protective against EPEC infection. Bottle feeding has been noted as a significant risk factor for EPEC infection. Explosive outbreaks in the community and in hospital nurseries were typical of the epidemiology of EPEC in Europe and North America through the 1950s, and mortality ranged up to 50% (19). These have largely disappeared, and this has been ascribed to improvements in sanitation and hygiene. In the developed world the current epidemiology of EPEC includes their implication as a cause of day-care center and occasional nursery outbreaks and community-acquired sporadic infantile diarrhea. Child care settings in the United States have been recognized as sites for EPEC-associated outbreaks (25,26), and chronic diarrhea was a prominent feature in these outbreaks. A recent study suggests that the frequency of EPEC as a cause of childhood diarrhea in the United States may be considerably underestimated (27). Although rare, outbreaks of EPEC infection in adults have been noted (28,29). The largest burden of disease due to EPEC is in the developing world. In some studies, EPEC accounted for one third of all infant diarrhea and rivaled rotavirus for the most common cause of diarrhea in the first year of life. The physical environment (linen, toys, scales, carriages, bathing area, dust) around an infant with EPEC diarrhea has been shown to be extensively contaminated (30). The route of transmission is likely a fecaloral one from contaminated foods or formula, from the contaminated hands of caregivers, or from soiled fomites. Respiratory transmission has also been postulated (30,31).

E. IsolationandIdentification EPEC have the same colony morphology on culture media and similar biochemical reactions as other E. coli. The definitive identification of EPEC-associated enteric disease is not possible with the diagnostic tools available in the average clinical laboratory. Serogrouping and serotyping are relatively uncomplicated but labor-intensive techniques, which were previously used as the basis to identify EPEC, but some strains have been described that do not fall into one of the classic EPEC serogroups (32). The current approach to identifying EPEC is to determine the adherence characteristics of the bacterial isolate and the absence of Shiga toxin production. Such testing may be performed either phenotypically or genotypically, with the latter based on the use of DNA probes and/or PCR assays. The A/E phenotype is demonstrated using the fluorescent actin stain (FAS) (33,34); this correlates with possession of e m gene sequences found within the locus of enterocyte effacement (LEE) pathogenicity island. Localized adherence (LA) to HEp-2 cells has generally correlated with the presence of the EAF plasmid and hfpA sequences (see below). Typical EPEC strains possess eae sequences and the EAF plasmid but do not produce Shiga toxin. Strains that lack the EAF plasmid but that otherwise have the key properties of EPEC (A/E phenotype and absence of Shiga toxin) are considered atypical EPEC. The intricacies of these diagnostic considerations have been recently reviewed in detail (15). F. Pathogenicity Despite the description of EPEC as the first recognized class of diarrheagenic E. coli, until recently our understanding of the sequence of events by which EPEC cause enteric disease has remained in a fairly primitive state in comparison to our understanding of the

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mechanisms of disease involved i n ETEC and EIEC infection. Recent reviews superbly summarize the explosion of work in this area (1 5,35). A distinct histopathological picture associated with human EPEC infection was first described by Polotsky et al. (36) in which the bacteria were observed to be very closely adherent to the epithelial cells and the surrounding microvilli were destroyed. Moon et al. applied the ternl, “attaching and effacing’’ first used by Staley et al. in 1969 (37), to describe the epithelial cell loss and cytoplasmic degeneration seen in the intestines of newborn pigs exposed to EPEC (38). Knutton st al. showed that EPEC attachment and effacement were associated with the accunlulation of actin within cultured cells at the point of EPEC attachment (33). Several other proteins of the cellular cytoskeleton have also been shown to accumulate beneath attaching and effacing EPEC strains (39,40). Studies on the molecular basis for the A/E lesion have now localized the genetic sequences for this distinctive histopathology to a large 35 kb chromosomal region called the LEE (41 ). Within the LEE are genes encoding several secreted proteins (espA, B, D), which appear to be involved in signal transduction and for which actual binding of the bacterium to the epithelial cell surface is necessary for them to effect signaling changes. Also contained in the LEE is the ecle sequence (E. coli attaching and effacing), which encodes intinin (42). This approximately 94 kb protein is involved in the intitnate adherence of bacteria to the cell surface and has 50% similarity to the invasin protein of Yersirzin. Another characteristic determinant in EPEC strains is their ability to adhere in localized microcolonies to epithelial cells in vitro (43). This feature is distinct from the diffuse adherence (or even nonadherence) seen with strains of ETEC, EIEC, or uropathogenic E. coli. This localized pattern of adherence was noted to be associated first in EPEC strain 0127:H6 with a 60 MDa plasmid designated pMAR2 (44) and subsequently in other EPEC strains with plasmids in the 55 to 70 MDa size range (45). These plasmids, termed “EPEC adherence factor” (EAF) plasmids, were noted to have a high degrees of homology at the DNA level. The basis for the bacterium-to-bacterium adherence in the localized adherence pattern is the bundle-forming pilus (BFP) (46), which has some sequence similarity to the toxinco-regulated pilus of Vibrio choler-ne.Whether BFP mediates bacterial adherence to the epithelial cell is unknown. A DNA probe was developed from a region of pMAR2 which was necessary for the localized adherence phenotype (47), and the EAF DNA probe has been utilized extensively in recent epidemiological studies detecting and characterizing EPEC (48-50). The importance of the 55 MDa plasmid in influencing both adherence phenotype and diarrheagenic potential can be seen in the following lines of evidence: first, nonadherent nonpathogenic laboratory strains of E. coli acquire the localized adherence trait with transfer of the plasmid; second, loss of the plasmid was correlated with loss of the capacity to exhibit localized adherence by several EPEC strains (51); and third, volunteers fed the EAF plasmid-cured strains had a much lower frequency of diarrhea than those receiving the parental EPEC strains that retained the plasmid (52). Not all EPEC strains hybridize with the EAF probe, although they have been implicated as diarrheal pathogens from epidenliological and volunteer studies and cause similar histopathological findings as EAF-positive strains. However, they have been given the designation atypical EPEC to distinguish them from the more common EAF probe-positive typical EPEC (50). Earlier descriptive studies of EPEC infection had shown intracellular bacteria by electron microscopy, suggesting that theorganisms could be invasive. However, the inability of EPEC strains to cause kerato-conjunctivitis in the guinea pig eye (Sereny test) and their lack of the very high molecular weight (140 MDaj plasmid of SkigeZZn and EIEC,

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which is associated with invasiveness, was taken as evidence that EPEC are not invasive. Revisiting the earlier observation of the intracellular location of EPEC, several investigators have recently shown that epithelial cells of various lineages can be invaded by EPEC strains (53,54) and the EAF plasmid increases invasion. How these events at the cellular level relate to the pathogenesis of EPEC diarrhea is not yet understood. The genetic basis of EPEC virulence, reviewed by Donnenberg and Kaper ( 3 3 , suggests a possible three-stage model for infection and disease production. Bacterial adherence to nlicrovilli is accomplished by a specific protein; subsequently the BFP allows bacteria to adhere to each other, contributing to the localized microcolonies seen in adherence studies. Both plasmid and chromosomal genes are required for this step. Secreted proteins contribute to signal transduction, with phosphorylation of several proteins involved in ion transport. Elevation of intracellular calcium levels is accompanied by microvillus effacement. Alteration of the cytoskeleton follows as the organisms adhere intimately and characteristic cuplike pedestals form. The epithelial cell may then be invaded by some of the closely associated surface EPEC organisms (35).

111.

ENTEROTOXIGENIC E. COLI

A.

HistoricalAspects

After EPEC, the second major group of the diarrheagenic E. coli to be recognized as a distinct pathogenic entity were the ETEC. A seminal observation was made by De et al. in the recognition of the similarity of findings induced in ligated ileal loops inoculated with either V. choleme or some serogroups of E. coli (55). Following De’s discovery of cholera toxinin 1959 (56), Taylor and Bettelheim noted that chloroform treatment of culture fluids of E. coli did not destroy their capacity to induce fluid secretion in the intestine, thus suggesting that enterotoxin(s) were made by these strains (57). The 1960s and 1970s saw the discovery of the enterotoxins, the formulation of a working definition of ETEC, and recognition of the profound importance of ETEC as causative agents of diarrhea in various settings. Smith and Gyles reported that two enterotoxins produced by E. coli could be distinguished on the basis of differential heat lability, a distinction that is reflected in the nomenclature of heat-labile toxin (LT) and heat-stable toxin (ST) (58). Gorbach et al. laid the groundwork for the appreciation of ETEC as important causes of human diarrhea in the tropics (59), while the further characterization of these strains by Sack validated their enlarging role as diarrheal pathogens (60). As noted in the section on EPEC, this body of work defining a basis for diarrhea from ETEC infection had the effect of casting doubt on the true pathogenicity of the EPEC, for which no mechanism had been defined. ETEC subsequently were shown to account for considerable proportions of infantile diarrhea in the developing world (61-65) in contrast to their relative unimportance in pediatric diarrhea in the developed world. Traveler’s diarrhea, a disorder of many sobriquets (“turista,” “Delhi belly,” “Rome runs,” “Aztec two-step.” “back door sprint,” “Montezuma’s revenge,” etc.), was added to the roster of settings in which ETEC were important etiological agents (66,67). ETEC also have been shown to cause diarrhea in military troops going into areas of lower hygiene and sanitation (68,69), including during the recent conflict in the Persian Gulf (70). Although generally uncommon in the United States, an outbreak of ETEC in the summer of 1998 in Chicago caused illness in more than 4000 people (71).

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B. Characteristics of the ETEC 1. Nomenclature By definition, ETEC strains produce either LT, ST, or both. However, enterotoxin production is not the sole determinant of pathogenicity; it also requires the presence of certain attachment factors called CAFs that permit adherence of bacteria to the human small intestine (see below). Depending on their complement of these attachment factors, ETEC may be pathogens of animals or humans or, rarely, both. 2. Serogroups Over 75 0 serogroups have been detected on ETEC strains, although half of the serogroups occurred only once (72). The most common ETEC serogroups are 06, 08, 025, 027, 078, 0128, 0153, 0148, and 0159 (72) (Table l). 0 serotyping is useful to identify ETEC causing outbreaks, but in endemic settings it isnot useful to identify ETEC because of the large proportion of ETEC with rare 0 serogroups. Further, ETEC 0 serogroups may overlap with nontoxigenic E. coli that commonly cause urinary tract infections or meningitis. However, 0 serogroup determination is useful for characterization of ETEC in endemic settings once they have been identified by other means. Over 30 H serogroups have been detected in ETEC. The most common are H9, H12, H16, H21, and H45. The role of flagella in ETEC pathogenesis is not clear (73), although by analogy to cholera it might be expected to play a role. Table 1 shows the association of H serotypes commonly associated with other factors. Attachment factors known as colonization factor antigens (CFA) can be considered a third serogroup. CFA/I, CFA/II, and CFA/IV have been found to be the most common (72) (Table l), but in some studies they occur on less than 30% of the ETEC (74-76). The terminology is somewhat confusing, because CFA/II may contain CS3, CS 1 and CS3, or CS2 and CS3. CFA/IV may contain CS6, CS4 and CS6, or CS5 and CS6. CS 1, CS2, CS3, CS4, CS5, and CS6 are distinct structures on the surface of bacteria. Attempts to find another common CFA have not been fruitful, although a number of CFA have been studied such as CS17, PCF020, PCFO159, PCFO166, and longus. To date, longus seems the most likely to be a common CFA (77), but more data are needed to assess these CFAs. The groundwork for an ETEC vaccine based on CFA types will require the combined epidemiological and molecular characterization of these antigens and continued determination of their relative distribution among the ETEC serogroups important in human disease.

Table 1 Common ETEC Phenotypes Toxin

Serotype 06:H16 08:H9 08:H9 027:H7 078:H12 0128:H12 0148:H28 0153:H45 0169:H-

CFA LTST LTST LT ST ST or LTST LTST ST ST ST

CFA/II (CS1 and CS3 or CS2 and CS3) CFA/II (CS3 alone) CS 17 CFA/IV (CS6 alone) CFA/I CFA/I CFA/IV (CS6 alone) CFA/I CFA/IV (CS6 alone)

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ETEC are common in environments with poor sanitation, and the common 0 and H serogroups and CFAs are all widespread throughout the developing world. In developed countries where ETEC infections rarely occur and no immunity exists among inhabitants, food and waterborne outbreaks of ETEC occur. In developing countries where ETEC is endemic and immunity to a wide variety of serotypes is acquired in early childhood, outbreaks rarely occur. The wide variety of serotypes increases the complexity of efforts to identify common antigens to include in vaccines against ETEC and to exploit for diagnostic assays.

C.Characteristics

of Disease

A noninflammatory diarrhea accompanied by nausea, mild to moderate abdominal cramping, and little if any fever is the typical illness produced by ETEC infection. Blood usually is not present inthe stool, and fecal leukocytes are absent. The diarrhea often starts abruptly 1-3 days after ingestion of ETEC. Because the mechanism of diarrhea production is so similar to that in V. cholerae infection, in its most severe form ETEC infection resembles cholera with severe dehydrating dian-hea and attendant high mortality. The course is somewhat variable depending upon the setting in which infection occurred. For most individuals with traveler's diarrhea, the illness is mild and usually lasts less than a week, but 5-10% may last longer. Regardless of the length of illness, the greatest impact is on the traveler's schedule (70,78). Under certain circumstances, as in military campaigns, even a mild illness may have dire consequences if a large proportion of the population is affected and is unable to perform scheduled duties. Because children are more sensitive to dehydration, ETEC infection can be severe in children (14,79). Frequent episodes of diarrheal disease can contribute significantly to malnutrition (62,80). Unlike EPEC, ETEC infections occur in nonimmune adults (travelers) as well as children. Fecal shedding of ETEC, either from symptomatic or asymptomatic infections, allows for continued transmission in areas of poor sanitation.

D. Epidemiology ETEC are not recognized as causative agents of diarrhea in Europe and North America (8 1,82), although occasional ETEC outbreaks have occurred (7 1,83-86). In the developing world ETEC is a major cause of childhood diarrhea. The attack rate increases with weaning, probably on a multifactorial basis that includes the withdrawal of factors protective against diarrhea found in breast milk, greater exposure to contaminated foods and water, and the decline in nutritional status that often accompanies weaning. Children in the developing world may sustain multiple clinically significant ETEC infections in the first 5 years of life as well as numerous asymptomatic infections (63,64,87). Although the attack rate declines after age 5, isolation of ETEC from asymptomatic people continues frequently, implying the development of immunity. Volunteer studies have shown that the inoculum required to produce illness with ETEC strains is fairly high, 108-10Joorganisms (88-91), an inoculum easily obtainable in unrefrigerated foods or contaminated water. Diminished host defenses such as decreased gastric acidity may allow illness to develop after receiving a smaller inoculum. For travelers, the risk of developing a diarrheal illness climbs proportionately with the length of stay in the geographic locale, with a 50% risk of illness being reached by

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a stay of 14 days. Studies have noted that the tnajority (20-75%) of traveler’s diarrhea is due to ETEC (70,92,93). The risk of ETEC infection appears to be highest with travel to Latin America and Africa, followed by travel to Asia (93). In contrast to people in endemic areas, asymptomatic infections with ETEC are infrequent in travelers.

E. Pathogenicity 1. Enterotoxins Two distinct enterotoxins, LT and ST (94), are the defining feature of ETEC strains. They may occur together or individually, and both cause secretory diarrhea. The names derive from their differing stability to heat: LT is a heat-labile toxin (inactivated after heating at 60°C for 30 minutes) and ST is stable after heating. A third enterotoxin called EAST has been found in some but not all ETEC strains (95). Evidence suggests that EAST alone tnay have pathogenic potential (29,96); it therefore may be a cofactor in ETEC. More data is needed to clarify the role of EAST in diarrheal disease. LT is a family of enterotoxins of varying similarity to each other and to cholera toxin (CT) of V. cholerue. The LT-I group includes LT of human origin (LTh) and porcine origin (LTp). LTh and LTp are very closely related to cholera toxin; they have 80% homology of amino acid sequences, the satne mechanism of action, and are neutralized by antibody to cholera toxin (94,97). Genes for both LTh and LTp are plasmid encoded. The LT-I1 group seems not to contribute to diarrhea in humans. LT holotoxin consists of one catalytically active A subunit and five B subunits, the latter being responsible for binding to a particular intestinal cell surface molecule, ganglioside GM,. Following binding to the GM, receptor, the A subunit of LT is internalized and enzymatically cleaved into the A1 and A2 subunits. The A1 subunit causes ADPribosylation of a regulatory protein, causing irreversible activation of adenylate cyclase. The resultant intracellular accumulation of cyclic adenosine monophosphate (CAMP)activates a protein kinase, causing absorption of sodium and chloride and secretion of chloride. Additional mechanisms may play a role in causing diarrhea. The watery diarrhea that results contains significant amounts of electrolytes. The discovery that neither CT nor LT impaired the transport of sodium from the intestinal lumen across the epithelial cells via a glucose and amino acid coupled pathway formed the basis for oral rehydration therapy (ORT) for diarrhea. ST differs from LT in size, structure, antigenicity, and tnechanism of action. ST resists heat inactivation, retaining activity even after boiling for 30 minutes (58), probably due to its heavy internal cross-linking by disulfide bonds. Two distinct forms of ST have been described: STa (also known as STA,STI, and ST1) and STb (also known as STII, STB, and ST2). Both are low molecular weight proteins that neither elicit an antibody response after natural infection nor are neutralized by antibody to LT or CT. STa is important in hutnan infection, whereas STb has been detected primarily in porcine strains. In contrast to the mechanism of LT, STa produces its effects immediately but irreversibly through the stimulation of guanylate cyclase with subsequent cyclic guanosine monophosphate (cGMP) accumulation (94,98j. The lack of commercial assays has limited the diagnosis of ETEC, but a GM,-based enzyme itnmunoassay (GM, ELISA), gene detection by hybridization, and PCR are assays in use in research settings. The detection of LT historically has been in tissue culture assays using Y-l adrenal cells or Chinese hamster ovary (CHO) cells, bothofwhich

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exhibit characteristic morphological changes following exposure to the toxin. Knowledge of the binding specificity of LT led to the development of GM, ELISA (99). Contrary to the relative ease with which LT can be detected, ST has not been detectable by tissue culture using nonintestinal cells. A suckling mouse model has been used for the detection of STa (98). After overcoming the lack of antigenicity of native ST, several ELISA methods have been developed (100- 102), including one that utilizes a monoclonal antibody (103). Specific DNA probes and primers now permit detection of LT and ST by hybridization (64,65,104-109) or PCR (107,110-113). ETEC strains may produce either LT or ST alone or in combination, and the genes for expression of these toxins are plasmid mediated. In many ETEC strains, one plasmid may carry genes for LT, ST, and a CFA. The distribution of toxin profiles among ETEC varies somewhat according to geographic region. In Thailand, Chile, and Mexico, colonization with strains expressing ST and LTST were associated with diarrhea (32,64,65), but more recently in Egypt, colonization with strains expressing only ST were associated with diarrhea (63). ETEC expressing only LT (which frequently lack CFAs) are isolated as frequently from symptomatic as asymptomatic volunteers. 2. Colonization Factors As noted above, enterotoxin production is one of two critical virulence determinants for enteric disease caused by ETEC; the other is possession of colonization factor antigens (CFAs) that permit the organism to attach to enterocytes in the proximal small intestine. The tissue specificity of the enterotoxins and the inefficient release of toxins requires that the bacteria be delivered at close range and attachment to the epithelial cell surface is the means to achieve this end. There has been intense interest in CFAs because of their potential development as vaccine components. CFA are long structures of repeated protein subunits on the surface of bacteria that promote attachment to receptors on epithelial cells of the intestine (1 14,115). As such they are fimbriae (also called pili) that are specific to ETEC. Electron micrographs show them as hairlike appendages projecting outward from all over the bacterial surface. Their structure somewhat resembles the flagellar antigens, but the CFA structures are much thinner and assembly mechanisms are much simpler than flagella. The repeating protein subunits are arranged in either a rigid structure 6-7 nm in diameter or a wiry, flexible structure that is thinner (2-4nm in diameter). Each structure may have thousands of subunits and reach lengths equal to the length of the bacterium. The CFA proteins are produced in culture at 37°C but not 18°C. The DNA sequences of many of these CFAs have been determined. Several ETEC colonization factor antigens have been described (1 14.1 15). CFA/I, CFA/II, and CFA/IV are the common colonization factors (see Table 1 and above). The nomenclature is inconsistent-those discovered after CFA/IV are named CS or PCF, and both CFA/II and CFA/IV are heterogeneous groups comprised of three distinct antigens; CFA/II expresses CS3 alone or in combination with CS l or CS2. CFA/IV expresses CS6 alone, but may express CS6 in combination with CS4 or CS6. To date, a predominant CFA has been described for all the main O:H serotypes of ETEC (Table l), but many ETEC isolates have been identified that do not contain any of the known CFAs, especially ETEC expressing LT alone, implying that many other CFA types may exist. The delineation of the full complement of CFA types and an understanding of their distribution among ETEC strains will determine the polyvalency of future ETEC vaccines based on colonization factors.

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CFAs exhibit striking host specificity, accounting in part for the lack of pathogenicity of many human ETEC strains in animals and vice versa. CFAs are plasmid encoded, often on the same plasmid as ST and LT, and are often associated with certain serotypes (Table 1) (72). The association of CFAs with diarrhea has been demonstrated a number of ways. In a study in Egypt, ETEC with CFAs were isolated more frequently from children with diarrhea than from controls, but ETEC without CFAs were isolated from both groups of children (63). Volunteer studies have shown that those receiving isogenic mutants of an ETEC strain lacking CFA/I did not develop diarrhea, while volunteers challenged with the parent strain did (89). In another study, volunteers immunized with an oral ETEC strain lacking LT and ST but containing the genetic sequence for its CFA developed much milder diarrhea and at a much lower rate compared to unimmunized controls when both were challenged with the parent strain (90). The most direct demonstration of the importance of CFAs to illness was a demonstration of passive protection from diarrhea by antibody to CFA/I (91).

IV.ENTEROINVASIVE

A.

E. COLI

Historical Aspects

EIEC are pathogenic E. coli that derive their virulence from their ability to invade epithelial cells. Not surprisingly, they originally were mistaken for Shigella, with which they share many biochemical phenotypes. The first association of EIEC with human disease was by Ewing and Gravatti in 1946 (1 16). These investigators analyzed a large sample of presumptive Shigellae from Allied soldiers serving in the Mediterranean during World War I1 who had received medical attention for dysentery. While most of the isolates were confirmed to be Shigellcre, a few strains were not easy to speciate but were described as “Shigella-like” because of their biochemical characteristics. In 1947, several dozen British school children were stricken with acute gastroenteritis, becoming manifest 14-24 hours after a common midday meal (1 17). Symptoms included fever, diarrhea, and vomiting. Dysentery was not noted. A “paracolon bacillus” was isolated from the stool cultures of the affected children, but not from any of the leftover food from the incriminated lunch. When fed to two human volunteers, an illness with symptoms resembling those observed in the children ensued. Subsequent serological and bacteriological assays demonstrated that the Shigella-like organisms of Ewing and Gravatti and the paracolon bacillus of Hobbs et al. were, in fact, E. coli of serotype 0124 (1 18).

B. Characteristics of Disease EIEC have a predilection for the colon, where they presumably exert their pathogenic effect by invasion. However, it is unknown if microscopic abnormalities are present in the small bowel, which would not be unexpected in view of the prominent vomiting from which many of the patients suffer. Despite their colonic venue and the invasive propensity of EIEC, dysentery seems to occur in only a small minority of patients in outbreaks (117,119). EIEC infection often begins with nonbloody diarrhea, which can be voluminous (120) and which is accompanied by abdominal pain, fever, and vomiting. Stools then

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become scanty, as is typical in dysentery due to Shigella. Most patients with EIEC diarrhea recover within 2 days without specific therapy. However, fluoroquinolones (121) in adults and ceftibuten and trimethoprim-sulfamethoxazole in children (122) have been used as therapy. The incubation period in outbreaks has been between 2 and 48 hours, with a median of 18 hours (117,119).

C. Epidemiology The epidemiology of sporadic cases of EIEC diarrhea and of less dramatic outbreaks than the ones described by Hobbs et al. ( l 17) and Marier et al. (1 19) is unknown. No doubt such situations exist (123- 126), and asymptomatic carriage can occur (127). The difficulty in determining the epidemiology of EIEC in general populations is the difficulty in identifying this organism in most clinical laboratories without an outbreak to prompt a more thorough investigation. EIEC in epidemics probably are foodborne, but few definite suspected vehicles have been implicated on microbiological and epidemiological grounds, because few outbreaks have been thoroughly investigated. The vehicles that have been identified include imported (to the United States) French cheese (1 19), old tofu (128), and guacamole (129). EIEC are a cause of endemic dysentery, albeit less frequently than Shigella, in developing parts of the world (130,13 1). EIEC have their highest age-specific incidence in children 3-5 years old (120) and are occasional causes of traveler’s diarrhea (132- 135). Person-to-person transmission also has been reported to occur (136).

D. Identification Like Shigella, EIEC are lysine decarboxylase negative (137) and are nonmotile. Early studies of EIEC strains, detected by differential biochemical reactions, suggested that most do not ferment lactose. More recent studies of EIEC using DNA probes as the format for detection have shown that the majority of isolates were lactose positive (120). No single biochemical test can be relied upon to identify EIEC specifically, and hence either lactosepositive or lactose-negative colonies that are confirmed to be E. coli and not Salmonella or Shigellu are EIEC candidates. Such strains can be typed in a reference laboratory to determine if they are in the classic EIEC serogroups (028ac, 029, 0112ac, 0124, 0136, 0143, 0144, 0152, 0164, and 0167) (138,139). EIEC serogroups do not overlap with those in the EPEC and ETEC. However, serogrouping may be complicated by the close antigenic relatedness of the 0 antigens of EIEC and Shigella. It is quite probable that EIEC not infrequently are misidentified as Shigella, an attribution that would not be considered aberrant clinically because of the similar clinical manifestations of EIEC infection and shigellosis. Invasion of cultured epithelial cells or positivity in the Sereny test (140) are the gold standards for confirmation of pathogenicity but are not practical for clinical laboratories. By the time these tests can be performed, the patient from whom a suspect EIEC strain was isolated usually has recovered. Serogroup 0167 does appear to produce both heat-stable and heat-labile enterotoxins (139). Despite the similarity of EIEC to Shigella, these strains have not been shown to produce Shiga toxin (Stx) I. or I1 (141), and they do not hybridize with a DNA probe that detects the gene sequences for these toxins (142).

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Cloned segments of the large, 140 MDa plasmid of EIEC have been used as a diagnostic probe (130,143), and polymerase chain reaction (PCR) amplification of other itlvasion-related sequences also has been described (144). Additionally, enzyme immunoassays that detect EIEC-specific secreted products, such as invasion-associated antigens (145,146), can be used toidentify these pathogens. These techniques are particularly valuable when seeking EIEC that do not ferment lactose and which would be overlooked on a standard stool culture. E. Pathogenicity As described above, the cardinal virulence trait of EIEC is the ability to invade epithelial cells. This trait is correlated with the presence of a large (140 MDa) plasmid in EIEC that also is found in Shigellcr (147). Many restriction endonuclease sites in these plasmids are not conserved, but the large plasmids nevertheless have considerable DNA homology to each other (147). Plasmidless EIEC do not invade, but the large plasmid of S. Jlemeri restores invasiveness (148). Much research has focused on the genetic structure and gene products of the 140 MDa plasmid in S. Jlexneri and, by extension, to EIEC (149). At least five genes spanning a distance of 20 kilobase pairs (kbp) appear necessary for the invasive phenotype (150). The four invasion plasmid antigens IpaA, IpaB, IpaC, and IpaD are encoded on the 140 MDa plasmid and are highly immunogenic. Mutations in ipaB, i p C , and i p D inhibit invasion. Recently, a provocative paper proposed that absence of loci in EIEC that encode proteins that might attenuate bacterial virulence (i.e., cadA, which encodes cadaverine) contributes to the pathogenicity of EIEC and Shigella (151). In addition to the Ipa gene products, a distant plasmid locus, \irF, encodes a gene product that is a positive regulator of ipuA-D (152). Also, virR, a chromosomal gene, can act in trans to suppress Shigella invasion (153). Additional putative virulence factors of EIEC include an exoribonuclease (Rnase R) (154) and a plasmid-encoded enterotoxin (155).

V.

E. coli 0157:H7 AND ENTEROHEMORRHAGIC E. COL/

A.

Historical Aspects

The known diarrheagenic groups of E. coli added a new member in 1982 with the discovery of E. coli serotype 0157:H7. This pathogen was the causative agent of two outbreaks of severe bloody diarrhea in Oregon and Michigan in whichtransmission occurred through consumption of contaminated fast-food hamburgers (156). Serotype 0157:H7 was considered rare because at the time only one prior isolation had been made in the United States, that being in 1975 from a woman in California with bloody diarrhea. Only six isolates had been noted in Canada from 1978- 1982 among more than 2000 E. coli isolates screened for enterotoxin and cytotoxin production (157). The term hemorrhagic colitis was used to describe the illness associated with E. coli 0157:H7 infection, and the term enterohemorrhagic E. coli (EHEC) evolved to designate serotype 0157:H7 and other serotypes that produce a similar clinical picture (4,158). In the decade since its discovery, E. coli 0157: H7 has been isolated with such an appreciable frequency in outbreaks of bloody diarrhea and in sporadic community-acquired diarrhea in western Europe and North and South America that it is no longer considered a "rare" serotype.

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A commonly asked question is whether E. coli 0157:H7 is truly a new (or newly emergent) pathogen or whether it simply has been present but notrecognized as a causative agent of disease. The retrospective assessment of large strain collections for this pathogen showed that it was veryinfrequently found among strains submitted for serotyping through the 1970s in the United States, Canada, and the United Kingdom (156,157,159). Population-based studies over the past 2 decades have suggested that the incidence of the hemolytic uremic syndrome (HUS) has been increasing, and HUS is now a well-recognized complication of E. coli 0157:H7 infection (see below). Taken together, these data along with evolutionary genetic analysis of E. coli 0157:H7 (160) suggest that this pathogen is both a new and an increasingly frequent causative agent of human illness. B. Characteristics of E. coli 0157:H7 and EHEC 1. MicrobiologicalFeatures There are no pathognomonic morphological features that distinguish serotype 0157:H7 or other EHEC serotypes from normal bowel flora cultured on solid media. All EHEC ferment lactose and have other biochemical reactions typical of E. coli (161). One distinguishing feature of serotype 0157:H7 is its lack of rapid (<48 h) fermentation of sorbitol. This has formed the basis of the most common screening procedure for this organism because 93% of E. coli isolated from humans do ferment sorbitol ( l 61,163). However, recently sorbitol-fermenting isolates of E. coli 0157:H7 have been reported (163,164). Serotype 0157:H7 also does not possess glucuronidase activity and, hence, cannot hydrolyze 4-methylumbelliferone glucuronide (MUG) to a fluorogenic product; it is therefore MUG negative (165,166). However, MUG positive variants have been isolated (163,164,167). Serotype 0157:H7 has been shown to grow well in broth media within the usual laboratory temperature range of 30-42°C and it survives freezing in ground beef quite well (168). At temperatures above 44-45°C serotype 0157:H7 grows poorly and as these temperatures are often used for the detection of E. coli in food samples, such conditions probably will negatively impact on the recovery of this serotype from food (168). Antibiotic susceptibility patterns of E. coli 0157:H7 isolates both from outbreaks and from sporadic cases had indicated that they are only rarely resistant and, even then, to very few antibiotics (169). This has seemed curious given the purported bovine source of these strains with transmission to humans via the food chain; this lack of resistance in E. coli 0157:H7 does not parallel the observations that have been made with salmonella in which an increasing frequency of multiple drug resistance has been observed (170). However, a multiply resistant isolate of E. coli 0157:H7 caused a large outbreak in Missouri (171); the outbreak strain was resistant to sulfisoxazole, tetracycline, and streptomycin, Resistance to these same three antimicrobials appeared in 13 of 176 (7.4%) strains from Washington state between 1989 and 1991, but it had not been seen in 30 isolates obtained between 1984 and 1987 (172). Antimicrobial resistance has been noted inseveral recent studies (173-176), but the data are too limited to assess whether there is a secular trend toward increasing antimicrobial resistance among these strains. Early efforts to place E. coli serotype 0157:H7 into one of the previously known groups of diarrheagenic E. coZi were unsuccessful. This serotype was not one of the traditional EPEC serotypes. Production of LT or ST could not be demonstrated, thereby precluding membership in the ETEC. The organism was not invasive, nor did it possess the invasion-associated plasmid that would have classified it as EIEC (156).

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Eventually serotype 0157:H7 was shown to produce verotoxin, a cytotoxic activity demonstrable on cultured Vero (African green monkey kidney) cells (177,178). and very rapidly thereafter this “verotoxin” was shown to be a family of cytotoxins of close structural and antigenic similarity to the Shiga toxin of SIzigellrr dyserzteriae (157,179,180). Excellent reviews of the family of Shiga toxins have been published detailing their history, genetics, and immunology ( 181,182).

2. Nomenclature The nomenclature of this group of diarrheagenic E. coli reflects the concurrent pathways of investigators who discovered cytotoxin production by E. coli 0157:H7, each of which chose a different name. Because of the recognition that these cytotoxins are related to one another with similar structure, functional activity, and cellular receptor, they have been designated Shiga-like toxins (SLT) or, more recently, Shiga toxin (Stx). Others have used the term Verotoxin, because the toxin activity was first observed as a cytotoxic effect on Vero cells (178). The terms “Shiga toxin” (or the older “Shiga-like toxin”) and “Verotoxin” are considered synonymous at this level of designation, although specific toxins (e.g., Stx2 and VT-2) may not be identical. Viewpoints for each nomenclature have been published (183,184). The designation Stx is used herein. Many strains of E. coli, including some EPEC strains, have been shown to produce Stx in varying amounts, but as most ofthese strains do not possess the other characteristics of the EHEC, they are not considered a member of this group. Thus, Stx production is necessary but not sufficient for strains to be considered in the EHEC category, and EHEC are one subset of the group of Shiga toxin-producing E. coli (STEC). All EHEC are considered human pathogens, whereas some but not all STEC are causative agents of disease. The term “EHEC” designates those serotypes of E. coli that often produce a clinical illness of severe bloody diarrhea (‘ ‘hemorrhagic colitis”) and that possess certain virulence determinants (Stx production, possession of a 60 MDa plasmid, and eliciting attaching and effacing histopathological lesions in an animal model). Levine et al. (185) proposed that serotype 026:Hll be considered an EHEC strain; however, an analysis of virulence properties of a collection of 026:Hll strains has shown that this serogroup is heterogeneous (186). Other EHEC serotypes include 026:H32, 055:H7, 0 1 13:H21, and 0 1 17:H14 (15). Serotype 0157:H7, as the most wellcharacterized, is considered the “prototypic” EHEC strain. C. Characteristics of Disease As one would expect, diarrhea is the hallmark of symptomatic infection with E. coli 0157: H7. Typically illness begins with a short prodrome of mild nonbloody diarrhea, which progresses within 24 hours to grossly bloody diarrhea, often associated with severe abdominal pain and cramping (187). Because of the copious amounts of frankly bloody rectal discharge, cases frequently have been mistakenly diagnosed as having a noninfectious cause, such as mesenteric infarction, inflammatory bowel disease, Meckel’s diverticulum, intussusception, and anatomical gastrointestinal bleeding. Hospitalization rates have been high (>50%), making hemorrhagic colitis a high-profile illness. Although the preponderance of cases reported in sporadic case series and the majority of cases in outbreaks have had bloody dialrhea, this may be due to ascertainment bias in that patients who are more severely ill or who are part of an outbreak may be more

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likely to have stool cultures for serotype 0157:H7 performed. It is now evident that nonbloody diarrhea can be seen with E. coli 0157:H7 infection (188-190) as well as with other serotypes (188). Patients with nonbloody diarrhea have been noted to be less severely ill ( 190). Many other enteric pathogens, such as Snlmorzelln, Shigella, and Camyylobncter, can be carried in the gastrointestinal tract asymptomatically, and E. coli 0157:H7 is no exception. Because persons without diarrhea rarely have stool cultures performed, documentation for such asymptomatic carriage has come from outbreak investigations and evaluations of family members in households with E. coli 0157:H7 infection (188,19 1193). The true frequency with which asymptomatic carriage occurs is unknown, and its importance in transmission is unclear (194). The hemolytic uremic syndrome (HUS) consists of an acquired Coombs’ negative hemolytic anemia, thrombocytopenia, and acute renal failure. It wasfirst described in 1955 (195), and small series and case reports had noted that mostpatients had a preceding diarrheal illness. A wide variety of microbiological and toxic agents had been described as “precipitants” of HUS, and it was assumed that HUS was the result of a final common pathway of injury resulting from a plethora of infectious and toxic insults. A few sporadic cases of E. coli 0157:H7 infection were noted to develop HUS (196,197), and both prospective and retrospective studies of HUS patients suggested a high frequency of infection with E. coli 0157:H7 or other STEC (198-200). Further studies have borne out this association (201-203). In one study (202), 100% of HUS patients cultured appropriately within 3 days of onset of illness had serotype 0157:H7 isolated; the detection rate fell if patients were cultured more than 7 days after onset, indicating that recovery of this serotype is highly time dependent. The proportion of patients infected with E. coli 0157:H7 who developed HUS has varied considerably (6-90%) among reported series and outbreak investigations. A complication rate of 8% has been observed in a recent prospective study of sporadic cases (204); however, the presence of various risk factors in the affected population may substantially increase this rate. The extremes of age, antimicrobial use, antimotility agents, and toxin type of the infecting strain all have been suggested to increase the risk of development of HUS after E. coli 0157:H7 infection. A few cases of thrombotic thrombocytopenic purpura (TTP) also have been described following E. coli 0157:H7 infection (205-207). This disorder consists of the cardinal features of HUS (hemolytic anemia, thrombocytopenia, and renal failure) along with fever and altered mental status. Prior series of patients with TTP have not indicated that many of these patients had antecedent gastrointestinal illness. The patients reported (205-207) all had bloody diarrhea and the overall features of their illness resembled those of severe HUS. Our current conceptual understanding of human infection with E. coli 0157:H7 is that a spectrum of clinical manifestations may result ranging from asymptomatic carriage to nonbloody or bloody diarrhea. Severe and life-threatening microangiopathic complications such as HUS and TTP may follow enteric illness with appreciable but variable frequency and thereby preclude volunteer studies with this pathogen. A rising incidence of HUS has been noted (208,209), suggesting either an increasing rate of human infection with E. coli 0157:H7 or an increasing rate of progression to this complication. Because HUS is a highly morbid disease with a fatality rate of5% and a considerable risk of serious long-term morbidity such as hypertension, chronic renal failure, and disability (210), a rising frequency of infection and/or its complications should provoke concern.

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Several helpful reviews on E. coli 0157:H7 (211,212) and HUS (213,214) have been published recently. D. Epidemiology Persons of all ages, ranging from one to 80 years, have been reported with E. coli 0157: H7 infection. In one population-based study in the Pacific Northwest, the highest agerelated incidence of infection was in children 0-9 years old (215). Serotype 0157:H7 is not an opportunistic pathogen, and it has produced severe illness in healthy individuals as well as those with chronic diseases. E. coli 0157:H7 has now been isolated on every continent except Antarctica (reviewed in Ref. 216). In North America, Canada and the tier of states along the U.S.Canadian border have tended to report both the largest number of outbreaks as well as sporadic cases. A striking seasonal pattern has been observed for both sporadic and community-acquired cases of E. coli 0 1 57:H7 infection, with a summertime predominance (187,216). Interestingly, a similar seasonality had been described for cases of HUS even prior to the "discovery" of E. coli 0157:H7. Doubtless this peak of symptomatic infection during the months of June through September represents some form of increased exposure to the agent, but whether this is because of periodic increases in contamination in the food chain or because of increased rates of exposure to a risk factor such as barbecuing are unknown. The mean incubation period for E. coli 0157:H7 infection as derived from outbreak investigations has ranged from 3.1 to 8 days (216). The wide variation in attack rates among outbreaks in which a food vehicle was implicated in transmission is probably due to differences in inoculum size, host susceptibility, and the presence of risk factors such as decreased gastric acidity and recent antimicrobial use. Person-to-person transmission has been noted, suggesting that the infectious dose is quite low, similar to that for Shigella infection. Quantitative recovery of E. coli 0157:H7 from foods involved in outbreaks indicates that the infectious dose can be 5100 organisms (217). Recently, outbreaks on an unprecedented scale have occurred in the United States involving 501 cases and 3 deaths (218), in central Scotland with 501 cases and 20 deaths (219). and in Japan with more than 5000 cases (320). These dratnatic outbreaks have strained the capacity of public health systems to investigate and control them, and the low infectious dose has necessitated a more stringent conceptual approach to food safety. Several facets of changing food animal production, large-scale food processing, and changes in consumer preferences have apparently provided a niche for the emergence of E. coli 0157:H7 as a human pathogen (221). With E. coli 0157:H7, as with many other pathogens, the major burden of disease is sporadic, rather than outbreak-associated. A new view of this situation was provided through prospective surveillance of E. coli in Minnesota during which all isolates were routinely subtyped using PFGE (332). Considerable heterogeneity was observed with >70 subtypes per year, but subtyping pertnitted the detection of outbreaks and small case clusters not recognized epidemiologically. These data could suggest that sporadic disease represents many "mini-outbreaks." Further work will need to define efficient and effective prevention strategies for outbreaks of both large and srnall scale.

E. IsolationandIdentification Virtually all procedures described to date for the primary isolation of E. coli 0157:H7 have been based on its lack of sorbitol fermentation (163,223). Colorless colonies on

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sorbitol Macconkey (SMAC) agar with morphology and biochemical reactions (indole positive, oxidase negative) typical of E. coZi may be serogrouped by slide, tube, or latex agglutination or by PCR assay for the rfb locus encoding the 0157 antigen (224) to detect the 0157 antigen. Serotyping can be completed by looking for immobilization in H7 antisera-containing motility media (162,223); however, some strains may be nonmotile (H-). Other strains of serogroup 0157 exist in nature, and although some are even sorbitol negative, none are of the H7 serotype (225). False-positive identifications of E. coli 0157: H7 have been reported (225-227). The recent description of sorbitol-positive strains may prove problematic for detection methods based on the lack of sorbitol fermentation if such strains are shown to occur elsewhere. Several techniques have been usedto subtype E. coli 0157:H7; those with the largest body of published experience include plasmid profile, toxin type, phage type (228), isoenzyme type (229), and analysis by pulsed field gel electrophoresis (230). Other less commonly used methods are ribotyping (23 1), insertion sequence subtyping (232), and restriction frequent length polymorphism using Stx probes (233). Isoenzyme profiling has been used to determine the placement of E. coli on the evolutionary tree of diarrheagenic E. coli and shows that the 0157:H7 group is a widely disseminated pathogenic clone, which has evolved relatively recently from an EPEC progenitor 0 5 5 strain (160). The other methods have mainly been used to "DNA fingerprint" strains to determine the interstrain relatedness, often in outbreak investigations. A superb overview has carefully delineated the published experience with these and related DNA-amplification subtyping approaches (234). Other methods for detection have been directed at demonstrating the presence of Stx, either directly in fecal filtrate or in culture supernatant from either pure or mixed cultures. Stx can be detected by tissue culture assay (direct cytotoxicity to Hela or Vero cells), enzyme-linked immunoassay, latex agglutination, colony immunoblot, and PCR assay (reviewed in Ref. 234). Although toxin-based detection methods can be extremely sensitive, their major drawback is that they may detect strains of unclear epidemiological and clinical significance (i.e., STEC not causally implicated in disease). As discussed in Sec. V.B above, not all STEC are EHEC and hence testing for additional virulence determinants is necessary for such strains. E. coli 0157:H7 produces a hemolysin, enterohemolysin (Ehly), whose phenotypic expression is visualized on washed sheep red blood cell agar (235). Ehly is distinct from a-hemolysin; E. coli 0157:H7 and some related EHEC possess only ehly sequences. The genes for Ehly are encoded on the large 60 MDa plasmid present in E. coli 0157:H7. DNA sequence-based methods (colony blot, PCR assay) have been described (236) and are particularly useful because not all strains express the ehly phenotype. Enterohemolysin production appears to be an interesting but imperfect correlate to pathogenic potential of the EHEC group as ehly-producing, non-Stx-producing E. coli have been found (234).

F. Pathogenicity Tremendous strides have been made in characterizing the molecular mechanism of action of the Stx family (for reviews see Refs. 181, 182). Both Stxl and Stx2 have a subunit structure consisting of one catalytically active A subunit and five B subunits responsible for binding to a specific cell surface receptor, globotriaosylceramide (Gb,). Both subunits are encoded on bacteriophage, and serotype 0 157:H7 produces either Stxl or Stx2 alone or in combination. Stxl differs by only one amino acid from Shiga toxin; Stx2 shares about 58% homology with Stxl. Some strains contain two copies of Stx2 genes, each

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copy with slightly different DNA sequences, and these differences probably account for the lack of cross-neutralization observed with certain antisera and for differences in biological activity on target cell lines (237,238). The biological significance of these differences is unclear. A third toxin, designated Stx2e (also known as SLT-IIe or VT2e) is produced by STEC strains of porcine origin (239). Although Stx2e has considerable DNA sequence homology to Stx2 (240), there are sufficient differences, particularly in the B subunit, that Stx2e binds to a different surface receptor than Stx2 (241). Stx2e is cytotoxic for Vero but not Hela cells. The role in human disease played by Stx2e-producing strains is unknown at present. These toxins function as protein synthesis inhibitors. Their RNA N-glycosidase activity leads to cleavage of a particular adenine residue in the 28s ribosomal RNA of the 60s ribosomal subunit (181). Both Stxl and Stx2 are extraordinarily potent, with cytotoxicity demonstrable from picogram quantities of toxin. Their biological activity is demonstrable on those cell lines such as Hela and Vero cells that express surface receptors for toxin; cell lines without these receptors are resistant. The exact role of these toxins in producing diarrhea or microangiopathy is presently unclear. Although a variety of animal models have been described that have employed various modes of toxin delivery, none have all the features of human infection, and in particular none duplicate the pathology seen in HUS. However, a substantial body of evidence has shown that for the parent toxin, Shiga toxin, both enterotoxic and cytotoxic activities are possessed by the same molecule, and these properties have also been shown for other members of the Stx family (242). The relationship of these biological activities to the clinical manifestations of diarrhea and dysentery was demonstrated in primate inoculation studies using toxin negative mutants of Shigella dysentericre 1 (243). Bloody diarrhea, the characteristic feature of dysentery, developed in animals fed the parent strain, and milder nonbloody diarrhea occurred in animals given the isogenic tox- mutant, thus implicating Shiga toxin in the colonic hemorrhage component of shigellosis. The compendium of evidence from several different animal models using a variety of bacterial constructs suggests that Stx expression by diarrheagenic E. coZi strains is responsible for the hemorrhagic aspect of diarrhea produced by other virulence determinants in those strains

(is). The pathological changes in HUS include endothelial cell damage, particularly in the glomerular endothelium in thekidneys. It has been hypothesized that systemic toxemia results from toxin release from the gastrointestinal tract with resultant damage to target endothelium. Support for this hypothesis largely comes from the lack of other obvious explanations such as bacteremia or cellular invasion to provide a connection between infection within the bowel lumen and end-organ damage in renal endothelium. Neutralizing antibodies to Stxl have been demonstrated in convalescent sera from patients with HUS (198). However, circulating toxin has never been demonstrated in patients with HUS or TTP, although this may bedue to rapid binding and internalization by cell types expressing surface receptors for toxin. Injection of toxin into laboratory animals has shown rapid uptake into tissues that are not typically affected (e.g., muscle) in HUS. Shiga toxin has been shown to be directly cytotoxic to human umbilical vein endothelial cells (244,245) directly cultured for short periods. Endothelial cells grown to confluence were demonstrated to be relatively resistant to Stx compared to Vero cells, a difference that may be ascribable to low levels of Gb3 in the endothelial cell membrane (246). The levels of cytotoxicity that could be demonstrated on these confluent endothelial cells were not aug-

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mented by lipopolysaccharide; a modest increase in cytotoxicity was seen with the addition of recombinant human tumor necrosis factor (246). Thus, a large body of mainly indirect evidence suggests that toxin is important in the pathogenesis of HUS but the pathophysiological sequence of events leading to microangiopathy distal to the bowel remains to be elucidated. A current area of active investigation is in the role of host-derived cytokines in HUS. Several studies have noted elevated serum levels of IL6 in HUS patients (247249); HUS patients showed higher levels of IL-10 and IL-IRa in comparison to hemorrhagic colitis patients (249). The extent to which cytokines alone versus those in combination with the effects of Stx contribute to both intestinal and extraintestinal disease is not clear at present. As would be expected by analogy with the other diarrheagenic E. coli, adherence properties of E. coli 0157:H7 are important in facilitating colonization. E. coli 0157:H7 exhibits an A/E phenotype like classical EPEC strains, not surprising given its origin from an EPEC progenitor serotype 055:H7. The molecular basis for A/E in 0157 strains has been elucidated (250) with identification and sequencing of the eae homolog in E. coli 0157:H7. The intimino157 protein encoded by the eaeo157gene has its greatest sequence divergence from the EPEC intimin in the C-terminal end, which binds the epithelial cell surface receptor. This in turn correlates with different sites of intestinal colonization for these pathogens-EPEC mainly colonize the small bowel, whereas E. coli 0157:H7 colonizes the large bowel (250). The sequence for the entire LEE of E. coli 0157:H7 was recently determined and compared to that for EPEC (251). For completely shared genes, the rate of divergence was greatest for those involved with bacteria1:host interactions, while genetic sequences for genes involved in other functions were highly conserved. This type of polymorphism of gene sequences specifically involved in pathogen-host interactions has been noted as an evolutionary strategy of several pathogens such as Borrelia burgdolferi and Streptococcus pyogenes (25 1).

VI.

ENTEROAGGREGATIVE E. COL!

A.

HistoricalAspects

The enteroaggregative E. coli (EAEC) are a relatively newly described, and somewhat controversial, cause of diarrhea. Their discovery illustrates the observation by Pasteur that "chance favors the prepared mind." Twenty, years ago, a particular type of adherence to HEp-2 cells had been described for EPEC (43). Using this assay, investigators intending to characterize the adherence phenotype of E. coli isolates from persons with diarrhea noted that some isolates exhibited a distinctly different form of adherence to HEp-2 cells than the localized adherence observed with EPEC strains (252,253). EPEC adherence is visualized as localized clusters of bacteria on the HEp-2 cell surface, appearing as a microcolony. Clearly distinguishable from this pattern was a palisading of bacteria in a stacked brick configuration, both on the cell surface as well as on the glass cover slip to which the HEp-2 cells adhered (254). This appearance was formed by "aggregative adherence" (AA), and E. coli strains exhibiting this phenotype were called enteroadherentaggregative E. coli or, later, enteroaggregative E. coli (EAEC). Aggregative adherence is distinguishable from diffuse adherence (DA); in the latter, bacterial cells are scattered across the surface of the HEp-2 cell, and there is no palisading observed on the glass cover slip (15).

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Subsequent epidemiological studies have more consistently isolated EAEC from persons with diarrhea compared to asymptomatic controls. although the frequency has varied considerably by geographic location (see below). For the diffusely adherent E. coli, there have been conflicting observations comparing the frequency of DAEC isolation from symptomatic persons compared with asymptomatic controls (254), and the DAEC are not yet fully accepted as a definite and distinct category of diarrheagenic E. coli. Although the EAEC were initially thought to be a type of EPEC, further support for classifying EAEC as a category separate from EPEC includes the lack of concordance of serotypes between the two groups and that EAEC strains are negative when tested with the EAF probe. The EAEC have caused diarrhea in both sporadic and outbreak settings as well as in volunteer studies (255). Thus a body of evidence has developed that supports the consideration of EAEC as a member of the diarrheagenic E. coli in a category distinct from the EPEC, ETEC, EIEC, and EHEC.

B. Characteristics of the EAEC 1. Serogroups Serogroups determination is not a basis for assigning isolates to the EAEC category. However, those serotypes characteristic of the EAEC include 03:H2,015:H18,044:H18,086: NM. 077:H18, Olll:H21, 0127:H2, and 0 nontypab1e:HlO (15). As previously noted, these serotypes are not overlapping with those of the EPEC category.

2. Identification As with the other categories of the diarrheagenic E. coli, there are no phenotypic or morphological features that distinguish EAEC on primary enteric isolation. The gold standard for assignment to the EAEC category is the demonstration in the HEp-2 adherence assay of the characteristic AA pattern. Varied culture conditions have been employed for preparation of the bacterial inoculum as well as performance of the adherence assay itself; however, at least one comparative study has indicated that the methods as originally described (43j yielded the best discrimination among adherence phenotypes (256). A DNA probe has been developed from a 1.0 kb fragment of a large plasmid from an EAEC strain (257). Nearly 90% of strains identified as EAEC by HEp-2 cell adherence hybridized with this DNA probe when the isolates had been obtained from Chile or south India (257.258); however, considerably fewer strains from Brazil, also identified on the basis of HEp-2 cell adherence, hybridized with the DNA probe (259). At present, DNA probe positivity is a helpful adjunct in categorizing EAEC strains: however, probe-negative strains that exhibit the AA phenotype in HEp-2 adherence are still potentially classifiable as EAEC strains. Ongoing epidemiological studies should help to define differences in pathogenic potential between probe-positive and probe-negative EAEC.

C. Clinical Manifestations A watery, noninflammatory diarrhea appears to be the most common clinical manifestation seen in outbreaks and in volunteer studies. In the latter, diarrhea has also been noted to be very mucoid. Several studies have associated EAEC with persistent diarrhea (259,260). EAEC have also been reported as a cause of diarrhea in an HIV-infected patient in the United States (261). EAEC have been detected more frequently in the stools of HIV-infected

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children in Zaire with diarrhea (41%) in comparison to HIV-uninfected children with and without diarrhea (30% and B%,respectively) (262).

Epidemiology D. EAEC have been isolated as causative agents of diarrhea in both the developing as well as the developed world. Rates of isolation have varied among locations (15,263). but essentially the EAEC appear to be widely dispersed geographically. EAEC outbreaks have been described in both the developed and developing world (260,264,265), and these have involved both healthy and malnourished individuals. In studies of pediatric diarrhea in India and Brazil, the EAEC are prominent causative agents of acute illness (258,266). In Brazil, persistent diarrhea was more frequently due to EAEC than to other pathogens (259). The importance of this finding is that there is greater overall workability with resistant diarrhea compared to acute diarrhea. Recently, an interesting but extremely disturbing observation noted the association of EAEC infection with growth retardation (267), and this was noted even for asymptomatic infection. These data indicate a much larger attributable morbidity due to EAEC infection than that of previous assessments based on acute diarrheal morbidity alone.

E. Pathogenicity At present, there is only a very incomplete picture of EAEC pathogenicity. Most of the work to date has been in studies of the AA phenotype and its genetic basis. A fimbrial structure designated aggregative adherence fimbriae I (AAF/l) has been identified, which appears to mediate adherence to HEp-2 cells (268). Excess mucus production has been identified in animal models and volunteer studies, and a role for excess mucus in enveloping bacteria proximal to the mucosa hasbeen postulated (15). Some degree of mucosal destruction has been noted in autopsy specimens, and cytotoxic effects on intestinal explants and cell lines has been noted by some, but not all, EAEC strains. A homolog of heat-stable toxin has been found in EAEC strains and designated EAST1 (269). Only half of EAEC appear to have this secretory toxin, which is also possessed by some EHEC strains. Using this available evidence, Nataro and Kaper have proposed a preliminary three stage model of EAEC pathogenesis (15). Initially, adherence to intestinal epithelial cells is mediated by AAF or other factors; subsequently enhanced mucus production entraps bacteria close to the cell surface, thus allowing delivery of toxin with resulting mucosal destruction.

VII.

ROLE OF FOOD AND WATER IN TRANSMISSION OF DIARRHEAGENIC E. COLI

Information on the source of transmission for the recognized groups of E. coli that cause diarrheal disease is derived mainly from outbreak investigations and prospective household-based studies (270). The former are more often carried out in the developed world largely as a consequence of improved recognition of outbreaks due to a lower background incidence of diarrhea and due to greater public resources. Foods important in transmission

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in the developed world may be essentially irrelevant in less developed countries due to nonavailability, nonacceptability, or differences in food-preparation practices. Prospective studies of food and water in the households of patients with diarrhea have more often been carried out in the developing world, and their major limitation is that often the food vehicles consumed by the index patient are no longer available. Despite the biases inherent in this imperfect data base, a few points are worthy of comment. ETEC are primarily transmitted via contaminated food and water, and person-toperson transmission is rare. This is most likely due to the relatively large inoculum needed ( lo8) to produce illness (68). Ingestion of nonrefrigerated foods and fecally contaminated water can result in intake of an inoculum of this magnitude; this is the mode of acquisition in ETEC-associated traveler’s diarrhea (271). Items considered at high risk for ETEC contamination include raw vegetables, such as in salads, untreated water, and ice (272). These observations have led to the dictum “Peel it, boil it, cook it, or forget it” as the main preventive measure for traveler’s diarrhea. The environmental conditions conducive to enterotoxin production in foodstuffs are almost never achieved, and hence, ingestion of preformed toxin does not seem to be an important means of becoming ill (273). Attempts to determine the source of infection in sporadic cases of ETEC diarrhea have not been highly successful. In Bangkok, different ETEC strains were isolated from market foods compared to isolates from children (274), and ETEC of the same serogroup as the index patient were detected in only two foods and two water sources in combined studies of over 100 households (275-278). Given the difficulties of such studies, as discussed above, these results should not be taken to preclude foodborne transmission of sporadic ETEC infection in an endemic setting. When EPEC-associated outbreaks of diarrhea in adults have been described, both food and waterborne transmission have been noted (28,279). Volunteer studies in adults showed that the inoculum necessary to produce disease was high, 10’ organisms, which would be consistent with that ingestible through contaminated food or water. It is possible, however, that infants are susceptible to lower inocula. The high secondary spread in hospital nursery outbreaks after the admission of an index case with EPEC gastroenteritis was invariably seen among the infants rather than the mothers, nurses, and family contacts (30,280). This finding is not explained by clinically inapparent infection as adult contacts of cases of EPEC diarrhea have not been shown to be colonized with these strains. All these findings together suggest that adults are fairly resistant to EPEC infection, but even such resistance can be overcome if the inoculum is high enough as has occurred in a few food- and waterborne outbreaks. The exact means of transmission of EPEC diarrhea among very young children is unclear. Large numbers of EPEC organisms are shed in the stool of infected infants, and the environment around these children becomes heavily contaminated (30j. The breadth of environmental surfaces that harbor EPEC and the ability of these strains to survive on dust and bedding create ample opportunity for the hands of caregivers to become contaminated and in turn perpetuate cross-infection. Aerosols containing EPEC have been shown in the immediate vicinity of patients with EPEC diarrhea (31) as has respiratory tract colonization in affected infants. This has raised the possibility that EPEC may be transmitted via an airborne route among young infants with ingestion of the inoculum following inhalation. Because a facile means of identifying EIEC in the routine clinical microbiology laboratory does not exist, information on transmission is drawn almost exclusively from outbreaks. Almost all such outbreaks have been foodborne, although at least one water-

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borne outbreak in Hungary has been reported (281). Volunteer studies showed that an inoculum of 10' organisms was necessary to cause disease in adults (68), a finding with which foodborne transmission is consistent. Person-to-person transmission has also been seen (136), not surprising given the similarity of EIEC to Shigella. Humans may be the main reservoir of the organism as they are for Shigella; evidence supporting this supposition is the lack of isolation of EIEC from animals and the linkage of outbreaks with either an infected food handler or fecally contaminated water, the latter contaminating the food item (Brie and Camembert cheese) during production in one large outbreak (1 19). Foodborne transmission of E. coli 0157:H7 has been repeatedly demonstrated since the original discovery in 1982 in which contaminated undercooked hamburger meat was the vehicle (156). In an almost eerie replay of the past, this same scenario was repeated a decade later in a large multistate outbreak of E. coli 0157:H7 infections associated with undercooked hamburgers from one fast food restaurant (218). The majority of outbreaks with E. coli 0157:H7 with a known vehicle of transmission have been due to contaminated beef (mainly hamburger), although less than half of all outbreaks have a known source. Consumption of undercooked ground beef has been shown to be a risk factor for illness in case-control studies of sporadic cases (282-283). Unpasteurized milk has transmitted E. coli 0157:H7 infection in several outbreaks in both the United States and the United Kingdom. The fairly common association of illness with bovine products has led to consideration that control of colonization in bovines would prevent most human disease. Given what is now known about the broad host range of E. coli 0157:H7, which includes wild animals such as deer, this approach is probably too simplistic. Other foods that have transmitted illness include dried venison (284) and sausage (216). Studies of retail meats not associated with human disease showed that E. coli 0157: H7 was recoverable from 3.7% of beef, 1.5% of pork, 2.0% of lamb, and 1.5% of poultry samples (285). Isolation rates may vary by geographic region as E. coli 0157:H7 was not detected in meat samples from Newfoundland or Ontario, Canada (286,287), or Thailand (288). Non-0157 STEC are frequently detected in raw meats (17% in one study) (289). However, many of these non-0157 STEC are of uncertain pathogenic significance, and there is controversy regarding the interpretation of the presence of STEC in foods (290,29 1). An unsettling development has been the recent instances of transmission of E. coli 0157:H7 infection by fresh produce such as lettuce (292), radish sprouts (220), alfalfa sprouts (293), and fresh unpasteurized apple juice or cider (294,295). These foods are eaten without additional cooking, posing a problem because of the extremely low infectious dose for E. coli 0157:H7. Sprouts are a special problem because the sprouting process itself is an amplification step; bacteria in the seeds can readily multiply to levels of lo7 cfu/g within 48 hours of sprouting the seed. E. coli 0157:H7 has remarkable ability to tolerate very low pH (<4) (296), and this acid tolerance probably contributes to its low infectious dose and prolonged survival in acid foods such as dry sausage and cider (216,297). Waterborne transmission of E. coli 0157:H7 has been described (171,298-300). Again reemphasizing the low infectious dose, in an outbreak due to a contaminated municipal water supply in a small town in Wyoming, some visitors became ill after consuming only small amounts of water during a several-hour stay (300). An interesting study from Chile has suggested that waterborne transmission may be less important than transmission by foods for all groups of diarrheagenic E. coli (301). Despite the access to clean water and toilet facilities within the home by nearly 70% of households, rates of infection with diarrheagenic E. coli remained high and suggested that

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transmission may bemore often foodborne than waterborne. Lack of adequate refrigeration was observed as probably contributing to illness by providing opportunity for bacterial multiplication in foods during the sumtner months when temperatures are high.

VIII. ISOLATIONFROMFOODS It is not within the scope of this chapter to provide a compendium of isolation techniques for the diarrheagenic E. coli, and the reader is referred elsewhere for comprehensive reviews (286,302). Several overall points are worth emphasizing here, however. Foodstuffs are obviously not sterile, and many bacteria other than E. coli may be present, complicating efforts to recover E. coli from the food item. One way to circumvent this problem is the use of an enrichment procedure that selects for the target E. coli group while discouraging the growth of other organisms. Such is the basis of isolation techniques such as that in the FDA Bacteriological Analytical Manual, which uses incubation temperatures of 44-45"C, which permit growth of some pathogenic E. coli while minimizing growth of other bacteria. There are several problems with this approach: (a) not all groups of the diarrheagenic E. coli are detectable with this method, e.g., the EHEC strain, E. coli 0157:H7, does not grow well at 44°C; (b) the phenotypic expression of those biochemical reactions that characterize E. coli may be poor at elevated temperatures; and (c) significant plasmid loss may occur during growth at an elevated temperature, thereby obviating the additional characterization of an isolate for plasmid encoded functions, e.g., enterotoxin production. Some of these difficulties have been surmounted by using an initial growth phase at 35°C in brain heart infusion broth to nonselectively permit growth and resuscitate injured or stressed microorganisms (303) before utilizing enrichment media such as tryptone phosphate (TP) broth (304) at 44°C to select for pathogenic E. coli. This procedure still does not allow for isolation of E. coli 0157:H7. Of all the diarrheagenic E. coli, only EHEC serotype 0157:H7 has a distinctive biochemical phenotype, allowing this to be the basis for initial screening procedures. This serotype does not ferment sorbitol rapidly, within 24-48 hours, but using this feature to discriminate E. coli 0157:H7 from other bacteria depends upon the background flora of similar phenotype in the original sample (305). Doyle and Schoeni (285,306) developed a method for direct isolation of E. coli 0157:H7 from foods with a sensitivity of 1.5 organisms per g, but the procedure has several steps and is time, labor, and materials intensive. A direct plating method (307), while simpler to perform, is several logs less sensitive, able to detect only 10'-104 organisms per g. Another method combining hydrophobic grid membrane filtration and using a monoclonal antibody has been reported with a sensitivity of 10 organisms per g (308) and was fairly rapid (24 hours) for negative samples, with positive samples requiring a further 2-3 days for analysis. A reactive disc blot ELISA technique has also been reported with similar turnaround times for positive and negative samples (309). Improved selective media incorporating cefixime and tellurite into sorbitol Macconkey media (310) have been combined with immunomagnetic bead separation (31 1) to provide a method very nearly equivalent to DNA amplification in sensitivity, with the advantage of providing a bacterial isolate available for additional subtyping. Regardless of method, all the aforementioned isolation procedures ultimately yield one or more E. coli isolates, which must be further characterized for the attributes that classify them as members of the EPEC, ETEC, EIEC, or EHEC. For EPEC, this entails

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0 and H serotyping, characterizing the strain for the presence of the A/E phenotype or e m gene and the EAF plasmid. Possible ETEC may be assayed by several methods for LT and/or ST production, but verification that an enterotoxin-producing strain is pathogenic for humans requires demonstration that the strain expresses a CFA type specific for humans. Candidate EIEC isolates can be characterized by combining serotyping with biochemical characterization, determining the presence of theinvasion-associated plasmid, and testing for invasion of Hela or HEp-2 cells in tissue culture or the ability to cause keratoconjunctivitis in the guinea pig eye. The prototype EHEC strain E. coli 0157:H7 is identified by 0 and H serotyping and Stx production or genotype. Other STEC are identified by the serotyping of isolates shown to possess the Stx gene, determination of the presence of the eae sequence and possibly Ehly. EAEC strains are categorized as such by serotyping and demonstration of the AApattern in tissue culture. All the methods noted above are dependent on recovery of a viable isolate and its subsequent growth in pure culture for further characterization and/or subtyping. Another methodology that circumvents the problems of viable organism recovery and labor- and time-intensive characterization procedures is the polynlerase chain reaction (PCR) assay. This technique allows for the rapid amplification of target DNA sequences to detectable levels within 24 hours. Depending on the specificity of the primers used in the reaction, the amplified DNA sequence can be organism specific (i.e., a conserved DNA sequence found only in E. coli allowing differentiation of E. coli from other Enterobacteriaceae), strain-specific (e.g., permitting amplification of SLT sequences used to delineate EHEC), or, even through the use of purposefully “cross-reactive” primers, the assay can be genus-specific (e.g.. allowing detection of members of the genus Mycobncteriunz without regard to species). PCR assays have been developed that allow for the rapid detection of target DNA sequences in pure isolates, thereby bypassing lengthy procedures that depend on the functional expression of such sequences, e.g., enterotoxin or cytotoxin production and detection. Validation that a genotype is always correlated with the previously described phenotype that characterizes a group of diarrheagenic E. coli, however, is necessary before PCR-based assays can supplant prior methods of strain characterization. PCR-based assays do not have inconsequential obstacles to overcome before they can become routine in almost any setting (3 12). They are exquisitely sensitive to contamination, although newer techniques have been developed that can overcome this problem. Their application to clinical specimens, human tissues, and environmental samples has been slowed by various inhibitors present in those specimens. Quality control in the individual assay itself as well as in the PCR laboratory is a very significant issue in order to ensure the scientific validity of the result. All nucleic acid amplification assays are limited by the fact that their end result does not yield a viable organism, thereby limiting further characterization such as the subtyping that plays an increasing role in epidemiological investigations. The diarrheagenic E. coli have particularly lent themselves to nucleic acid-detection methods because they have been among the first pathogens whose genetic basis of virulence has been defined. As such there has been a virtual explosion of publications describing PCR assays for their detection in foods and clinical and environmental samples. These are too extensive to be reviewed here. Many of these, however, were developed using artificially “spiked” samples and do not have side-by-side comparisons with other techniques, particularly in prospective studies. Nucleic acid amplification nlethodology has proven to be a very helpful and reasonably reliable means to categorize the diarrheagenic E. coli when pure cultures have been

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available. Its routine use on clinical and environmental samples and foodstuffs will need the types of validation studies mentioned previously. Those investigations coupled with focused epidemiological studies should enlarge our understanding of the ecological niche of the diarrheagenic E. coli and provide a more solid understanding on which to base disease-prevention strategies. This may be the means by which one of the most important achievements of public health in this century, safer food (313,314), is taken forward into the next millennium.

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Neil1 et al. clinical features of infection. epidemiology and pathogenesis. Curr. Clin. Top. Ilfect. Dis., 151230-252. Cobeljic. M.. Miljkovic-Selitnovic. B.. Paunovic-Todosijevic, D., et al. (1996). Enteroaggregative Esclzerichirr coli associated with an outbreak of diatrhoea in a neonatal nursery ward. Epidenriol. Infect., 117:ll-16. Eslava, C., Villaseca, J., Morales, R., Navarro. A., and Cravioto A. (1993). Identification of a protein with toxigenic activity produced by enteroaggregative Escherichia coli (abstract B-l 05). In Abstracts of the 93rd Gelzeral Meetirzg of the Americait Socieh for Microbiology, American Society for Microbiology. Washington, D.C., p. 44. Bhan, M. K., Raj. P., Levine, M. M., et al. (1989). Enteroaggregative Eschet-iclziacoli associated with persistent diarrhea in a cohort of rural children in India. J. hfect. Dis., 159:10611064. Steiner. T. S., Lima, A. A., Nataro, J. P., and Gue~rant,R. L. (1998). Enteroaggregative Escherichia coli produce intestinal inflammation in growth impairment and cause interleukin8 release from intestinal epithilial cells. J. Infect. Dis., 177:88-96. Nataro, J. P., Deng. Y., Maneval. D. R.. et al. (1992). Aggregative adherence fimbriae I of enteroaggregative Eschericlzia coli mediate adherence to HEp-2 cells and hemagglutination of human elythrocytes. Infect. I m m n . , 602297-2304. Savarino, S. J., Fasano, A., Watson, J., et al. (1993). Enteroaggregative Escherichia coli: heat-stable enterotoxin 1 represents another subfamily of E. coli heat-stable toxin. Proc. Natl. Acad. Sci. USA, 90:3093-3097. Echeverria. P., Serichantalergs, O., Changchawalit, S., and Sethabutr. 0. (1992). Escherichia coli gastroenteritis. In Food urd Waterborne Irzfections (A. Tu. ed.), Marcel Dekker. New York, pp. 71-101. Wood, L. V., Ferguson, L. E., Hogin, P., Thurman, D., Morgan, D. R., DuPont, H. L., and Ericsson, C. D. (1983). Incidence of bacterial enteropathogens in foods from Mexico. Appl. Em+-on. Microbiol., 46:328-332. Blaser, M. J. (1986). Environmental interventions for the prevention of traveler's diarrhea. Rely. Irfect. Dis., 8(Suppl 2): 142- 150. Doyle, M. P. and Padhye. V. V. ( 1989). Escherichia coli. Tn Foodborne Bacterial Pathogens (M. P. Doyle, ed.). Marcel Dekker, New York, pp. 236-281. Black, R. E., Merson, M. H., Rowe, B., et al. (1981). Enterotoxigenic Escherichia coli diarrhea: acquired immunity and transmission in an endemic area. Bull. WHO. 59263268. Echeverria, P., Seriwatana, J., Leksomboon, U., Tirapat, C., Chaicumpa, W., and Rowe, B. (1984). Identification by DNA hybridization of enterotoxigenic Escherichia coli in homes of children with diarrhea. Lancet. 1:63-65. Echeverria, P., Seriwatana, J., Taylor, D. N., Tirapat, C., Chaicumpa. W., and Rowe. B. (l 985). Identification by DNA hybridization of enterotoxigenic Escherichia coli in a longitudinal study of villages in Thailand. J. Itlfect. Dis.. 151:124-130. Echeverria, P., Seriwatana, J., Taylor, D. N., Yanggratoke, S., and Tirapat, C. (1985). A comparative study of enterotoxigenic Escherichia coli, Shigella, Aeromoms, and Vibrio as etiologies of diarrhea in Northeastern Thailand. Am. J. Trop. Med. Hyg., 34547-554. Echeverria, P., Taylor, D. N.. Seriwatana, J., Leksomboon, U., Chaicumpa, W., Tirapat, C., and Rowe, B. (1987). Potential sources of enterotoxogenic Escherichia coli in homes of children with diarrhea in Thailand. Bull. WHO, 65207-215. Costin, I. D., Voiculescu, D., and Gorcea, V. (1964). An outbreak of food poisoning in adults associated with Esclzerichia coli serotype 86:B7:H34. Pathol. Microbiol.. 27:68-78. Hinton, N. A., and MacGregor, R. R. (1958). A study of infections due to pathogenic serogroups of Escherichia coli. Can. Med. Assoc. J., 79:359-364. Lanyai B., Szita. J.. Ringelhann B., and Kovach K. (1959). Awaterborne outbreak of enteritis associated with Escherichia coli serotype. Acta Sci. Hzmg., 6:77-84.

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282. Slutsker, L., Ries, A. A., Maloney, K., et al. (1998). A nationwide case-control study of Escherichia coli 0157:H7 infection in the United States. J. Infect. Dis., 177:962-966. 283. Parry, S. M., Salmon, R. I., Willshaw. G. A., et al. (1998). Risk factors for and prevention of sporadic infections with vero cytotoxin (shiga toxin) producing Escherichia coli 0157. Lcrizcet, 351:1019-1022. 284. Keene, W. E.. Sazie, E., Kok, J.. et al. (1997). An outbreak of Escherichia coli 0157:H7 infections traced to jerky made from deer meat. JAMA, 227:1229-1231. 285. Doyle. M. P., and Schoeni, J. L. (1987). Isolation of Esclzericlzirr coli 0157:H7 from retail fresh meats and poultry. App. Erzviron. Microbiol., 53:2394-2396. 286. Ratnam, S., and March, S. B. (1986). Sporadic occurrence of hemorrhagic colitis associated with Escherichia coli 0157:H7 in Newfoundland. Can. Med. Assoc. J.. 134:43-46. 287. Read, S. C., Gyles. C. L., Clarke, R. C.. et al. (1990). Prevalence of verocytotoxigenicEscherichia coli in ground beef, pork and children in southwestern Ontario. Epidemiol. hlfect., 105:ll-20. 288. Suthienkul, O., Brown, J. E., Seriwatana, J., et al. (1990). Shiga-like toxin-producing Escherichia coli in retail meats and cattle in Thailand. Appl. Emiron. Microbiol., 56: 1135-1 139. 289. Samadpour, M., Ongerth, J. E., Liston, J., et al. (1994). Occurrence of Shiga-like toxin-producing Escherichia coli in retail fresh seafood, beef. lamb, pork, and poultry from grocery stores in Seattle, Washington. Appl. Erwiron. Microbiol., 60: 1038-1040. 290. Ta-r, P. I., and Neill, M. A. (1996). Perspective: the problem of non-O157:H7 shiga toxin (verocytotoxin)-producing Eschericlzia coli. J. Infect. Dis., 174: 1136- 1139. 291. Paton, J. C., and Paton. A. W. (1998). Pathogenesis and diagnosis of shiga toxin-producing Esclrerichia coli infections. Clin. Microbiol. Rev., 11:450-479. 293,. Ackers, M. L., Mahon, B. E., Leahy. E., et al. (1998). An outbreak ofEscherichia coli 0157: H7 infections associated with leaf lettuce consumption. J. Infect. Dis., 177:1588-1593. 293. CDC. (1997). Outbreaks ofEsrhet-iclziacoli 0157:H7 infection associated with eatingalfalfa sprouts-Michigan and Virginia, June-July 1997. MMWR, 46:741-734. 294. CDC. (1996). Outbreak of Escher-ichict coli 0157:H7 infections associated with drinking unpasteurized commercial apple juice-British Columbia, California, Colorado, and Washington, October 1996. MMWR, 45:975. 295. CDC. (1997). Outbreaks of Escherichitr coli 0157:H7 infection and cryptosporidiosis associated with drinking unpasteurized apple cider-Connecticut and New York, October 1996. MMWR. 46:4-8. 296. Leyer, G. J., Wang, L. L., and Johnson, E. A. (1995). Acid adaptation of Escherichio coli 0157:H7 increases survival in acid foods. A$,. Em~iron.Microbiol., 61:3752-3755. 297. Zhao, T., Doyle. M. P., and Besser, R. E. (1993). Fate of enterohemorrhagic Eschericlzia coli 0157:H7 in apple cider with an without preservatives. Appl. Emit-on. Microbiol., 59: 2526-2530. 398. Keene, W. E., McAnulty, J. M.,Hoesly. F. C., et al. (1994). A swimming-associated outbreak of hemorrhagic colitis cause by Escherichia coli 0157:H7 and Shigella sonnei. N. Engl. J. Med.. 331:579-584. 299. Blake, P. et al. (1998). Escherichia coli 0157:H7 outbreak among visitors to a water park. C h . Infect. Dis.. 27: 1033. 300. Olsen, S. J., Miller. G.. Breuer, T.. et al. (1998). A waterborne outbreak of E. coli 0157:H7 infections: evidence for acquired immunity. Abstracts of the 36th anma1 westing,Infectious Disease Socieo of America, November 12-15. 1998. Denver, Abstract 783. 301. Levine, M. M., Ferreccio, C., Prado. V., et al. (1993). Epidemiologic studies of Escherichia coli diarrheal infections in a low socioeconomic level peri-urban community in Santiago, Chile. A m . J. Epidemiol., 138:849-869. 302. Meng, J., Feng. P., and Doyle, M. P. Pathogenic Escherichicr coli. In: Compendium of Methods for the Microbiological Examination of Foods. American Public Health Association, Washington, D.C., 3000 (in press).

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303. Hitchins, A. D., Feng, P., Watkins, W. D., Rippey, S. R., and Chandler, L. A. (1992). Escherichia coli and coliform bacteria.In FDA Bacteriological Analytical Manual, 7th ed. Association of Official Analytical Chemists, Arlington. VA, pp. 27-49. 304. Mehlman, I. J., and Romero, A. (1982). Enteropathogenic Escherichia coli: methods for recovery from foods. Food Technol., 36(3):73-79. 305. Tarr, P., Tran, N. T., and Wilson, R. A. (1999). Escherichia coli 0157:H7 in retail ground beef in Seattle: results of a one-year prospective study. J. Food Pr-ot. 62:133-139. 306. Padhye, N.V., and Doyle, M. P. (1991). Rapid procedure for detecting enterohemorrhagic Escherichia coli 0157:H7 in food. Appl. Environ. Microbiol., 572693-2698. 307. Szabo, R. A., Todd, E. C. D., and Jean, A. (1986). Method to isolate Escherichia coli 0157: H7 from food. J. Food Prot., 49:768-772. 308. Todd, E. C. D., Szabo, R. A., Peterkin, P., Sharpe, A.N., Parrington, L., Bundle, D., Gidney, M. A. J., and Perry, M. B. (1988). Rapid hydrophobic grid membrane filter-enzyme-labelled antibody procedure for identification and enumeration of Escherichia coli 0157 in foods. Appl. Environ. Microbiol., 54:2536-2540. 309. Okrend, A. J. G., Rose, B. E., and Matner, R. (1990). An improved screening method for the detection and isolation of Escherichia coli 0157:H7 from meat, incorporating the 3M Petri film test kit-HEC-for hemorrhagic Escherichia coli 0157:H7. J. Food Prot., 53:936940. 3 10. Zadik, P. M., Chapman, P. A., and Siddons, C. A. (1993). Use of tellurite for the selection of verocytotoxigenic Escherichia coli 0157. J. Med. Micr-obiol., 39:155-158. 311. Chapman, P. A., Wright, D. J.. and Siddons, C.A. (1994). A comparisonof inmunomagnetic separation and direct culture for the isolation of verocytotoxin-producing Escherichia coli 0157 from bovine faeces. J. Med. Microbiol.. 40:424-427. 3 12. Persing, D. H. (1991). Polymerase chain reaction: trenches to benches. J. Clin. Microbiol., 29~1281-1285. 313. CDC. (1999). Ten great public health achievements-United States. 1900-1999. MMWR, 48:241-243. 314. CDC. (1999). Safer and healthier foods. MMWR, 48:905-913.

10 Listeria monocytogenes Catherine W. Donnelly University of Vermont, Burlington. Vermont

I. Introduction 213 A. History and characterization of Listeria B. Distribution 215

214

11. Listeriosis in Humans 216

111. IV.

V. VI.

VII.

1.

A. Characterization of the illness listeriosis 216 B. Mechanisms of defense against Listeria in normal, healthy hosts 216 C . Listeriosis in immunocompromised hosts 218 Pathogenesis of Listericr monocytogenes 219 Foodborne Transmission of Listeriavzonocytogenes 221 A. Major foodborne disease epidemics 221 B. Sporadic cases of listeriosis with foodborne etiology 224 C. Other reports of foodborne listeriosis: epidemic and sporadic 225 Sources of Listeriu in Foods and Food-Processing Environments 226 Detection of Listeria in Foods 229 A. Selective procedures 229 B. Detection of sublethally injured Listeria 231 Summary 234 References 235

INTRODUCTION

Listeria mor~ocytogenes,the causative agent of the disease listeriosis, was first discovered almost 100 years ago. Within the past 15 years this organism has emerged as a foodborne pathogen of major significance. Prior to 198l, Listeria was recognized primarily as an animal pathogen, and it was suspected that humans acquired listeriosis through direct contact with infected animals. We now know that consumption of foods contaminated with Listeria can cause both sporadic illness as well as foodborne disease epidemics. In order to prevent foodborne listeriosis, it isnecessary to understand the disease, susceptible persons, distribution of the organism within the environment, and behavior of the organism in foods. This chapter is designed to summarize current knowledge with respect to the 213

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foodborne role of Listeria, with special emphasis on improvements in isolation procedures designed to detect the presence of Listeria in foods.

A.

History andCharacterization of Listeria

Listeria mor1ocytogenes is asmall (1.0-2.0 p X 0.5 p), gram-positive, facultatively anaerobic, rod-shaped bacterium, which is widely distributed in nature. The organism can exist in an intracellular state within monocytes and neutrophils, and its name is derived from the fact that large numbers of tnonocytes are often found in the peripheral blood of monogastric animals that this organism infects (1). Listeria is recognized as a causative agent of the disease listeriosis, a zoonotic illness that affects animals and humans. Reports of organisms resembling Listeria first appeared in the scientific literature in 1891 when Hayem of France observed small gram-positive rod-shaped organisms in human tissue (1 ). This observation was repeated 2 years later (1893) by Henle, who was working in Germany. Hulphers, working in Sweden in 191l , reported the presence of gram-positive rod-shaped organisms in the livers of rabbits and designated the organism Bacillus hepatis. Complete characterization of the organism now recognized to be Lister-in rzlorlocytogelles was achieved by Murray et al. in 1926 (2). These investigators, working at Cambridge University, observed infection of an animal colony caused by an etiological agent that induced pronounced monocytosis and caused hepatic lesions. The name given to this agent was Bacterium nzorlocytogenes. Pirie (3), working in South Africa one year later, isolated a small gram-positive bacillus from the livers of gerbils, which he designated Listerella hepntoliticcr, in honor of Lord Lister (4). In 1940, the genus/species designation Listeria nlotzocytogerzes was proposed for this organism. A detailed historical chronology can be found in the classic review article on Listeria and listeric infection published by Gray and Killinger (1 ). Presently included within the genus Listeria are the species L. rnonocvtogenes, L. illnoem, L. seeliger-i,L. welslzimeri, L. ivnrzovii, and L. grclyi (Table 1). DNA base composition and DNA-DNA hybridization studies led Stuart and Welshimer ( 5 ) to propose a new genus M~wraycrto include M. g r q i and M. grayi subsp. murrrryi. However, more recent examination of the genomic relatedness of L. gmyi and L. rmrrayi based upon DNA/DNA hybridization and multilocus enzyme electrophoresis reveals that these organisms should be considered as members of the single species L. grnyi (6). Members of the

Table 1 Characterization of Listeria Species Characterlstic P-Hetnolysis CAMP S. ourells R. rqrri Fermentation of mannitol xylose rhamnose Extremely Extremely Pathogenic YesIn humans

L. monocyrogenes

L. i1mncwii

L. seeligeri

L, imocua

L. rr-elsllimeri

+ +

+

+

-

-

-

+

-

-

-

-

~

-

+

-

-

-

-

-

+

+

-

-

-

+-

+

No rare3 cases

rare1 case

+ t No

Listeria monocytogenes

215

genus Listeria can be easily differentiated on the basis of the following biochemical reactions: reduction of nitrates to nitrites, P-hemolysis; acid production from mannitol, Lrhamnose, and D-xylose; and the CAMP test (Table 1). L. nzorzocytogenes and L. irzrzocrrcr are very closely related, and recent studies have confirmed that within the 16s rRNA, only 2. of 1281 base pairs differ between the two species (73). Only one species within the genus Listeria is generally regarded as capable of causing illness in humans, this species being L. monocytogenes. Three reports of human infection caused by L. ilwnovii exist in the scientific literature (9,10), along with one report of a case of human illness caused by L. seeligeri (1 1,12). Thus, L. ilwzovii and L. seeligel-i are extremely rare causes of illness in humans, and the main species of concern in human health is L. vzonocytogenes. L. irzrzocua and L. welshirnel-i are not capable of causing illness. These nonpathogenic species are of interest from a food microbiology standpoint since their presence indicates the potential for the presence of thepathogenic L. morzocytogenes. Although 16 serotypes of L. monocvtogeizes have been identified, three serotypes (4b, 1/2a, 1/2b) are responsible for 96% of human infections in the United States (13). A similar survey of strains from cases of human listeriosis in Britain between 1967 and 1984 revealed 1/2, 3, and 4 as the predominant serogroups causing human infection (14). Serotyping has poor discriminating power and is therefore of limited value as a subtyping method when compared to more advanced methods of genetic analysis. Of all available phenotypic and genotypic typing methods, pulsed-field gel electrophoresis (PFGE) is the preferred method for subtyping of L. nzonocytogenes strains (15), offering a high degree of discrimination of Listeria strains as well as reproducibility.

B. Distribution L. nzonocytogenes is very widely distributed in nature. This organism can be readily isolated from soil, water, sewage, green plant material, decaying vegetation, and numerous species of birds and mammals, including humans (1). Although widely distributed within mammalian species, cattle, sheep, and goats are the domestic mammals most frequently afflicted by listeriosis (16,17). A close relationship between onset of listeriosis in ruminants and feeding of contaminated silage has long been recognized ( l 7-20). Palsson (2 l ) reported that in Iceland, the relationship between silage feeding and onset of listeriosis is so strong that the disease has been referred to as "votheysveiki," or silage sickness. Presence of Listeria in silage is strongly influenced by pH, and samples having a pH in the range of 5.0-6.0 or above are far more likely to be sources of L. monocytogerzes than silage where the pH is below 5.0 (19,22-24). The tnost common disease syndrome of listeriosis in ruminants is encephalitis, leading to observations of nervous system involvement in cattle and sheep. Afflicted animals have been observed as disoriented, causing them to circle endlessly in one direction or another depending upon the direction in which their head is drooped. For this reason, listeriosis in ruminants is often referred to as "circling disease'' (425). Previous investigations have identified sheep as a major reservoir of Listel-icr in nature. In one study alone, 88% of tested sheep were identified as carriers of some member of the genus Listeria (26). Gray (17) used serotyping techniques to demonstrate the relatedness of isolates obtained from listeric sheep and the oat silage that they consumed. More recent studies have used strain-specific ribotyping to support the link between on-farm sources of Listeria (silage) and subsequent contamination of dairy-processing environments (27). Infected animals displaying symptoms of listeric infection may

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excrete L. rnorzocvtogerzes in milk, blood, and feces. High excretion rates of L. monocytogenes in milk from asymptomatic cows and goats have frequently been reported (28).

II. LISTERIOSIS IN HUMANS

A. Characterization of the Illness Listeriosis Because of its ubiquity in the environment, humans frequently come into contact with L. rrlorlocytogenes.Mere exposure to this organism does not necessarily dictate that infection will result. L. rnonocytogerzes is frequently shed in the stools of healthy humans who otherwise show no signs of the illness, thus confirming the high exposure rate to this organism (29). It is estimated that 5% of healthy humans harbor L. Inonocytogenes in their gastrointestinal tract. The first case of human listeriosis was described in 1929 (30), and since that time listeriosis has been recognized as a rare but often fatal illness. In adults, the disease listeriosis is characterized by the onset of severe symptoms including meningitis, septicemia, primary bacteremia, endocarditis, nonmeningitic central nervous system infection conjunctivitis, and flu-like illness (fever, fatigue, malaise, nausea, cramps, vomiting, and diarrhea) (3 1). Gastrointestinal symptoms are observed in approximately one third of documented cases of listeriosis (32). Whether this is due to a concurrent illness that disturbs the gastric mucosa, allowing invasion of L. nzoIzocytogems, or it is a nonspecific response to systemic infection, or it is actually part of the disease spectrum of listeriosis is unknown (29). Humans shown to be at high risk for acquiring listeriosis include pregnant women, neonates, the elderly, organ transplant recipients, or those receiving immunosuppressive therapy. In the latter case, treatment of patients with corticosteroids or antimetabolites renders a suppressed immune system. Persons suffering from chronic disorders such as alcoholism, malignancy, diabetes, heart disease, or acquired immunodeficiency syndrome (AIDS) have also been shown to be at risk. Additional underlying factors that have been reported in association with listeriosis include sarcoidosis, chronic otitis, collagen-vascular disease, idiopathic thrombocytopenic purpura, asthma, ulcerative colitis, and aplastic anemia (3 1). Age has been shown to be a predisposing factor in listeriosis. An 11% casefatality rate is documented in persons age 40 or under; a 63% case-fatality rate is recorded for persons over age 60 (29). Age-related reasons for increased incidence of listeriosis may include a decline of the immune system as a function of age, increased prevalence of immune-suppressive disorders, and increased dependence on immune-suppressive medications (29). Although the above-listed conditions may predispose patients to acquiring listeriosis, it should be noted that persons showing no apparent immunocompromising conditions have been shown to acquire listeriosis. The most recent estimates indicate that on an annual basis, approximately 1092 cases and 248 deaths occur due to listeriosis in the United States (13). This illness, while rare, has a case-fatality rate ranging from 23 to 35% (29,33).

B. Mechanisms of Defense Against Listeria in Normal, Healthy Hosts Cell-mediated immunity (CMI) plays an important role in dictating the resistance or susceptibility of a human host to infection by L. monocytogenes (34-36). Listeriosis occurs

Listeria monocytogenes

217

most often in persons with impaired CMI. CM1 is dependent upon the activity of mononuclear phagocytes as early response, nonspecific effectors, and specific T cells as a secondary response to infection. Since the response of T cells to infection takes time, resistance expressed at an infection site is most likely T-cell independent and is mediated by resident macrophages, which limit early bacterial proliferation (34). Thereafter, the CM1 response becomes sufficiently developed to effect control and ultimately eradication of listeriosis (37). In early phase host defense, macrophages function as primative scavenger phagocytes; later, macrophages function as final effector cells in lymphokine-mediated cellular immunity and in antibody-dependent cell-mediated cytotoxicity (38). Specific response to infection results in an increase of macrophages at the site of infection due to either the proliferation of resident macrophages or circulating blood monocytes, which are attracted to the infection site by substances produced by T cells (39-42). Polymorphonuclear leukocytes (PMNs) also contribute to phagocytosis and killing of Listeriu (34), and in the acute stages of infections by facultative intracellular bacteria, infected tissues are infiltrated by PMNs (43). As infection progresses, the number of macrophages at the infected site increases. With recognition of a microbial (listerial) antigen, macrophages become "activated," resulting in enhanced antimicrobial activity against that specific antigen as well as against nonrelated pathogens (44,45). Activated macrophages contain more lysosomes, display increased oxidative metabolic activities, and are more phagocytic (42,46). The accumulation of macrophages at the site of infection results in lesions structured in granulomas that contain mostly lymphocytes and macrophages. Within granulomas, the cells are tightly packed and their closeness allows cell to cell interactions, enhancing the destruction of Listeria (34). Two other roles of macrophages involve the production of interleukin-. 1 (leukocyte-activating factor), which specifically alters T-cell function, and the processing and presentation of immunologically active molecules to the lymphocytes (45). In work with murine models, nonspecific host defense mechanisms act against L. monocytogerzes in the first 2 days following challenge with Listeria (47). Within 10 minutes of injecting mice with a sublethal dose of L. monocytogenes, the organisms are trapped by the liver and spleen. Resident tissue macrophages in the liver destroy most Listeria, but surviving bacteria multiply within liver and spleen macrophages and are not inactivated until development of acquired cellular resistance. The main function of Listeria-sensitized T cells is to attract, focus, and activate macrophages at infective sites (43) by liberation of macrophage inhibitory factor (MIF), which encourages circulating blood monocytes to localize at the infection site, and by liberation of macrophage activating factor (MAF), now called y-interferon, which activates macrophages to enhance their phagocytic activity (34,48,49). Activation of T lymphocytes leading to acquired cellular immunity involves three distinct stages (50). Stage one involves the priming of prekiller T cells and the generation of Listeria antigen-specific helper T cells. In the second stage, helper T cells are stimulated by Listeria antigens to release a soluble substance or lymphokine (interleukin-2), which is a lymphocyte-activating factor. This causes helper cells to grow and proliferate and stimulates cytotoxic types (48). In the final phase, interleukin-2 converts the prekiller T cell into a L. nlorrocytogenesdependent cytotoxic T lymphocyte, which rapidly destroys the organism. Cytotoxic suppressor cells appear to be more important in eradication of Listeria than helper T cells (43). T-cell-deficient nude (athymic) mice have been shown to limit listeriosis to a chronic infection by relying on resident macrophage populations (37).

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In summary, primary infection with L. nlonocytogenes in a healthy human causes colonization of the liver and spleen. The dissemination from the infection site occurs through the lymphatic pathway. There is a short period of bacterial growth, which is stopped by the arrival of activated macrophages from the blood and peritoneal cavity. Thymus-derived leukocytes enhance the macrophage action, which results in elimination of bacteria and establishment of a cell-mediated resistance to further infections.

C. Listeriosis in lmmunocompromisedHosts Alteration of T-cell or macrophage function due to immunosuppression would result in an impairment of cell-mediated immunity allowing a listeric infection to occur after the primary infection or after further contact with Listerin. Patients with cancer (51) or undergoing treatment with steroids or cytotoxic drugs (46,52), pregnant women or neonates (1,53,54), renal transplant recipients, and elderly or alcoholic patients (29,31) are well known to be the primary target for listeriosis. The risk of complications due to L. nzonocytogenes infection has long been recognized in CMI-compromised conditions such as Hodgkin’s lymphoma and other hematological malignancies (55). North (40) suppressed cell-mediated immunity of mice to L. monocytogems by administration of an antimitotic drug and induced a listeric infection. Golnazarian et al. (56) showed that the L. monocyfogenes infectious dose for mice immunocompromised by administration of hydrocortisone acetate was much lower than for normal resistant mice. Studying the regulation of macrophages in mice with combined immunodeficiency (lack of T- and B-cell function), Bancroft et al. (57) demonstrated that, although numbers of macrophages were almost normal after primary infection, mice developed chronically high loads of bacteria. The same state of chronic listeriosis was noted in an experiment done by Newborg and North (37). 1. Pregnancy and Listeriosis During pregnancy, selective factors of CM1 become depressed to prevent rejection of the fetus by the mother. However, depression of these selective factors may result in decreased maternal resistance to L. nlorlocytogenes infections and thereby increase the maternal or fetal risk to onset of listeriosis (58). Such selective factors include shifts in levels of hormones or serum factors that affect lytnphocyte or macrophage synthesis, activation, or function during pregnancy (54). Plasma levels of hydrocortisone increase during pregnancy to levels three to seven times higher than those found in nonpregnant humans (54). Corticosteroids are known to suppress both lymphokine activation and phagocytic activity of macrophages. Low levels of IgM and decreased activity of the classic complement pathway during the neonatal period also occurs and demonstrates the importance of opsonization in the immune response to Listeria (29j. In humans, listeriosis occurs most often during the third trimester of pregnancy. Three outcomes are normally followed: an asyptomatic maternal infection and a resulting infected infant, a severely ill mother who enters premature labor and delivers a stillborn or severely ill infant, or an unaffected fetus with death of the mother (54). In most perinatal cases of listeriosis, the mother is usually mildly affected, exhibiting flu-like symptoms, but neonatal morbidity and mortality are common. In early-onset neonatal listeriosis, transplacental infection results in a syndrome known as granulomatosis infantisepticum, a necrotic disease of the internal organs (59). Spontaneous abortion of the fetus or stillbirth of the neonate are most common, but if the fetus infected in utero is born alive, recovery is not likely ( 1,59). Late-onset listeriosis occurs several days after birth, and infants are generally full-term and healthy at birth. Late-onset listeriosis is more likely than early-

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onset listeriosis to present as meningitis, and case fatality rates are lower than for earlyonset infection. Of late-onset cases reported in Britain during 1967-1985, 93% of cases presented evidence of central nervous system infection (60). Buchdahl et al. (61) described several cases in which complications due to listeriosis arose during the course of pregnancy. In one case, a mother at 32 week’s gestation developed a flu-like illness and developed irregular uterine contractions. Spontaneous membrane rupture occurred with release of meconium-stained amniotic fluid. The infant, delivered by ceasarean section, was found to have blood and cerebrospinal fluid contaminated with L. nzorzocytogerzes. Although the infant survived, neurological handicap was evident. The mother recalled consumption of a soft-ripened French cheese 9 days prior to delivery. 2. Listeriosis in Renal Patients Listeriosis is well recognized as a complication of renal transplantation. Most patients become ill while they are receiving immunosuppressive therapy, which increases their susceptibility to listeriosis (62). Meningitis is recorded as the most common presentation of listeriosis in renal transplant patients, and the fatality rate for listeria1 meningitis in renal transplant recipients is 38%. However, pneumonia due to L. mor~ocytogerzeshas also been observed in renal patients, suggesting a possible respiratory route of transmission (62). In a study of healthy renal transplant recipients, fecal carriage of L. rnorzocytogenes in 8 of 37 patients was documented (62). In a review of 83 cases of listeriosis in renal patients, Stamm et al. (62) found that one third of patients had been treated for acute rejection. Nieman and Lorber (31) have reported that hemodialysis is not a predisposing factor for most patients in acquiring listeriosis. However, Mossey and Sondheimer (63) reported four cases of L. monocytogerzes bacteremia associated with long-term hemodialysis and transfusional iron overload. None of these patients were receiving immunosuppressive therapy. Many surveys document a higher incidence of listeriosis in the tnonths from July to October, and the same seasonal variation has been reported for renal transplant recipients (62). 3. Listeriosis and Patients with AIDS Patients with AIDS exhibit an impairment of T-cell-mediated immune response and would therefore be expected to be at high risk for listeriosis. In early studies of the incidence of listeriosis in AIDS patients, L. morlocytogenes was rarely implicated as an agent affecting persons with AIDS (47). Reasons given for this surprising finding included the fact that AIDS patients who displayed frequent gastrointestinal tract infections were given multiple courses of antibiotic therapy, thus decreasing exposure to Listericr (47,64). Five cases of listeriosis in Los Angeles county between January 1985 and March 1986 occurred in patients with AIDS. Prior or concurrent gastrointestinal illness was recorded in three of the patients, and four patients had no history of prior antibiotic administration. This and subsequent investigations have shown that while listeriosis is a rare infection in patients exhibiting HIV infection, persons with AIDS have a 300- to 1000-fold increased risk of acquiring listeriosis compared with the general population (29,65). Persons with AIDS are therefore advised to refrain from ingestion of food items associated with listeriosis (64).

111.

PATHOGENESIS OF LISTERIA MONOCYTOGENES

The production of sulfhydryl-activated hemolysin, listeriolysin 0 (a-listeriolysin), is associated with the pathogenic potential of L. monocytogenes (66-70). Listeriolysin 0 is simi-

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lar to streptolysin 0 and pneumolysin, and antigenic cross-reactivity with these hemolysins as well as with hemolysins produced by L. ivanovii and L. seeligerihas been demonstrated (69,71). The virulence of Listeria species has also been associated with the ability to survive and grow intracellularly (72,73). Gaillard et al. (67,68) studied the role of hemolysin (listeriolysin 0)in pathogenicity of L. morzocytogenes. Working with transposon Tn1545, these investigators inactivated a genetic determinant for hemolysin production and obtained nonhemolytic mutants from hemolysin-producing strains. The loss of hemolysin production was shown to be associated with loss of virulence in a mouse model. The spontaneous loss of the transposon restored hemolysin production and virulence to the revertants (67). Further studies by Gaillard et al. (68) found that L. monocytogerm and L. ivnnovii invaded a continuous gut epithelial cell line, whereas L. seeligeri, L. i~nocua,and L. welskimeridid not. A nonhemolytic mutant of L. monocytogenes invaded these gut enterocytes at the same rate as the hemolytic wild type (67). This finding, which has been corroborated by others in a fibroblast 3T6 continuous cell line (74), demonstrates that listeriolysin 0 is not involved in invasion. Studies have been done that identified an extracellular protein (p60) that may be involved in the process of attachment and invasion of L. mojzocytoge1zes (75). Both listeriolysin and p60 are produced by all virulent L. morzocytogenes strains and are involved in the virulence of L. nrorzocytogenes(74,75). Under heat-shock conditions, listeriolysin is synthesized, whereas production of p60 no longer occurs (66). Protein p60 is found as both a major secreted protein, as well as on the cell surface of all L. monocytogems isolates. In addition, this protein possesses murein hydrolase and is involved in cell division. Rough mutants of L. nzor1ocytogenes that lack p60 form long chains of cells, which fail to separate. These mutants also show reduced uptake by 3T6 fibroblast cells (75). Factors other than hemolysin have recently been defined as essential virulence factors for Listeria monocytogenes. Hof and Rocourt (12) found that a construct of a virulent L. nzonocytogems EGD with selective blockade of phospholipase C production became avirulent. Tilney and Portnoy (76) demonstrated that L. r~onocytogenesis capable of bypassing the humoral immune system by remaining in an intracellular state and spreading from cell to cell. Following phagocytosis by host macrophages and escape from the phagocytic vacuole, Listeria are coated with actin filaments, form a pseudopod, dissolve the phagocytic vacuolar membrane, presumably by use of hemolysin, and repeat the cycle. It was postulated from this and other studies (72-74) that once Listeria enters macrophages, listeriolysin 0 is needed to lyse phagosomes, thereby releasing Listeria into the cytoplasm so that it can multiply. Listeria that lack hemolysin fail to grow in vivo because of an inability to dissolve the endosomal membrane and failure to escape from the endosome into the cytoplasm (73,76). L. rnonocytogenes can enter host cells in two distinct ways: through active ingestion by phagocytic cells such as macrophages, or through the production of specific gene products that control ingestion by normally nonphagocytic cells (68,76). Studies conducted by Gaillard et al. (77) identified a surface protein of L. monocytogerles, intemalin, which mediates bacterial invasion of epithelial cells. Once internalized, the life cycle of L. monocytogenes within both phagocytic and nonphagocytic cells is similar. The majority of virulence genes that produce products associated with the intracellular life cycle of L. rnonocytogenes reside on a region of the chromosome known as the PrfA-dependent gene cluster (78). This region is comprised of the genes prfA, plcA, hly, mpl, actA, and plcB. The prfA gene product is a positive regulatory factor, a 27 kDa regulatory factor that controls all virulence genes of the virulence gene cluster. The plcA product is phosphati-

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dylinositol-specific phospholipase C (PI-PLC), which contributes to vacuole escape in cells such as bone maTow-derived macrophages. The plcB gene encodes for a phosphatidylcholine-specific phospholipase C (PC-PLC), which, together with metalloprotease, the mpl gene product, enables listeriolysin-o independent escape of L. rnorzocytogerzes from primary vacuoles in human epithelial cells. Metalloprotease (mpl) pennits bacterial movement from the cytosol to the host surfaces and the ensuing cell-to-cell spread (76). The act A gene locus is responsible for the accumulation of actin around Listeria in the cytosol. The lack of one of these determinants has been shown to interfere with the pathogenicity of L. morlocytogerzes (1 1). Cowart (79j showed that hemolysin activity is stimulated in iron-deprived medium; cytolytic activity of hemolysin is maximally expressed at pH 5.5. Therefore, it can be assumed that when L. nlonocytogenes are engulfed in phagosomes that do not contain iron and have a pH value around 5.5, hemolysin production is maximized, allowing destruction of internal membranes surrounding them. An additional mechanism of intracellular survival is dependent upon theability of L. molzocytogenes to resist killing by oxidizing agents produced by phagocytes. The production of superoxide disrnutase (SOD) and catalase by L. rnonocytogenes has been associated with intracellular survival (72,80). Bortolussi et al. (72) demonstrated that resistance of L. monocytogelzes to hydroxyl radical (OH) during the log phase of growth was due to the production of sufficient amounts of catalase to inactivate this product. Welch et al. (80) found that catalase-negative strains of L. monocvtogerzes possessed at least twofold greater SOD activities than catalase-positive strains. Not all strains of L. monocytogenes are pathogenic. Further, within L. rnotzocytogenes strains, there may be particular serotypes that possess enhanced virulence potential. A survey conducted by Pinner et al. (81) showed that foods containing L. molzocytogenes serotype 4b were four times as likely to contain strains identical to patient strains than were foods containing serotypes 1/2a or 1/2b. These and other observations suggest that serotype 4b strains may have an enhanced capacity to cause human disease (81,82). Comparative virulence of Listerin strains is typically assessed on the basis of mouse lethal dose (LD,O)following intravenous or intraperitoneal inoculation (83,84). Numerous investigators have attempted to develop in vitro assays to measure virulence potential. Farber and Speirs (85) found that culture filtrates of L. morzocytogenes and L. ivarzovii were cytotoxic to eight tested continuous cell lines, whereas cell filtrates from L. innocucr had no effect on these cell lines. Pine et al. (83) found that use of human continuous cell line Caco-2 stained with trypan blue could differentiate virulent from avirulent L. r7lonocytogenes.

IV. FOODBORNETRANSMISSIONOF LISTERIA MONOCYTOGENES A.

MajorFoodborneDiseaseEpidemics

Listeria nzorlocytogenes has emerged as a foodborne pathogen of major significance within the last decade. A number of foodborne disease outbreaks worldwide have been linked to consumption of food products contaminated with L. morzocytogerles. This information has been summarized and is presented in Table 2. In 1979, listeriosis was diagnosed in at least 23 hospitalized patients in the Boston area (86). The vehicle of infection in this outbreak was linked to hospital food, and patients who had consumed lettuce, carrots, and

2

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9

5 S:

a

h

=!

3

a

c e Tt

e

2 3

t! CS t N

.d

a

d-

m

\o

S

9 4

m

Y

W

.4

3

5 m m m 00

m m 3

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radishes were more likely to contract the illness. Isolates from 20 of 23 cases were identified as serotype 4b. Symptoms reported by the afflicted patients included bacteremia or meningitis. Fifty percent of the patients involved in this outbreak were immunosuppressed because of cancer, chemotherapy, or steroid treatment. Curiously, 60% of the afflicted patients had reported the use of antacids or the antiulcer medication cimetidine. Cimetidine is a histamine-2 antagonist that blocks the H? effects of histamine, thereby decreasing gastric acid secretion (87), whereas antacids neutralize gastric acid. Itwas found that patients who had consumed antacids or cimetidine were more likely to develop hospitalacquired infection caused by L. monocytogenes. It was hypothesized that gastric acid neutralization following use of antacids or cimetidine predisposed humans to acquiring listeriosis as a result of ingestion of this organism via a foodborne vector. This finding has been corroborated in later studies, where patients were more likely than controls to have used antacids, laxatives, or H,-blocking agents prior to onset of listeriosis (88). In 1981. an outbreak of listeriosis occurred in the Maritime Provinces of Canada (89). The vehicle of transmission was identified as commercially prepared coleslaw. Cabbages used to prepare coleslaw were traced to a sheep farm where an outbreak of listeriosis had killed several sheep. Use of manure from infected sheep was suspected as a factor in this outbreak. Thirty-four cases of listeriosis in pregnant women resulted in spontaneous abortions, stillbirths, or live birth of ill infants. Seven nonpregnant adults who showed no evidence of immunosuppression had symptoms of meningitis, aspiration pneumonia, and sepsis. The overall mortality rate for this outbreak was 41%. All patient isolates were identified as serotype 4b, and L. monocytogenes isolates from unopened packages of coleslaw were also identified as serotype 4b. In 1983,49 patients in Massachusetts were diagnosed with listeriosis (90). Epidemiological evidence pointed to a strong association between consumption of pasteurized whole and 2% milk and onset of the illness. Forty-two patients were characterized with underlying illnesses such as cancer or alcoholism, and several patients were undergoing corticosteroid therapy. Seven of the cases involved fetuses or infants. The overall casefatality rate in this outbreak was 29%. Of 49 isolates available for serotyping, 32 were identified as serotype 4b. Despite numerous attempts, the epidemic serotype of L. monocytogerzes responsible for this outbreak was never recovered from the incriminated milk. The consumption of soft ripened cheese accounted for two major illness outbreaks of listeriosis in the 1980s. The first documented link between cheese consumption and an outbreak of listeriosis was reported in Orange County, California, in 1985 (91). In this outbreak, consumption of Jalisco-brand Mexican-style cheese was linked with the onset of 142 cases of listeriosis. The outbreak occurred during the period January l-August 15, 1985. Ninety-three (65.5%) of the cases were recorded in pregnant women or their offspring, while 49 cases were reported in nonpregnant adults. In all, 48 deaths resulted from this outbreak. A majority of the afflicted individuals (62%) were pregnant Hispanic women. In this outbreak, the cheese was most likely manufactured from a combination of raw and pasteurized milk, and the cheese plant that manufactured the incriminated cheese was found to be contaminated with Listeria. The epidemic strain was identified as L. moizocJjtogenesserotype 4b, and this serotype was recovered from unopened packages of Queso Fresco and Cotija Mexican-style cheese. A second outbreak of foodborne listeriosis linked to cheese was reported by Bula and coworkers (92). This outbreak occurred in Vaud, Switzerland, and was linked to consumption of Vacherin Mont D'or cheese. A total of 122 cases occurring during the period 1983-1987 were reported. The normal endemic rate of listeriosis in Switzerland is 5-10

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cases per million persons. During the outbreak period, the rate of listeriosis rose to 50 cases per million persons. Sixteen cases were reported in 1983,24 cases in 1984, 13 cases in 1985, 28 cases in 1986, and 41 cases in 1987. A mortality rate of 28% was associated with these cases. Of clinical isolates available from the epidemic period, 111 of 120 (93%) were serotype 4b of two unique phage types, and 85% of these strains matched the epidemic phage types isolated from Vacherin Mont D'or cheese. Dalton et al. (93) reported on an outbreak of listeriosis linked to chocolate milk served at a picnic. Forty-five individuals developed illness due to L. morzocytogenes characterized by fever and gastroenteritis without progression to invasive disease. Further, none of the patients reported immune deficiency or chronic illness and only one patient was pregnant and delivered a healthy baby. The median dose of Listerin consumed by patients may have been as high as 2.9 X 10" CFU per person. Proctor et al. (94) used PFGE to link four additional sporadic cases of invasive listeriosis to recalled 1% low-fat chocolate milk responsible for the previously mentioned outbreak. At the time of this writing, two active multistate outbreak investigations involving L. nzo;?ocytogerzes were reported by the Centers for Disease Control and Prevention (95,96). The first investigation of an 1l-state outbreak involving hot dogs and deli meats identified over 50 illnesses occurring during the period August 1998-January 1999 due to a single strain of L. monocytogerzes. This outbreak prompted a major nationwide recall of incriminated product. A second multistate outbreak involved a different strain of L. nzonocytogenes (serotype 4b), which was identified in 11 cases involving four states. These cases of illness occurred during August 1998-December 1998.

B. Sporadic Cases of Listeriosis with Foodborne Etiology Unlike the epidemics described previously, human listeriosis occurs most often as a sporadic illness (29). Information on whether the majority of these sporadic cases result from foodborne transmission is being provided by ongoing and active disease surveillance. For instance, from September to June 1987, the CDC conducted a population-based active surveillance for L. nlonocytogenes infections (97). This surveillance involved 154 patients from six regions of the United States (New Jersey, Missouri, Oklahoma, Tennessee, Washington, and Los Angeles). From this data, it was estimated that approximately 1700 cases of listeriosis occurred in the United States in 1986, for an annual incidence rate of 7.1 cases per million persons. Epidemiological evidence suggested that consumption of contatninated foods accounted for 30 of the 154 cases (20%) of listeriosis reported. Two foodborne sources were epidemiologically linked with onset of illness, these being uncooked hot dogs and undercooked chicken. As a result of this active surveillance, a recall of turkey franks commenced after being linked with the death of a patient in Oklahoma (98). L. r7zorzocytoge;zes serotype 1/2a strains of identical isoenzyme types were isolated from the patient as well as unopened packages of turkey franks (98). Between November 1, 1988, and December of 3 1, 1990, the Centers for Disease Control (CDC) conducted a second major case-control study in order to identify dietary risk factors for sporadic listeriosis (33). The population base in this active surveillance was in excess of 18 million persons distributed within five geographic regions of the United States. Cases were enrolled from patients identified through active surveillance. Underlying patient conditions included pregnancy, steroid therapy, cancer, renal dialysis, diabetes, HIV infection, liver disease, and organ transplant recipients. Three hundred and

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one cases of listeriosis were confirmed in this study. Foods in the refrigerators of patients were examined for presence of Listeria. Sixty-four percent of refrigerators (79 out of 123 examined) yielded a L. nzonocytogenes isolate. Of 2229 foods examined, 11% were positive for L. monocytogenes (81). Serotypes 4b, 1/2a, and 1/2b accounted for 95% ofL. monocytogenes isolates recovered from foods. Of the L. nzonocytogenes-positive foods, 33% matched the patient isolates. Sixty-seven percent of dairy isolates matched patient strains, implicating dairy products as sources of L. monocytogenes. Specific dairy product sources included Mexican-style cheese, feta cheese, and commodity cheeses. Thirty-two percent of sporadic cases of listeriosis could be attributed to eating foods purchased from delicatessan counters, Mexican-style and feta cheeses, and undercooked chicken (33). Preliminary CDC data for 1991 suggested a decrease of 30-40% in the number of cases compared with 1989-90. The annualized sporadic incidence for listeriosis was found to be 7.4 cases per million population, with an overall case fatality rate of 23%. Serotypes 1/2a (23%) and 1/2b (36%) together accounted for 59% of the cases; serotype 4b was isolated from 37% of the patients (33). Tapper0 et al. (13) reported a 44% decrease in rates of invasive listeriosis and a 48% decrease in the numbers of deaths due to listeriosis in the United States from 1989 to 1993. There were 1092 cases of listeriosis reported in 1993, resulting in 248 deaths, for an overall annual incidence of 4.2 cases per million population. Case-fatality rates remained similar (25% in 1989 compared to 23% for 1993). The decreased incidence rate was attributed to food industry efforts, sustained prevention efforts, and continued active surveillance.

C. OtherReports of FoodborneListeriosis:Epidemic and Sporadic Consumption of pat6 was cited as a likely cause of an epidemic of human listeriosis in the United Kingdom and the Republic of Ireland between 1985 and mid-l989 (99,100). The endemic rate of listeriosis in this region for the period between 1967 and 1982 was fewer than 100 cases annually. In 1987, the number of cases increased to 258; in 1988, 291 cases were reported, and in 1989, 250 cases were reported. The number of cases reported in 1990 showed a dramatic decrease to 90 cases, indicating that the outbreak period had ended. Food histories obtained during patient interviews revealed a significant association between pat6 consumption and onset of infection by L. monocytogenes. Surveys of the incidence of L. morzocytogelzes in pat6 revealed that pat6 from a single plant was more likely to be contaminated at a rate (48% positive) higher than that of other producers (4% positive). Two subtypes of L. monocytogenes, serotype 4b phage type 6,7 and serotype 4bX, accounted for the majority (30-54%) of the human cases. These subtypes accounted for 96% of all isolates of L. monocvtogenes from the pat6 produced by a single manufacturer. Raw fish and shellfish were epidemiologically linked to an epidemic of perinatal listeriosis in Auckland, New Zealand (101). A point source foodborne outbreak was epidemiologically linked with consumption of shrimp (102). Most patients suffered mild illness, with the exception of one woman who suffered miscarriage as a result of infection. An additional case of sporadic listeriosis was linked to consumption of contaminated fish (103). In 1983, a cluster of cases of perinatal listeriosis was reported in nine Hispanic mother-neonate pairs in Houston, Texas (104). All patient isolates from this outbreak were

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identified as serotype lb. The cause of this outbreak was unknown. This case was suspected to be of foodborne etiology, but attempts to identify a common source of exposure were unsuccessful. In Vermont, a case of listeriosis in a 76-year-old female patient was reported (105j. The patient presented mild respiratory congestion and chronic renal failure secondary to pyelonephritis upon admission to the hospital. Raw milk from the patient’s farm was incriminated as the vehicle of infection in this case of sporadic listeriosis. Isoenzyme typing and ribosomal RNA typing demonstrated that the raw milk isolates from the farm were indistinguishable from the patient isolates, and all isolates were confirmed as L. nlonocytogerles serogroup 112a. Two common source outbreaks of listeriosis were identified in Denmark between 1989 and 1990, which were epidemiologically linked to consumption of Danish blue-mild and hard cheeses (106). Jellied pork tongue was linked to a 1992 outbreak of listeriosis reported in France. This outbreak involved 279 cases, which resulted in 63 deaths and 22 spontaneous abortions (107). One year later (1993), a second meat-related outbreak was reported in western France linked to consumption of pork “rillettes.” A single strain of Listeria from this outbreak was linked to 39 cases of illness (108). In 1995, an epidemic of listeriosis occurring during the period January through July was linked to consumption of Brie de Meaux cheese (109). Bibb et al. (1 10) developed a multilocus enzyme electrophoresis (MEE) system for L. rnonocyfogerzes,which has proved useful in linking foodborne and clinical isolates of L. rnonocytogems. Baloga and Harlander (1 11) found that patient and product isolates were not distinguishable by serotyping, ribotyping, or multilocus enzyme electrophoresis. DNA fingerprinting proved to be the only method capable of proving relatedness of pairs of patient-product isolates. As mentioned previously, although phage typing has been successfully applied in epidemiological investigations, not all strains can be typed by this method. Pulsed-field gel electrophoresis is accepted as the preferred method to confirm relatedness of patientlfood product isolates (15 ) .

V.

SOURCES OF LISTERIA IN FOODS AND FOOD-PROCESSING ENVIRONMENTS

A host of unique properties possessed by Lisferiu render this a difficult organism to control in foods. Listeria can grow over a wide range of temperatures (- 1.5 to 45-50°C) (1 12114) and pH ranges (4.3-9.6) (115,116), survives freezing (117,118), and is relatively resistant to heat (90,119-127). Minimal water activity levels for growth of L. nzor1ocytog e r m and L. imzocna range from 0.90 to 0.97 (128,129). Shahamat et al. (130) reported survival of L. morzocytogerlesfor 132 days at 4°C in trypticase soy broth containing 25.5% NaCl. Lister-io is a psychrotrophic pathogen, and growth at temperatures as low as -0.1 to -0.4”C in chicken broth and pasteurized milk (13 1) and at - 1.5”Cin vacuum-packaged meat (114) has been recorded. The ability of L. morlocyfogenes to resist the heating temperatures used during milk pasteurization continues to be debated in the scientific literature (90,119-127). Fleming et al. (90), in their studies of an outbreak of listeriosis in Boston, concluded that “intrinsic contamination of the milk and survival of some organisms despite adequate pasteurization is both consistent with the results of this investigation and biologically plausible.’’ Fleming et al. reached this conclusion based upon the fact that milk involved in the outbreak came

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from farms where outbreaks of listeriosis had occurred, there was no evidence of improper pasteurization or postpasteurization contamination of the processed milk, and evidence in the scientific literature demonstrated the ability of L. rnonocytogenes to survive pasteurization (119,123,132). Comprehensive studies conducted by the U.S.Food and Drug Administration (FDA) and the U.S. Department of Agriculture (USDA) (121,122) and by Health and Welfare Canada (125) have shown that Listeria is unable to survive normal conditions of milk pasteurization. Knabel et al. (127) found that growing L. monocytogenes at 43°C prior to heat inactivation caused an increase in thermotolerance, but a study conducted by Farber et al. (125) demonstrated that even under worst-case scenario conditions, which included cultivation of L. monocytogenes populations at 43°C prior to inactivation, pasteurization would render a 4.5 to 6.2 D process. Lovett et al. (133) and Beckers et al. (134) estimate that extremely low levels of L. nzonocytogerzes (0.5- 1.O ListerialmL) exist in commercial bulk tank raw milk. Therefore, while populations of L. monocytogems have been shown to survive minimum pasteurization treatments of 7 1.1“C/ 16 S in various laboratory studies, survival under actual conditions of commercial milk pasteurization and processing is unlikely. Listericr contamination of processed dairy products is most likely a function of postpasteurization from the dairy plant environment, and numerous surveys (1 35-138) document presence of Listeria within the dairy plant environment. Sources of Listeria within the dairy plant environment include floors in coolers, freezers, and processing rooms, particularly entrances; cases and case washers; floor mats and foot baths; and the beds of paper fillers (135). Pritchard et al. (139), in a study of dairy-processing facilities, found that those processing plants having a farm contiguous to the processing facilities had a significantly higher incidence of Listeria contamination than farms without an onsite dairy farm. Arimi et al. (140) used ribotype analysis to demonstrate the link between on-farm sources of Listericr contamination (dairy cattle, raw milk, and silage) and subsequent contatnination of dairy-processing environments. Raw milk is a well-recognized source of Listeria, and for this and numerous other microbiological reasons, consumption of raw milk should be avoided (105,134,141). Studies by Ryser and Marth (142-144) examined the fate of L. monocytogenes during the manufacture of cheddar, Camembert, and brick cheese. Rapid growth of Listeria to populations of 5 X lo7 CFU/mL is observed in Camembert cheese, which has a pH that increases during ripening, thereby creating a favorable growth environment for Listeria (143). In contrast, Listeria populations show a marked decline in viable population levels during ripening of cheddar cheese. However, population levels do not decline to undetectable levels. Current U.S. regulations call for cheese made from raw or subpasteurized milk to be ripened at 1.7”C (35°F) for at least 60 days prior to sale. Ryser and Marth have shown that aging alone will not ensure the production of Listerin-free cheddar cheese (142). Madec et al. (145) used the “Bactocatch” process (tangential membrane filtration) to eliminate L. monocytogelzes from raw milk. Treatment of skim milk at 50°C in conjunction with the “Bactocatch” process resulted in a 99% decrease in Listeria populations. This technique may enable elimination of Listeria during cheddar cheese manufacture. Genigeorgis et al. (146) evaluated the ability of 24 types of market cheeses to support growth of L. tnonocytogerzes. Cheeses able to support growth of L. monocvtogenes included soft Hispanic-type cheeses, Ricotta, Teleme, Brie, Camembert, and cottage cheeses (pH range 4.9-7.7). Cheeses not supporting growth and that resulted in gradual death of L. rnorzocytogelzes included Cotija, cream, blue, Monterey jack, Swiss, cheddar, colby, string, provolone, Muenster, feta, and Kasseri (pH range 4.3-5.6). A correlation was ob-

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served between growth of Listeria in cheeses having a pH of greater than 5.5 and in cheeses manufactured without a starter culture. Ryser et al. (147) examined the fate of L. monocytogenes during manufacture and storage of cottage cheese. L. molzocytogenes survived in both creamed and uncreamed cottage cheese during 28 days of storage at refrigeration temperatures and was recovered in higher numbers from creamed (pH 5.32-5.45) versus uncreamed (pH 5.12-5.22) cottage cheese. Hicks and Lund (148) examined the ability of L. monocytogenes to survive in creamed cottage cheese when stored at 4, 8, or 12°C for 14 days. The organism survived, but failed to increase in numbers, during storage in product with a pH range of 5.064.53. Chen and Hotchkiss (149), however, found that L. monocytogenes grew in cottage cheese stored at 7°C for 16 days or 4°C for 63 days, but would not grow under these conditions when modified CO2 packaging conditions were used. Conner et al. (150) investigated the effects of temperature, NaC1, and pH on the growth of L. monocytogenes in cabbage. Results indicated that cabbage juice provided a good substrate for growth of Listeria. The organism was found to survive well at 5°C even in the presence of 5% NaC1, and the organism could grow and tolerate a pH of less than 5.6. This study, together with the findings of Schlech et al. (89), confirms that cabbage can serve as a vector of transmission of L. monocytogenes to humans and demonstrates the potential for L. monocyfogenes to persist and proliferate on vegetables and in brines used to ferment vegetables. Potatoes and radishes have been identified as sources of L. monocytogenes during retail food surveys (15 1). Additional studies have confirmed growth and survival of L. monocytogenes on asparagus, broccoli, cauliflower, corn, green beans, lettuce, and radishes (152,153). During initial studies to prove efficacy of the USDA testing procedure for recovery of L. monocytogenes in meat and poultry products, McClain and Lee (154) recovered L. monocytogenes from 40% of analyzed frozen ground beef samples and from 52% of sampled pork sausage. Subsequent surveys (155) confirmed the presence of L. monocytogenes in 14 of 49 (28.6%) samples of domestic cooked beef, roast beef, and cooked corned beef. Glass and Doyle (156) examined the ability of L. monocytogerzes to grow on a variety of processed meat and poultry products. Growth and survival correlated well with product pH. Summer sausage (pH 4.8-5.2) supported poor growth of Listeria; cooked roast beef (pH 5.8) supported only slight growth of L. monocytogenes. L. monocytogenes grew well on those products having a pH near or above 6.0 (ham, bologna, and bratwurst). L. monocytogenes populations grew extremely well on sliced chicken and turkey, increasing by lo3lo5 CFU/mL during 4 weeks of storage at 4.4"C. Raw poultry is a well-recognized source of L. monocytogenes, and numerous surveys have confirmed the presence of L. monocytogenes in retail poultry samples (157,158). Bailey et al. (159) recovered L. monocytogenes from 23% of sampled broiler carcasses, the most prevalent serotype isolated being 1/2b. Ready-to-eat poultry products have been implicated as vectors of transmission of listeriosis to humans. Cooked-chilled chicken and turkey frankfurters were confirmed as vehicles of Listeria infection in England and the United States, respectively, during 1988 and 1989 (98,160). Gilbert et al. (161) confirmed the presence of L. monocytogenes in 12% of precooked, ready-to-eat poultry products collected from London-area retail establishments between mid-November 1988 and midJanuary 1989. Survival of L. wonocytogenes on chicken breasts processed by moist and dry heating methods has been demonstrated (162). Wenger et al. (163) examined a production facility that manufactured turkey franks in order to determine sources and incidence of contamination. L. monocytogenes was

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isolated from only 2 of 41 environmental samples from the plant, which included a cooler room floor and a conveyer belt attached to a peeler. Yet L. monocytogenes was isolated from six of seven retail lots of product produced over a 37-day production period. Product samples taken at the production line post peeler were more likely (12 of 14 samples positive) to be contaminated than samples from other production locations (2 of 40 samples positive). Therefore, product contamination was found to occur at a single point during the peeling process prior to packaging of product. Seafood is recognized as a source of L. nzonocytogenes.Weagant et al. (164) documented presence of L. monocytogenes in frozen seafood samples, which included shrimp, crabmeat, lobster tail, fin fish, and surimi-based products. Farber et al. (165) isolated L. monocytogenes from ready-to-eat shrimp, crab, and smoked salmon, and further laboratory studies demonstrated growth at 4°C of L. molzocytogenes on cooked crabmeat, lobster, shrimp, and smoked salmon. Jemmi (166), upon examination of 377 samples of smoked and marinated fish, found L. nzonocytogenes in 47 samples. A survey in Newfoundland conducted by Dillon et al. ( 167) also revealed the presence of Listeria in smoked seafood products.

VI.

DETECTIONOF LISTERIA IN FOODS

A.

SelectiveProcedures

Most selective enrichment and isolation procedures developed to date for the detection of Listeria take advantage of the resistance of this organism to selective compounds, which suppress growth of background contaminants. Selective agents commonly used in selective enrichment/plating media include acriflavine, naladixic acid, lithium chloride, moxalactam, and phenylethanol (1 33,141,168,169). Detection of L. nzonocytogenes in food products or food-processing environments is accomplished by use of a variety of standard or rapid microbiological procedures. Among the most widely used are protocols devised by the U.S. Department of Agriculture-Food Safety Inspection Service (USDA-FSIS) for the detection of Listeria in meats (154,169- 171) and FDA for the detection of Listerin in dairy products (133,172). The FDA procedure initially involves enrichment of 25 g or 25 mL of sample into 225 mL of enrichment broth, M52 (172), which consists of a trypticase soy broth/yeast extract base supplemented with: monopotassium phosphate (anhydrous), 1.35 g/L; disodium phosphate (anhydrous) 9.6 g/L; acriflavin HC1 10 mg/L; nalidixic acid sodium salt 40 mg/L; cycloheximide 50 mg/L; and pyruvic acid (sodium salt 10% w/v aqueous solution) 11.1 mL. Samples are enriched without selective agents for 4 hours at 30°C. Following addition of selective agents (acriflavin, nalidixic acid, and cycloheximide), samples are incubated for an additional 44 hours for a total incubation period of 48 hours at 30°C. This procedure is a modification of the original procedure, which called for sample enrichment for 1 and 7 days at 30°C (141). The original broth was modified by increasing its buffering capacity, thereby positioning this media to be used successfully in conjunction with DNA probe and other methods, which are more sensitive than conventional cultural procedures. After 24 and 48 hours, EB cultures are streaked onto OXA (173) and Listerin plating medium (LPM) agar (170) or LPM plus esculin/Fe3' agars, both of which have replaced the originally recommended modified McBride agar (MMA) as the selective isolation media. PALCAM agar (174) may beused in place of LPM agar. This substitution brings the method into closer alliance with methodology used outside the United States

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and decreases reliance on the Henry technique. OXA and PALCAM plates are incubated (with optional use of a C02-air atmosphere) at 35°C for 24-48 hours, with LPM plates incubated at 30°C for 24-48 hours. LPM plates can be viewed using Henry illutnination or alternatively, esculin and ferric iron salt may be added to LPM to eliminate the need for Henry illumination. With OXA and PALCAM, Listeria colonies develop a black halo. It is recommended that five or more typical colonies be picked from OXA and PALCAM or LPM and transferred to TSAYE for confirmation of purity and typical isolated colonies. The selection of five colonies ensures that multiple species of Listeria, if present, will be identified. TSAYE plates are incubated at 30°C for 24-48 hours (35°C incubation may be utilized if colonies are not being used for wet tnount motility confirmation). Purified isolated are subjected to a series of standard biochemical tests, with a total of 10-1 1 days being required to isolate and confirm the presence of Listeria in food satnples via the FDA procedure. This procedure was specifically developed to optimize Listeria detection in milk and dairy products. The USDA-FSIS selective enrichment protocol for isolation of Listeria from raw meat and poultry was developed by McClain and Lee (154). The USDA-FSIS procedure for isolation of Listeria, while originally designed to detect the presence of Listeria in meats, has been subsequently used with success for isolation of Listeria from dairy products and environmental samples. The USDA scheme differs from the FDA procedure primarily in the selective enrichment and plating media used, along with the size of the sample tested. The revised USDA procedure (171) differs from the original method in that (a) LEB I1 has been replaced by Fraser Broth (175) as the secondary enrichment medium, (b) LPM agar has been replaced by Modified Oxford agar (MOX), and (c) the regulatory sample size has been increased to 25 g. Fraser broth and Modified Oxford Agar will both blacken during incubation due to the ability of Listeria spp. and other contaminants to hydrolyze esculin, with colonies of Listeria exhibiting black halos on Modified Oxford agar following 24-48 hours of incubation. MOX is also more selective than LPM or Oxford agar, with staphylococci and streptococci both generally unable to grow on MOX. Inadequacies in the original (176) FSIS procedure related to the use of Fraser broth as the secondary enrichment broth have been reported. False-negative results due to reliance on Fraser broth darkening and a 24-hour secondary enrichment have been reported by a number of laboratories (177,178). Kornacki et al. (178) compared recovery of L. rnonocytogerzes from Fraser broth incubated for 26 versus 48 hours. L. morzocytogerles was isolated from 60 of 1088 meat product and environtnental swabs from meat and dairy plants. A false-negative rate of up to 6.7% was recorded, attributed to the failure of L. nzorzocytogenes to be detected in Fraser broth at 26, but not at 48 hours, and by the failure of Fraser broth to blacken. Further, when primary enrichments were streaked directly to selective enrichment media, failure to recover L. monocytogerzes in eight samples was recorded. As a result of this study, a 48-hour incubation of Fraser broth was recommended. Thus, all Fraser broth enrichment cultures should be streaked regardless of color following 24-26 hours of incubation. Once cultures have been streaked to MOX, Fraser broth cultures should be reincubated at 35°C for an additional 24-hour enrichment period. MOX plates streaked from 24- to 26-hour Fraser broth enrichment cultures should be examined for presence of colonies displaying typical Listeria morphology. If present, isolation should proceed. If absent, a second MOX plate should be streaked from the 48-hour enrichment culture.

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A detection method widely used in Europe is the Netherlands Government Food Inspection Service (NGFIS) method, developed by Van Netten et al. (174). These authors have reported increased sensitivity with this method over that of the USDA procedure when used to examine foods containing L. monocytogenes at levels of less than 10 CFU/g. Food samples are enriched in L-PALCAMY enrichment broth for 48 hours at 30°C. After 24 and 48 hours, 0.1 mL of L-PALCAMY enrichment broth is plated onto PALCAM agar. Plates are incubated at 30°C for 48 hours under microaerophilic conditions (5% oxygen, 7.5% carbon dioxide, 7.5% hydrogen, and 80% nitrogen). Numerous studies have been conducted to compare the efficacy of these and other widely used detection/isolation protocols. Hayes et al. (179) compared use of the USDAFSIS procedure with cold enrichment as a means of identifying L. monocytogerzes in suspect food samples. Both procedures were able to identify L. nzonocvtogenes in 28 of 51 positive samples. The USDA-FSIS procedure identified 2 l samples missed by cold enrichment while the cold enrichment procedure identified an additional 2 samples that the USDA-FSTS procedure missed. A comparison of three enrichment methods was also made by Hayes et al. (180) when examining foods obtained from the refrigerators of patients with active clinical cases of listeriosis. Two thousand two hundred and twenty-nine foods were examined in the study, 1 % 1 of whichwere positive for L. ~)~onocytogenes. A comparative evaluation of three microbiological procedures was conducted on 899 of the examined foods. The USDA-FSIS (176), FDA (133), and Netherlands Government Food Inspection Service (NGFIS) (172) methods were not statistically different in their ability to isolate Listeria from the 899 samples. The FDA procedure detected L. monocytogelzes in 65% of foods shown to bepositive, while theUSDA-FSIS and NGFIS procedures detected L. monocytogerzes in 74% of foods shown to be positive. Thus, none of these widely used conventional methods proved to be highly sensitive when used independently for analysis of Listeria contamination in foods. It was noted, however, that use of a combination of any two methods improved detectability from 65-74% for individual protocols to 8791% for combined protocols. B. Detection of SublethallyInjured Listeria Most conventional and rapid methods for detection of Listeria in food product and environmental samples use highly selective enrichment media to facilitate growth over competitive background flora. These highly selective enrichment procedures do not account for recovery of sublethally injured Listeria, which could exist within a variety of heated, frozen, and acidified foods, or heated, frozen, and sanitized areas within food-processing environments. It is well recognized that Listeria can be injured as a result of exposure to a variety of processing treatments, which include sublethal heating and freezing, drying, irradiation, or exposure to chemicals (sanitizers, preservatives, acids) ( 122,142,181- 193). Under ideal conditions in food systems, injury is reversible and injured Listeria can repair sublethal damage. Repair of heat-injured L. nzonocytogems has been shown to take place in whole and 2% milk stored at 4°C (194). Several investigators have attempted to improve the sensitivity of current detection systems by recognizing that Listeria may exist in an injured state in food products and food-processing environments. All current detection procedures, withthe exception of cold enrichment, involve selective enrichment and/or selective plating. Cold enrichment is not feasible for routine sample analysis because several months may be necessary to

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record positive results. By failing to consider recovery of injured Listeria, current methodologies underestimate the true incidence of Listerin. Several previous studies have reported on the ability of commonly used plating media to recover injured Listeria. Among the compounds examined, phenylethanol, acriflavin, polymixin-acriflavin, and sodium chloride were found to inhibit recovery of thermally stressed and nonstressed Listeria (186,195- 198). It should be noted that these compounds comprise media that are routinely used for detection of Listeria. When examined for ability to quantitatively recover thermally stressed Listeria on solid media, Listeria enrichment broth (LEB) agar, modified McBride's agar (MMA), lithium chloride phenylethanol moxalactam (LPM) agar, and FDA enrichment broth agar showed significantly impaired abilities to recover injured cells. Warburton et al. (199) recently examined the ability of the modified FDA and USDA methods for isolating stressed and low levels of L. rnonocytogenes in food and environmental samples. These authors found the modified FDA and USDA methods to be comparable in their abilities to isolate stressed and low-level populations of L. wonocytogenes. However, the authors failed to assess the degree of injury within bacterial populations following exposure to sublethal stress. The percent injury existing within a population of bacterial cells can profoundly affect results of comparisons of media performance. Thus, it is difficult to determine whether valid conclusions can be drawn from these studies. Busch and Donnelly (184) developed an enrichment medium capable of resuscitating heat-injured Listeria. This medium, Listeria repair broth (LRB), permits complete repair of injured Listeria within 5 hours at 37°C. LRB is composed of the following constituents (per liter of distilled water): trypticase soy broth, 30.0 g; glucose, 5.0 g: yeast extract, 6.0 g; magnesium sulfate, 4.94 g; ferrous sulfate, 0.3 g; pyruvic acid (sodium salt), 10.0 g; MOPS-free acid, 8.5 g; and MOPS-sodium salt, 13.7 g. In studies that compared the efficacy of LRB in promoting repair/enrichment of heat-injured Listeria with that of existing selective enrichment media, repair was not observed in FDA enrichtnent broth (133), phosphate-buffered Listeria enrichment broth (PEB; Gene-Trak Systems, Framingham, MA). or UVM enrichment broth (l 69). Final Listeria populations in selective enrichment media following a 24-hour incubation were l .7-9.1 X lo8CFU/mL, compared with populations in LRB, which consistently averaged 2.5-8.2 X 10" CFU/mL. Studies with LRB were extended to examine the potential for repair of freeezeinjured and sanitizer-injured L. rnonocytogenes (193,200). Although variation in the susceptibility of L. nzonocytogerm strains was recorded, in general, L. wzorzocytogerzes is not highly injured by freezing. Golden et al. (1 17) compared the extent of freeze injury in four strains of L. rnof1ocytogenessubjected to freezing at - 18°C for 7 and 14 days. Percent injury was found to vary between 72 and 80%. El-Kest and Marth (201) recorded 65% injury and 55% death after one day of storage in phosphate buffer at - 18°C. Sallam and Donnelly (193) examined the ability of four commonly used dairy plant sanitizers to induce injury in L. monocytogenes when exposed at sublethal concentrations. UVM broth failed to support the growth of sanitizer-injured cells, while LRB medium permitted recovery of tested strains. Flanders et al. (202) examined the efficacy of using a repair step to increase sensitivity of recovery of injured Listeria from environmental sponge samples obtained from dairy processing plant environments. The USDA-FSIS Listeria isolation protocol using UVM-modified Listeria enrichment broth was compared with a modified USDA-FSIS format, which utilized LRB as the primary enrichment medium. UVM and LRB broths were also used in conjunction with rapid methods. Of 80 sites positive by any method, UVM and LRB showed similar recovery rates (87.5% and 88.8%, respectively). However, cultural methods combined with either rapid method from each broth increased

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sensitivity of detection to 97.5-98.8% when data from UVM and LRB enrichment was combined (202). Flanders et al. (203) also examined incorporation of ceftazidime into LRB (LRBC) to increase recovery of Listeria in dairy plant environmental samples. LRB, UVM, and LRBC enrichment media were evaluated for their abilities to identify Listeriapositive environmental samples. While no single broth was able to detect all Listeriapositive sites, LRBC detected 67 out of 89 positive sites (75.3%), and LRB and UVM each detected 60 out of 90 positive sites (66.7%). Combining results from any two broths increased recovery percentages from 66.7-75.3% to 82.2-94.4%. The combination of LRBC and UVM detected 94.4% of positive samples, and combining LRBS and LRBC recovered 9 1.1% of positive samples. In yet another study, Pritchard et al. (204) compared performance of UVM, LRB, and LRBC for isolation of Listeria from the dairy plant environment. Of eighty positive samples identified, 54 samples came from UVM medium, 56 were from LRB, and 57 of the positive samples came from LRBC medium. A total of 26 samples (32.5% of positive samples) were identified by either LRB or LRBC but not by UVM media. The use of UVM combined with either LRB or LRBC was shown to substantially increase the number of positive samples identified. When results of UVM and LRB are combined, 65 of 80 (8 1.3%) positive samples were identified. The combined use of UVM and LRBC resulted in identification of 74 of 80 (92.5%) positive samples. Despite improved recoveries when combined use of media is employed, these results illustrate the severe limitations associated with the regulatory procedures currently used to assure absence of Listeria in foods and food-processing environments. Ryser et al. (205) evaluated the ability of UVM and LRB to recover different strainspecific ribotypes of L. nzonocvtogenes from meat and poultry products. Forty-five paired 25 g retail samples of ground beef, pork sausage, ground turkey, and chicken underwent primary enrichment in UVM and LRB (30°C/24 h) followed by secondary enrichment in Fraser broth (35"C/24 h) and plating on modified Oxford agar. A 3-hour nonselective enrichment period at 30°C was used with LRB (with tested food) to enable repair of injured Listeria prior to addition of selective agents. Of 180 meat and poultry products tested, LRB identified 73.8% (133/180) and UVM 69.4% (124/180). Although there was not a statistically significant difference in these results, combining UVM and LRB results increased overall Listeria recovery rates to 83.3%. These results demonstrate that use of LRB for repair/enrichtnent of samples in conjunction with the USDA/FSIS method has the potential to improve recovery of Listeria from meat and poultry products. In this study, Listeria isolates were genetically discriminated using the automated RiboprinterTM Microbial Characterization System. A total of 36 different Listeria strains comprising 16 L. morzocytogelzes (including four known clinical ribotypes), 12 L. innocucI, and 8 L. welshimeri ribotypes were identified from selected positive samples (15 samples of each product type, 2 UVM and 2 LRB isolates per sample). Twenty-six of 36 (13 L. monocytogenes) ribotypes were detected using both UVM and LRB; whereas 3 of 36 (1 L. nzorlocytogenes) and 7 of 36 (3 L. morzocytogenes)Listeria ribotypes were observed using only UVM or LRB, respectively. Ground beef, pork sausage, ground turkey, and chicken yielded 22 (8 L. 111onocytogei1es), 21 (12 L. monocytogenes), 20 (9 L. nzonocytogenes), and 19 (1 1 L. nzonocytogenes) different Listeria ribotypes, respectively, with some Listeria ribotypes confined to a particular product. More importantly, striking differences in both the number and distribution of Listeria ribotypes, including previously recognized clinical and nonclinical ribotypes of L. monocytogenes, were observed when 10 UVM and 10 LRB isolates from five samples of each product were ribotyped. When a third set of six samples per product type was examined from which two Listeria isolates were obtained using only

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one of the two primary enrichment media, UVM and LRB failed to identify L. rnonocytogerzes (both clinical and nonclinical ribotypes) in two and four samples, respectively. These findings illustrate the complex microbial ecology of Listerin in foods and the limitations of existing detection procedures to fully represent the total distribution of Listeria isolates in foods. Furthermore, two of the L. vlonocytogenes ribotypes missed using UVM were known clinical isolates of serotype 1/2a, both of which were responsible for sporadic and epidemic cases of human listeriosis in England and Scotland (14). Continuing work (206) on enrichment of dairy environmental samples in UVM and LRB has shown that combining these two primary enrichment media into a single tube of Fraser broth for secondary enrichment yields a significantly higher 0, < 0.05) percentage of Listeria-positive samples than when either LRB or UVM are used alone. These findings, combined with reports of L. inlzocua being able to outgrow L. rvonocytogems in UVM and Fraser broth (207,208), suggest that different genetic types of L. rno1zocytogerzes may vary somewhat in their nutritional requirements or in abilities to compete with other genetic types of Listeria. Refinement of existing systems should consider the nutritional needs associated with specific genetic types (unique riboprints) of Listeria strains distributed in foods. Roth and Donnelly (192) assessed the survival of acid-injured L. morzocytogenes in four different acidic food systems, and they examined the efficacy of two different enrichment media, LRB and UVM, to recover acid-injured Listerin from acidic foods. Populations of L. nzorzocytogenes F5069 serotype 4b were injured in lactic (pH 3.0) and acetic (pH 3.5j acids. Two levels of injury were produced and monitored-one population with 99.99% injury and the second with approximately 95% injury. Four acidic food systems were used as models. Foods studied were fresh apple cider (pH 3.3), plain nonfat yogurt (pH 4.2), fresh coleslaw (pH 4.4), and fresh salsa (pH 3.9). Acid-injured Listeria were added to each acidic food system and monitored by selective and nonselective plating methods. Simultaneously, samples were tested using LRB and UVM enrichment procedures followed by standard isolation/identification procedures. Additionally, the survival of healthy L. monocvtogenes was also monitored. These experiments were conducted at 4°C (storage temperature) and 30°C (abuse temperature). Results indicated that acid-injured Listeria failed to repair in the foods tested, but survive in the tested foods for over a week. Storage temperature was found to affect survival, with 4°C storage having bacteriostatic effects, whereas 30°C imparted bacteriocidal effects. Parameters involved in the survival of acid-injured Listeria included the degree to which the bacterial population was injured (percent injury), storage temperature, and the pH of the food. At time points where differences were detected, LRB proved to be superior, detecting 22 of 54 samples, compared to UVM, which detected only 3 of 54 positive samples.

VII.

SUMMARY

Despite reductions in disease incidence due to Listeria morzocytogelzes, this organism remains the leading cause of death due to a foodborne pathogen (207). Recent multistate outbreaks of illness and death highlight the need for renewed collaboration among industry, university, and governmental agencies to control this dangerous but interesting foodborne pathogen. Improvements in testing methods are also needed to ensure adequate

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sensitivity of detection of regulatory procedures used to identify and ultimately control Listeria.

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162. Harrison, M. A., and Carpenter,S. L. (1989). Survival of large populations of Listeria monocytogenes on chicken breasts processed using moist heat. J. Food Prot., 52:376-378. 163. Wenger, J. D., Swaminathan. B., Hayes, P. S., Green, S. S., Pratt, M., Pinner, R. W., Schuchat, A., and Broome, C. V. (1990).Listeria rnonocytogerlescontanination of turkey franks: Evaluation of a production facility. J. Food Prot., 53:1015-1019. 164. Weagant. S. D., Sado, P. N., Colburn, K. G., Torkelson, J. D., Stanley, F. A., b a n e , M. H.. Shields, S. C., and Thayer, C. F. (1988). The incidence of Listeria species in frozen seafood products. J. Food Prot., 51:655-657. 165. Farber, J. M. (1991). Listeria rnonocytogems in fish products. J. Food Prot.. 54:922-923. 166. Jemni, T.(1990). Actual knowledge ofListeria in meat and fish products. Mitt. Geb. Lebewsmittel. Hyg., 81:144-157. 167. Dillon, R., Patel, T., and Ratnum, S. (1992). Prevalence of Listeria in smoked fish. J. Food Prot.. 55~866-870. 168. Donnelly, C. W., and Baigent, G. J. (1986). Method for flow cytometric detection ofListeria nzorzocytogenes in nilk. Appl. Environ. Microbiol.. 52689-695. (1989). FSIS method for the isolation and identification of 169. McClain, D., and Lee, W. H. Listeria r~lonocytogerzesfrom processed meat and poultry products. Lab Comm. No. 57, Revised May 24, 1989. U.S. Dept. Agric., FSIS Microbiol. Div., Beltsville. MD. 170. Lee, W. H., and McLain. D. W. (1986). Improved Listeria rnonocytogenes selective agar. AppI. Erz\iron. Microbiol., 52:1215-1217. 171. Johnson. J. L. (1998). Isolation and identificationof Listeria nzorzoc?ltogenesfrom meat, poultry and egg products. In USDA-FSIS Microbiology Laborator? Guidebook, 3rd ed., Vol. 1. Washington, D.C., pp. 8-1-8-18. 172. Hitchins, A. D. (1995). Listeria monocytogenes. In Food and Drug Adrrzinistration Bacteriological Analytical Manual, 8th ed., AOACInternational,Gaithersburg.MD,pp.10.0110.13. 173. Curtis, G. D. W., Mitchell, R. G., King, A. F., and Griffen, E. J. (1989). A selective differential medium for the isolation of Listeria nzorzocytogenes. Lett. Appl. Microbiol., 8:95-98. 173. Netten, Van, P., Perales. I., Van de Moosdijk, A., Curtis, G. D. W., and Mossel, D. A. A. (1989). Liquid and solid selective differential media for the detection and enumeration of L. ntorzocytogenes and other Listeria spp. M. J. Food Microbiol., 8999-316. 175. Fraser, J. A., and Sperber, W.H. (1988). Rapid detection ofListeria spp. in food and environmental samples by esculin hydrolysis. J. Food Prot., 51:762-765. (1989). Method for the Isolution and Identification 176. Carnevale, R. A., and Johnston, R. W. oj*Listeria monocytogenes fr-om Meat ancl Poultry Products. United States Department of Agriculture Food Safety and Inspection Service, Laboratory Communication No. 57, Revised May 24, USDA-FSIS, Washington, DC. for the simultaneous 177. Bailey, J. S., and Cox, N. A. (1992). Universal preenrichment broth detection of Scrlnzonella and Listeria in foods. J. Food Prot., 55:256-259. 178. Kornacki, J. L., Evanson, D. J., Reid, W., Rowe, K., and Flowers, R. S. (1993). Evaluation of the USDA protocol for detection of Listeria rnonocytogerzes. J. Food Prot., 56:441-433. 179. Hayes, P. S., Graves, L. M., Ajello. G. W., waminathan. B., Weaver. R. E., Wenger, J. D., Schuchat,A.,Broome. C. V.,andthe Listeria studygroup.(1991).Comparison of cold enrichment and the U.S. Departmentof Agriculture methods for isolatingListeria monocytogenes from naturally contaminated foods. Appl. Erniron. Microbiol., 57:2109-2113. 180. Hayes, P. S., Graves, L. M., Swaminathan, B., Ajello, G. W.. Malcolm, G. B., Weaver, R. E., Ransom, R.. Deaver, K.. Plikaytis, B. D.. Schuchat, A., Wenger, J. D., Pinner, R. W., Broome, C. V.. and theListeria study group. Comparisonof three selective enrichment methods for the isolation of Listericr monocytogenes from naturally contaminated foods. J. Food Prot., 55:952-959. and Marth, E. H. (1990). Acid-injury of Listeria rnonocytogenes. J. Food Prot., 181. Ahamad. N., 53126-29.

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182. Beuchat, L. R., Brackett, R. E., Hao, D. Y.-Y., and Conner, D. E. (1986). Growth and thermal inactivation of Listeria ~nonocytogenesin cabbage and cabbage juice. Can. J. Microbiol., 32~791-795. 183. Bunduki, M. M.-C., Flanders, K. J., and Donnelly, C. W. (1994). Metabolic and structural sites of damage in heat- and sanitizer-injured populations of Listeria morrocytogenes. J. Food Prot., 581410-415. 184. Busch, S. V., and Donnelly, C. W. (1992). Development of a repair-enrichment broth for resuscitation of heat-injured Listeria monocytogenes and Listeria innocua. Appl. Environ. Microbiol., 58: 14-20. 185. Cassiday, P. K., Brackett. R. E., and Beuchat, L. R. (1989). Evaluation of ten selective direct plating media for enumeration of Listeria rttortocytogenes in ham and oysters. Food Microbiol., 55:113-125. 186. Crawford. R. G., Beliveau, C. M., Peeler, J. T.. Donnelly, C. W., and Bunning, V. K. (1989). Comparative recovery of uninjured and heat-injured Listeria nzortocytogenes cells from bovine nlilk. Appl. Environ. Microbiol., 55: 1490-1494. 187. Curtis, G. D. W., Nichols, W. W. and Falla, T. J. (1989). Selective agents for Listerin can inhibit their growth. Lett. Appl. Microbiol., 8:169-172. 188. Garayzabel, J. F., and Genigeorgis, C. (1990). Quantitative evaluation of three selective enrichment broths and agars used in recovering Listeria microorganisms. J. Food Prot., 53: 105-1 10. 189. Golden, D. A., Beuchat, L. R., and Brackett, R. E. (1988). Evaluation of selective direct plating media for their suitability to recover uninjured, heat-injured and freeze-injured Listeria nronocytogenes from foods. Appl. Environ. Microbiol., 54: 1451-1456. 190. Golden, D. A., Beuchat, L. R., and Brackett, R. E. (1988). Inactivation and injury of Listeria monocytogenes as affected by heating and freezing. Food Microbiol., 5:17-23. 191. McCarthy. S. A., Motes, M. L., and McPhearson, R. M. (1990). Recovery of heat-stressed Listeria nronocytogenes from experimentally and naturally contaminated shrimp. J. Food Prot., 53:22-25. 192. Roth, T. T. and Donnelly, C. W. (1995). Injury of Listeria rnonocytogenes by acetic and lactic acids: mechanisms of repair and sites of sublethal damage. IF” Ann. Mtg. Book of Abstracts 81D-1, p. 246. 193. Sallam, S. S. and Donnelly, C. W. (1992). Destruction, injury and repair of Listeria species exposed to sanitizing compounds. J. Food Prot., 55:771-776. 194. Meyer, D. H. and Donnelly. C. W. (1992). Effect of incubation temperature on repair of heat-injured Listeria in milk. J. Food Prot., 55:579-582. 195. Leasor, S. B., ‘Abbas, C. A., and Firstenberg-Eden, R. (1990). Evaluation ofUVMas a growth medium for Listeria morrocytogenes. Abstr. Ann. Mtg. Amer. Soc. Microbiol. P39: 284. 196. Smith, J. L., and Archer, D. L. (1988). Heat-induced injury in Listeria rnonocytogenes. J. Zndust. Microbiol., 3: 105-1 10. 197. Warburton, D. W., Farber, J. M., Armstrong, A., Caldeira, R., Hunt, T., Messier, S.. Plante, R., Tiwari N. P., and Vinet, J. (1991). A comparative study of the “FDA” and “USDA” methods for the detection of Listeria monocytogenes in foods. Znt. J. Food. Microbiol., 13: 105-1 18. 198. Warburton, D. W., Farber. J. M., Armstrong, A., Caldeira, R., Tiwari, N. P., Babiuk, T.. Lacasse P., and Read. R. (1991). A Canadian comparative study of modified versions of the “FDA” and “USDA” methods for the detection of Listeria rnonocytogenes. J. Food Prot., 669-676. 199. Warburton, D. W., Farber, J. M., Powell, C., Tiwari, N. P., Read, S., Plante, R., Babiuk, T., Laffey, P., Kauri. T. Mayers, P., Champagne, M.-J.. Hunt, T,. Lacasse. P., Viet. K., Smando, R., and Fran Coates. (1992). Comparison of methods for optimum detection of stressed and low levels of Listeria monocytogenes. Food Microbiol., 9: 127-145.

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200. Flanders, K. P. (1991). Injury. resuscitation and detection of Listeria spp. From frozen environments, M. S. thesis, University of Vermont, Burlington, VT. 201. El-Kest, S. E. and Marth. E. H. (1992). Freezing of Listeria mortocytogenes and other microorganisms: a review. J. Food Prot., 55:639-648. 202. Flanders, K. J., Pritchard, T. J., and Donnelly, C. W. (1995). Enhanced recovery of Listeria from dairy plant processing environments through combined use of repair enrichment and selective enrichment/detection procedures. J. Food Prof., 58:404-407. 203. Flanders, K. J.. Beliveau, C. M., Pritchard T. J., and Donnelly. C. W. (1994). Enhanced recovery of Listeria from dairy plant environments using modified selective-enrichment media. IFT Ann. Mtg. Technical Program: Book of Abstr. 59C, p. 166. 204. Pritchard, T. J.. Flanders, K. J., and Donelly. C. W. (1995). Comparison of the incidence of Listeria on equipment versus environmental sites within dairy processing plants. Znt. J. Food Microbiol., 26:375-384. 205. Ryser, E. T., Arimi. S. M., Bunduki M. M.-C., and Donnelly, C. W. (1996). Recovery of different Listeria ribotypes from naturally contaminated, raw refrigerated meat and poultry products with two primary enrichment media. AppI. Emiron. Microbiol., 62: 1781- 1787. 206. Pritchard, T. J. and Donnelly. C. W. (1995). Combined secondary enrichment of UVM and LRB primary enrichment broths increases the sensitivity of Listericr detection. IFT Ann. Mtg. Book of Abstracts, 34-2, p. 96. 207. Curiale, M. S. and Lewus, C. (1994). Detection of Listeria monocytogenes in samples containing Listeria inrlocurr. J. Food Prof., 57: 1048-1051. 208. Petran, R. L., and Swanson, K. M. J. (1993). Simultaneous growth of Listerin mortocytogenes and Listeria inrzocua. J. Food Prof., 56:616-618. 209. United States General Accounting Office. May 1996. FoodSafety: hlforntntion on Foodborne Zlbzess. Report to Congressional Committees. B-270753, pp. 1-31.

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11 Bacteriology of Salmonella Robin C. Anderson and Richard L. Ziprin U.S. Departwwnt of Agriculture. College Stcrtioiz, Te.ucrs

247 247 11. History andNomenclature 111. IsolationandCultivation 2-58 IV. Serotyping 25 1 V. Biotyping 252 VI. PhageTyping 253 253 VII. MolecularTyping I. Introduction

VIII.

Conclusions References

I.

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INTRODUCTION

Salnlorlella are gram-negative bacteria belonging to the family Er1terobacteriaceae (1). These bacteria are widely distributed throughout various ecological habitats, having been isolated from soil, water, foods, andthe intestinal tracts of humans and animals. The propensity of all Salrmlzelln to cause systemic or enteric infections in humans and animals makes them important pathogens, and despite many hygienic improvements in food production, these remain among the most important causative agents of foodborne disease (2-4j. Manifestation of disease can be varied depending on the serotype and host. For instance, certain host-adapted serotypes cause disease almost exclusively in their specific hosts, whereas other serotypes exhibit a broad host range specificity. Details regarding pathogenicity and disease in humans and animals have been more appropriately presented elsewhere in this text (see Chapters 12 and 13).

II. HISTORYANDNOMENCLATURE The study of salmonellae began with Eberth's first recognition in 1880, and the subsequent isolation by Gaffky (see Ref. 5 ) , of the bacillus causing human typhoid fever (now known 247

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as Sdmonella (ser.) Qphi). Shortly thereafter, Salmon isolated the bacterium then thought, but later disproved, to be the etiological agent of hog cholera. The genus was later named SnlmoneZZn by Lignikres in 1900 in honor of D. E. Salmon (see Ref. 5). Other salmonellae were soon isolated, and it became common for each new isolate to be named by the medical and veterinary fields for the diseases they caused or the species of animal afflicted. For instance, names such as Salrnorzelln abortus ovis, Salrnonellll bovis rzrorbijicms, Scrlrnor~ellayullormr, and Sdrnonelln gallinarml were used by the veterinary profession to designate bacteria afflicting specific hosts (i.e., sheep, cattle, and poultry). Names such as Snlrnonella Qylzi, Sdrnonel/a vphosa, and Sulmorzella pcrrcroplzi were used by the human medical profession to designate bacteria associated with a specific disease. More recently, each new isolate was often named for the geographic location from which it was isolated (e.g., Salrno~zellayantrnzcr, Salr.zronellu saintpnul ). Historical accounts of some of the early isolations are available (6,7). The use of many of the above-mentioned names continues to this day, because it provides a practical and established nomenclature, although to do so incorrectly implies that each is a different species. Since 1987, all Snlnlonella within the genus were classified as a single species based on their close relatedness as determined by DNA hybridization (8). The species initially named Salmorrella cholemesuis by LeMinor (9) was later renamed Salmonella entericn in order to avoid confusion of the former with an already named serotype (8). Scdnzonelln erztericnis divided into six subspecies: erzterica, scrlavrae, nrizonae, dinri,-orzae, houtenae. and indica, with each ascribed formal taxonomic status by the WHO Committee (10,ll). A second species, S. bongori, has now been proposed by Reeves et al. (13), who, using multilocus enzyme electrophoresis, showed that S. erlterica ssp. bow gori is sufficiently different from the others to warrant such classification. Publication of this new genus and species combination in the Irzter~ratiorzalJounrnl qf Systerncttic Bacteriology (13) has made the name available for use in bacteriological nomenclature. Such publication, however, ascribes no taxonomic rank, and thus S. bongori has been slow to be accepted as a second species by some microbiologists. Contemporary nomenclature, which we shall use throughout the remainder of our text, now appropriately conserves traditional connotations associated with important disease-causing salmonellae simply by presenting the serotype in Roman rather than in italic typeface thus negating any implication of formal taxonomic status (10,14-15). For example, the serotype formerly written as SalnzonelZn choleraesuis is more appropriately written as S. enterica subspecies erztericcr serotype (ser.) Choleraesuis, or less cumbersomely, as Salrnonella (ser.) Choleraesuis, Sdn?onekr Choleraesuis, or Choleraesuis. This method applies only to serotypes within the subspecies entericn, of which greater than 99% of salmonellae isolated from humans belong; the remaining subspecies are not named but rather are designated by their antigenic formulas preceded by their subspecies designation ( 15).

111.

ISOLATION AND CULTIVATION

Salmonellae are mesophilic heterotrophs, needing only simple inorganic salts containing nitrogen, phosphorus, sulfur, a source of divalent cations, and an organic substrate for carbon and energy to sustain life (5,16). Wild-type salmonellae are fully capable of synthesizing all their biochemical machinery from the components of simple defined media corn-

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posed of ammonium sulfate, phosphate buffer, a source of magnesium, and sodium citrate or glucose (4). While salmonellae grow best at moderate temperatures (35-37"C), they can grow over a much wider temperature range, with growth occurring at temperatures as low as 4°C (17) and as high as 48°C (18). Growth is also best at moderate pH (6.57.5) in conditions of high water activity and low osmolarity (4). Extensive descriptions of the genus have been published (14,16,19), and these provide the ultimate criteria for identification of isolates. Scdrrrorzella are nonsporeforrning, oxidase-negative, faculative anaerobes. They do not produce indole, do not hydrolyze urea, and do not deaminate phenylalanine or trytophan. Most reduce nitrate to nitrite, and most ferment a variety of carbohydrates with concomitant production of acid. D-Glucose is fermented via the mixed acid pathway, thus yielding a positive methyl red reaction; acetyl methyl carbinol is not produced by Salmonella, and they therefore yield a negative Voges-Proskauer reaction. Most Salmonella are motile via means of peritricious flagella; however, Salmonella serotypes Gallinarum and Pullorum are notable exceptions (16). Other prominent characteristics of salmonellae are that most produce abundant hydrogen sulfide, most decarboxylate lysine, arginine, and ornithine, and, with few exceptions (notably those belonging to subspecies arizonae and diariconae),most do not ferment lactose. These latter characteristics have been the basis for the development of numerous selective and differential media for the culture and presumptive identification of salmonellae (20). Examples of such media include Rambach agar (RA) xylose lysine decarboxylase (XLD) agar, ScllnloneZln-Shigella (SS) agar, brilliant green agar (BGA), brilliant green sulfite (BGS) agar, Hektoen enteric (HE) agar, Macconkey's agar, lysine iron agar, triple sugar iron (TSI) agar, dulcitol lactose iron agar, dulcitol lactose sucrose agar, Kliger iron agar, Levine eosin-methyline blue (EMB), and desoxycholate citrate lactose agar (2023). Samples to be cultured for Sdrnonella often contain too few cells to allow detection via direct plating on the selective and differential agars. Thus, in order to increase the sensitivity for detection it is necessary to increase the number of cells via enrichment and perhaps even via a prior preenrichment (24). Preenrichrnent is a process in which the sample is first cultured in a nonselective growth medium such as buffered peptone water or tryptic soy broth with the intent of allowing the growth of any viable bacteria, whether SalmorleZZcr or not. Preenrichment often is useful in allowing injured cells to recover and increase in number and sometimes provides a way to dilute any potential growth suppressors (24). Because of the nonselective nature of preenrichment, it is often necessary to diminish the proportion of competing bacteria via enrichment. Enrichment, in contrast to preenrichment, is a process by which growth of a desired bacterial species is favored and (or) growth of undesired bacteria is inhibited (25). A very simple form of enrichment may be used for some organisms. For instance, Listeria monocytogenes can grow at 4°C while most other bacteria of medical importance cannot. Thus, the former can be enriched via growth at refrigeration temperatures (4). In the case of Salmorzelln, enrichment is usually achieved by culturing samples in media containing inhibitors to restrict the growth of undesired bacteria. Following the enrichment period, the enriched cultures are spread onto selective and differential agar plates, and then after appropriate incubation the plates are examined for the presence of colonies exhibiting morphologies typical of Salmonella (25,26). Confirmation of colony type is typically confirmed via biochemical tests and serological examination. Both of these confirmation procedures are relatively easy toperform and interpret. For instance, suspect colonies are inoculated into triple sugar iron agar, lysine iron agar,

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urease test broth, and indole nitrate medium. Additional tests such as the ability to ferment a variety of carbohydrates or to utilize citrate may be used to further distinguish serotypes of major importance such as Snlrnorlella Choleraesis, Schorzelln Enteritidis, Scrlrnonelln Paratyphi, and Snlrnorlellcr Typhi (14,16). Ultimately, the final determination relies on the ability of the organism to react immunologically against antisera raised against particular antigenic determinants expressed on the cell surface (see next section). Enrichment media commonly used to enrich Scdrnorzellcr include the tetrathionate broth medium of Muller (27), Muller-Kauffman tetrathionite brilliant green (28), Leifson’s selenite cystine broth (29), and the more recent but now widely preferred RappaportVassilidis broth (30-32). The latter medium uses both malachite green and magnesium chloride as selective agents and has been proven to be very useful and more sensitive than the Muller-Kauffmann tetrathionate brilliant green. An added advantage of the RappaportVassilidis medium is that it can be formulated both as a broth or a semisolid medium (33-38). Comparisons of various multistep isolation protocols describing the use various enrichment and plating media are available (20,39-46). Just recently, a new medium component, ferrioxamine-E, has been reported to substantially enhance the selectivity of enrichment media for recovery of salmonellae (47). Some Scrlrnonella do not exhibit the typical biochemical characteristics of the genus and these present special problems diagnostically because they are easily tnissed on comtnonly used differential plating media. For instance, about 1% of the many Salr~zorwlla serotypes submitted to the Centers for Disease Control (CDC) ferment lactose, and hydrogen sulfide production can be quite variable (4). Bismuth sulfite agar has been used to culture and detect biochemically atypical Snlnzordn, but its sensitivity to storage and minor variations in preparation make it difficult and unpopular to use (22). A selective and differential medium devised by Devenish et al. (22) has gained widespread acceptance by many laboratories because it allows for detection of many lactose-fermenting and non-lactose-fermenting Saltnorrelln. This medium contains brilliant green as a selective agent for Sulnlorzella, ferric ammonium citrate for hydrogen gas detecton, novobiocin as an antimicrobial agent active against coliforms and Proteus (but not Snlmorlellcr), and glucose (the only carbohydrate used by all Schorzellr) as the carbon and energy source. Unfortunately, Snlt.zlonellcr Typhi grows poorly on this novobiocinbrilliant green-glucose agar. Moreover, it is important to note that none of the selective and differential media presently available are 100% reliable and foolproof. Thus, when dealing with samples of critical importance, it is recommended that more than one plating media be used. When screening for Snlmorwllcl Typhi or Snlrrlorlellcr Paratyphi, it is imperative that bismuth sulfite agar or bismuth sulfite brilliant green agar be used (48). The processes of preenrichment, enrichment, isolation on selective differentiation agars, and biochemical confirmation normally take several days, with each step often requiring overnight incubation. Consequently, there is considerable interest in the development of more rapid methodologies, particularly for diagnostic purposes. Presently, a variety of conlbination media exist that simultaneously test for several phenotypic characteristics [e.g., o-nitrophenyl-P-D-galactopyranoside-urease-indole broth (49), L-pyrrolidonyl peptidase activity (50), and malonate-dulcite-lysine agar (5l)]. Moreover, rapid test strip systems such as API System S.A. (bioMerieux Vitek, Inc., Hazelwood, MO) (521, MICRO-ID (General Diagnostics, Morris Plains, NJ) (53-55), Minitek (BioQuest Division, Becton Dickinson and Co., Cockeysville, MD) (56), RapID onE (57), and automated systems (58,59)have also been developed. Numerous immunological (e.g., enzymelinked immunoassays, immunodiffusion, or immunomagnetic separation) and nucleic

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acid-based (e.g., PCR amplification, in situ hybridization) procedures for detecting Salmonella have now been developed and commercialized. We refrain from mentioning all of the numerous methods, but rather refer the reader to an excellent review (60) and a few comparative studies illustrating that some of these procedures are as sensitive or even more so than conventional culture methods (6 1-69). Nevertheless, bacteriological cultivation of specimens remains the most often used and most trusted detection method.

IV. SEROTYPING Following the initial isolations of numerous different salmonellae from a wide range of habitats, it soon became apparent that a method was needed to classify this "one great group" of organisms (see Ref. 25). Serological typing arose out of that necessity as a powerful tool for differentiating salmonellae and has proven extremely useful in the epidemiological tracking of various outbreaks. This procedure, which involves the grouping of bacteria based on immunological reactions (agglutinations) of antisera raised against specific antigens expressed on the bacterial surface, allows for the differentiation and classification of serotypes that otherwise may be indistinguishable by biochemical methods. Development of our modern-day scheme is credited primarily to the work of Kauffmann and White, although notable contributions were made by Schutze, Andrewes, Felix, Smith, and Reagh (see Ref. 25). Officially recognized by the World Health Organization, the Kauffmann-White diagnostic scheme involves the primary subdivision of the bacteria into serogroups and further delineation into serotypes according to the specific somatic (0)or flagellar (H) antigens expressed on their cell surface. There are currently more than 2300 Snlnlonella serotypes (lo), and by convention each is now denoted by an antigenic formula with the major 0 antigen listed first, then the phase 1 H antigen(s), and then the phase 2 H antigen(s). The phase 1 H antigens are designated by lowercase letters and the phase 2 H antigens by Arabic numerals or, in some cases, by components of the e or z series (25). For instance, Salmonella Choleraesuis is 6,7:c: 1,5. Many microbiologists still refer to some serogroups by their traditional letter designation, i.e., group C, for Salmonelkz Choleraesuis (25). Serological specificity of the 0 antigens, which are part of the cell wall lipopolysaccharide (LPS), is determined by the type and order of various carbohydrate moieties making up side chains and the chemical nature of terminal groups attached to a lipopolysaccharide core. Presently, 0 antigens are designated by Arabic numerals from 1 up to 67, although the series isnot continuous because some 0 antigens previously assigned to bacteria now known not to be Scrlnlorzella have been deleted from the scheme (25). A particular Salrzlo1zeIla isolate is grouped into one of 46 possible 0 serogroups depending on which major 0 antigen is expressed on the cell surface (1 1,25,70). These 46 serogroups can be arranged into 17 chemotypes based on the polysaccharide composition. Each chemotype contains lipopolysaccharide composed of the same five sugars, plus up to three additional sugars, although differences in cross-linking patterns between the sugars in various chemotypes accounts for the antigen variation. The Kauffmann-White typing system is capable of detecting minor differences in the polysaccharide constitution of the bacterial cell, and in the case that more than one 0 antigen is expressed, only one is considered major-the others are considered minor (25). The commercial availability of antisera raised against 0 antigens now makes serogrouping a task readily performed at practically any laboratory; however, it is important

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for those doing the tests to be aware of certain pitfalls. For instance, cross-agglutinations can occur between some serotypes that share certain minor 0 antigens (25). Moreover, the polysaccharide component of Snlnzonella lipopolysaccharide is variable and can be influenced upon acquisition of genetic elements (e.g., plasmids) (11,15,71). Phage conversion can also alter the sugar composition of the lipopolysaccharide core, thereby changing the serotype and virulence attributes of a particular salmonellae (72-76). Nnalue et al. (72,73) have shown that phage conversion by the lysogenic bacteriophage P14 increased the length of the 0-antigen polysaccharide chains and increased mouse virulence. Chart et al. (76) have shown that phage conversion can change the virulent Salmorzella Enteritidis phage type 4 into the less virulent phage type 7 and that the change involves an alteration in the lipopolysaccharide component of the cell envelope. Some typhoid strains of Salnzonella and some strains of Salnzonellu Dublin and Scrlmonella Paratyphi C possess a distinct capsular antigen, named Vi in reference to virulence, that renders the 0 antigen resistant to agglutination by the anti-0 antibody (77). Thus, an organism that expresses phenotypic characteristics of SaZnzonella may react negatively with polyvalent anti-0 antisera because of the masking effect of the Vi antigen. Under such circumstances, it is imperative that the organism be tested with antisera specific to the Vi antigen, and then, following a boiling procedure to remove the Vi capsular material (0 antigens, being lipopolysaccharide in nature, are heat-stable), the organism should be retested for the presence of the 0 antigen. Results from such testing will delineate whether or not the organism in question in indeed a Scdnzonelln. Further differentiation of bacteria into serotypes is much more complicated than serogrouping due to possible phase variations that may be encountered among many of the Salwzonella serotypes. These variations not only make interpretation of the serological tests difficult but make preparation of antisera specific enough to discriminate between the various H antigens laborious and wrought with possible pitfalls (25). Thus, serotyping is usually done at public health laboratories that possess the necessary materials, antisera, and trained personnel.

V.

BlOTYPlNG

While serotype is the primary characteristic by which salmonellae are classified, certain variations in phenotype within a given serotype may also be used to further differentiate one organism from another. Organisms expressing different phenotypes are considered a different biotype, and it can be that these differences can be associated with differences in virulence. For example, Salmorzelln Pullorum and Sahzonel/a Gallinarum are considered the same organism by some authors and are treated as such in Bergey 'S Mar1ual of Systematic Bacteriology (16), e.g., as Salnzorzellc~Gallinarum-Pullorum. Yet these can be differentiated based on biotype (78,79) and have historically been treated as different bacteria that cause different disease syndromes in chickens. Salmonella Pullorum possesses ornithine decarboxylase activity and produces gas from glucose, whereas Scrlmorzellu Gallinarum does not. Other differences also exist between these two bacteria, notably the propensity of Snln~orzellaPullorum to cause pullorum disease, which is a septicemia of embryos and chicks, whereas Salmonellrr Gallinarum causes "fowl typhoid," which affects adult chickens (80,81). Biotyping schemes have been been devised to help differentiate other Salnlorlella serotypes such as Salnzorzella Agona (82), Snlmonella Livingstone

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(83), Salmonella Montevideo (84), Salmonella Senftenburg (85) and Salmonella Typhimurium (86-88).

VI.

PHAGETYPING

Salrnonella serotypes can be differentiated according to their susceptibilities to lytic bacterial viruses (bacteriophage) via a process called phage typing. A phage-typing system for Vi antigen positive salmonellae was developed in 1938 by Craigie and Yen, and Felix and Callow later developed phage-typing systems for Sulmonellu Paratyphi B and for Salmonella Typhimurium (see Ref. 89). Phage-typing systems have since been developed for several other Salrlzorzella serotypes (90-99). Phage typing can be extremely useful for epidemiological investigation (89,99). By the mid-1950s there were 33 recognized Viphage types, and their usefulness as epidemiological tools was well established. Similarly, there were more than 30 distinguishable phage types of Salmonella Paratyphi B. Bacteriophages useful for typing are distributed by the World Health Organization via collaboration with the Central Public Health Laboratory at Colindale in the United Kingdom, and an international phage-typing system for tracking human Salmonella infections is underway in Europe ( 100). Phage typing has proven useful regarding distribution of salmonellae in the North American poultry industry. For instance, Poppe et al. (101) examined Canadian broiler flocks for Salmorzellc~Enteritidis and found the flocks infected with fifty different Salmonella serotypes. Of the nine flocks infected with Schzonella Enteritidis, phage type 8 was found in seven flocks while phage type 13a was found in two flocks. Scdmonella Enteritidis phage type 4 is of particular interest to the poultry industry because these organisms seem to be especially virulent for chicks and may findtheir way to newly hatched chicks through transovarian transmission (102). Unfortunately, Salmonella Enteritidis phage type 4 has become so prevalent in many areas that information obtained via phage typing is of limited use when trying to determine the source of this bacterium in examined samples (103). When dealing with phage typing, it is important to remember that phage conversions can occur naturally when a particular bacterium becomes infected with bacteriophage or acquires new plasmids. For instance, acquisition of incN incompatability group R-plasmids by Salmonella Enteriditis 4 results in a phage conversion of this bacterium to phage type 24 (104). Moreover, acquisition of lysogenic phage can alter mouse virulence (76).

VII.

MOLECULARTYPING

Numerous typing methods based on the analysis of genetic elements have now beendeveloped, and while commonly used to compliment traditional typing methods, these have not yet replaced serotyping as the method of choice for typing salmonellae (15). Deternlinations of the plasmid size and copy number within a particular serotype enables the construction of plasmid profiles that are useful for monitoring infection patterns and possible transmission routes (105,106). The sensitivity of the typing method may be increased by creating a “plasmid fingerprint’’ via cleavage of the plasmid with selected endonucleases ( l 07). Threlfall et al. (108) have observed nine plasmid profiles among a strain of Salmonella Enteritidis phage type 4. They found sufficient variation in plasmid profiles among 13 other phage types to conclude that plasmid profiles are ‘an effective adjunct

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to phage typing for the subdivision of Sdmonelln Enteritidis" (108). Similarly, Wachsmuth et al. (109) found 16 distinct plasmid profiles among two phage types of Sulmollellcr Enteritidis, Colindale 8, and 13a. Plasmid analysis has been used in the epidemiological investigation of an instance of Scrhzone~/ncontaminated chocolate (1 10). in tracing an outbreak of salmonellosis to contaminated marijuana (11l), and to confirm that a single strain was responsible for recurrent infection in an immunocompromised patient (1 12). Plasmids are not always useful as epidemiological tools since not all strains contain plasmids. In one study of sahnorzella Typhi isolates obtained during a typhoid outbreak in Chile and Pent, only 17 of 141 isolates contained plasmids, and the plasmid profiles were not useful for subdividing the major Vi-phage groups (99). Thirteen of17 isolates contained identical plasmids suggesting a common origin among the 13 isolates (99), but the absence of plasmids among the other 124 isolates is clear evidence that plasmids are not required for virulence by organisms in this particular serotype and that virulence by Sdnlomdln Typhi is determined by the chromosomal genes. It thus seems likely that by being highly adapted to human beings, Scrlmolwlln Typhi has retained virulence genes for humans but lost genes needed for a wider host range. Plasmid analysis has also proven useful in elucidating the role of plasmids in virulence of other Salmonella serotypes (113). Helmuth and colleagues (114) reported that 90% of 337 isolates originating from 29 different countries contained serotype-specific plasmids. Of the seven serotypes represented in this study, four (Typhimurium, Dublin, Enteritidis, and Choleraesuis) contained plasmids that were associated with a lower mouse virulence (approximately 1 million-fold) as interpreted from LDS,,(lethal dose 50%) values. Plasmid contents were found to be unrelated to virulence in the other three serotypes (Infantis, Panama, and Heidelberg) (1 14). It has been proposed that the virulence plasmids of Salrl?onella Typhimurium and Dublin plus large plasmids (54 and 76-kbp) contained by Snlnlorlella Abortusovis, Salrnorzelln Enteritidis, and Snlrtlordlcr Paratyphi C comprise a family of related virulence plasmids (1 15). These plasmids were absent in strains of the following serotypes: Agona. Bovismorbificans, Heidelberg, Infantis, Panama, Paratyphi A, Paratyphi B, Saintpaul, Seftenberg, and Typhi. Moreover, virulence plasmids of Srrlmorzella Choleraesuis, Scrlmonella Dublin, and Scrlsnorwlln Enteritidis and the cryptic plasmids of Sdnlorwllcr Copenhagen and Sdmorzella Sendai are thought to belong to the same incompatibility grouping (1 16,117). Sulmorzella Typhimurium also contains high molecular weight plasmids (8590 kbp),which play a role in extraintestinal dissemination and replication of the pathogen within the tissues of mice (118). Similar large plasmids have been found necessary for the virulence of Salmonella Gallinanlm-Pullorum strains. Sabnolzella Typhimurium cells cured of their plasmids, thereby rendered avirulent in the mouse virulence assay, regain virulence by reintroduction of the virulence plasmid from Scrlrnorlella GallinarumPullorum ( 119). Beninger et al. (120) have shown that plasmids of different size and endonuclease restriction patterns found in Snlrnorlellrr Enteritidis and Scdnlolwllcr Choleraesuis often share a common 4 kb Eco-R1 restriction fragment with the 80 kbp virulence plasmid of Scdnzonella Dublin, pSDL2. Plasmid pSDL2. is required for the development of a lethal systemic infection in the mouse virulence test, and portions of pSDL2 are homologous to a virulence plasmid, pIB 1, of Yensillin species (121). Mouse virulence studies combined with molecular genetics studies of 22 common serotypes have demonstrated the essential role of plasmids containing the 4 kbp Eco RI restriction fragment for development of systemic infection in mice (121). Naturally occurring strains of the serotypes most often

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isolated from nontyphoid human systemic salmonellosis, serotypes Choleraesuis, Dublin, and Enteritidis, typically carry the virulence plasmids. It is possible that the 4 kbp region of the virulence plasmid is partly responsible for the ability of these serotypes to cause human systemic infections. However, virulence plasmids will not confer mouse virulence on all Sulmorzelka serotypes, e.g., Salnrorzellu Typhi and Paratyphi A, and this fact suggests that virulence also requires chromosomal determinant(s) (122). We are now beginning to understand the mechanisms by which plasmids operate to confer virulence. In some cases, they encode for outer membrane proteins that are necessary for replication within the host tissues (123-125). Plasmids may also be responsible for a reduced host immune response (126). Molecular analysis of chromosomal DNA sequences can also be usedto differentiate Salmonellcl; however, depending on the sequence analyzed and the method employed, the discriminatory power of these methods may be lacking (103,127). Thus, analysis of chromosomal DNA sequences are often performed in conjunction with another typing technique. Some of the DNA regions targeted include portions of the 16s rRNA gene (128,129). thefEiC flagella gene (130), virulence genes (1 3 1,132), and insertion sequences (133,134). Protocols involving polymerase chain reaction (PCR) amplification of selected nucleotide sequences have been developed to increase the sensitivity of these molecular techniques, but at present these often employ preenrichment cultural procedures of varing duration. Fingerprinting protocols involving electrophoretic separation of DNA fragments produced via digestion of chromosomal DNA with endonucleases (referred to as restriction fragment length polymorphism, RFLP) and(or) via PCR amplification have proven useful as tools for differentiating various Sall~orzelluserotypes that are not particularly amendable to phage or plasmid typing ( 103,135- 137). Use of pulsed field gel electrophoresis has been used to resolve fragments generated from the genome, and this enhanced the power to discriminate strains within serotypes (138,139) or phage types (140,141).

VIII. CONCLUSIONS Sdr~~onella are important pathogens that cause disease in humans and animals. Ubiquitous in nature, a few serotypes exhibiting host specificity tend to infect only their respective hosts, while many others exhibit broad host range specificity. Techniques, including nucleic acid and immunologically based methods, have been developed to detect and/or isolate Sullnonella from various specimens, but bacteriological cultivation remains the most used method. Serotype, biotype, and phage type are all determinants of host-range and virulence. Individual isolates vary in virulence, biochemistry, susceptibility to bacteriophage, and plasmid content. As alluded to previously, acquisition of additional genetic elements such as lysogenic phage (temperate phage or prophage) and extrachromosomal elements (plasmids) by a particular bacterium can bring about changes in phage type, serotype, and virulence.

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81. Snoeyenbos G. H. (1972). Pullorun~disease. In Diseases of Poultq, 6th ed. (M. S. Hofstad, B. W. Calnek, C. F. Helmboldt, W. M. Reid, and H. W.Yoder Jr., eds.), Iowa State University Press, Ames, pp. 83-114. 82. Barker, R., and D. C. Old (1982). Differential typing of Sahnonella agona: type divergence in a new serotype. J. Hyg. Camb., 88:413-423. 83. Odongo, M. O., McLaren, I. M., Smith, J. E., and Wray, C. (1990). A biotyping scheme for Salmonella livingstone. Br. Vet. J., 146:75-79. 84. Old, D. C., Munro, D. S., Reilly, W. J., and Sharp, J. C. M. (1985). Biotype discrimination of Salr~onellamontevideo. Lett. Appl. Microbiol., 1:67-69. 85. Tuchili, L. M., McLaren, I. M., Smith, J. E., and Wray, C. (1991). Differentiation of Salmonella senftenberg into biogroups. Vet Rec., 129530-531. 86. Anderson, E. S., Ward, L. R., de Saxe, M. J., Old, D. C., Barker, R., and Duguid, J. P. (1978). Correlation of phage type, biotype and source in strains of Salmorzella typlzimuriunz. J. Hyg. Camb., 81:203-217. 87. Brandis, H. (1970). Die Lysotypic von Salmonellen der Enteritisgruppe mit besonderer Beriicksichtigung von S. typltinrurinm. Zentralbl. Brrberiol., 222232-244. 88. Duguid, J. P., Anderson, E. S., Alfredsson, G. A., Barker, R., and Old, D. C. (1975). A new biotyping scheme for Salmonella typlzimul-ium and its phylogenetic significance. J. Med. Micl-obiol., 8:149-166. 89. Anderson, E. S., and Williams, R. E. 0. (1956). Bacteriophage typing of enteric pathogens and staphylococci and its use in epidemiology. J. Clin. Patlzol., 9:94-127. 90. Jayasheela. M., Singh, G., Sharma, N. C., and Saxena, S. N. (1 987). A new scheme for phage typing Salmonellae bareilly and characterization of typing phages. J. Appl. Bacterid., 62: 429-432. 91. Laszlo, V. G., and Csorian, E. S. (1988). Subdivision of common salmonella serotypes: phage typing of S. virchow, S. mawhattan, S. tlzonlpson, S. oranienburg, and S. bareilly. Acta Microbial. Hung., 35:289-294. 92. Vieu, J. F., Jeanjean, S., Tournier, B., and et Klein, B. (1990). Application d’une sirie unique de bactiriphages i la lysotypic de Sah?lorzel/askrovar Dublin et de Salmonella sirovar Enteritidis. Mbd. Malad. Iitfect., 20:229-233. 93. Ward, L. R., de Sa, J. D. H., and Rowe, B. (1987). A phage-typing scheme for Salmonella enteritidis. Epidemiol. Itgect., 99:29 1-294. 94. Hicknlan-Brenner. F. W., Stubbs, A. D., and Fanner 111, J. J. (1991). Phage typing of Salmonella enteritidis in the United States. J. Clin. Microbiol., 29:2817-2823. 95. Lilleengen, K. (195 1). Typing of Salmonella gallinarum and Salmonella pullor~mby means of bacteriophage. Acta Path. Microbiol. Scand., 30: 194-202. 96. de Sa, J. D.H., Ward. L. R., and Rowe, B. (1980). A scheme for phage typing of Salntonella hadar. FEMS Microbiol. Lett., 9:175-177. 97. Gershman, M. (1977). A phage typing system for Salrnonella heidelberg. J. Food. Prot., 40: 43-44. 98. Laszlo, V. G., Csak, K., and Csorian, E. S. (1988). A phage typing system for Salntonella infantis. Acta Microbiol. Hung., 3555-69. 99. Maher, K. O., Morris Jr., J. G., Gotuzzo, E., Ferreccio, C., Ward, L. R., Benavente, L., Black, R. E., Rowe, B., and Levine. M. M. (1986). Molecular techniques in the study of Salmonella typlti in epidemiologic studies in endemic areas: comparison with Vi phage typing. Am. J. Trop. Med. Hyg., 35:831-835. 100. Killalea, D., Ward, L. R., Roberts, D., de Louvois, J., Sufi, F., Stuart. J. M., Wall, P. G., Susman, M., Schwieger, M., Sanderson, P. J., Fisher, I. S. T., Mead, P. S., Gill, 0. N., Bartlett, C. L. R., and Rowe, B. (1996): International epidemiological and microbiological study of outbreak of Salmonella agona infection from a ready to eat savoury snack-I: England and Wales and the United States. Br. Med. J., 313:1105-1107.

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101. Poppe, C., Irwin, R. J., Messier, S., Finley, G. G., and Oggel, J. (1991). The prevalence of Snln~onellr~ enteritidis and other Salntonella spp. among Canadian registered commercial chicken broiler flocks. Epidentiol. Infect., 107:201-211. 102. O’Brien, J. D. P. (1988). Salmonella enteritidis infection in broiler chickens. Vet. Rec., 122: 214. 103. Olsen, J. E., Brown, D. J., Skov, M. N., and Christensen, J. P. (1993). Bacterial typing methods suitable for epidemiological analysis. Applications in investigations of salmonellosis among livestock. Vet. Q., 15:125-135. 104. Frost, J. A., Ward, L. R., and Rowe, B. (1989). Acquisition of a drug resistance plasmid converts Salnlonella enteritidis phage type 4 to phage type 24. Epiderniol. Infect., 103:243238. 105. Threlfall, E. J., and Frost, J. A, (1990). The identification, typing and fingerprinting of Salmonella: laboratory aspects and epidemiological applications. J. Appl. Bacteriol., 685-1 6. 106. Platt, D. J., Brown, D. J., Old, D. C.. Barker, R. M., Munro, D. S.. and Taylor, J. (1987). Old and new techniques together resolve a problem of infection by Salntonella tyl,’l?imurium. Epidemiol. Iilfect., 99:137-142. 107. Platt, D. J., Chesharn, J. S.. Brown, D. J., Kraft, C. A., and Taggart, J. (1986). Restriction enzyme fingerprinting of enterobacterial plasmids: a simple strategy with wide application. J. Hyg. Carnb., 97:205-210. 108. Threlfall, E. J., Rowe. B., and Ward, L. R. (1989). Subdivision of Salmonella enteritidis phage types by plasmid profile typing. Epidemiol. Itlfect., 102:459-465. 109. Wachsmuth, I. K., Kiehlbauch, J. A., Bopp, C. A., Cameron, D. N., Strockbine. N. A., Wells, J. G., and Blake, P. A. (1991). The use of plasmid profiles and nucleic acid probes in epidemiologic investigations of foodborne. diarrheal diseases. Int. J. Food Microbiol., 1277-90. 110. Kapperud, G., Lassen, J., Dommarsnes, K., Kristiansen, B. E., Caugant, D. A., Ask, E., and Jahkola. M. (1989). Comparison of epidemiological marker methods for identification of Salrnonellu t?.lyhinzuriunrisolates from an outbreak caused by contaminated chocolate. J. Clin. Microbiol., 272019-2024. 111. Taylor, D. N., Wachsmuth, I. K., Shangkuan. Y.-H., Schmidt, E. V., Barrett, T. J., Schrader, J. S., Scherach. C. S., McGee, H. B., Feldtnan, R. A., and Brenner, D. J. (1982). Salmonellosis associated with marijuana: A multistate outbreak traced by plasmid fingerprinting. N. Engl. J. Med., 306:1249-1253. 112. Mayer, K. H., and Hanson, E. (1986). Recurrent Salrnonellct infection with a single strain in the acquired immunodeficiency syndrome. Confirnlation by plasmid fingerprinting. Dingn. Microbiol. Infect. Dis., 4:71-76. typhimurittm and other salmonellae. 113, Gulig, P. A. (1990). Virulence plasmids of Sul~~onella Microb. Pathog., 8:3-11. 114. Helmuth. R., Stephan, R., Bunge, C., Hoog, B., Steinbeck, A., and Bulling, E. (1985). Epidemiology of virulence-associated plasmids and outer membrane protein patterns within seven conmon Salmonella serotypes. Infect. Immun., 48: 175-1 82. 115. Popoff, M. Y., Miras, I.. Coynault, C., Lasselin, C., and Pardon, P. (1984). Molecular relationships between virulence plasmids of Salmonella serotypes typlzirnuriunz and dublin and large plasmids of other Salmonella serotypes. Anrl. Microbiol. (Paris), 135A: 389-398. 116. Ou. J. T., Baron, L. S., Dai. X., and Life, C. A. (1990). The virulence plasmids of Salmonelln serovars typlzirnurizun,choleraesuis, dublirt, and enteritidis, and the cryptic plasmids of Salmonellt serovars copenhaget1 and sendai belong to the same incompatibility group, but not those of Salmonella serovars hcrban, gallinarurn, give, irlfantis and pullorunt. Microb. Pathog., 8:lOl-107. 117. Ou, J. T., and Baron, L. S. (1991). Strain differences in expression of virulence by the 90 kilobase pair virulence plasmid of Salmonella serovar Typhimurium. Microb. Pathog., 10: 247-25 1.

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118. Caldwell, A. L., and Gulig, P. A. (1991). The Salmonella t y h i n r w i t m virulence plasmid encodes a positive regulator of a plasmid-encoded virulence gene. J. Bacteriol.. 173:71767185. 119. Barrow, P. A.,andLovell, M. A. (1989). Functionalhomologyofvirulenceplasmidsin Srrllnonella gnllinasrcnr, S. pullort~nz.and S. ~phinturircnr.Irlfect. Imrnun.. 57:3 136-3 141. 120. Beninger, P. R., Chikami, G., Tanabe, K., Roudier, C., Fierer, J., and Guiney, D. G. (1988). Physical and genetic mapping of the Sultnonelln dzdblin virulence plasmid pSDL2. Relationship to plasmids from other Salr~ronellastrains. J. Clin. h e s t . , 81:l341-1347. 121. Krause, M., Harwood, J., Fierer, J., and Guiney, D. (1991). Genetic analysis of homology between the virulence plasmidsof Salnzonelln dublin and Yersinia pseudotuberculosis. Illfect. Imnun., 59:1860-1863. 122. Roudier, C., Krause, M., Fierer, J., and Guiney, D. G. (1990).Correlation between the presence of sequences homologous to the vir- region of Strlmonelln dublin plasmid pSDL2 and the virulence of twenty-two Saltnonelln serotypes in mice. Zrfect. Zmrnm., 58:1180-1185. 123. Taira, S., Riikonen, P., Saarilahti, H., Sukupolvi, S., and Rhen, M. (1991). The mkaC virulence gene of the Scrllnonellrr serovar Typhimurium 96 kb plasmid encodes a transcriptional activator. Mol. Gen. Genet.. 228:381-384. 124. Taira, S.. Baumann, M., Riikonen. P., Sukupolvi, S., and Rhen. M. (1991). Amino-terminal sequence analysis offour plasmid-encoded virulence-associated proteins ofSalmonella 8phinruriunz. FEMS Microbiol. Lett., 77:319-323. 125. Valone, S. E., and Chikami, G. K. (1991). Characterization of three proteins expressed from thevirulenceregion of plasmidpSDL2in Sal~no~~ellc~ dzcblin. hfect. I r n m m . , 59:35113517. 126. Hoertt, B. E., Ou. J., Kopecko, D. J., Baron, L. S., and Warren, R. L. (1989).Novel virulence properties of theSnblrorzelln typlzimwium virulence-associated plasmid: immune suppression and stimulation of splenomegaly. Plmnid, 21 :48-58. 127. Threlfall, E. J., Powell, N. G.,and Rowe, B. (1994). Differentiation of salmonellas by molecular methods. PHLS Micr-obiol. Dig.. 11:199-202. 128. Esteban, E., Snipes. K.. Hird, D., Kasten, R., and Kinde, H. (1993). Use of ribotyping for characterization of Scrlnronella serotypes. J. Clin. Microbiol., 3 1233-237. of 129. Nastasi, A., and Mammina, C. (1995). Epidemiological evaluation by PCR ribotyping sporadic and outbreak-associated strains of Suhnonella entericn serotype Typhimurium. Res. Microbiol., 146:99-106. 130. Kilger, G., and Grimont, P. A. D. (1993). Differentiation of Salmonella phase I flagellar antigen types by restriction of the amplified p i c gene. J. Clin. Microbiol., 31 : l 108-1 110. 131. Rexach, L., Dilasser, F., and Fach, P. (19931. Polymerase chain reactionfor Snlrnonellrr virulence-associated plasmid genes detection: a new tool in salmonella epidemiology. Epidenziol. Infect., 112:33-43. 132. Rhan, K., De Gandis, S. A., and Clarke, R.C. (1992). Amplification of an invA gene sequence of Srrlnlorzellcr Dphirnuriunz by polymerase chain reaction as a specific method of detection of salmonella. Mol. Cell Probes, 6:271-279. 133. Soria, G., Barbe, J., and Gibert, I. (1994). Molecular fingerprinting of Salnronelln typphinzuriurn by IS200-typing as a tool for epidemiological and evolutionary studies.Il4icrobiologia, 10:57-68. 133. Stanley, J., and Saunders, N. (1996). DNA insertion sequences and the molecular epidemiology of Salmonella and Mycobncteriurn. J. Med. Microbiol.. 35236-25 1. 135. Tompkins. L. S., Troup. N., Labigne-Roussel, A., and Cohen. M. I. (1986). Cloned, random chromosomal sequences as probes to identify Snlrnonella species. J. Infect. Dis., 154:156162. 136. Lin. A. W., Usera, M. A., Barrett, T. J., and Goldsby, R. A. (1996). Application of random amplified polymorphic DNA analysisto differentiate strains ofSnlrnonellrr enteritidis. J. Clin. Microbiol., 34:870-876.

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137. Olsen, J. E., Skov, M. N., Threlfall. E. J., and Brown. D. J. (1994). Clonal lines of Salnronellu entericn serotype Enteritidis documented by TS200-, ribo-. pulsed-field gel electrophoresis and RFLP typing. J. Med. Microbiol., 40: 15-22. 138. Suzuki, Y., Ishihara, M.. Matsumoto, M., Arakawa, S., Saito, M., Ishikawa, N., and Yokochi, T. (1995).Molecular epidemiology ofSnlmo~~ellu ewteritidis. An outbreak and sporadic cases J. Zrzfect., 31211-217. studied by means of pulse-field gel electrophoresis. 139. Thong, K.-L., Cheong, Y.-M., Puthucheary, S., Koh, C.-L., and Pang. T. (1994). Epidenliologic analysis of sporadicSulnlonelln hphi isolates and those from outbreaks by pulsed-field gel electrophoresis. J. Clill. Microbiol., 32:1135-1141. 130. Powell, N.G., ThreIfall, E. J., Chart, H., and Rowe, B. (1994). Subdivlsion of Sal~no~ellrr enteritidis PT 4 by pulsed-field gel electrophoresis; potential for epidetniological surveillance. FEMS Microbiol. Lett., 119: 193-198. 141. Powell, N. G., Threlfall, E. J., Chart, H.. Schofield. S. L., and Rowe, B. (1995). Correlation of change in phage type with pulsed field profile and 16s rrn profile in Solmorzellu enteritidis phage type 4, 7 and 9A. Epidemiol. Infect., 114:403-411.

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12 Salmonellosis in Animals David J. Nisbet and Richard L. Ziprin U S . Department of Agricrrlture, College Stutiorr, Texas

I. Sulmonelln in Companion Animals 265 11. Salnzorudlu in Cattle and Dairy Cows 267 111. Subuonella in Poultry 269 IV. Salmonella in Swine 274 References 275

Salmonella bacteria can colonize or infect a wide variety of domestic animals, both companion and agricultural animals. When colonized animals are asymptomatic, they may become an insidious source of inoculum for foods and, ultimately, humans. Rodents, pests, and reptiles may act asreservoirs and vectors for the transmission of Salmonellct (1). Insect pests have also been incritninated as Salmonella carriers (2). Symptomatic salmonellosis in animals, as in humans, can be a relatively benign illness with only minor gastrointestinal symptoms, or it can bea severe and life-threatening illness, Each animal species responds differently to specific serotypes, and therefore the nature of salmonellosis varies with the species infected, the infecting serotype, and the general condition of the infected animal, i.e., age and overall health status.

1.

SALMONELLA IN COMPANIONANIMALS

Salmonellosis has been observed in common pets such as cats (3- lo>and dogs (1 1-17) and in less common companion animals such as the horse (18-30). Salmonellosis in cats, dogs, and horses is particularly instructive because the full range of disease variability is readily seen in these animals. The percentage of asymptomatic Salmonella carriers among cats has been variously estimated at between 1% and 18% (3,4,6). Serotypes found in cats include Salmonella Derby, Salnzonella Typhimurium, SulrnoneZla Anatum, Salmonella Enteritidis, and Salmonella Newport. While SaZmoneZZn-infected cats are usual asymptomatic caniers, disease symptoms of salmonellosis in cats can include conjunctivitis and bacteremia. Feline salmonellosis may also present as a chronic febrile illness resembling human enteric fever (typhoid fever). 265

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The incidence of asymptomatic carriage in dogs has been estimated at between 0.6 and 27.6% of randomly sampled animals. depending on the study population. Fifty-three serotypes were found in one study of 8000 dogs in Florida (1 3).In 1996 Alaskan sled dogs participating in the Iditarod Trail Sled Dog Race were sampled for the presence of salmonellae. Salnnonelln was isolated from 18 of 26 (69%)normal asymptomatic dogs prior to the race during routine prerace veterinary inspection. Of these isolates 13 were Saltnorzella Typhimurium or Snlrnorlelln Typhimurium (Copenhagen); other serotypes isolated were Sulmorzella Infantis and Salrnolzella Reading. Two dogs were infected with both Salmonella Typhimuriunl (Copenhagen) and S c h o l d l n Infantis. During the race 30 dogs with diarrhea were sampled, 19 (63%) of which were Snlmorzelln positive: additionally 23 nondiarrahetic dogs were sampled, of which 13 (57%) were Salnzonelln positive. In this study no difference in the incidence of Salmonella was found between the prerace dogs and dogs sampled during the race (17).However a surprisingly high incidence of Salmonella in these Alaskan sled dogs was found compared to the prevalence reported in other dogs. Salmonellosis is also common in racing greyhounds. A study performed in 1993 found Snlnlonella in 11% of asymptomatic greyhounds and 76% of diarrhetic greyhounds (15).In that study a high prevalence of Snlnzor?ella was also found in the dogs' food that consisted of raw meat obtained from rendering plants (16).The increased prevalence of Scrlnzonella found in these working dogs may be associated with not only feed but also the stresses associated with rigorous training. The serotypes commonly isolated from dogs include those commonly isolated from humans. S a Z ~ ~ ~ o ~Typhimurium ~elln was isolated from 40% of the Scrlr.llonell~~-positive dogs. Salmonellu Enteritidis, Sdnlonelln Infantis, and Snlmor~ellnParatyphi B have all been transmitted to humans through an association with illdogs. Interestingly, Salmonella are not usually found in dog biscuits, dog candy, flakes, or kibbled products because these are heat-processed pelleted or extruded foods and the heat and pressure are sufficient to kill the bacteria. Salmonellosis in symptomatic dogs is similar to the disease in other mammals-fever, malaise, diarrhea, or abortion following uterine infection. In severe cases, symptoms may include hemorrhagic gastroenteritis and ultimately death (5,l 1,12,14). Salmonella Ohio has been observed to survive in dried canine fecal matter (1 1.6% moisture content) for at least 3 months (14). Similarly, horses may be asymptomatic carriers of Sulrnomlln. or they may experience clinical disease with dialrhea lasting 5-10 days. Fever may reach extretnes in instances of necrotizing colitis (106"F),and the infection may become a fatal septicemia. Young animals, under 4 months of age, may face a mortality rate as high as 50%. Snlmonella Abortus-Equi is a cause of abortion in pregnant mares. When the foals of infected mares are live-born, they commonly die within a few hours. When they do survive they may develop a septic arthritis (18).Although often reported in Asia and Africa, until recently Salrnonelln Abortus-Equi was no longer isolated from Europe and the United States (31).However, a recent outbreak was reported in Croatia in a herd of 38 horses, of which 26 were pregnant mares. In this outbreak 21 mares aborted suddenly and Salmonelln Abortus-Equi was isolated as the single causative agent in each case (32). Studies of the prevalence of salmonellae in asymptomatic horses have suggested carriage rates in the 0.2-15% range (33,34),and Salnzonelln Agona, which in 1975 was the fifth most frequently isolated serotype from humans, is commonly found in horses (1 1,3536). However, the incidence of asymptomatic carriage may be much higher. While the 0.2-15% rate is based on fecal or cecal cultures, enrichment culture of mesenteric lymph nodes from healthy horses demonstrated that 35 of 50 horses examined had asymptomatic SalnlonelZa infections (37).Fifteen different serotypes were recovered from the

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35 infected horses. These serotypes included the common serotypes found in human infections: Sctl~rrorzellc~ Reading, Scrlmonellcr Cerro, and Scdmolzelln Munchen (37). This high incidence of recovery of Salrzlor~ellerfrom lymph nodes establishes the status of horses as true carriers. It is well known among veterinary clinicians that acute clinical equine salmonellosis tends to occur when the animals are stressed. Smith et al. (34) list shipping, antibiotics, anthelminthic drugs, surgery, and prednisone as stresses associated with equine salmonellosis. The mechanisms by which stress causes asymptomatic carriage to transform into a clinical illness are largely unknown. Antibiotic treatments may simply allow the development of a large intraluminal Schnonelln population as a consequence of disruption of the normal intestinal microflora. Stresses such as shipping and veterinary treatment with immunosuppressive doses of corticosteroids undoubtedly impact in a negative wayon imnlunological defense mechanisms (38). Hospitalized horses are also at risk of becoming infected with Snlmorzellcr. A case-control study involving nosocomial cases was performed during an 1 l-yearperiod at the Veterinary Medical Teaching Hospital, University of California, Davis. This study revealed that horses in which nasogastric tubes were passed had a 2.9-fold greater risk of having Snbuolzellc1 isolated, compared with horses that did not undergo this procedure. Horses treated with antibiotics parenterally had a 6.4-fold greater risk, and those treated with antibiotics orally and parenterally had a 40.4-fold greater risk of developing salmonellosis, compared to horses not receiving such treatment. Additionally, horses admitted because of colic were 4.2 times more likely to have Snlmorzellcr isolated as those admitted for other reasons. Breed, age, and type of surgery did not appear to be risk factors (39). Recently the Large Animal Veterinary Teaching Hospital at Colorado State University was temporarily closed due to possible nosocomial infections of animals with Sabnomdla Infantis. The original source of the organism responsible for the outbreak was never determined, however, possible sources included hospital patients, hospital personnel, pigs housed in the facility used for teaching and research, wildlife (rodents or birds), and feed used for large animal patients (40). During the past several years there have been several reports of Salmonella outbreaks in veterinary teaching hospitals (33-35). Horses in such facilities may be particularly susceptible to Snlmonellcr infections due to factors such as long-distance transportation, dietary changes, anesthesia, surgery, concurrent disease, and treatment with antimicrobials (36-38). It is unfortunate that the horse is an expensive animal and is generally not well suited to use as a laboratory animal, as it might otherwise be a very useful model for studying the effects of stress on resistance mechanisms.

II. SALMONELLA IN CATTLEANDDAIRYCOWS Salmonellosis wasfirst observed in cattle as early as 1902, but infection of cattle by Snlrnolzelln had been considered rare as recently as the early 1960s (41,42). At that time a 5-year study at Cornel1 University of 731 Scdmorlelln Typhimurium cultures isolated from animal sources revealed that only 2 isolates originated in cattle (42). According to Jensen and Mackey (42), one study of 7365 salmonellosis outbreaks revealed that only 39 of these involved cattle. In 1965, a study of salmonellosis on Michigan farms revealed 39 outbreaks during a 20-month period with a 23% mortality rate among calves (43). Bovine salmonellosis is now considered an economically important disease to producers and continues to increase as a public health problem (44-52). Snlrno?zellnin dairy cattle

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is also of particular importance due to both the economic losses in animal production and because both milk and meat from infected cattle can pose serious public health risks. Salmonella infections of cattle range in severity from asymptomatic carriage to fatal bacteremia. Young animals have high mortality rates, but salmonellosis can be fatal in adult animals as well (53). The most frequently identified serotypes recovered from cattle are Scrlnrorlella Typhimurium, Salmonella Dublin, and Salmonella Typhimurium (Copenhagen) (50,5 1,54-57). Additionally, Salrnorzellcr Enteritidis (58) and Sulrnorlelln Muenster have also been isolated in milk and traced to cows with subclinical mammary infections (59). Corrier et al. (60) collected fecal samples from a herd of 200 feeder-calves and determined the prevalence of Snlrnonella carriage and the serotypes of the isolates. They monitored the herd from assembly at an auction market through the shipping process and at the feed-lot. Salmonellae were not isolated from samples collected at the farm or the auction market, but Salmonella-positive samples were found at the feed-yard. After 30 days at the feed-lot approximately 8% of the calves were infected, and the authors suggested that stressors immediately prior to arrival at the feedlot or during feedlot confinement may be contributing factors to the increase in fecal excretion of Salmonella. Serotypes recovered included Sabtzouella Anatum, Salnzonella Cerro, Saltnonellcl Newbrunswick, Salmor1ella Typhimurium (Copenhagen), and Subnonella Reading. Saltnorzellu Dublin was not isolated during this study. Salnlorlella Dublin is of particular concern. It causes septicemia in calves and abortion in adult dairy cattle. The feces of infected animals that have survived their infection may become persistently contaminated with Salrnorzella (61). Asymptomatic carriers also shed Scrlmonella Dublin in their milk. Concentrations of the organism in raw milk may approach 100,000 cells/mL, and epidemics of human salmonellosis have been traced to consumption of contaminated unpasteurized milk and dairy products (62,63). Salnzonella Dublin has a reputation for being very virulent to humans. It tends to infect older persons and patients suffering from chronic debilitating illness. Bacteremia has been observed in 75% of infected individuals, and mortality rates as high as 35% have been observed (62). In one case, an otherwise healthy 27-year-old, having sustained a minor abdominal injury during a fight, suffered a splenic rupture due to salnlonellosis caused by Salmorlella Dublin. The patient had consumed raw milk 12 days before his injury and had not had any gastroenterological symptoms or other significant symptoms of salmonellosis prior to his injury (64). Because the Dublin serotype is of special concern to public health and because there is a need to identify asymptomatic carrier cattle, considerable effort has gone into the development of immunological methods for determining the carrier status of individual animals. Wray and Callow (65) have developed a fluorescent antibody method, while Smith and colleagues (66,67) have worked to develop an enzyme-linked immunoassay (ELISA). The principle of the test is based on the fact that infected cattle develop antibodies against salmonellae, and these antibodies, which are present in milk, will react with Salmonella Dublin antigens bound to the solid surface of the microtiter plates. The bound antibodies are detected with horseradish peroxidase conjugated goat anti-bovine antisera (66,67). ELISA has been used to measure specific IgG titers in cattle and has proven useful for assessing vaccine responses, infection rates, carrier status, and epidemiology of bovine salmonellosis (68). Cattle often persistently shed Salmolzella Dublin in their milk, suggesting the presence of chronic mastitis. It has recently been discovered that bovine mastitis is accompanied by changes in the serum concentration of an acute phase protein, haptoglobin, which

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Animals

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is known to increase during mastitis (69,70). To these authors’ knowledge, haptoglobin measurements have not been used to identify possible carrier cows. There have been several efforts to immunize dairy cattle against Dublin. Besides reducing morbidity in cattle, the goal is to protect human health by reducing the incidence of Dublin-infected animals and hence Dublin-infected dairy products (7 1-79). Reported results have been mixed and inconclusive, but there have been successes. For example, Segall(71) reported that a vaccine strain of S. Dublin protected against the Dublin serovar but not against challenge with Typhimurium.

111.

SALMONELLA IN POULTRY

There is an extremely extensive literature on avian salmonellosis, and much of this literature has been compiled and interpreted in veterinary texts (80,81). Pullorum disease and fowl typhoid are acute lethal diseases of chickens caused by Salmonella GallinarumPullorum. While these diseases are of considerable interest and economic importance to poultry producers, Salrnonella Gallinarum-Pullorum does not have human public health significance. Chickens may also become infected with many of the other Salmonella serotypes. Usually only young chicks become ill, and disease among young hatchlings can result in high death rates with concomitant significant economic loss. Mortality rates may vary from negligible to 80% in unusually severe outbreaks (82j. Turkey poults are exceptionally susceptible to salmonellosis, and mortality rates may become extreme. Chicks seem in general to be more resistant than turkey poults. Chicks rapidly develop resistance to system salmonellosis during the first few days of life, especially between day 3 and day 5. By the seventh day it is nearly impossible to cause acute systemic illness, even with massive doses of bacteria given by direct intraperitoneal injection. This acquisition of resistance is tied to maturation of the immune system and the onset of cell-mediated immunity (8386). Usually the presence of salmonellae in a chicken flock has no effect on production whatsoever and goes unnoticed. Salmonella is therefore of no particular concern to the individual farmer. There are, however, two public health issues related to asymptomatic carriage: contaminated broiler carcasses and Snlrzlonella-contaminated shell eggs. Both are significant sources of salmonellae and pose serious public health issues. Snlmonellu bacteria are present on the skin, feathers, and in the feces of a small proportion of broiler chickens at the time of slaughter, and conditions in the slaughterhouses tend to allow the spread of these bacteria among the carcasses so that eventually a high percentage of the carcasses are contaminated to some degree; estimates range upward from 21% (87-89). The USDA’s Food Safety and Inspection Service in the early 1990s conducted one study at a chicken-processing plant located in Puerto Rico. Eight hundred samples were collected during 20 consecutive 8-hour shifts at five sites along the processing line. Seventy-seven percent of the “postautomatic cut’’ samples were culture positive for salmonellae, and 58% of the “preevisceration” carcasses were found to be contaminated with salmonellae (90,9 1). There have been several studies of the contamination incidence at different points during broiler production. However, with the initiation of the new Hazard Analysis Critical Control Points (HACCP) system for quality control in poultry-processing plants (92,93), the incidence is likely to decrease dramatically. Chicks may initially become infected in the hatchery (94), at the broiler house from con-

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taminated feed (95j, and from the droppings of animal pests (95). However, in one study, contamination of broiler carcasses was shown to mainly occur during transport and processing (89). It is not possible to obtain an accurate estimate of the proportion of human salmonellosis cases attributable to poultry. A given case could be attributed to any of many possible sources in the home, including pets, but the patient may only associate the illness with food recently consumed. Tauxe (96) has discussed some of the difficulties that the CDC faces when trying to estimate the proportion of human salmonellosis cases attributable to poultry. Estimates vary from 7.5 to 37% depending upon the methodology used and the assumptions underlying the methodology. Fortunately, careful attention to common household hygiene and cooking procedures and attention to food production hygiene and cooked food handling are sufficient to deal with health threats due to contaminated broiler carcasses. Nonetheless, the high rate of Sch?orzelln contamination among broiler carcasses has resulted in some considerable anxiety among the consuming public (96-99). Proper cooking of poultry as well as other food products that may be contaminated will certainly ensure that Scrlnzor~ellainfection does not occur due to ingestion of the cooked product. Improper handling of the product prior to cooking can lead to contamination of surfaces and utensils used during food preparation, and infections can occur due to contact with these surfaces or items. A somewhat different situation exists with regard to Sc~lnzolzelZn-contan7inatedshell eggs. First, the Salrnolzellcr Enteritidis serotype that is commonly associated with shell eggs appears to be more virulent for humans than other serotypes. Second, ordinary foodpreparation methods for egg-containing recipes may not always be adequate to control the organism. Third, while one may safely assume that for practical purposes all meat has some microbial contanlination and must be thoroughly cooked, the same assumption does not apply to eggs. Often egg-containing foods such as hollandaise sauce, mayonnaise, and bernaise sauces are prepared with uncooked or only partially cooked egg yolk (96,100104), and some people like to eat “3-minute” eggs in which the yolk is not fully cooked. Fourth, as regards the occurrence of large nosocomial (institutional) outbreaks (105-107), the nature of institutional food service operations associated with hospitals and nursing homes provides opportunity for dissemination of Snlvzonellcr Enteritidis among large populations of debilitated individuals. Indeed, during the 4-year period of 1985-1 988 there were 30 deaths among 4976 cases of Salmolzella Enteritidis food poisoning, 27 of which were among hospital and nursing home patients. From 1975 through 1987 there were 115 outbreaks of foodborne disease in nursing homes that involved nearly 5000 patients. Fiftytwo percent of these outbreaks and 82% of the deaths were attributable to Salrnorwlln of various serotypes, but Sulnzonellct Enteritidis was responsible for 56% of the deaths due to salmonellosis (96,103,108,109). The largest nosocomial outbreak due to Snlnzordln Enteritidis was investigated by the New York City Department of Health and involved 404 of 965 patients at one hospital. The outbreak resulted in nine deaths. Investigation traced the outbreak to raw eggs. traced back the eggs to the producing farm, and traced the infection to the ovary of a hen on the producing farm ( l lOj. Snlnzolzellu Enteritidis and sometimes other serovars (1 1 1,112jwill infect the ovaries of laying hens ( 113-1 16). The role of ovarian transmission of SalnlorzelZcr Enteritidis to the egg and the role of such a process in the natural dissemination of the organism is now generally accepted. Snlmonellrr can also penetrate and enter the egg through an intact shell as well as through minor cracks in the eggshell, and therefore it is not certain that all instances of outbreaks due to Scrlmonelle~contaminated eggs are the result of transovarian infection. Also, there is some evidence that while Scrlnzorder Enteritidis phage type 8 can

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penetrate the eggshell, it cannot enter the egg contents (1 17), as the inner membrane structure of the egg acts as a mechanical barrier protecting the egg contents. Invasive strains of Salmonella Enteritidis such as phage type 4 have been identified as the most likely source of egg contamination due to transovarian transmission and eggshell penetration (118,119). The presence of Salmonella Enteritidis (phage type unreported) has been reported in environmental samples collected from broiler fmns in the United States (120), but Saln1onella Enteritidis phage type 4 had not been isolated in U.S. poultry flocks until recently. In 1996 this especially virulent strain was isolated from a layer flock in the United States ( 121). Egg yolks obtained from Salnzonellcr Enteritidis-infected chickens contain antibodies directed against serogroup D antigens, suggesting that it may be possible to detect infected chickens, and possibly the eggs from infected chickens, by serological means (1 1 1,122- 125). Efforts are being made to improve upon the specificity of serological detection methods by focusing upon bacterial antigens that are limited to a very few serotypes, gm flagellin isolated from a highly motile strain of phage type 8 Sdmozzella Enteritidis, for example (126). There have been several proposals to vaccinate chickens against salmonellae. Recently, a live avirulent Salmonello vaccine was shown to induce excellent protection against intestinal, visceral, reproductive tract, and egg colonization, invasion, and/or contamination by Salnlorzella. The duration of this protection was for 11 months at which time the experiment was terminated (127). One disadvantage of a vaccination program would be the interference of vaccine-induced antibodies in serological testing for pullorum disease and interference in the serological detection of Salrnonellu Enteritidisinfected birds. Not all outbreaks of SaZmolleZZa Enteritidis are traceable to the consumption of contaminated eggs. In one large outbreak traced to a fast food restaurant, it was determined that an employee who had been ill with gastroenteritis contaminated the restaurant food. This employee continued working to the end of his shift even after experiencing the initial symptoms of illness and was probably responsible for infecting several other employees as well as thepatrons served during that shift. The source of his illness was not determined (128). Another restaurant-related outbreak was traced to sauces prepared from eggs. Eighty-one patrons were infected, 24 cases were culture-proven, and l 1 patients required hospitalization. Grade A intact extra-large eggs were the source of infection, and the eggs were traced to a farm at which Sulnzonelln Enteritidis was found. The isolate had plasmid profiles and antimicrobial susceptibility patterns indistinguishable from the outbreak isolate (103). In another series of restaurant-related outbreaks, scrambled eggs prepared from a large batch of raw egg mixture was implicated. At one of the restaurants the scrambled eggs were deliberately undercooked and 1 13 patrons developed gastrointestinal illness lasting more than one day. Of these 113 victims, 17 required hospitalization and 2 suffered Salmonella sepsis (129). Although these outbreaks are dramatic, restaurant food is usually quite safe. It has been estimated that nearly 46 tnillion people eat at more than 138,000 fast food restaurants per day, and there are remarkably few incidents of salmonellosis attributable to these establishments (128). Contaminated turkey has also been implicated in outbreaks of human salmonellosis (96). Salmonella Reading, Snl?nonellrHeidelberg, and Salmonella Saint-Paul are the three serotypes most frequently isolated from the birds (54,55), and these serotypes account for approximately 42% of the turkey isolates. Because salmonellosis is economically important to the turkey producer and because the turkey production system in the United States is unusually well vertically integrated, the turkey industry has greater means for Salmonella control than other animal industries (130). Critical control points have been identified

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that cover all phases of turkey production, turkey feed production, and whole carcass processing (130). A 5-year epidemiological study of Salmonella infections in turkeys has demonstrated the importance of preventing environmental contamination of breeding premises and revealed the importance of contaminated feed as a source of Salmonella infection (131). Surveys of animal-rendering facilities have demonstrated the need for additional hygiene practices in the animal feed industry. One survey of 83 rendering facilities revealed that 27% of analyzed samples were Salmonella positive (132). It is possible to produce Salmonellr-free turkeys if the feed is Scrlmonella-free, and ordinary prudent hygiene and pest control practices are followed. In one 4- to 5-year study, 2.5 million Salmonella-free poults were produced from 7500 hens and 600 toms. The producer used pelleted feed that did not contain animal protein, a frequent source of Salmonella, and samples of the feed were cultured upon delivery to the farm in order to ensure that the feed did not contain Snlnzorda (133). (The advantage of pelleting in the preparation of pet food has been previously mentioned.) Because of the importance of animal feed as a source of salmonellae to both turkey and chicken producers, the Animal Protein Producers Industry (APPI) has established an educational program for their members, which is designed to help them reduce the Salmonella contamination of their products. This Salnzonella Education-Reduction plan is based on the premise that careful attention to simple hygiene practices can help the animal feed producer produce feed free from salmonellae. Another approach to animal feed sanitation is the use of feed additives with possible antibacterial/anti-Salmonella properties: typically these are proprietary formulations of formic or propionic acid (134- 140). Ideally we wish to eliminate Salmonella from live birds so that the organisms do not reach the processing plant and cannot be disseminated during processing. It is possible to prevent introduction of Salmonella into a flock, and it may be possible to produce Salmonella-free chickens through a combination of relatively simple procedures-provided that they are made mandatory. In Sweden, a mandatory Scdmonella control plan has been in place for more than a decade for farms with flock populations above 5000 birds. The essentials of the plan are simple. Feed is produced in very well-regulated sanitary mills that are operated with the intent of producing Salmonella-free feed. All imported feed is tested and rejected if found to be contaminated. When salmonellae are found at the farm, the premises are quarantined and the birds are sent to special slaughter facilities designed to deal with contaminated birds. When flocks are found in which more than 5% of the birds are contaminated, the birds are destroyed and there is an indemnification program for the farmer. All breeding birds are routinely screened for salmonellae, and all dead birds on a farm are tested for salmonellae. The Nurmi competitive exclusion concept is utilized; that is, procedures are followed that aid the rapid establishment of normal intestinal flora in young birds. Young chicks with a normal “adult” intestinal flora resist colonization by salmonellae (14 1- 147). Recently there has been considerable research interest in improving the efficacy of competitive exclusion. Cox et al. (148) have attempted to ensure the early colonization of chicks by applying competitive exclusion bacteria it2 ovo, though this proved largely ineffective. And there have been attempts to improve control by manipulating the competitive exclusion flora in various ways (149-151). More recently, research interest was focused on defining the bacteria responsible for colonization control, and some studies have pointed to lactobacilli as important contributors to colonization control (152- 154). Other studies have shown that competitive exclusion cultures developed from a single strain of bacteria or from mixtures of strains from the same genus have not provided consistent

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protection against Sdmonella challenge (155), and it has been suggested that efficacious defined competitive exclusion cultures will need tobe more complex and contain a mixture of several bacterial isolates from different genera (156,157). Undefined competitive exclusion cultures contain mixtures of several hundred different bacterial isolates and have been shown to be efficacious, but cannot be used in many countries because they have not been approved by regulatory agencies. There are public health issues that arise any time food animals are deliberately exposed to undefined cultures that may contain pathogens or potential pathogens. In the United States these types of product are considered undefined drugs, and the U.S. Food and Drug Administration (FDA) has not yet given them regulatory approval. At least one undefined competitive exclusion culture has been shown to be efficacious in commercial field trials (158). An obvious alternative to undefined competitive exclusion cultures would be the development of an efficacious defined competitive exclusion culture. Historically, mixed defined competitive exclusion cultures have been difficult to develop due to lack of criteria for isolation, identifying, or maintaining the cultures in vitro (156,157), and this difficulty has hindered the development of a commercial product. Continuous-flow culture systems were recently used for the first time to maintain normal mixed microflora from the cecae of adult chickens in steady-state continuous culture (159,160). Subsequently this technique was utilized to develop a competitive exclusion culture characterized to contain 29 different bacterial isolates that was shown to control Sdmonelln colonization in experimental studies (16 1) and commercial field trials (162). Additionally, in an FDA-approved, doubleblinded, clinical trial, chicks treated with the culture at day-of-age were cecal Sulmonellu free at end of grow-out (163). In early 1998 this competitive exclusion culture (trade name PREEMPT@')was approved by FDA for use in commercial poultry production in the United States. Another method that has been investigated for use in controlling Salrnonella colonization in poultry is the use of various sugars provided to chickens in either feed or water. When chicks are given feed containing lactose, arabinose, or galactose, adherence of SnlInonella Typhimurium to the chick cecal surface is reduced (164), and numerous studies have now demonstrated that dietary lactose can sometimes be an effective adjunct to competitive exclusion (165-169). The mechanism by which lactose operates in conjunction with cecal microflora to control colonization is largely unknown. Because the chicken lacks the enzyme lactase, most of the dietary lactose is passed through the intestinal tract and becomes available for fermentation by lactose fermenting bacteria in the chicken cecae. Also, it has been suggested that sugars may compete with Salmonella for binding sites on the intestinal mucosal surface (170). Competition for binding sites on intestinal mucosal surfaces is, in our opinion, probably not a realistic mechanism, and there is very good circumstantial evidence that control is accomplished by other mechanisms including bacteriostatic/bactericidal volatile fatty acids produced in situ by the intestinal flora. The mechanism by which bacteriostatic volatile fatty acids may reduce colonization by intestinal pathogens has been well studied in chickens, and this mechanism may also operate to reduce Salmonella colonization within the intestinal lumen of swine (171). There is one report in which the Salmonella incidence was compared between pig farms that raised swine on liquid whey (naturally high in lactose) and farms that used ordinary drinking water. Salmonella bacteria were found at a lower percentage in swine on farms using whey than those using water. On farms where the premises were known to be contaminated, whey treatments reduced the proportion of pigs found to be culture positive. There was a highly significant statistical reduction in theproportion of culture-positive pigs given

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whey compared to those given water (172). The use of lactose to control Salrnonelln in commercial poultry flocks has not been adapted by the industry, most likely due to production economics and inconsistent experimental results.

IV.

SALMONELLA IN SWINE

We have already noted that Salmorzelln erzterica Choleraesuis has been a known swine pathogen since the mid-1880s. Approximately 1000 of the 1400 Scdrnor~ellaisolates from swine Sulrnorzella serotyped between July 1989 and June 1990 by the National Veterinary Services Laboratories, Ames, Iowa, were Salrrzonelln Choleraesuis. The remaining isolates were distributed more or less randomly among the other serotypes (54). Of 277 Sc~ln~or~elle~ isolates obtained from swine in Kansas during the 1979-1 983 period, 66% were Salmonella Choleraesuis, 8.6% were Salrnorzelln Typhimurium, and none of the remaining 25 serotypes isolated comprised more than 4% of the total. Of the top 10 clinical serotypes recovered from swine in 1996 as reported by National Veterinary Services Laboratories (173) and the top 10 serotypes recovered from humans as reported by the Centers for Disease Control in 1994 (174), three serotypes-Salmonella Typhimurium, Sahnomllcr Heidelberg, and Snlr~lonellaAgona-appear on both lists. Salrnonellu Agona has not been recognized as a significant pathogen of swine despite its importance in the human population (175). The USDA, Animal Plant Health Inspection Agency, Veterinary Service analyzed 6655 samples from 152 grower/finisher farms in a 1995 study. The sample and herd prevalence rates were 6.2% and 38.2%, respectively. The 10 most common serotypes recovered were Derby (33.5%), Agona (13.0%), Typhimurium (Copenhagen) (lO.l%), Bradenburg (8.0%), Mbandaka (7.7%), Typhimurium (3.6%), Heidelberg (3.6%), Anatum (1.9%), Enteritidis PT13A (1.7%), and Worthington (1.7%). Among the 58 Salmonellapositive farms, 15.8% were positive for Snlmonellrr Derby, while 6.6% of the farms were positive for Snlnzomlln Agona (176). Ferris and Miller reported that from July 1996 to June 1997, Snlnzonelln Choleraesuis (Kuzendorf) was the most frequently isolated serotype from swine (177). Although it has been well known that Sdmonelln Choleraesuis is highly pathogenic for swine in that it invades the tissues and establishes a carrier state in surviving swine, it has only recently been demonstrated that other serotypes can invade and persist within the lymphatic system of apparently healthy swine (178). Salrnorzelln Heidelberg has been isolated from clinically healthy swine, and the organism has been found to be aninvasive pathogen capable of dissemination from the intestines to the tissues with the concomitant establishment of a prolonged carrier state (179). Similarly, experiments with both Salnzonelln Newport and Salrnorzellcl Typhimurium demonstrated that these serotypes were able to establish themselves within swine tissues where they persisted throughout a 28-week study (180,181). Salmonellcr can be recovered from the gut even when the gastrointestinal tract is not the portal of entry. It has been demonstrated that the organism can be recovered from the gastrointestinal tract 3 hours after respiratory exposure, indicating that the respiratory tract is a portal of entry for salmonellae (182). The full significance of this method of infection has yet to be determined. Carrier pigs infected with salmonellae pose a potential public health problem when meat from infected animals is sold (183-1 85), and therefore disease prevention at the farm level is important. As with chickens, the use of clean Scrlrnorlellu-free feed is an important component of colonization control at the farm (1 86).Slaughterhouse sanitation is also an important additional component of disease prevention (187), and preslaughter handling at the abattoir will also influence the Snhnorzellu isolation rate (188,189). Fortu-

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nately, human infection with Salmonella Choleraesuis is rare, as this serotype is especially virulent and there is ahigh case fatality rate among people infected with Salrnonellcl Choleraesuis. The lower incidence of human salmonellosis attributable to pork may be a result of the fact that the public seems to understand that pork needs to be well cooked or processed in order to prevent the transmission of other historically significant diseases associated with undercooked pork products, such as trichinosis. Besides the potential public health issue posed by carrier pigs at slaughter, the animals themselves are prone to develop overt salmonellosis when stressed, and it has been estimated that nationwide swine salmonellosis costs producers $100 million yearly (190). For this economic reason, there is considerable interest in developing intervention methods (such as competitive exclusion and vaccination) and evaluating animal husbandry practices (such as all in all out vs. continuous flow rearing systems) that may help limit the spread of the Salnzonellcr problem at the farm level. Recently competitive exclusion cultures have been developed and efficacy in Scdmonellrr control evaluated. Initial results suggest that this might be a helpful method that will aide in the reduction of salmonellae on the farm (191,192). Live attenuated Salmonella vaccines have also been efficacious in reducing mortality and morbidity associated with salmonellosis (189,193,194), however, carrier animals still exist within the population, suggesting that other strategies are needed.

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157. Stavric, S. (1992). Defined cultures and prospects. Int. J. Food Microbiol., 55:245-263. 158. Blankenship, L. C., Bailey, J. S., Cox, N. A., Stem, N. J., Brewer, R.,and Williams, 0. (1993). Two-step mucosal competitive exclusion flora treatment to diminish salmonellae in commercial broiler chickens. Poult. Sci., 73: 1667- 1672. 159. Nisbet, D. J., Corrier, D. E., and DeLoach, J. R. (~1993).Effect of mixed cecal microflora maintained in continuous culture and dietary lactose on Salmonella hphirnurium colonization in broiler chicks. Avian Dis., 37528-535. 160. Nisbet, D. J., Ricke, S. C., Scanlan, C. M., Hollister, A. G., and DeLoach, J. R. (1994). Inoculation of broiler chicks with a continuous-flow derived bacterial culture facilitates early cecal bacterial colonization and increases resistance to Salmonella iypkimurium. J. Food Prot., 57:12-15. 161. Corrier, D. E., Nisbet, D. J., Scanlan, C. M., Hollister, A. G., Caldwell, D. J., Thomas, L. A., Hargis, B. M., Tomkin, T., and DeLoach, J. R. (1995). Treatment of commercial broiler chickens with a characterized culture of cecal bacteria to reduce salmonellae colonization. Poult. Sci., 74:1093-1101. 163,. Corrier. D. E.. Nisbet, D. J., Scanlan, C. M.. Hollister, A. G., and DeLoach, J. R. (1995). Control of Salntonellu Qphinzrwizcnt colonization in broiler chicks with a continuous-flow characterized mixed culture of cecal bacteria. Poult. Sci.. 74:916-924. 163. Corrier, D. E., Nisbet, D. J., Byrd 11, J. A., Hargis. B. M., Keith, N. K., Peterson, M., and DeLoach. J. R. (1998). Dosage titration of a characterized competitive exclusion culture to establish Salmonella colonization resistance in broiler chickens during growout. J. Food Prot.. 61:796-801. 164. McHan, F., Shotts. E. B., and Brown. J. (1991). Effect of feeding selected carbohydrates on the in vivo attachment of Salntonella typlrintzwizrm in chick ceca. AvianDis., 35:328331. 165. Conier. D. E., Hinton Jr., A. H., Ziprin, R. L., Beier, R. C., and DeLoach, J. R. (1990). Effect of dietary lactose on cecal pH, bacteriostatic volatile fatty acids, and Salmonella typhimuritun colonization of broiler chicks. Alian Dis., 34:617-625. 166. Corrier, D. E., Hinton Jr., A. H., Kubena, L. F., Ziprin, R. L., and DeLoach, J. R. (1990). Decreased Salmonella colonization in turkey poults inoculated with anaerobic cecal microflora and provided dietary lactose. Poult. Sci., 70:1345-1350. 167. Ziprin, R. L., Corrier, D. E., Hinton Jr., A. H., Beier, R. C., Spates, G. E., DeLoach, J. R., and Elissalde, M. H. ( 1990). Intracloacal Salr~lonellafyphirmriunt infection of broiler chickens: reduction of colonization with anaerobic organisms and dietary lactose. Avian Dis.,34:749753. 168. Ziprin, R. L., Elissalde, M. H., Hinton Jr., A. H.. Beier, R. C., Spates, G. E., Corrier, D. E., Benoit, T. G., and DeLoach, J. R. (1991). Colonization control of lactose-fermenting salmonellu typhinturizm in young broiler chickens by use of dietary lactose. Am. J. Vet. Res., 52: 833-537. 169. Corrier, D. E.. Hinton, Jr., A. H.. Ziprin, R. L., Beier, R. C., and DeLoach, J. R. (1991). Effect of dietary lactose and anaerobic cultures of cecal flora on Salmonella colonization of broiler chicks. In Colonization Control of Hurnan Bacterial Enteropathogens (L. C. Blankenship, ed.), Academic Press, Inc., New York, pp. 299-308. 170. Oyofo, B. A., DeLoach, J. R.. Corrier, D. E., Norman, J. O., Ziprin, R. L., and Mollenhauer, H. H. (1989). Effect of carbohydrates on Salmonella iyphinturiunz colonization in broiler chicks. J. Avian Dis., 33:531-533. 171. Prohaszka, L., Jayarao, B. M., Fabian, A., and Kovacs, S. (1990). The role of intestinal volatile fatty acids in the Salnlonella shedding of pigs. Zentralbl Veterinarmed [B] 37:570574. 172. Van Schie, F. W., and Overgoor, G. H. A. (1987). An analysis of possible effects of different feed upon the excretion of Salmonella bacteria in clinically normal groups of fattening pigs. Vet. Q., 9:185-188.

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173. Ferris, K. E., and Miller,D. A. (1996).Salmonella serotypes from animals and related sources reported during July 1995-July 1996. In Proc. U.S. Aninz. Health Assoc. Ann. Mtg., Little Rock, AR, 1996, pp. 505-526. 174. CDC Annual Summary, Salmonella Surveillance, 1994. 175. Williams, J. L. P. (1980). Salmonellosis. In Bacterid, Rickettsial, m d Mycotic Diseases (J. H. Steele, ed.), CRC Press Inc., Boca Raton, FL, pp. 570-583. 176. Bush, E. (1995). The 1995 NAHMS Swine Grower/FinisherStudy. In Proc. Lisestock Cons. Inst., Kansas City, April 5-7, pp. 207-208. from animals and related sources 177. Ferris, K. E., and Miller, D. A. (1997). Salmonella serotypes reported during July 1996-June 1997. In Proc. of the IOP' United States Animal Health Association Annual Meeting, Louisville, KY, 1997, pp. 423-443. 178. Keteran, K., Brown, J.. and Shotts. E. B. (1982). Salrlrorda in the mesenteric lymph nodes of healthy sows and hogs. Am. J. Vet. Res., 43:706-707. 179. Reed, W. M.,Olander,H. J., andThacker,H. L. (1985).Studies on thepathogenesis of Salrnonella heidelberg infection in weanling pigs. Am. J. Vet. Res., 46:230@-2309. 180. Wood. R. L. (1989). Swine as a reservoir of Salmonella: persistent infection with Salmonella typhimurium and Salmonella newport to market age. In Proceedings 93rdAnn. Meet. of the U.S. Animal Health Assoc., Las Vegas, NV, 1989, pp. 513-516. 181. Wood, R. L., Rose, R., Coe, N. E., and Ferris, K. E. (1991). Experimental establishment of persistent infection in swine with a zoonotic strain of Salmonella newport. Am. J. Vet. Res.. 521813-819. 182. Fedorka-Cray, P. J., Kelley, L. C., Stabel, T. J., Gray, J. T., and Laufer, J. (1995). Alternate routes of invasion may affect the pathogenesis of Snhonelln fyphirnuriunl in swine. Infect. Irnn~un.,632658-2664. 183. Oosterom, J., Dekker, R., deWilde, G. J. A., van Kempen-de Troye, F., and Engels, G. B. (1985). Prevalence of Car~lpylobacterjejuni and Salmonella during pig slaughtering. Vet. Q., 7:31-34. 184. Oosterom, J. (1987).Epidemiological studies of Sal~nonellnand Canpdobacter jejuni.Vet. Q., 9~348-354. 185. Mafu, A. A., Higgins, R., Nadeau, M., and Cousineau, G. (1989). The incidence of Salmonella, Campylobacter, and Yersinia enterocolitica in swine carcasses and the slaughterhouse environment. J. Food Prot., 52:642-645. 186. Linton, A. H. (1979). Salmonellosis in pigs. Br. Vet. J . . 135:109-112. 187. Childers, A. B., Keahey, E. E., and Kotula, A. W. (1977). Reduction ofSalmonella and fecal contamination of pork during swine slaughter. J. Am. Vet. Med. Assoc., 171:1161- 1164. 188. Craven, J. A., and Hurst. D. B. (1982). The effect of time in lairage on the frequency of Salmonella infection in slaughtered pigs. J. Hyg. Camb., 88: 107-1 11. 189. Morgan, I. R., Krautil, F. L.. and Craven, J. A. (1987). Effect of time in lairage on caecal and carcass Srdmonella contamination of slaughter pigs. Epidemiol. Itlfect., 98:323-330. 190. Schwartz. K. J. (1990). Salmonellosis in midwestern swine. In Proc. 94'l Ann. Meet. sf the U.S. A n i n d Health Assoc., pp. 443-449. 191. Fedorka-Cray, P. J., Bailey, J. S., Stern, N. J., and Cox, N. A. (1997). Mucosal competitive exclusion to reduce Salmonella in swine. In PI-oc. 2nd I d . S y p . on Epidenz. m d Control of Sahnonella in Swine, Copenhagen, pp. 164-166. 192. Nisbet, D. J., Anderson, R. C., Buckley,S. A., Fedorka-Cray, P. J., and Stanker, L. H. (1997). Effect of competitive exclusion on Sulmonella shedding in swine. In PI-oc. 2nd Intl. Synp. on Epidenl. and Control of Snlnlonella in Swine, Copenhagen, pp. 176-177. 193. Curtis, I.R., Porter, S. B., and Munson, M. (1991). Nonrecombinant and recombinant avirulent Salmonella live vaccinesfor poultry. In Colonization Control of H~martBacterial Enteropathogens i n f'ozdtry (L. C. Blankenship, ed.), Academic Press, San Diego, pp. 169-198. 194. Kramer, T. T., Roof, M. B., and Matheson,R. R.(1992). Safety and efficacyof an attenuated strain of Salmonella cholemszsis for vaccination of swine. Am. J. Vet. Res.. 53:434-348.

13 Human Salmonellosis: General Medical Aspects Richard L. Ziprin and Michael H. Hume U S . Department of Agriculture, College Station, Texas

I. Salmonella Serovars of Significanceto Humans 286 A. Host adaptation 286 Nontyphoid B. serovars 286 11. Typhoid Fever

287

Key A. features 287 Incidence B. rate 287 of S. Typhi 287 C. Diagnostic characteristics D. Typhoid fever 288 E. Incidence and drug resistance 289 F. Connection betweentyphoid fever and carcinoma290 G. Treatment and antibiotic resistance 290 111. Nontyphoid Salmonellosis 291 Key A. B. C. D. E. F. G. H.

features 291 Enterocolitis/Gastroenteritis 291 Nontyphoid bacteremia/septicenlia/enteric fever 292 Bacteruria 294 Asymptomatic carriage/nosocomial outbreaks 294 Multidrug-resistant strains 294 Treatment 295 Arthritis 296

IV. Mouse Model V.

298

Vaccines 299

A. Vaccination to prevent typhoid fever 301 B. Gal E mutants: alive-attenuated vaccine 301 C. Genetically "engineered"typhoid fevervaccine strains D. Vi antigen vaccines 303 VI. Conclusion 304 References 304

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I. A.

SALMONELLA SEROVARS OF SIGNIFICANCE TO HUMANS HostAdaptation

There are three host adaptation patterns among the Salmolzella serovars: highly host adapted, somewhat host adapted (or intermediate), and non-host adapted (1) [more simply stated, host adapted and ubiquitous (2)]. Human host-adapted serovars tend to cause the most severe illness. In contrast, the highly host-adapted chicken pathogens Scrlrnor~ella Pullorum and Salmonella Gallinarum are not human pathogens. While host adaptation patterns are somewhat linked with the severity of human illness, non-host-adapted serovars (ubiquitous serovars) often do cause severe illness (3). The most highly host-adapted organism of interest in human medicine is Scrlmonella Typhi, the cause of human typhoid fever. Typhoid fever is sometimes referred to as enteric fever, but the term enteric fever can be used to describe typhoid-like illness caused by other serovars. There is no animal source of S. Typhi and no evidence that the S. Typhi host range extends to animals other than human beings. S. Typhi is adapted totally to humans. Therefore, S. Typhi is always contracted from water or food that has been contaminated in some manner by another human being (3-8). There are very rare exceptions to human-to-human transmission, which occur as laboratory accidents (9- 12).

B. Nontyphoid Serovars Other Snlmorzelln serovars are host-adapted animal pathogens and sources of zoonotic infections, i.e., etiological agents of diseases of animals that are secondarily transmitted to human beings (13). Srdnzorzella Choleraesuis is a pathogen of swine and also causes severe systemic infections in humans (3,14). S. Choleraesuis is especially virulent. There is a high case fatality rate among infected individuals. Mycotic aneurysms (infection and weakening of arterial walls) may occur as a result of S. Choleraesuis infection and are sometimes fatal (3,14). Fortunately, the actual incidence of human S. Choleraesuis infection is rare. This rarity of incidence in humans can be attributed to the fact that most people are quite aware that pork products must be well cooked or properly processed to prevent transmission of other diseases such as trichinosis. Salmonella Dublin may cause septicemia in cattle and be transmitted to human consumers of raw milk or other unpasteurized dairy products. As happens with many infectious agents, S. Dublin tends to infect older persons, the very young, inmune-compromised individuals, and those suffering from chronic debilitating disease. S. Dublin can be highly invasive and virulent, and infections result in a high case fatality rate (3,1517). S. Dublin causes mycotic aneurysms (17) and has infected a prosthetic vascular graft (16). There is at least one citation that Dublin sometimes produces Vi antigen, the antigen believed to cause virulence in S. Typhi (18). Snlrr~orzellnEnteritidis and Snlrr~onellaSenftenberg are somewhat host adapted to chickens and turkeys. respectively (3). Both are capable of causing severe human illness, including death. S. Enteritidis is of particular public health concern, because chicken eggs destined for human uses are sometimes contaminated by S. Enteritidis. If the Contaminated eggs are not properly cooked or if eggs are used in a food containing raw or undercooked egg, the human consumer will become infected. S. Enteritidis is one of the major problem areas in human bacterial diseases.

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S. Senftenberg is generally associated with turkeys and has been known to find its way from turkey and turkey products into human food. It is a relatively rare cause of human illness. Nevertheless, S. Senftenberg has caused a serious nosocomial outbreak at a major medical/hospital center (1). The likely cause of the outbreak was cross-contamination of kitchen equipment by turkey meat. S. Senftenberg has been transmitted from a mother’s breast milk to her baby (19). There has been a recent report of a lung abscess due to Senftenberg (20). This incident is notable in part because the infection did not respond to conventional antimicrobial therapy and is one of numerous examples of the growing threat of multi-drug-resistant salmonellae in our environment. Multidrug-resistant strains have changed antimicrobial treatment protocols for systemic salmonellosis, as an ever-expanding list of antimicrobials must be used (6). Most Sch?oneZlu serovars (except as noted above) are not host adapted. Instead, they are found in a wide array of animal products, fruits, vegetables, and processed foods. Although these serovars tend to be somewhat less virulent than the host-adapted serovars (i.e., they are less likely to cause enteric fever-like disease), they are responsible for the overwhelming number of incidents of human salmonellosis (90%0), primarily uncomplicated enterocolitis (3,643).

II. TYPHOIDFEVER A.

KeyFeatures 1, Etiological agent: Sulnzonelln Typhi 2. Source of infection: human carrier (food or water contaminated with feces from a carrier, symptomatic or asymptomatic) 3. Nature of the infection: systemic infection, bacteremia 4. Onset of illness: slow (incubation period of 7-21 days, thenan incremental increase in fever to 103-104°F occurring during the first 1-2 weeks of illness) 5. Mortality: 10% in untreated cases, less than 1% when treated 6. Treatment: antimicrobial therapy, traditionally with chloramphenicol, ampicillin, trimethoprim-sulfamethoxazole; more recently gentamicin, fluoroquinolones and cephalosporins. Multidrug-resistant strains exist, especially in underdeveloped countries.

B. IncidenceRate S. Typhi causes true typhoid fever. Worldwide, typhoid fever is responsible for between 12 and 33 million incidents each year and a mortality of 500,000-600,000 persons (21,22). Fortunately, true typhoid fever is a relatively rare illness within developed countries and the United States in particular. C.DiagnosticCharacteristics

of S. Typhi

Bismuth sulfite agar, though it has a reputation for being tricky to use, is the culture media of choice for isolating S. Typhi (23). Once a putative SnZmoneZZn is isolated, an attempt should be made to determine its serogroup, as serogrouping will also confirm that the organism is salmonellae. S. Typhi is a serogroup D organism, which has two important serological differences from S. Enteritidis: S. Typhi has a ‘d” phase I flagella antigen, L

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whereas Enteritidis has the phase I antigenic formula “g,m.” S. Typhi produces a somatic antigen, the Vi antigen (3,4,24), which was discovered early in this century when it was realized that some Salmonella strains failed to agglutinate with specific antiserum. Vi antigen masked the somatic antigens, thus interfering with the agglutination reaction. The name Vi antigen was chosen to denote the fact that the antigen was present in virulent strains (25). There is a practical implication of Vi antigen’s presence on the surface of S. Typhi. Any time an isolate has the usual phenotypic characteristics of salmonellae yet fails to react with polyvalent anti-0 antiserum, the possibility that the isolate is S. Typhi must be considered. The suspect organism must be tested with antiserum directed against the Vi antigen, and additionally the culture should be boiled to remove the Vi antigen and unmask the somatic antigens. The organism should then be retested with anti-0 antiserum. There are strains of Vi antigen-negative S. Typhi that cause typhoid fever. Typically illness due to these Vi-negative strains is not as severe as that associated with Vi-positive strains (26,27).

D. Typhoid Fever Although all human ailments caused by salmonellae share common features, typhoid fever is somewhat different from nontyphoid salmonellosis. Onset of typhoid fever is much slower (3,543). There is usually a brief gastrointestinal disturbance during the first day or two after ingestion of S. Typhi followed by a prolonged incubation period prior to the onset of serious symptoms. These may last from 1 to 3 weeks. Typhoid fever is characterized by a remittent fever. The fever increases over a period of days by stepwise increments. Many patients will develop “rose spots,” a skin rash characteristic of typhoid fever (4-8.22). Left untreated, fever may last for 2 or more weeks and the illness may cause death. The patient may experience intestinal perforation and hemorrhage, coma, delirium, and seizures. But, even in the preantibiotic era, 90% of patients eventually recovered. The case fatality rate is less than l% among individuals who have received appropriate therapy. Survivors often become constant asymptomatic carriers of S. Typhi. The organism remains present in the gallbladder and intestines. These asymptomatic carriers are most likely to be unknowing vectors of the disease (3-8). This carrier state causes a substantial public health hazard if the carrier happens to be a food handler. Carrier status is a feature of salmonellosis that is not limited to those who have recovered from typhoid fever. Indeed any Snlmonellcr infection can produce varying degrees of a postinfection carrier state (1,343). Typhoid fever involves a general invasion of the body by S. Typhi. In contrast, most other SalnzonelZa serovars usually cause only anenterocolitis with diarrhea. Systemic infections by nontyphoid serovars are relatively infrequent, whereas bacteremia is a constant feature of typhoid fever. The gastrointestinal tract is the portal of entry in typhoid fever. Organisms spread from the gastrointestinal tract to other organs-the lymphatics, the blood, the spleen, the liver-and produce a generalized infection. Then, all the pathophysiological effects of the ScdnloneZZr endotoxin (lipopolysaccharide) become apparent. With the organism causing a systemic infection, all major organ systems and tissues may become infected by S . Typhi. There is splenic enlargement, ulceration of the intestinal walls (sometimes with perforation leading to fatality), infection of the gallbladder, the bones (osteomyelitis), and brain or meningeal involvement. None of these features is unique to typhoid fever, and each may occur after infection by almost any Salmor~ella serovar.

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Because typhoid fever is a very serious health hazard and hygienic conditions cannot be assured during combat situations, it was recognized early on by military authorities that troops needed to be protected from typhoid fever. Military leaders mandated that their troops be vaccinated. Military vaccination against typhoid fever was first accomplished in 1904 with British troops and civilian travelers headed to India and was introduced to U.S. troops in 1911 (28-31). Early vaccines were merely saline suspensions of the organism inactivated by heating at 55-56°C for 1 hour. During the past two decades there have been extensive efforts to develop high-tech vaccines (3) (see Sec. V).

E. Incidenceand Drug Resistance Modern sanitation, hygiene, and public health measures (monitoring food handlers) have dramatically reduced the incidence of typhoid fever within the United States. Typhoid was very common during the pioneer days in North America. During the decade 19851994, the Centers for Disease Control and Prevention (CDC) received reports of only 2445 domestic typhoid cases-200 cases per year on average. Fortunately, only 10 of these incidents resulted in death (32). The overwhelming number of cases were clearly acquired through recent international travel (72%). Among the more recent cases, 12% of the Typhi isolates were resistant to at least three of the previously most effective antimicrobials-ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole. This increased antimicrobial resistance is very worrisome, since typhoid fever, unlike Snlmonella enterocolitis, is a systemic infection that must be effectively treated with antimicrobial agents. As a consequence of increased resistance to traditional treatment, the cephalosporins and fluoroquinolone antibiotics are sometimes required for the treatment of typhoid fever. Most typhoid cases in the United States and the economically developed world are linked to international travel, but there are domestic cases. Approximately 20% of 55 cases in the Chicago area were in patients with no history of foreign travel. Of 55 cases reported to the Chicago and Cook County Health Departments during a 7-year period, 3 1% of the isolates were resistant to both ampicillin and trimethoprim-sulfamethoxazole. Forty percent of the cases involved contact with either food handlers or health care workers (33). There are relatively few outbreaks (as opposed to cases reported) of typhoid fever in the United States each year: only six were reported between 1980 and 1989. In Canada, there were 43 small outbreaks involving 116 persons during the decade 1983-1992 (34). One outbreak in the United States involved 60 persons who attended both a family picnic and a Latin food festival. Of the 60 attendees at the family picnic, 40% became ill and 16 cases were confirmed by culture. The likely source of organisms was a potato salad prepared with "intensive handling" and without adequate temperature control by a recent immigrant from El Salvador who was an asymptomatic carrier. This individual had elevated Vi antibodies in her serum and S. Typhi in her stools, strongly suggesting a carrier state. All of this indicates that typhoid fever, while rare, remains a serious concern as a foodborne disease (35). Another incident is similarly instructive. An 8-year-old girl was infected on two separate occasions with S. Typhi. The infective source was her grandmother, who was a known carrier (36). In this instance the child fortunately responded well to treatment with trimethoprim-sulfamethoxazole and the grandmother was treated successfully with the fluoroquinolone ciprofloxacin. A take-home lesson from these cases is that typhoid fever differs from other incidents of salmonellosis in part by the fact that the source of the

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infection is another human who is a carrier. As there are no nonhuman reservoirs of S. Typhi, the source of infection is radically different from the nontyphoid salmonelloses. Typhoid fever is not a zoonotic disease. One incident illustrates the role of a human source in the dissemination of S. Typhi. In 1981 there was a large outbreak of typhoid fever among guests at a New York resort hotel. Forty-three guests were eventually determined to have culture-confirmed cases of typhoid fever, while another 24 guests were ill, probably with typhoid fever, though it was not culture-confirmed. Twenty-one guests were hospitalized, two with bowel perforations. The source of infection was determined to be a 208 liter vat of orange juice prepared by the kitchen staff, one of whom was an asymptomatic carrier (37).

F. ConnectionBetweenTyphoidFeverandCarcinoma Recently, there has been some interest in the relationship between chronic microbial infections and carcinoma. It is well established that untreated Helicobacter pjdori infections may lead to gastric carcinoma (38-42). There is a sitnilar suspicion concerning chronic carrier states with salmonellae, in particular S. Typhi. Both typhoid fever and carcinotna of the gallbladder are endemic in India. The gallbladder is of course the site colonized by S. Typhi after an acute episode of thedisease. A thousand bile specimens were collected from cases of carcinoma of the gallbladder, cholelithiasis (stones), and other forms of biliary pathology. Samples were cultured and both S. Typhi and Snlrr1oneZla Paratyphi were detected in a significantly higher number of patients with carcinoma of thegallbladder than among controls with cholelithiasis (43,44).

G. TreatmentandAntibioticResistance When S. Typhi is susceptible to chloramphenicol, this drug remains the treatment of choice. In the United States there is reluctance to use chloramphenicol because of a 1 in 30,000 chance that a rare bone marrow aplasia will occur. Therefore, other antimicrobials are used, specifically ampicillin, amoxicillin, trimethoprim-sulfamethoxazole, and ceftriaxone (22). Because untreated asymptomatic carriage of S. Typhi can continue for years, effective sewage systems and surveillance of food handlers are necessary for keeping the incidence of typhoid fever low. Fortunately, these measures are very effective and have reduced the incidence of typhoid fever in the United States to negligible levels. Chronic carriage after recovery from enteric fever is, however, very difficult to eliminate. Sometimes surgical removal of the gallbladder is required to rid the patient of focal infection that does not respond well to medical treatment. When the infecting organisms are resistant to antimicrobials of first choice, the fluoroquinolone ciprofloxacin and other quinolones are efficacious, shorten the acute phase, and eliminate the carrier phase (45).However, the fluoroquinolones are not recommended for use in children (22). Instead the cephalosporins ceftriaxone, cefotaxime, and cefoperazone may be used (22). Multidrug-resistant strains sometimes fail to respond adequately to antimicrobial treatment. There have been isolates containing large plasmids that confer resistance to chloramphenicol, trimethoprim-sulfamethoxazole, ampicillin, and gentamicin. During the past decade these multidrug-resistant (MDR) S. Typhi have been responsible for numerous outbreaks throughout the world (37,46-48). For these MDR strains, aztreonam, cefixime, ampicillin-sulbactam, and furazolidone may be effective (22).

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The worldwide occurrence and concomitant vast useland abuse of antimicrobials that typhoid fever necessitates create a selective pressure for the accumulation and transfer of resistance genes among the various serovars. Hence, indirectly, the worldwide prevalence of typhoid fever is potentially significant to us here in the United States. Slowly, but certainly, and for a variety of reasons, MDR salmonellae of all serotypes are becoming serious public health problems. Vaccination programs in endemic areas are essential to reduce the worldwide incidence and protect society at large (see Sec. V).

111.

NONTYPHOIDSALMONELLOSIS

A.

Key Features Etiological agent: all Snbnorzelln serovars except S . Typhi Onset: rapid, hours to a few days, dose dependent Symptoms: enterocolitis and diarrhea, sometimes systemic infections Source of infection: food, water, animals and animal products, human feces Mortality: possible, especially with more virulent serovars (severe disease more probable in very young, aged, debilitated, and immunologically compromised individuals) 6. Treatment: antimicrobial therapy with chloramphenicol, ampicillin, trimethoprim-sulfamethoxazole, gentamicin, fluoroquinolones, and cephalosporins (there is an ever-increasing incidence of MDR strains throughout the world, in both developing and developed countries) 1. 2. 3. 4. 5.

B. Enterocolitis/Gastroenteritis Whereas the onset of typhoid fever is insidious, the onset of nontyphoid salmonellosis tends to be dramatic. Onset is rapid, occurring within 12 hours (perhaps even earlier) to a few days after consumption of contaminated food or beverage. The initial symptoms are a dramatic diarrhea, which is sometimes accompanied by abdominal pain, nausea, vomiting, headaches, chills, myalgia, and low-grade fever. The diarrhea is due to invasion of the intestinal mucosa and a concomitant inflammatory response that leads to a secretory diarrhea mediated by activation of cyclic adenosine monophosphate (49,50). Some strains of Salmonella might produce enterotoxins that are biochemically/immunologically related to cholera toxin, E. coli heat-labile toxin, and Shiga toxin (50-52). But evidence that Salmonelln produces enterotoxins is marginal. Work with a real host, the calf, has shown that enterotoxins have a limited role in the causation of diarrhea. Instead, the invasion associated type I11 export system encoded on Salmonella pathogenicity island 1 has been shown to mediate fluid accumulation and PMN influx (53). Virulent organisms may cause bacteremia or nontyphoid enteric fever, and high fever should be a warning that the organism has invaded from the intestines. When invasion occurs, virulent organisms may establish themselves at focal loci within various tissues and organs throughout the body. However, healthy adults usually endure only a self-limited, uncomfortable diarrhea that resolves without treatment in 2 days to a week. Except when invasive disease is suspected, gastrointestinal salmonellosis requires no treatment beyond fluid and electrolyte replacement (6). Drugs to treat the diarrhea generally are not indicated. If used, bismuth subsalicylate (Pepto-Bismol") is preferable to antimotility drugs such as loperamide, which may increase the odds that the infection will become a systemic one. [Bismuth salts are compo-

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nents of culture media selective for salmonellae, so this author questions the antimicrobial benefit to the use of bismuth in the treatment of Salmonella enterocolitis. However, we have not thoroughly researched this question. A MedlineTMsearch, 1985 to present, using salmonellosis and bismuth as keywords returned only one citation (54),which does support the use of bismuth for its antisalmonella activity.]

C.

NontyphoidBacteremia/Septicemia/Enteric Fever

In most cases of Scrlr~zonelluenterocolitis/diarrhea, the organisms remain confined to the intestines and the infection is self-limiting. Typically the infection is foodborne, though some cases occur by direct transmission from an asymptomatic carrier to another human. Interestingly, it is possible for individuals to acquire a mixed infection, that is to coincidentally suffer from both salmonellosis and campylobacteriosis. A nursing home outbreak involved 119 of 580 residents ill with either Salnzonella Heidelberg, Cmzpylobacter jejuni, or both. Both organisms were recovered from 27% of the ill residents (55). Regardless of the identity of the infecting serovar, the lines of demarcation between diarrhea, gastroenteritis, enterocolitis, and a wider systemic infection may be blurred. One leads to the other as bacteria exit the intestines and establish within the organs. The severity of the resulting illness is influenced by a wide array of factors. The infecting dose, the serovar causing infection, the host’s general health, and the antibiotic resistance patterns of the invading organism all contribute to the course of the infection. The literature contains many reports of fatal salmonellosis among both previously healthy adults and those with predisposing factors: infants, elderly, immunocompromised, and otherwise ill or debilitated individuals. Some recent examples are given (56-63). A case of S. Enteritidis enterocolitis in a healthy 66-year-old man with one predisposing factor, rheumatoid arthritis, has been reported (56). The patient was hospitalized for diarrhea and abdominal pain and had been receiving predisolone for rheumatoid arthritis. Perhaps this anti-inflamlnatory agent was a factor in reducing the individual’s resistance. The patient died of hemorrhagic necrotic enteritis localized to the ileum. S. Enteritidis infections are often contracted from consumption of undercooked and improperly stored egg or egg products. S . Enteritidis is, therefore, of commercial and economic interest to the poultry industry. It is among the two or three most common causes of human nontyphoid salmonellosis. In 1994, contaminated eggs were responsible for an outbreak at a hospital in Mexico City. Ninety-eight employees who ate at the hospital cafeteria became ill. Cultures from the kitchen staff were negative, so the kitchen help was not the source of the outbreak. The source was traced to an egg-covered meat meal (64). Green and Vinker (59) reported a high incidence of Sdnonella infections among patients who suffer from systemic lupus erythematosus. As with the incident given in the previous paragraph, anti-inflammatory drug treatment was a predisposing factor contributing to illness. In the case reported, the patient had recurrent episodes of salmonellosis with two serovars, S. Typhimurium and S . Enteritidis. The S. Enteritidis infection was ultimately fatal. Four other cases of S. Enteritidis have been described in which the patients suffered from predisposing conditions: neoplasms, splenectomy, and hemophilia (60). All recovered from their illness, so the outcome is by no means necessarily bleak. Unfortunately, there are incidents of endovascular infection of atherosclerotic arteries. These infections of arterial walls are very dangerous as they can lead to fatality when the weakened artery

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fails. Fortunately improved medical and surgical care can save these patients. A case of S. Enteritidis mycotic aneurysm in a 65-year-old man has been described (one of many similar incidents). The patient presented with a protracted fever, weight loss, and thigh pain. Blood cultures were negative as were serological results, but a CAT scan revealed the aneurysm. A surgical repair was accomplished and S. Enteritidis was recovered from the arterial wall and the surrounding fluid (61). Sickle cell disease is yet another predisposing factor, and salmonellosis in sickle cell patients is especially dangerous. It is believed that capillary occlusion due to the sickling devitalizes the gut, permitting invasion. Then, reduced liver and splenic function and other reticuloendothelial system abnormalities suppress clearance of the bacteria from the blood. Salmonella osteomyelitis is a frequent complicating factor in such individuals and septic arthritis (active infection in a joint/synovial fluid), when it occurs, has a poorer prognosis. Aggressive surgical intervention is necessary (65). Somewhat surprisingly, despite frequent bouts of bacterial infections, septic arthritis is rare among HIV-positive patients. When it occurs, it is possible to culture the offending organism from the synovial fluid (66,67) (see Sec. 1II.H). Salmonella infections may occur in infants and even premature infants (68). It is possible for a pregnant woman to be the source of infection for an infant. There is a report of a premature infant delivered by cesarean acquiring a fatal Enteritidis infection from the mother. The child died shortly after birth, and S. Enteritidis was recovered from blood cultures and swabs of the child and from the placenta. The author describing the above case argues that antibiotic treatment should be given to women to treat Salmonella infections occurring during pregnancy. This procedure reduces the risk of transplacental infection of the fetus (57). Reports of salmonellosis during pregnancy or in the immediate neonatal period are not unusual (69-75). There may be drastic outcomes for the fetus or infant (70,71). At least one salmonellosis outbreak has occurred in a maternity and prenatal intensive care unit (73). In another case, the mother had contracted S. Enteritidis gastroenteritis (enterocolitis) one week before delivery. Antimicrobial treatment of the infant was described (75). In a study of 74 consecutive cases of salmonellosis, S. Typhimurium was the most common strain causing septicemia. S. Typhimurium infections are more frequently fatal during the neonatal period and in the presence of an associated disease such as congenital heart disorders or underlying immunodeficiency disorders (76). Nontyphi salmonellosis is 20 times more frequent in the HIV-positive population than in the general population, and a Snlmonella infection in AIDS patients may remain remarkably persistent (63). Increased susceptibility to salmonellosis among HIV-positive individuals is possibly due to both immunodeficiency and a deficiency in gastric acid secretion (77). Gastric acid deficiency allows Snlrnonelln entry past the stomach to the intestines and ultimately into the body (78-80). Judged from the amount of television advertising and the number of antacids found on pharmacy shelves, it is obvious that huge amounts of antacids are consumed each year. Omeprazole (PrilosecB) is advertised on television and prescribed as part of the format treatment for ulcers due to H. pylori infection and for common gastric acid reflux disorders. Omeprazole, like other potent antacids, predisposes the individual to salmonellosis (among other infections) by reducing the protective barrier offered by gastric acid secretions. Two cases of severe S. Enteritidis infections have been reported to have occurred in patients 4-5 weeks after the onset of omeprazole therapy (80). The difficulty with antacid use is

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that salmonellae survive at pH 4, whereas they die off rapidly at pH 2-3. Thus, normal gastric acidity is one of the mechanisms protecting the intestines from colonization (81). Chronic use of antacids makes it possible for salmonellae to pass through the stomach and into the intestinal tract. Once there, replication to large numbers occurs and the organism causes diarrhea and may invade the rest of the body. Not all fatal or complicated cases of salmonellosis involve the common serovars S. Typhi, S. Enteritidis, S. Dublin, S. Choleraesuis, or S. Typhimurium. Indeed, each of the many thousands of other serovars may cause serious illness. As just one example, Saltnonelkcl Weltevreden, certainly not a common isolate, caused a fatal salmonellosis in an individual whose condition prior to infection was one of relative good health. The infection was acquired during travel to Indonesia (82). Snln~or~ella infections can occur at any site in the body. Typically, the portal of entry is oral and invasion occurs at the ileum from whence the organisms travel and infect other sites. Sometimes the infections can occur in very unusual locations. Twelve cases of Salmonellu osteomyelitis of the skull have been reported. Two cases reported by one author involved extradural S. Typhimurium infections. Treatment included antibiotic therapy and surgical removal of the affected area (83). D.

Bacteruria

Twenty-eight cases of Salmonella bacteruria (bladder infections’)have been reported. Sixteen cases involved S. Enteritidis and 5 involved S. Typhimurium. Twenty-one patients were symptomatic and 7 were asymptomatic. Of the symptomatic patients, 16 hadcystitis, 3 pyelonephritis, and 2 suffered renal abscesses. Sixteen of the patients were immunocompromised, while the remainder had urological abnormalities (84).

E. AsymptomaticCarriage/NosocomialOutbreaks Chronic carriage poses a significant public health threat, especially when such carriage occurs in food handlers or hospital employees. We have already mentioned a typhoid fever outbreak at a resort hotel where orange juice became contaminated by a worker (37). In Italy an outbreak of SnlmonelZa Hadar occurred at a construction site canteen. In this large outbreak there were 448 symptomatic cases and 22 asymptomatic excretors of S. Hadar. The index case was a cook who had acute symptoms for 5 days before the onset of the episode (85). A nosocomial S. Enteritidis phage type 4 outbreak occurred in 1995. No common or continuing food source was located, but seven asymptomatic excretors of S . Enteritidis were identified among the patient population. The outbreak was likely due to personto-person transmission within the hospital itself (86). In another instance of nosocomial salmonellosis, fecal screening revealed a high carriage rate among the catering staff. The economic cost was staggering at 33,000 Pounds (approximately $70,000) (87). F. Multidrug-ResistantStrains Multidrug-resistant S . Typhinlurium has been around since the 1980s (88,89), and MDR strains of S. Typhi have been part of the typhoid fever landscape for more than two decades. These are in no way unusual. By the 1990s, 19% of the Typhi isolates in Britain were resistant to chloramphenicol and 92% of the isolates were resistant to trimethoprim,

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ampicillin, and gentamicin as well (3,46,90-92). Multidrug-resistant Typhi has found its way to the United States and overall where these isolates are not unusual (93-97). More recently a strain of S. Typhimurium known as DT104 has emerged, primarily in England, and found its way to the United States. DT104 stands for definitive type 104, and the organism is MDR to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracyclines (98). The prevalence of S. Typhimurium isolates with the five-drug pattern of resistance increased from 0.6% in 1979-1 980 to 34% in 1996 (98-100). The organism is associated with beef and other animals, including house pets (101-105).

G. Treatment Appropriate treatment for both acute salmonella enterocolitis and asymptomatic carriage is still unclear. Conventional wisdom argues against treating the acute infection except in cases where the infection translocates outside the guts. Antibiotics are not needed for treating uncomplicated Salmonellu enterocolitis. Fluid and electrolyte replacement are essential, but antimicrobials can usually be avoided except in high-risk patients (3,6). Bacteremia and focal infections indicative of invasion of salmonellae from the intestinal tract require antimicrobial therapy. Focal infections may also require surgery to remove the infected tissue and, in the case of aneurysms, to prevent a calamity (3,6). Chronic asymptomatic carriage of salmonellae after recovery from acute symptoms of salmonellosis is often difficult to treat. There has always been some concern about the effectiveness of antimicrobials for reducing the time to clearance of the carrier state. The effectiveness of antimicrobials for this purpose is somewhat problematic (106-1 lo), and it is generally heldthat long-term carriage rate is unaffected by such treatment (106,111-113). For treating systemic infection, effective therapeutics should be able to enter the phagocytic cells and have direct access to the microorganisms at their site of multiplication. Salmonella spp. penetrate into host cells and reside and even multiply there. A cure is achieved only when these intracellular bacteria are eradicated. Clearing the organism from the gut is not sufficient. Quinolones are accumulated in host cells and act upon bacteria in this peculiar intracellular environment. Ciprofloxacin is particularly useful because high concentrations may be achieved at the proper site. Clinical experience shows that in most instances a rapid amelioration of the acute disease is achieved. Thus, for microbiological, pharmacological, and clinical reasons ciprofloxacin is considered appropriate for the treatment of systemic infections with Salmonella spp. (114). However, studies of ciprofloxacin use in acute diarrhea showed that it did not reduce pathogen carriage after the acute episode (1 15,116). Nonetheless, there is some evidence that newer quinolones can work effectively (1 17). Oral doses of perfloxacin appear to have cleared the organism from affected children (117). Fifteen children who were excluded from school or nurseries due to asymptomatic convalescent-phase nontyphoid Salmonella carriage were treated with perfloxacin, and all were culture negative within 10 days of treatment onset (118). Overall the fluoroquinolones appear to shorten the duration of treatment. Nonetheless, two potential pitfalls have to be kept in mind: the risk of selecting for quinoloneresistant mutants and the risk of enhancing persistent carriage (1 19). Yet another pitfall is that quinolones are generally contraindicated in children and pregnant women because they may injure developing bones (22). This problem is not insurmountable. Perfloxacin may be especially useful in children (120). Moulin et al. (120) studied 42 patients (21 girls and 21 boys) aged 1 month to 12 years (mean 3.3 years) who were admitted to an

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hospital for severe Salmonella infections. Some of these patients had recovered from an initial acute episode of enterocolitis, became asymptomatic carriers, and then suffered a relapse of acute symptoms. Twenty-five of the patients were given perfloxacin. Among perfloxacin-treated patients, diarrhea and fever disappeared within 2 days of treatment. None developed side effects following its administration. Perfloxacin may be an alternative choice for treating severe Salmonella infections in children. It cleared the acute infection and reduced by 50% the number of asymptomatic carriers. Another report of the use of perfloxacin concluded that it is effective at eliminating the carrier state (121). Similarly, there continue to be encouraging studies of various new forms of the quinolones (122). Hof and Kretschmar (1 14) wrote, ‘‘One prerequisite for an effective treatment of a Salmonella enteritis with antibiotics is a reliable direct antibacterial activity of the drug. In comparison to most of [sic] all other usual antibiotics quinolones, especially ciprofloxacin, fulfill these conditions, because its activity is high and resistance of Salmorzella against quinolones is extremely rare. Since quinolone resistance is never plasmid coded, there will be even in the future no obvious risk of spreading of resistant strains.” Unfortunately this statement is not quite correct. Bacteria do develop resistance to fluoroquinolones. The parent compound affects DNA gyrase, and it is possible to develop mutations in the gyrA (123) and gyrB (124) genes, which increase resistance to nalidixic acid and the quinolone antibiotics in general (125,126). The reality that resistance to the quinolones is both possible and has already occurred in nature is fundamentally a scary one. A six-drug multiresistant organism, e.g., DT104 with quinolone resistance, would be extraordinarily difficult to treat because there are few if any antimicrobials left to which the organism might respond. For this reason it is important to monitor resistance patterns in new isolates and to control the spread ofany strains containing resistance to all six antimicrobials. Unfortunately, the prediction that fluoroquinolone resistance may become a problem is well founded. In England, DT104 started out as a problem in cattle but then rapidly spread to other livestock, including pigs and chickens. The use of olaquinodox in pig farms (in the United Kingdom since 1982) and enrofloxacin in poultry led to the appearance of DT104 isolates resistant to ciprofloxacin (98). Fourteen percent of the five-drug-resistant isolates were resistant to a fluoroquinolone (ciprofloxacin) (127,128). Other examples of six-drug-resistant strains have been reported (129).

H. Arthritis When bacteria find their way to a hostile environment, e.g., the high oxidative environment within phagolysosomes, they produce stress response proteins. Salmonella normally synthesize more than 30 proteins within the phagolysosome of a macrophage in response to the intracellular environment conditions (1 30-132). These proteins are involved in postinfection reactive arthritis. Reactive arthritis is a documented sequel to Salrnorlella infection that results from an inappropriate immunological response to Salmonella proteins. Typically, the illness starts a few days after an episode of enterocolitis, usually within less than 3 weeks from the onset of the gastrointestinal disturbance, and it tends to afflict those patients in whom the diarrheal symptoms persisted longer than usual. Reactive arthritis tends to afflict patients with a particular genetic predisposition-the HLA-B27 tissue type. Patients who develop reactive arthritis typically have mononuclear cells present in their synovial fluid. These cells react with a proliferative response to particular 65 kDa heatshock proteins (environmental stress response proteins) and to the antigens present on heat-killed Salmonella (3).

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The underlying immunological mechanisms responsible for reactive arthritis are unknown. Theories have included such ideas as anti-Salmonella antibodies reacting directly with the tissues, bacterial antigen initiation of proliferative responses, altered T-cell responses, changes in cytokine production within the joint, and so on. There is to date no satisfactory explanation of the role of the HLA-B27 tissue type, although several theories have been propounded and explored. Reactive arthritis triggered by salmonellosis is occurring more often than in the past, but the long-term prognosis for patients is unclear (133). Postinfection immunopathological reactions to salmonellosis may occur at sites other than the joints, for example, uveitis (inflammation of the vascular middle coat of the eye), iritis (inflammation of the iris of the eye), and urethritis (inflammation of the urethra). Postinfection reactions may include Reiter's syndrome, which is a triad of urethritis, conjunctivitis (inflammation of the conjunctiva of the eye), and arthritis. Reiter's syndrome is probably caused by an abnormal immune reaction to infectious agents (134). A retrospective study of patients hospitalized for salmonellosis at one facility during a 15-year period (1970-1986) found 63 patients with salmonellosis-related arthritis-of these 88% were HLA-B27 positive (133). The relationship between salmonellosis and arthritis is evident from a study in which 919 individuals suffered enterocolitis after eating at a single restaurant. All were sent questionnaires, 321 of which were returned. Twenty-three respondents reported persistent joint symptoms. Joint symptoms were reported most frequently by persons who suffered a diarrhea of long duration. Of these 23 respondents, 66% reported the extra-articular symptoms of Reiter's syndrome (135). Although the relationship between salmonellosis, reactive arthritis, and Reiter's syndrome seems well established, efforts to compare the presence of anti-S&zonelZa antibodies in the serum of patients and controls have failed to show a difference in the prevalence ofanti-SulnzonelZa antibodies between the two groups (136,137). Nor is the mechanism by which salmonellosis causes reactive arthritis atall clear, especially with regard to the role of the HLA-B27 antigen. The humoral response, as just mentioned, does not seem to explain the immunopathogenesis, but bacteria-specific T cells have been consistently found in the synovial fluid of individuals with reactive arthritis ( l 38). These cells may function as antigen-presenting cells, and antigen presentation by T cells may modify the antibacterial immune response (138). There is considerable circumstantial evidence that nlicrobial antigens locally present within the inflamed joints have a key role in the etiology of reactive arthritis. Synovial fluid has been found to have a powerful microbicidal activity, which likely accounts for the general failure to recover viable bacteria from the synovial fluid of individuals with reactive arthritis (139). Indeed, it is not possible to recover viable bacteria, and PCR studies have shown that bacterial DNA is not present in the joints of reactive arthritis patients. These two observations suggest that the disorder is essentially one of autoimmune dysfunction (140). Notwithstanding the above, it has long been speculated that persistence of causative microbes or their structures within the body is part of the mechanism of reactive arthritis. It has been postulated that HLA-B27-positive individuals develop complications eliminating the microbe after initial infection. There is some experimental evidence for this. It has been shown that the expression of HLA-B27 in cultured U937 cells does not influence their uptake of S . Enteritidis, but it remarkably impairs the elimination of the organism: "The impaired elimination of ReA-triggering microbes by HLA-B27+ monocytes may explain the persistence of ReA-triggering microbes in susceptible HLA-B27 + individuals" (141). S. Enteritidis survives within mouse fibroblasts transfected with HLA-B27, suggesting as above that the bactericidal effect is impaired (142). Further, nitrous oxide

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production is reduced in cells transfected with the HLA-B27 gene. All of this suggests a persistence by intracellular salmonellae in the HLA-B27 individual that does not occur in others. However, experimental models are extremely artificial, and as mentioned above there is no evidence of bacterial persistence, at least in the joints, which is where the pathology occurs (140). Another interesting hypothesis concerning reactive arthritis suggests that the pattern of cytokines normally present in the joints is somehow disturbed. Mononuclear cells were recovered from synovial fluid and assayed for interferon-?, tumor necrosis factor, interleukin-10, and interleukin-4. The cells were then stimulated with bacteria, and it was observed that stimulated cells secreted low amounts of interferon-y and tumor necrosis factor-a, but high amounts of IL-10. Exogenous IL- 12 enhanced interferon-? and tumor necrosis factor secretion. It was suggested that IL-10 and IL-12 secretion is involved in the regulation of the cytokine pattern in patients with reactive arthritis (143). In brief, there are many scientifically “sexy’ ’ ideas about the pathophysiology of reactive arthritis. In reality, there is little confirmatory evidence to support anyof the numerous hypotheses. In a classic understatement, it has been written that “the association between arthritis-causing bacteria and HLA-B27 positive cells is a complex event” (144).

W. MOUSE MODEL From experience with AIDS patients and others with disturbed immune function, it is known that natural resistance to Scdmo~zellnrequires normal cell-mediated defenses (63,77,145). T-cell defects, tnacrophage defects, neutrophil defects, metabolic defects of immune regulation, cytokine physiology, and atypical arachidonic acid metabolism all increase the chances that an individual will contract a systemic Sdnzorwlla infection. These phenomena are best studied with the mouse model, though it has well-recognized limitations. The dogma that the mouse has to be the model for all types of Salmonella infections, including unfortunately typhoid fever, has overstayed its usefulness. It is a poor model for studying diarrhea, which is best studied in other animal models (53,146). Medline@and Biosis‘@data bases searched with the Dialog“’ System search terms (mouse or mice) and (salmonellosis or Snlmonella) located 5455 and 6030 citations, respectively, from the 1960s through July 1998. These manuscripts deal with an extraordinary array of subject matter related to salmonellosis in the mouse: susceptibility and resistance factors, bacterial virulence factors, immunity, and vaccine development. One reason the mouse model is used so extensively is that no animal other than humanbeings contracts true typhoid fever [one abstract indicates infection of swine with S. Typhi (147j1. While the mouse is relatively resistant to S. Typhi, it is highly susceptible to some strains of S. Typhimurium. When infected with S. Typhimurium, the mouse develops an illness quite similar to human typhoid fever, murine typhoid fever. (For an excellent description of the mouse typhoid fever model and a presentation showing a correlation between in vivo reactions to various S. Typhimurium strains and an in vitro model of invasiveness, see Ref. 148.) From this manuscript we note that only a few cells of virulent S. Typhimurium are sufficient to cause murine typhoid fever, whereas avirulent strains of S. Typhimurium do not become lethal until mouse challenge doses are as high as lo7 cells per mouse. Similarly, S. Typhi does not kill mice until the challenge dose is in the 106-107 range ( 149).

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Human experience, veterinary medical experience, and the mouse model show that the general events involved in host resistance to salmonellae and the pathogenesis of salmonellosis are extremely complex. Effectively reviewing and organizing this literature is a monumental task and beyond the scope of this manuscript. The extensive mouse model literature contains highly complex experiments. conflicting and confounding data, and is almost ''intellectually indigestible." Still, the information obtained from mouse experiments paves the way for treatment strategies, for an understanding of pathophysiological events, and (most importantly) for development of prophylactic or preventive methodologies-vaccine development. Using the mouse model, we have learned a great deal about survival of salmonellae in the gastric environment, growth and replication of salmonellae within the gut (150), adhesion of salmonellae to intestinal epithelial cells, and interactions between salmonellae and the intestinal mucosa. We have learned about intracellular transport of salmonellae across the epithelial lining (151), toxin production, and numerous virulence factors (131,132,152-161). There exist a smorgasbord of citations relevant to the above subject matter, mostly citations of murine experiments, but a few from human or nonmurine studies (50-52,7881,150.156,162-204). Other subjects investigated with the tnouse model include, but are certainly not limited to, transport of salmonellae from the gut throughout the body by way of the circulatory system, growth and replication within phagocytic cells, intracellular residence and replication within nonphagocytic cells, and safe sites where salmonellae hide from host defenses (27,177,178,205-238). The mouse model has shown that the general series of events involved in resistance to salmonellosis include acid inactivation of ingested organisms within the gastric environment and the hostile intracellular environment of phagosomes (239,240), presence of secretory immunoglobulins within the lumen and wall of the intestines (241-243), chemotaxis of phagocytic cells to the site of infection (188,244,245), translocation and intracellular killing of internalized salmonellae (206,225-227,246,2471, antigen processing (233,248), T-cell responses and cell-mediated immunity (168,223,249-264), and humoral immune responses (168,252-254,256259,261,265,266). Lessons learned from studies of murine salmonellosis have led directly to significant developments in typhoid fever and nontyphoid salmonellosis prophylaxis, i.e., modern vaccines.

V.

VACCINES

Perhaps the most useful application of the mouse model has been in the realm of vaccine development. Mouse, animal, and human clinical experience have revealed that live-attenuated vaccine strains of salmonellae usually provide better protection than either killed salmonellae or purified antigens (3,267,268). Many experiments have been conducted to determine the relative roles of humoral and cell-mediated responses in resistance to salmonellosis (see citations immediately above). The consensus opinion is that both forms of immunity are important and active immunity is best accomplished with a vaccine that stimulates both humoral and cell-mediated phenomena. This goal is best accomplished with a live-attenuated Snlnlonellcl vaccine strain. The primary advantage of live-attenuated vaccines is that the live organisms are transported from the gastrointestinal tract into the body, where their presence initiates a strong immune response. The secondary advantage of live-attenuated vaccines is that

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mechanical injection is not necessary-the individual merely swallows the vaccine. This advantage is obvious for humans, who find injections objectionable and who sometimes suffer adverse responses at the injection site. Oral dosage is also a labor-saving advantage in agricultural practice, where injection is a costly procedure compared to adding a substance to drinking water or to feed. Currently there are at least two vaccines against typhoid fever on the market in the undeveloped world. One is a live attenuated vaccine and the other a purified injectable form ofVi antigen. To understand these vaccines it is best to first look at the parallel situation in the animal and agricultural world. Once vaccines are discussed in that context, it will be easier to understand the status of typhoid fever vaccines. Also, it must be noted that in the developed world there are too few human salmonellosis cases to justify mass immunization of the population. Vaccination is primarily appropriate for agricultural use. In nations with inadequate hygiene and public health systems the situation is reversed. The primary need is for vaccines suitable for human use against typhoid fever and nontyphoid enteric fever. Salmonellosis is an economic problem for segments of the agricultural industry. Turkey producers sometimes suffer economic losses due to salmonellosis in newlyhatched turkeys (269,270). Pigs are susceptible to salmonellosis and often develop systemic salmonellosis affecting growth and weight gain, etc. Pigs are especially susceptible to the serovar Choleraesuis. Pigs are also potential sources of salmonellosis for the consumers of pork products. The reason that pigs are not a primary human source of salmonellosis is probably that most people understand pork must be well cooked. Dairy cows may be infected with S. Dublin, but routine pasteurization usually protects the general public from this particular serovar (3). The most important agricultural need for Snlmorzelln vaccines is in the chicken industry. Broiler chickens are common sources of salmonellae, which find their way to the human food supply. Chickens themselves are very susceptible to the SnZw1onelln Gallinarum and S. Pullorum. S . Enteritidis infections in laying hens provide an active source of S. Enteritidis-infected table eggs that are a direct health hazard to the human community. A live-avirulent vaccine that prevents SnlnzoneZla Enteritidis infection of laying hens has been described, and this subject has been thoroughly reviewed (271). The usefulness of live-avirulent (live-attenuated) vaccines is based on three properties of the vaccine strain: an ability to invade from the gastrointestinal tract, an ability to initiate an effective immune response, and an assured inability to uncontrollably replicate within the body-avirulence. To achieve these goals, microbial geneticists developed avirulent vaccine strains containing double deletion mutations. Deletion mutations are irreversible, and the presence of two mutations assures that virulence cannot be regained by reverse mutation. The specific mutations were carefully chosen to assure that they adversely affected the ability of the vaccine strain to repeatedly replicate in animal or human tissue. The mutations introduced two requirements for exogenous metabolites-p-aminobenzoic acid and dihydroxybenzoic acid (272,273). As these are not present in host tissues, viable cells will invade and replicate for a few rounds but will not survive long within the host (274-280). These ’ ‘aromatic-deficient” (AnroA) strains had the distinct advantage that molecular geneticists could introduce foreign antigens into the vaccine strain. The vaccine strain was then able to serve as a carrier of additional antigens. Hence it became possible to simultaneously immunize against salmonellosis and another agent (254,25631-286). Through the years there have been a number of studies of AnroA vaccines in chickens (166,287-292). Results have not always been consistent. Some results have been very

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positive. Findings have included development of solid humoral immunity (287), reduced fecal shedding (288), protection against gut colonization and organ invasion (289), and serotype-specific responses (290). Serotype specificity was confirmed by Methner et al. (291), but protection against fecal shedding was not confirmed (292). Investigations of the properties of AaroA double deletion strains have continued for nearly two decades since the original work was published. During this time a competing vaccine strain was developed (174,27 1,293-295). The basic concept is the same, but the deleted genes are different. Adenylate cyclase and cyclic AMP receptor protein genes are deleted-Acyu, Acrp. This particular avirulent strain appears to be more efficacious in chickens than the AaroA, although this issue of efficacy is not entirely clear, Assuming the correctness of the assertion, it is both obvious and rational that part of the art of creating genetically altered vaccine strains is maintenance of immunogenicity for the target host species. As is the case with AaroA strains, Acyn, Acrp tnutants can be constructed containing DNA coding for unrelated antigens (202,255,256,296-298).

A. Vaccination to Prevent Typhoid Fever Early efforts to develop an attenuated or avirulent vaccine strain against S. Typhi did not involve high-tech genetic manipulations. Instead, random mutations were introduced into salmonellae with general mutagens, e.g., nitrosoguanidine. Virulence, protective activity, and the stability of avirulence was assessed with mouse virulence assays. While avirulent strains were readily obtained, avirulent strains generally contained multiple mutations that adversely affected properties such as intestinal colonization, invasion, and immogenicity. Sometimes the mutations responsible for avirulence were spontaneously reversible, i.e., the avirulent strain could regain virulence. So for these reasons, random mutagenesis did not appear appropriate for vaccine development. Nonetheless, the first live-attenuated S. Typhi strain fully developed for human vaccination against typhoid fever, comtnercialized, licensed, and actually put into widespread use was obtained by nitrosoguanidine mutagenesis.

B. Gal E Mutants: A Live-Attenuated Vaccine Smooth strains of S. Typhi are virulent and highly immunogenic in man. Similarly, smooth strains of S. Typhimurium tend to be more virulent than rough strains in the mouse typhoid fever model. It has been possible to expose a virulent wild-type strain of S. Typhi, Ty2, to a general mutagen and recover bacteria with mutations at multiple genetic loci. These cells have unusual phenotypic properties that lend themselves well to the specific purposes of a typhoid fever live-attenuated vaccine. They are known as gal E mutants (149,299). The gal E mutants are avirulent, but become virulent and immunogenic in the presence of galactose. When exposed to galactose, gal E strains synthesize a cell envelope component, which they cannot make in the absence of exogenous galactose and which contributes to virulence. However, the gal E mutants also have a very limited ability to survive in the presence of galactose. In practice, an avirulent strain is given as a vaccine. In vivo, the organisms are exposed to galactose. They then synthesize a cell wall component that is highly immunogenic. Indeed, these cells would also be virulent. However, as the cells accumulate galactose in vivo, they are killed by bacteriolysis: "the properties of the gal E in vivo are dependent upon two tnechanisms acting in opposite direction: a virulence-

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and immunogenicity-increasing biosynthesis of cell wall lipopolysaccharide and a vimlence-lowering galactose induced bacteriolysis” (299). In 1975 Germanier and Furer (299) reported that they were able to introduce the galactose epimerase mutations into a wild-type strain, S. Typhi Ty2. The mutantwas named Ty2 l a, and its safety and efficacy as a potential vaccine strain were initially evaluated using the mouse model of typhoid fever and the “mouse protection test.’. Mice were immunized with 10‘ viable Ty2 la, and it was observed that the vaccine organisms were cleared from their livers and spleens within 3 days. In contrast, Ty2 was still present in mouse tissues 15 days after inoculation even though the initial dose contained only lo4 viable cells. Four weeks after this initial ilnmunization with Ty2la, the tnice were challenged with an i.v. injection containing 5 X lo7viable Ty2 cells. (Remember that while S. Typhi is not especially virulent in mice, very high doses will cause mortality.) The vaccinated mice survived this high challenge dose and an even higher subcutaneous challenge (149). Ultimately Ty’la became a successful human vaccine strain (300). Interestingly, though the above explanation for the effectiveness and avirulence of gal E is frequently cited in the literature, the explanation is neither complete nor accurate. It turns out that while Ty2la has a mutation in gnl E , and while the nature of the mutation appears to adequately explain the avirulence of this organism, gal E mutations may not be the reason for the strain’s attenuation (301). Other potential typhoid fever vaccines have been developed by genetically altering Ty2, Ty21a, and by transferring virulence genes from Ty2 to avirulent S. Typhimuriunl vaccine strains. In general, it is too early to know how successful these will be as substitutes for Ty2la. Further, there are several properties of Ty2la that have contributed to its success as a vaccine, and it is not totally clear that thevarious modern genetic constructs can do as well as the original gcd E mutant.

C. Genetically Engineered Typhoid Fever Vaccine Strains Two genes are involved in the production of Vi antigen, via A and vin B. Some serovars of Snlnzonella besides S . Typhi contain only one or the other of these genes but not both and hence are Vi negative. The S. Typhi vic~B gene from Ty2 has been cloned into an S. Typhimuriunl Acya Adelto crp, which is an attenuated mutant of S. Typhitnurium. This recombinant was able to produce the Vi capsular polysaccharide and was also able to colonize the small intestines of mice and to invade through the mesenteric lymph nodes. Mice treated with the recombinant developed secretory antibodies to the Vi antigen and delayed hypersensitivity reactions by footpad testing. Mice were completely protected against challenge doses of virulent S. Typhi Ty2 (302). Ty541 and Ty 543 are nroA deletion mutants derived from Vi-positive and Vinegative strains of S. Typhi. Thirty-seven adult human volunteers received inoculations with doses as high as 10“’ vaccine organisms, and these individuals developed strong cellular immune responses. However, the humoral responses were weak. Additional deletion constructs have been developed (300). Genes involved in the regulation of virulence have been disrupted (303-306). Alteration of transcriptional regulator (PhoP) and environmental sensor (PhoQ) genes inhibits appendage formation necessary for invasion of the intestinal epithelium (307). After deleting the Phop/PhoQ virulence genes from S. Typhi Ty2, the resultant strain was a live attenuated typhoid fever vaccine believed suitable for testing in a limited number of hu-

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mans. Volunteers did not develop bacteremia or side effects and did develop solid IgA responses to S. Typhi lipopolysaccharides. Volunteers also developed IgG against S. Typhi. Humoral immune responses were measured and compared with those of control vaccinees who received four oral doses of S. Typhi Ty21a. In the most sensitive assays, 9 of 11 volunteers and 5 of 8 Ty2la control vaccinees had evidence of IgG directed against S. Typhi antigens. Ty800. the above genetic construct, apparently is safe and highly immunogenic in humans (308,309). There are many parallels between the requirements for typhoid fever vaccines and cholera vaccines. Many of the same genetic tricks have been applied to the development of oral attenuated vaccines against typhoid fever and cholera. S. Typhi Ty2la and Vibrio choleme CVD 103-HgR have been licensed for human use. Many studies have been conducted concerning the best way to deliver these, to store them, and to evaluate if they can be co-administered. The Ty2la and CVD 103-HgR (V. cholerae) vaccines have been coadministered to humans, alone and in combination with antimalarial drugs, polio vaccine, and yellow fever vaccine. Antimalarial drugs reduced the efficacy of the cholera vaccine but notthe typhoid fever vaccine and both vaccines could safely be co-administered (310-313). There are tnany other genetic constructs of various S. Typhi and S. Typhimurium strains that have been reported as avirulent and immunogenic. But a host of factors must be considered before any of these can be placed in prelitninary human trials and move on to field trials in the undeveloped world. An excellent review of the general subject has been written (300).

D. ViAntigenVaccines Although the bulk of research effort and practical experience has involved the live attenuated vaccine strain TY21a, some forms of typhoid fever vaccines do not employ live attenuated strains. Rather, purified Vi antigen is utilized as the immunogen. The earliest efforts to use Vi antigen as a vaccine immunogen occurred in 1954, but the highly purifiedVi antigen used inthis early study did not protect human volunteers. It was later found that the antigen had become denatured during manufacture. After manufacturing methods were changed to produce a nondenatured Vi antigen, there was anactive review of all studies involving the newly licensed vaccine composed of the Vi capsular polysaccharide. The Vi polysaccharide vaccine became licensed outside the United States, and studies were conducted among populations from the undeveloped world. Twenty-five micrograms of the purified polysaccharide was given by intramuscular injection, and it was observed that the vaccine was well tolerated. An antibody response lasting 3 years occurred in about 90% of subjects including children as young as 2 years of age. The Vi polysaccharide vaccine compared well with other typhoid vaccines with regard to safety, immunogenicity, and efficacy (3 14). Vi polysaccharide is a well-standardized antigen that is effective in a single parenteral dose, is safer than whole-cell vaccine, and may be used in children 2 years of age or older. Safety and efficacy were evaluated in at-risk populations, and a commercial Vi antigen vaccine was licensed for use in Chile, the Congo, Ivory Coast, Korea, the Netherlands, Peru, Philippines, Togo, and the United Kingdom. The Vi antigen has been conjugated to tetanus toxoid, diptheria toxoid, and cholera toxin in an effort to both increase cell-mediated immunity and co-vaccine against other important diseases (300).

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Initial trials indicated that purified Vi antigen vaccines protected humans for about 17-21 months, at which time surveillance was stopped. Additional follow-up studies showed that anti-Vi antibodies could be recovered from vaccinated subjects 3 years after vaccination. Repeat vaccination caused some additional local and systemic side effects. In efforts to improve the Vi polysaccharide vaccine, preparations consisting of a conjugate of the Vi polysaccharide and either heat-labile E. coli toxin or Pseudomonas aeruginosa exoprotein A were examined. These conjugates elicited higher antibody levels than the native Vi polysaccharide (3 15). The primary difficulty with Vi polysaccharide vaccines is the fact that there are Vi-negative strains of S. Typhi that are virulent. Even allowing for this problem, the Vi polysaccharide vaccines are not as efficacious as Ty2 1 a. The Ty2la protective effect lasts longer and protects a higher percentage of participants. Considering the disadvantages and inconvenience of an injectable vaccine versus an oral dose (Vi polysaccharide vs. Ty2la), the apparent current edge is toward oral administration of the Ty2la (26).

VI.

CONCLUSION

Typhoid fever, nontyphoid enteric fever, and Salnzonella enterocolitis are extraordinarily important human diseases. Each is acquired by ingestion of salmonellae and stands as a foodborne illness. Although these various forms of salmonellosis are strikingly similar to each other, there are important distinctions with regard to transmission, source of infective material, nature of the disease, preventive measures, vaccination potential, treatment, and sequelae. It is all too easy to overlook these differences and think of salmonellosis as a single disease caused by identical bacteria, Sdmonella choeraesuis (Salnlonella erzterica). In actuality the nature of salmonellosis is determined by numerous host factors, the infecting serovar, and an array of virulence factors which may or may not be present in any given serovar. Treatment varies according to the specifics of the disorder and the antimicrobial resistance patterns of the offending Salmonella strain. Similarly, the consequences and sequelae of an episode depend on the nature of the offending Sulmonella strain and host factors. Salmonellosis remains an important and common human illness, even in developed countries with well-developed food and water hygiene measures.

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