Pathogenic Escherichia coli in Latin America

Pathogenic Escherichia coli in Latin America  

Editor ALFREDO G. TORRES

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CONTENTS Foreword

i

Preface

ii

Contributors

iv

CHAPTERS 1. Overview of Escherichia coli A.G. Torres, M. Arenas-Hernandez and Y. Martinez-Laguna

1

2. Evolution and Epidemiology of Diarrheagenic Escherichia coli N. Williams, A.G. Torres and S. Lloyd

8

3. Enteropathogenic Escherichia coli (EPEC) T.A.T. Gomes and B. Gonzalez-Pedrajo

25

4. Enteroaggregative Escherichia coli F. Navarro-Garcia, W.P. Elias, J. Flores and P.C. Okhuysen

48

5. Shiga Toxin Producing Escherichia coli B. Guth, Valeria Prado and M. Rivas

65

6. Enterotoxigenic Escherichia coli J. Flores and P.C. Okhuysen

84

7. Detection and Subtyping Methods of Diarrheagenic Escherichia coli Strains R.M.F. Piazza, C.M. Abe, D.S.P.Q. Horton, E. Miliwebsky, I. Chinen, T.M.I. Vaz and K. Irino

95

8. Clinical Management of Escherichia Coli Cases (The Latin America Experience) H.A. Repetto

116

9. Host Responses to Pathogenic Escherichia coli C. Ibarra and M. Palermo

122

10. Diarrheagenic Escherichia coli in Argentina M. Rivas, N.L. Padola, P.M.A. Lucchesi and M. Masana

142

11. Escherichia coli Situation in Brazil B.E.C. Guth, C.F. Picheth and T.A.T. Gomes

162

12. Shiga Toxin Producing Escherichia coli in Chile R.M. Vidal, A. Oñate, JC. Salazar and V. Prado

179

13. Epidemiology of Diarrheagenic Escherichia coli Pathotypes in Mexico, Past and Present A. Navarro and T. Estrada-Garcia

191

14. Diarrheagenic Escherichia coli in Children from Uruguay, Colombia and Peru G. Varela, O. Gomez-Duarte and T. Ochoa

209

15. Escherichia coli Animal Reservoirs, Transmission Route and Animal Disease A.F. Pestana DE Castro, A. Bentancor, E.C. Mercado, A. Cataldi and A.E. Parma

223

16. Host-Pathogen Communication M.P. Sircili, C.G. Moreira and V. Sperandio

249

17. Future of Escherichia coli Research in Latin America T.A.T. Gomes, C. Ibarra, F. Navarro-Garcia, M. Palermo, V. Prado, M. Rivas and A.G. Torres

256

Index

262

i

FOREWORD In November 1946, Gerardo Varela, the most prominent Mexican bacteriologist of his time, published a paper in the bulletin of the Children’s Hospital in Mexico City [1] describing the identification of a “new” type of Escherichia coli isolated from a child who had died from a severe diarrhea in the same hospital. His collaborators were Alejandro Aguirre, a young pediatrician, and Julio Carrillo, who had personally performed the microbiological studies during the autopsy and who had isolated the bacteria and kept it for further studies. A few months earlier, Bray had published similar results from a group of children in the Great Britain who were dying from diarrhea in a town in England [2]. Considering that the Second World War had just ended, there was no communication between these two groups at the time. However, once published, a heated discussion began on both sides of the Atlantic with most people reluctant to accept that a bacterium like E. coli, which until then had been considered as an organism that was not associated with disease, could be the cause of these children’s deaths. Although, both Bray and Varela were able to show that the serum obtained from the children infected with these putative pathogens were able to agglutinate the bacteria in vitro, Varela’s approach went a step further. One of his main interests was to study the cross reactions between different enteric organisms when tested against an antiserum raised in rabbits. For these particular assays, he primarily used antisera prepared against somatic and flagellar antigens of different types of Salmonella, which was the dominant pathogen of interest at the time. As reported in his publication [1], an antiserum prepared against the somatic antigen of Salmonella adelaide was able to agglutinate the E. coli isolated from the feces of the child who had died. A similar reaction was found with another E. coli isolated from a different child who had also died from severe diarrhea, and from a cook working in the kitchen of the hospital who had been sampled during a study to determine sources for such infections. The cross reaction tests allowed Varela and his colleagues to demonstrate that these E. coli, usually considered as a part of the normal intestinal flora, were somehow different from other E. coli found in feces from humans without diarrhea. These two seminal studies in the mid-40’s led to major discussions about the role of E. coli as a pathogen, while remaining the most modest inhabitant of the intestine of humans and animals. The discussions prompted a deluge of new research in laboratories around the world that in one form or another had found similar results. Over the next few years, groups in Britain, the United States, Brazil and Mexico sent strains of E. coli isolated from children with severe diarrhea to Copenhagen, where Fritz Kauffmann had set up a serological system in his laboratory to identify the somatic and flagellar antigens of these organisms. The most interesting finding that comes out of Kauffmann’s serological studies was that E. coli isolated in different parts of the world was restricted to a few somatic and flagellar antigen combinations, called serotypes. All of these initial studies provided the catalyst for a whole new field of research that over the past 60 years has allowed us to understand how bacteria interact with intestinal cells, and how they are able to cause diarrhea. Researchers, either born or working in Latin America, have contributed extensively and consistently to this field over the years. Under the dynamic leadership of Alfredo Torres, who has been able to convince and cajole his friends working all over Latin America to put into writing their most recent work, this unique and interesting volume follows the tradition started by Varela and others in the 1940’s and shows the developments made since those early days in the scientific and clinical study of E. coli. I am sure that this book will help us involving in teaching microbiology and infectious diseases, and I hope it will encourage new questions and better answers in a field that, in spite of improved knowledge and increased understanding, is still looking for the necessary tools to prevent young children from dying from diarrhea around the world.

Alejandro Cravioto, M.D. International Centre for Diarrhoeal Disease Research Dhaka, Bangladesh

ii

REFERENCES [1] [2]

Varela G, Aguirre A, Carrillo J. Escherichia coli-Gomez, nueva especie aislada de un caso mortal de diarrhea. Bol Med Hosp Inf Mex 1946; 54: 623-6. Bray J. Isolation of antigenically homogenous strains of Bact. coli neapolitanum from summer diarrhea of infants. J Pathol Bacteriol 1945; 57: 239-47.

iii

PREFACE In 2009, during a session at the 7th International symposium on Shiga Toxin (Verocytotoxin) – Producing Escherichia coli Infections in Buenos Aires, Argentina, I was sitting at the back of the auditorium and realized that a large proportion of the attendees were young Latin American students, postdoctoral fellows and investigators, and many of them were participating for the first time in an international meeting where the world experts in pathogenic E. coli research discussed the “state of the art” in the field. I also observed that many of them were current and former trainees of Latin American laboratories and institutions with a long tradition in E. coli research, and those laboratories have not only contributed to the understanding of Shiga toxin-producing E. coli infections, but played a pivotal role in the identification and characterization of other categories of pathogenic E. coli. At that moment, I realized that it was the to organize a group that helps promoting the research of the scientist in this region and as a first task to write a comprehensive text on pathogenic E. coli summarizing and reviewing the accumulated knowledge generated by these Latin American investigators, which had make a significant impact on our understanding of these important human pathogens. In the first 10 years of the 21st century, the different categories of pathogenic E. coli have been reviewed extensively in review articles and some books, representing the vast body of literature on this bacterium, making pathogenic E. coli the best reviewed organism in the field of bacterial pathogenesis and infectious diseases. Even though, thousands of investigators around the world have been studying different aspects of the pathogenic attributes of E. coli for more than 60 years, pathogenic E. coli remains an important cause of diarrhea and death in infants in developing countries. Intestinal infections caused by E. coli remain as an important health problem in all Latin American countries and there was a need to publish an overall review of all the studies conducted in this region that have shown, the appearance of serotypes not previously associated with disease and the evolution of some categories of E. coli, which have become the predominant pathogenic E. coli in some of these countries. This body of knowledge produces by these investigators needed a critical review that was comprehensive and integrate all the different countries and all the researchers. Why this book is different from other books which are already published on pathogenic E. coli? Most prior volumes concentrated on the basic and clinical research progress performed by laboratories in North America, Europe, Australia or Japan, and none of them covered the situation in Latin America. The book “Pathogenic Escherichia coli in Latin America” is a unique, comprehensive analysis of the most common categories of E. coli associated with diarrheal illness in Latin America. The aim of the book is to allow leading investigators in this region to discuss molecular mechanisms of E. coli pathogenesis followed by chapters on diagnosis, clinical management, host immune responses, animal reservoirs and epidemiology. In addition, the authors discuss the current situation of E. coli in representative countries, including Argentina, Brazil, Chile, Colombia, Mexico, Peru and Uruguay. This ebook presents timely and vital information to understand the current work on pathogenic E. coli in Latin America and presents future research in this region. The book is divided into 17 parts. The first 2 parts introduce the foundations of E. coli and the evolution and epidemiology associated with this pathogen. Parts 3-6 review the 4 most important categories of intestinal pathogenic E. coli in Latin America. Parts 7-9 are an overview of the current knowledge regarding diagnosis, clinical management and host responses to E. coli. Part 10-14 present the current situation of E. coli infections in 7 Latin American countries. Part 15 discuss the animal reservoirs, transmission and animal disease. Part 16 introduce a relative new area of investigation regarding communication mechanisms between host and pathogen. Finally, part 17 is an assay by top investigators in the region discussing future directions of E. coli research in Latin America. I hope this book becomes a useful textbook for current and future generations of investigators and serves as a reference for the E. coli community to understand the past and present of research in Latin America.

Alfredo G. Torres, PhD Galveston, Texas

iv

CONTRIBUTORS Cecilia M. Abe

Laboratório de Bacteriologia, Instituto Butantan, São Paulo, SP, Brazil

Margarita M.P. Arenas-Hernandez

Centro de Investigaciones en Ciencias Microbiológicas. B. Universidad Autónoma de Puebla, Puebla, México

Adriana Bentancor

Universidad de Buenos Aires, Argentina

Angel Cataldi

INTA-CONICET, Argentina

Isabel Chinen

Servicio Fisiopatogenia, Departamento de Bacteriología, Instituto Nacional de Enfermedades Infecciosas “Carlos G. Malbrán”, Buenos Aires, Argentina

Alejandro Cravioto

ICDDR,B, Dhaka, Bangladesh

Waldir P. Elias

Laboratory of Bacteriology, Instituto Butantan, São Paulo, SP, Brazil

Teresa Estrada-Garcia

Department of Molecular Biomedicine, CINVESTAV-IPN, Mexico City, Mexico

Jose Flores

Division of Infectious Diseases, The University of Texas at Houston Medical School, Houston, Texas, USA

Tania A.T. Gomes

Departmento de Microbiologia, Imunologia, e Universidade Federal de São Paulo, São Paulo, Brazil

Oscar G. Gómez-Duarte

International Enteric Vaccines Research Program, Division of Infectious Diseases, Department of Pediatrics, University of Iowa Children’s Hospital, USA

Bertha Gonzalez-Pedrajo

Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México, D.F., Mexico

Beatriz E.C. Guth

Department of Microbiology, Immunology, and Universidade Federal de São Paulo, São Paulo, Brazil

Denise S.P.Q. Horton

Laboratório de Bacteriologia, Instituto Butantan, São Paulo, SP, Brazil

Cristina Ibarra

Departamento de Fisiología, Facultad de Medicina, Universidad de Buenos Aires, Argentina

Kinue Irino

Seção de Bacteriologia, Instituto Adolfo Lutz, São Paulo, SP, Brazil

Sonja J. Lloyd

Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, U.S.A.

Paula M.A. Lucchesi

Laboratorio de Inmunoquímica y Biotecnología, Facultad de Ciencias Veterinarias, Universidad Nacional del Centro, 7000 Tandil, Prov. de Buenos Aires, Argentina.

Ygnacio Martinez-Laguna

Centro de Investigaciones en Ciencias Microbiológicas. B. Universidad Autónoma de Puebla, Puebla, México

Parasitologia,

Parasitology,

v

Marcelo Masana

Instituto Tecnología de Alimentos. Centro de Investigación de Agroindustria, Instituto Nacional de Tecnología Agropecuaria, INTA. B1708WAB Morón, Prov. de Buenos Aires, Argentina.

Elsa C. Mercado

Instituto Nacional de Tecnología Agropecuaria (INTA), Argentina

Elizabeth Miliwebsky

Servicio Fisiopatogenia, Departamento de Bacteriología, Instituto Nacional de Enfermedades Infecciosas “Carlos G. Malbrán”, Buenos Aires, Argentina

Cristiano G. Moreira

University of Texas Southwestern Medical Center, Department of Microbiology, Dallas, USA

Armando Navarro

Departamento de Salud Pública. Facultad de Medicina, Universidad Nacional Autónoma de Mexico. Mexico City, Mexico

Fernando Navarro-Garcia

Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), México DF, Mexico

Theresa Ochoa

Instituto de Medicina Tropical “Alexander von Universidad Peruana Cayetano Heredia, Lima, Perú

Pablo C. Okhuysen

Division of Infectious Diseases, The University of Texas at Houston Medical School, Houston, Texas, USA

Angel Oñate

Department of Microbiology, Faculty of Biological Sciences. Universidad de Concepción. Chile

Nora Lía Padola

Laboratorio de Inmunoquímica y Biotecnología, Facultad de Ciencias Veterinarias, Universidad Nacional del Centro, Prov. de Buenos Aires, Argentina.

Marina Palermo

Departamento de Inmunología, Academia Nacional de Medicina and ILEX-CONICET, Buenos Aires, Argentina

Alberto E. Parma

Universidad Nacional del Centro-CICPBA, Argentina

Antonio F. Pestana de Castro

University of São Paulo, Brazil

Roxane M.F. Piazza

Laboratório de Bacteriologia, Instituto Butantan, São Paulo, SP, Brazil

Cyntia F. Picheth

Department of Medical Pathology, Federal University of Paraná, Curitiba, Brazil

Valeria Prado

Microbiology Program, Institute of Biomedical Sciences, Faculty of Medicine, Universidad de Chile, Santiago, Chile

Horacio A. Repetto

Department of Pediatrics, Faculty of Medicine, and Hospital Nacional Prof. A Posadas, University of Buenos Aires, Buenos Aires, Argentina

Marta Rivas

Branch of Physiopathogenesis, Department of Bacteriology, Instituto Nacional de Enfermedades Infecciosas-ANLIS "Dr. Carlos G. Malbrán", Buenos Aires, Argentina

Humboldt”,

vi

Juan C. Salazar

Institute of Biomedical Sciences, Faculty of Medicine Universidad de Chile

Marcelo P. Sircili

Laboratório de Bacteriologia, Instituto Butantan, São Paulo, SP, 05503900, Brazil

Vanessa Sperandio

University of Texas Southwestern Medical Center, Department of Microbiology, Dallas, TX 75390-9048, USA

Alfredo G. Torres

Department of Microbiology and Immunology, Department of Pathology and the Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, Texas, U.S.A.

Gustavo Varela

Departamento de Bacteriología y Virología. Instituto de Higiene “Arnoldo Berta”. Facultad de Medicina. Universidad de la República. Montevideo, Uruguay

Tânia M.I. Vaz

Seção de Bacteriologia, Instituto Adolfo Lutz, São Paulo, SP, Brazil

Roberto M. Vidal

Institute of Biomedical Sciences, Faculty of Medicine Universidad de Chile

Nina D. Williams

Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, U.S.A.

Pathogenic Escherichia coli in Latin America, 2010, 1-7

1

CHAPTER 1 Overview of Escherichia coli Alfredo G Torres1,*, Margarita MP Arenas-Hernández2 and Ygnacio Martínez-Laguna2 1

Department of Microbiology and Immunology, Department of Pathology and the Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, Texas 77555-1070 and 2Centro de Investigaciones en Ciencias Microbiológicas. B. Universidad Autónoma de Puebla, Puebla, Puebla 72570 México. Abstract: Escherichia coli are Gram-negative bacteria found as normal commensal flora in the gastrointestinal tract. As a pathogen, E. coli are the most frequent causes of bacterial infections, including urinary tract infections, diarrheal disease, and other clinical infections such as neonatal meningitis, pneumonia and bacteremia. At least six different categories of pathogenic E. coli causing enteric infections have been identified and further characterized. In Latin America, as well as many other developing countries, diarrheal infections caused by E. coli remain an important cause de infant morbidity - mortality. Due to the appearance of the highly virulent strain of E. coli of serotype O157:H7 in the US and Canada in the 1980’s, and subsequently in other Latin American countries, there is an increase need for accurate testing for this and other pathogenic E. coli strains, substantially enhancing detection of virulent strains and, therefore, facilitating identification of sporadic E. coli infections and outbreaks.

ESCHERICHIA COLI: THE ORGANISM The genus Escherichia is named after the German pediatrician Theodor Escherich, who isolated the type species of the genus in 1885 [1]. E. coli are facultative anaerobic bacteria with a type of metabolism that is both fermentative and respiratory. They are either non-motile or motile by peritrichous flagella. E. coli strains are a major facultative inhabitant of the large intestine, widely distributed in the intestine of humans and warm-blooded animals and it is the predominant facultative anaerobe in the intestine and part of the essential microbiota that maintains the physiology of the healthy host [2]. E. coli is a member of the family Enterobacteriaceae [3], and although most strains of E. coli are not regarded as pathogens, they can be opportunistic pathogens that cause infections in immunocompromised hosts. Physiologically, E. coli is versatile and well-adapted to its characteristic habitats. It can grow in media with glucose as the sole organic constituent. Wild-type E. coli has no growth factor requirements, and metabolically it can transform glucose into all of the macromolecular components that make up the cell [4]. The bacterium can grow in the presence or absence of O2. Under anaerobic conditions it will grow by means of fermentation, producing mixed acids and gas as end products. However, it can also grow by means of anaerobic respiration, since it is able to utilize NO3, NO2 or fumarate as final electron acceptors for respiratory electron transport processes. In part, this versatility is what gives E. coli its ability to adapt to its intestinal (anaerobic) and its extraintestinal (aerobic or anaerobic) habitats [4]. E. coli is used as an indicator of fecal contamination because the organism is abundant in human and animal feces and not usually found in other niches. Furthermore, since E. coli could be easily detected by its ability to ferment glucose (later changed to lactose), it is easier to isolate from contaminated food or water than contain other known gastrointestinal pathogens. Due to the presence of other enteric bacteria like Citrobacter, Klebsiella and Enterobacter, which can also ferment lactose, the term "coliform" was coined. These enteric organisms are similar to E. coli in phenotypic characteristics and are not easily distinguished. Therefore, a broad definition indicates that the coliforms are a group of Gram-negative, facultative anaerobic rod-shaped bacteria that ferments lactose to produce acid and gas within 48 h at 35°C, and which are an indicator of contamination [5]. Further, the coliforms are well adapted to mammalian intestines, e.g. different strains of E. coli grows best in vivo or at the higher temperatures characteristic of such environment, rather than the cooler temperatures found in soil and other environments. Within the coliform group, the fecal coliforms consists mostly of E. coli (the indicator species), but some other enterics, such as Klebsiella, is also important indicator because they can also ferment lactose at temperatures between 44.5-45.5°C. *Address correspondence to: Alfredo G. Torres, Department of Microbiology and Immunology, University of Texas Medical Branch, 301 University Blvd, Galveston, Texas 77555-1070; Tel (409) 747-0189. E-mail: [email protected] Alfredo G. Torres (Ed) All rights reserved - © 2010 Bentham Science Publishers Ltd.

2 Pathogenic Escherichia coli in Latin America

Torres et al.

Detection and enumeration of coliforms is used as an indicator of sanitary quality of water or as a general indicator of sanitary condition in the food-processing environment [6]. Almost all the methods used to detect E. coli, total coliforms or fecal coliforms are enumeration methods that are based on lactose fermentation [7]. Colony-forming units and Most Probable Number (MPN) are two methods commonly used to assess the threat of pathogen contamination. For example, the MPN method is a statistical, multi-step assay consisting of presumptive, confirmed and completed phases. In the assay, serial dilutions of a sample are inoculated into broth media and the number of gas positive (fermentation of lactose) tubes is scored, from which the other 2 phases of the assay are performed using the combinations of positive results to consult statistical tables to estimate the number of organisms present. Typically only the first 2 phases are performed in coliform analysis, while all 3 phases are done for E. coli [8]. As a result of this type of analysis, fecal coliforms remain the standard indicator of choice for shellfish and shellfish harvest waters; and E. coli is used to indicate recent fecal contamination or unsanitary processing [5, 9]. One useful property of coliforms is that they are very easily differentiated from others by growing them in lactose– peptone–nutrient medium (e.g., Mac–Conkey broth) at 37°C for 24-48 h and then checking if they produced acid and gas. For further differentiation of fecal coliforms, the samples can be grown in lactose–peptone–eosin–methyl blue (EMB) agar medium. After incubating the medium at 37°C for 24-48 h, E. coli develops into blue black colonies with light reflecting metallic shine, whereas Enterobacter forms reddish slimy colonies. For E. coli O157:H7, the stool specimen are normally tested on sorbitol–MacConkey (SMAC) agar. To perform the complete differentiation of fecal contaminant (e.g., E. coli) and the non–fecal contaminant (e.g., Enterobacter), a series of traditional biochemical tests are still in use, which are collectively known as IMViC test [10]. In these tests, indol production from tryptophan (indol test), production of strong acid causing red color in methyl red indicator (methyl red test), production of acetoine (Voges–Proskauer test), and use of citrate as the only carbon source (citrate test) are conducted. E. coli shows positive reactions for the first two tests whereas Enterobacter aerogenes for the last two tests. Significance in Determining E. coli as a Contaminant of Food Products and Water As described above, coliforms are found in the soil, in water, in muck and all over the natural environment. However, E. coli strains are specifically adapted to live in the guts of warm blooded animals. E. coli is used for detection because it makes up about 10 percent of intestinal microorganisms of human and animals; consequently, there are a lot more coliforms in human feces than there are pathogens. Therefore, E. coli is considered a contaminant risk not only in water, but in food products as well, and in recent years there have been an increasing number of food recalls because of E. coli contamination [11, 12]. As a water contaminant, E. coli was chosen several years ago as an "indicator" of the amount of human fecal matter level in the water [13]. Comparing the number of coliform/E. coli with the standardized coliform index, the water quality can be graded and recommended for certain use or none. However, caution is recommended as it can be misleading to use E. coli alone as an indicator of human fecal contamination, because there are other environments in which E. coli grows well. Monitoring the levels of E. coli contamination is important because differences between non-pathogenic and pathogenic E. coli strains are often detectable only on the molecular level; however, many of these differences cause changes in the physiology or life cycle of the bacterium, leading for example to the different pathogenic lifestyles. New strains of E. coli arise all the time from the natural biological process of genetic variability (i.e. mutation, horizontal transfer genes), and some of those strains develop characteristics that can be harmful to their host animal. Although in most healthy adult individuals, such a strain would probably cause no more than a diarrheal episode or might produce no symptoms at all; in young children, people who are or have been recently immunocompromised, or in people taking certain medications, such virulent strain can cause serious illness and even death. A recent example of the evolution of a virulent strain is represented by E. coli O157:H7, which possess the stx-phages, which carry the genes encoding Shiga toxin (Stx). Shiga toxins have driven and are driving the emergence of Stx-producing pathogens and since the emergence of E. coli O157:H7 as a cause of significant human disease, more than 500 different serogroups of E. coli have been reported to produce Shiga toxin, as well as a few other organisms [14, 15]. ESCHERICHIA COLI AS A COMMENSAL Commensal intestinal microbiota (normal microbiota, indigenous microbiota) consists of those micro-organisms, which interact with epithelial cells and are exposed to the external environment [16]. The adult gastrointestinal tract

Overview of Escherichia coli

Pathogenic Escherichia coli in Latin America 3

acquires at least 17 families of bacteria yielding 400 to 500 different microbial species with regional variation of bacterial composition within the gastrointestinal tract. In general, there is a qualitative and quantitative increase in complexity from the stomach to the colon with the colon as the primary site for commensal bacterial colonization in humans and animals. Commensal bacteria co-evolved with their hosts, however, under specific conditions they are able to overcome protective host responses and exert pathologic effects [17]. E. coli is part of the commensal flora and a normal inhabitant of the human gut, but is also the Gram-negative bacillus most frequently isolated in cases of human infection [18]. It has been postulated that commensal enteric E. coli may be the natural reservoir of pathogenic strains, because this normally harmless commensal needs only to acquire a combination of mobile genetic elements to become a highly adapted pathogen capable of causing a range of diseases, from gastroenteritis to extraintestinal infections of the urinary tract, bloodstream and central nervous system [19]. Indeed, intestinal or extraintestinal E. coli infections are caused by strains harboring numerous virulence factors located on plasmids, bacteriophages, transposons and pathogenicity islands [20]. The ubiquitous commensal population constitutes an enormous reservoir from which pathogenic strains continually emerge. The ability to E. coli to exist as a humanadapted commensal compounded with its natural tendency for frequent genetic exchange, its ubiquitous presence, and the enormous, diverse, and largely uncharacterized reservoir of genetic variation found within the species genomes, contribute to the emergence of new pathogenic strains and potentially resistant to antimicrobial drugs. Several studies have shown that pathogenic E. coli strains may be derived from commensal strains by the acquisition of chromosomal or extra-chromosomal virulence loci [21], “pathoadaptive mutations” which are genomic deletions that enhance pathogenicity [22]; or random point mutations that increase adaptation for pathogenic environments [23]. These strategies of genome plasticity for a commensal strain to become virulent has led to the hypothesis indicating that the fecal E. coli population may influence the occurrence and etiology of extraintestinal and intestinal infections because E. coli populations have a clonal structure [24]. Phylogenetic analyses have shown that E. coli strains fall into four main phylogenetic groups (A, B1, B2, and D [25], and these classification are utilized to perform rapid and simple classification of pathogenic E. coli [26]. The assignment of E. coli clones to one of these four groups is the basis of phylogenetic studies of the species [27]. For example, it has been found that Shigella clones are derived from E. coli outside the phylogenetic groups B2 and A [28], while Shiga toxin-producing E. coli O157:H7 clones belong to phylogenetic group D [26]. In contrast, the clones responsible for human extraintestinal infections frequently belong to the anciently diverged B2 phylogenetic group [29]. Recent evidence indicates that commensal and pathogenic bacteria can also participate in the pathogenesis of the inflammatory bowel diseases [17]. Although there is no evidence that a single pathogen causes Crohn's disease or ulcerative colitis, it has been observed that increased numbers of mucosa-associated E. coli are observed in both major inflammatory bowel diseases. As a result, a new pathovar of E. coli, designated Adherent-Invasive E. coli (AIEC) has been found associated with ileal Crohn's disease. AIEC strains colonize the intestinal mucosa by adhering to intestinal epithelial cells, displaying the ability to invade them via a macropinocytosis-like process, and to survive and replicate intracellularly. Within macrophages, AIEC strains survive and replicate extensively without inducing host cell death and induce the release of high amounts of TNF [30]. All these virulence properties designate AIEC as a possible pathogen potentially able to induce persistent intestinal inflammation, further supporting the idea that commensal E. coli are a natural reservoir of pathogenic strains. ESCHERICHIA COLI AS AN ANTIBIOTIC RESISTANCE RESERVOIR IN THE MICROBIOME The human microbiome substantially impacts human health and plays beneficial roles in dietary processing and prevention of pathogen intrusion [31-33]. The widespread use of antibiotics in human medicine and agriculture has likely induced substantial responsive changes in this community. Many commensal bacterial species, which were once considered relatively harmless residents of the human microbiome, have recently emerged as multidrugresistant disease-causing organisms [34]. E. coli, as indicator bacteria, it is useful because this microorganism acquires antimicrobial resistance faster than other conventional bacteria. Thus, changes in the resistance of this species may serve as a good indicator of resistance in potentially pathogenic bacteria [35, 36]. Plasmids are genetic elements, not virulence factors per se, that can be transmitted between bacteria. Plasmids encode genes for a variety of factors that contribute to pathogenesis, including antibiotic resistance, fimbriae, toxins, secretion systems, and invasion factors. Transmission of plasmids plays a large role in the growing problem of antibiotic resistance [37]. An overview of the major

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plasmid families that are currently emerging in multidrug-resistant Enterobacteriaceae strains isolated worldwide among those conferring resistance to clinically relevant antibiotics, such as extended-spectrum cephalosporins, fluoroquinolones, and aminoglycosides has been recently published [38]. Acquisition of antibiotic resistance genes by non-pathogenic bacteria is detrimental for two reasons. First, these bacteria will constitute a reservoir of antibiotic resistance genes (and antibiotic resistance vectors) that may be transferred to virulent bacteria. Second, antibiotic-resistant bacteria can protect the susceptible ones (eventually pathogenic) from the action of antibiotics [39]. Conversely, acquisition and further spread of antibiotic resistance genes among pathogenic bacteria is a phenomenon that has occurred just in the last 50 years as a consequence of extensive antibiotic use for human therapy and animal farming. At first glance, pathogenicity and resistance should be unlinked phenomena. However, several examples indicate that this is not the situation for several bacterial pathogens. Antibiotic resistance and virulence genes can be linked (and then co-selected) in the same replicon, or eventually a single determinant can be involved in both virulence and resistance [39]. For example, the EHEC virulence plasmid pO26-CRL contains a complex antibiotic-resistance gene locus located between virulence determinants, such as the enterohemolysin operon (ehxCABD) and the STEC-specific extracellular serine protease (espP) [40]. This region encompasses a 22,609 bp Tn21 derivative encoding resistance to trimethoprim, streptomycin, sulfathiozole, kanamycin, neomycin, -lactams, and mercuric chloride. Plasmid pO26-CRL is nonconjugative but is mobilizable and raises the concern that antibiotic use could be co-selected with the virulence determinants, leading to increase disease potential in both commensal and pathogenic E. coli populations [40]. The widespread use of antimicrobial agents that are regarded as critically or highly important for use in humans creates a reservoir of resistant bacteria and antibiotic resistance genes, which adds to the burden of antimicrobial resistance in human medicine and may shorten the time that these valuable antimicrobial agents will be available for effective treatment of human infections. Humans may obtain antimicrobial-resistant E. coli or antibiotic resistance genes of animal origin directly, via contact with animals, food of animal origin, or the environment. These bacteria may subsequently colonize humans or may transfer resistance genes to other bacteria during passage through the intestinal tract. Although the carriage of antimicrobial-resistant E. coli in the intestine is not a human health hazard itself, it might give rise to bacterial infections with limited therapeutic options and an increased risk of treatment failure. The contribution of the animal reservoir to the burden of antimicrobial resistance in humans has not been quantified; however, the use of antimicrobial agents considered as a critical or highly important for humans use should be avoided or minimized in food animals, to preserve the efficiency of these antimicrobial agents for treatment of infection in humans [41]. Evidence is accumulating to support the hypothesis that intestinal bacteria not only exchange resistance genes among themselves but might also interact with bacteria that are passing through the colon, causing these bacteria to acquire and transmit antibiotic resistance genes. By significantly expanding comparative genomics to a population scale, we will peer into the E. coli population, with previously unattainable resolution, and identify the genetic pathways leading to the emergence of human-adapted, pathogenic strains. ESCHERICHIA COLI AS AN ENTERIC PATHOGEN In the United States, for example, E. coli is the leading cause of both community-acquired and nosocomial Urinary Tract Infections (UTI). E. coli also causes 12-50% of nosocomial infections and 4% of cases of diarrheal disease. In tropical countries, in contrast, E. coli infections are one of leading causes of diarrhea, responsible in some situations for up to 40% of cases of infant or traveler's diarrhea. These infections are traditionally acquired after the consumption of contaminated meat obtained from a variety of animal species, other food products and water. Historically, serotyping was important in distinguishing the small number of strains that actually cause diarrheal disease. Some serotypes of these enteric organisms have been related to emergent zoonotic infections in developed and developing countries. Currently, with over 700 antigenic types (serotypes) of E. coli (which are recognized based on O, H, and K antigens) and with increasing number of serotypes associated with disease, the pathogenic E. coli are now also classified based on their unique virulence factors and adherence properties. Analysis for pathogenic E. coli usually requires that the isolate first be identified as E. coli by testing for metabolic characteristics and virulence markers before the serotype is determined. When an outbreak is suspected, it is necessary to differentiate the pathogenic E. coli isolates from commensal E. coli, because they are indistinguishable at the

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biochemical level and, therefore, additional tests to those traditionally performed in the clinical laboratory are required to identify the specific isolate [42, 43]. For the diagnostic of pathogenic E. coli, some of the following methodologies are required: a) serotypification; b) adherent assays on HEp-2 cells; c) FAS test (Fluorescent Actin Staining); d) different molecular biology techniques to amplify genes encoding specific virulence factors [42-44]. One of the phenotypic diagnostic test is the adherence assay on HEp-2, which allows the identification of distinct pattern of bacterial adherence on the cells, namely, Localized, Localized Adherence-Like, Diffuse and Aggregative adherence [45, 46]. The second phenotypic assay is the FAS test, which is an alternative technique utilized in epidemiological studies and basic research [47]. In this assay, the accumulation of host cell cytoskeleton actin underneath the adherent bacteria is observed, and this accumulation is due in response to bacterial secreted factors. The FAS test can be utilized in: a) direct way on intestinal biopsies in patients with diarrhea and where is suspicion of a E. coli infection; b) with tissue cultured cells (HEp-2, HeLa, or Caco-2) infected with an E. coli strain isolated from infected feces [42, 47, 48]. The adherence assay and the FAS test are sufficient to identify some of the categories of E. coli listed in the subsequent chapters, however, additional molecular test to identify specific virulence factors are required to have a complete idea about the pathogenic capabilities of the strains. As a cause of enteric infections, different pathogenic mechanisms of 6 different categories of E. coli have been reported [14, 42]. Enterotoxigenic E. coli (ETEC) is a major cause of travelers’ diarrhea in adults from industrialized countries and children in developing countries worldwide. Enteropathogenic E. coli (EPEC) is a cause of infant diarrhea in developing countries. Enterohemorrhagic E. coli (EHEC), a food-borne pathogen of worldwide importance, can cause a non-bloody diarrhea but the most serious manifestation of disease is bloody diarrhea that can progress to a fatal illness due to acute kidney failure (hemolytic uremic syndrome [HUS]), particularly in children. Enteroaggregative E. coli (EAEC) were originally recognized as predominant etiologic agents of persistent diarrhea in developing countries and still remain an important cause of acute as well as protracted diarrhea in several parts of the world, including industrialized countries. Enteroinvasive E coli (EIEC) cause a watery diarrhea and dysentery in humans and, interestingly since EIEC are closely related to Shigella spp, the knowledge regarding EIEC virulence has been mainly extrapolated from the studies in Shigella. Finally, diffusely adhering E. coli (DAEC) strains are characterized by their diffuse adherence pattern on cultured epithelial cells, however, as compared with the other categories; little is known about the mechanism of DAEC pathogenesis. In the human intestine, ETEC, EPEC, EAEC colonize the small intestine, while EIEC and EHEC preferentially colonize the large bowel prior to causing diarrhea. Because of a large number of DAEC serotypes are associated with this category; the exact location for intestinal colonization of this pathogen has not been defined. ESCHERICHIA COLI: THE INTEND OF THIS BOOK In Latin America, acute gastroenteritis remains to be an important cause of morbidity in adults and a major cause of morbidity and mortality in children. A child under 5 years of age belonging to a low income segment of the Latin American population will develop 5 to 10 episodes of diarrhea every year [49]. Even with the impressive progress done to understand pathogenic mechanisms of enteric bacterial pathogens, at least one-third of all diarrheal cases in this region are still associated with the different categories of pathogenic E. coli. The rapid expansion of this field has been fueled by the continual emergence and re-emergence of new E. coli strains as a global public health problem; indeed, few infectious diseases have generated more sustained attention from the scientific and, notably, the lay media because the ability of some of the strains (e.g. E. coli O157:H7) to cause important outbreaks. The authors of this chapter believe that the field of pathogenic E. coli in Latin America was in great need of, the comprehensive review that this book represents. Although other books have been written about pathogenic E. coli, their focus generally has been the research progress in other parts of the world; this is the first volume, to our knowledge, with a nearly complete coverage of the pathogenesis, epidemiology, diagnostic, therapeutics, animal reservoirs, mechanism of action, host-pathogen interactions, and other aspects associated with E. coli intestinal infections, with special emphasis to the situation in Latin America. This book combines and illuminates several years of tenacious study of pathogenic E. coli by multiple research groups in Latin America. The goal was to integrate the diverse aspects of the E. coli research performed in the majority of the countries in this region toward a unified view of how these E. coli infections continue been such a serious threats to humans.

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Escherich T. Die darmbakterien des neugeborenen und sauglings. Fortshr Med 1885;3:5-15-522,47-54. Conway PL. Microbial ecology of the human large intestine. In: Gibson GR, Macfarlane GT, editors. Human colonic bacteria: role in nutrition, physiology and pathology. Boca Raton, FL.: CRC Press; 1995. p. 1-24. Ewing WH. Identification of Enterobacteriaceae. 4th ed ed. Edwards, Ewing, editors. New York: Elsevier; 1986. Shiloach J, Reshamwala S, Noronha SB, et al. Analyzing metabolic variations in different bacterial strains, historical perspectives and current trends - example E. coli. Curr Opin Biotechnol. 2010;Jan 28 [Epub ahead of print]. Leclerc H, Mossel DA, Edberg SC, et al. Advances in the bacteriology of the coliform group: their suitability as markers of microbial water safety. Annu Rev Microbiol. 2001;55:201-34. Simpson JM, Santo Domingo JW, Reasoner DJ. Microbial source tracking: state of the science. Environ Sci Technol. 2002;36:5279-88. APHA. Compendium of Methods for the Microbiological Examination of Foods. 3rd ed ed. Washington, DC: American Public Health Association; 1992. APHA. Standard Methods for the Examination of Water and Wastewater. 20th ed ed. Washington, DC: American Public Health Association; 1998. Wohlsen T, Bates J, Vesey G, et al. Evaluation of the methods for enumerating coliform bacteria from water samples using precise reference standards. Lett Appl Microbiol. 2006;42:350-6. Huang SW, Chang CH, Tai TF, et al. Comparison of the beta-glucuronidase assay and the conventional method for identification of Escherichia coli on eosin-methylene blue agar. J Food Prot. 1997;60:6-9. CDC. Diagnosis and management of foodborne illnesses: a primer for physicians and other health care professionals. MMWR. 2004;53:352-6. Erickson MC, Doyle MP. Food as a vehicle for transmission of Shiga toxin-producing Escherichia coli. J Food Prot. 2007;70:2426-49. Pearson H. The dark side of E. coli. Nature. 2007;445:8-9. Kaper JB, Nataro JP, Mobley HL. Pathogenic Escherichia coli. Nat Rev Microbiol. 2004;2:123-40. Zhou Z, Li X, Liu B, et al. Derivation of Escherichia coli O157:H7 from its O55:H7 precursor. PLoS One. 2010;5:e8700. Yan F, Polk DB. Commensal bacteria in the gut: learning who our friends are. Curr Opin Gastroenterol. 2004;20:565-71. Packey CD, Sartor RB. Commensal bacteria, traditional and opportunistic pathogens, dysbiosis and bacterial killing in inflammatory bowel diseases. Curr Opin Infect Dis. 2009;22:292-301. Duriez P, Clermont O, Bonacorsi S, et al. Commensal Escherichia coli isolates are phylogenetically distributed among geographically distinct human populations. Microbiology. 2001;147:1671-6. Croxen MA, Finlay BB. Molecular mechanisms of Escherichia coli pathogenicity. Nat Rev Microbiol. 2010;8:26-38. Mühldorfer I, Hacker J. Genetic aspects of Escherichia coli virulence. Microb Pathog. 1994;16:171-81. Finlay BB, Falkow S. Common themes in microbial pathogenicity revisited. Microb Mol Biol Rev 1997;61:136-69. Torres AG. The cad locus of Enterobacteriaceae: more than just lysine decarboxylation. Anaerobe. 2009;15:1-6. Sokurenko EV, Hasty DL, Dykhuizen DE. Pathoadaptive mutations: gene loss and variation in bacterial pathogens. Trends Microbiol. 1999;7:191-5. Selander RK, Levin BR. Genetic diversity and structure in Escherichia coli populations. Science. 1980;210:545-7. Herzer PJ, Inouye S, Inouye M, et al. Phylogenetic distribution of branched RNA-linked multicopy single-stranded DNA among natural isolates of Escherichia coli. J Bacteriol. 1990;172:6175-81. Clermont O, Bonacorsi S, Bingen E. Rapid and Simple Determination of the Escherichia coli Phylogenetic Group. Appl Environ Microbiol. 2000;66:4555–8. Pupo GM, Karaolis R, Lan R, et al. Evolutionary relationship among pathogenic and non pathogenic Escherichia coli strains inferred from multilocus enzyme electrophoresis and mdh sequence studies. Infect Immun 1997;65:2685-92. Pupo GM, Lan R, Reeves PR. Multiple independent origins of Shigella clones of Escherichia coli and convergent evolution of many of their characteristics. Proc Natl Acad Sci USA. 2000;97:10567-72. Picard B, Sevali-Garcia J, Gouriou S, et al. The link between phylogeny and virulence in Escherichia coli extraintestinal infection. Infect Immun. 1999;67:546-53. Rolhion N, Darfeuille-Michaud A. Adherent-invasive Escherichia coli in inflammatory bowel disease. Inflamm Bowel Dis. 2007;13:1277-83. Gill SR, Pop M, Deboy RT, et al. Metagenomic analysis of the human distal gut microbiome. Science. 2006;312:1355-9. Turnbaugh PJ, Hamady M, Yatsunenko T, et al. A core gut microbiome in obese and lean twins. Nature. 2009;457:480-4. Jia W, Li H, Zhao L, et al. Gut microbiota: a potential new territory for drug targeting. Nat Rev Drug Discov. 2008;7:123-9.

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Marshall BM, Ochieng DJ, Levy SB. Commensals: Underappreciated Reservoir of Antibiotic Resistance. Microbe. 2009;4:231. Kijima-Tanaka M, Ishihara K, Morioka A, et al. A national surveillance of antimicrobial resistance in Escherichia coli isolated from food-producing animals in Japan. J Antimicrob Chemother. 2003;51:447–51. Von Baum H, Marre R. Antimicrobial resistance of Escherichia coli and therapeutic implications. Int J Med Microbiol. 2005;295:503–11. Prats G, Mirelis B, Miro E, et al. Cephalosporin-resistant Escherichia coli among summer camp attendees with salmonellosis. Emerg Infect Dis. 2003;9:1273-80. Carattoli A. Resistance Plasmid Families in Enterobacteriaceae. Antimicrob Agents Chemother. 2009;53:2227–38. Martínez JL, Baquero F. Interactions among Strategies Associated with Bacterial Infection: Pathogenicity, Epidemicity, and Antibiotic Resistance. Clin Microbiol Rev. 2002;15:647–79. Venturini C, Beatson SA, Djordjevic SP, et al. Multiple antibiotic resistance gene recruitment onto the enterohemorrhagic Escherichia coli virulence plasmid. FASEB J. 2010;24:1160-6. Hammerum AM, Heuer OE. Human Health Hazards from Antimicrobial-Resistant Escherichia coli of Animal Origin. Clin Infect Dis. 2009;48:916–21. Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11:142-201. Rodríguez-Ángeles G. Principal characteristics and diagnosis of the pathogenic groups of Escherichia coli. Salud Publica Mex. 2002;44:464-75. Cravioto A, Vasquez V. Escherichia coli: pathogenic mechanisms and enterohemorrhagic strains. Bol Med Hosp Infant Mex. 1988;45:196-7. Cravioto A, Gross RJ, Scotland SM, et al. An adhesive factor found in Escherichia coli belonging to the traditional infantile enteropathogenic serogroups. Microbiology. 1979;6:3427-37. Torres AG, Zhou X, Kaper JB. Adherence of diarrheagenic Escherichia coli strains to epithelial cells. Infect Immun. 2005;73:18-29. Knutton S, Baldwin T, Williams PH, et al. Actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli. Infect Immun. 1989;57:1290-8. Knutton S, Lloyd DR, McNeish AS. Adhesion of enteropathogenic Escherichia coli to human intestinal enterocytes and cultured human intestinal mucosa. Infect Immun. 1987;55:69-77. Prado V, O'Ryan ML. Acute gastroenteritis in Latin America. Infect Dis Clin North Am. 1994;8:77-106.

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CHAPTER 2 Evolution and Epidemiology of Diarrheagenic Escherichia coli Nina D Williams1, Alfredo G Torres1,2 and Sonja J Lloyd1,* 1

Department of Microbiology and Immunology and 2Department of Pathology and the Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, Texas 77555-1070. Abstract: The emergence and evolution of pathogenic Escherichia coli strains associated with diarrheal diseases have become a topic of active investigation in recent years due to the emergence of more virulent strains and the association of new serotypes with disease. Outbreak studies indicate that most patients with an intestinal E. coli infection develop mild, uncomplicated diarrhea. However, a significant risk exists that infections caused by highly virulent E. coli isolates, such as the enterohemorrhagic E. coli O157:H7, develop into serious and potentially life-threatening complications, such as hemolytic uremic syndrome. The relative contribution of recombination events in the generation of new categories of pathogenic E. coli varies among the E. coli population, and it is represented by the wide variety of mobile elements found in different diarrheal strains (e.g. pathogenicity islands, phages, transposons, pathoadaptive mutations, etc). Understanding the population structure of pathogenic E. coli is important, since it impacts the effectiveness of molecular epidemiological studies. Such studies are needed to understand the increasingly recognized diversity of enterotoxigenic E. coli, a leading cause of pediatric and travelers’ diarrhea. In addition, factors underlying the emergence of enteroaggregative and atypical enteropathogenic E. coli strains associated with persistent diarrhea are unknown. Horizontal transfer of genetic elements that affect virulence of diarrheagenic E. coli strains and changes in global agricultural processes, as well as movement of humans and animals, may contribute to the complex natural history of diarrheagenic E. coli.

INTRODUCTION TO E. COLI EVOLUTION Biologists have long considered the mechanisms behind genetic variation and how it arises and persists. Organisms must have a balance between robustness and evolution capability, between an individual’s physiological responses to change and the changes by which a population of genomes continuously updates information about past experiences and how future generations should respond to those influences [1]. Adaptation has been historically viewed as a gradual process. Early studies led to two generalizations concerning the emergence and persistence of this variation. First, competition for the same limiting resources selects for the one fittest variant. Second, variation arising from mutations is subject to “periodic selection,” which leads to a succession of clones each more fit than its predecessor [2]. Now, experimental evidence demonstrates how one clone of Escherichia coli adapts to a particular environmental factor and suggests that multiple genotypes can arise from a single ancestral clone and can co-exist over time – that in other words, out of one comes many [2]. Empirical evidence has been found for alternating periods of stasis and rapid evolution[3]. Environmental changes are an insidious part of an organism’s life, and the mechanisms that allow adjustment to environmental conditions will compensate for the effects of the mutations required to produce that phenotype. Selection may favor mutants better adapted to particular regions or those that are better able to colonize niches at the boundaries of these regions. Selection may also favor clones that can better scavenge limited resources or more efficiently use those resources for essential processes. The outcome is dependent upon the founding ancestral clone, the pathways which lead to the different adaptive strategies, the influence of differential gene regulation on the evolutionary process, and the likelihood that key steps along these pathways will actually occur (i.e. mutations). Variation in the adaptation rate may be as a result of environmental changes, the invasion of new habitats, and other circumstances which either promote or inhibit gene flow[3]. Evolution at the molecular level is now known to have arisen from many directions: single base changes; loss, duplication, or rearrangement of genes; and, importantly, the horizontal transfer of genes [4]. Current ideas concerning bacterial evolution center on the idea that pathogenic diversity is the result of the acquisition of pathogenic genes, or virulence determinants, through horizontal gene transfer. E. coli is a good model *Address correspondence to: Sonja J. Lloyd, Department of Microbiology and Immunology, University of Texas Medical Branch, 301 University Blvd, Galveston, Texas 77555-1070; Tel (409) 747-2424. E-mail: [email protected] Alfredo G. Torres (Ed) All rights reserved - © 2010 Bentham Science Publishers Ltd.

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for addressing this question as it is the best known member of the normal microbiota of the human intestine and is the most intensively studied and best understood of all bacteria. The reference strain K-12 and its derivatives have been vital in the advancement of the fields of genetics, molecular biology, and physiology. Investigations of E. coli virulence have revealed a wealth of information regarding the emergence and evolution of these pathogens. Comparison of the genomic sequences of the non-pathogenic laboratory strain K-12 with that of E. coli O157:H7 has shown that these strains share a common DNA backbone, with numerous islands of DNA that were apparently acquired over an extended period of time through horizontal gene transfer [5]. The ongoing and stepwise evolution of E. coli allows it to adapt to constantly changing conditions and environments and ensures the emergence of new pathogenic clones. In a study to better understand the genetic relationships of commensal and pathogenic E. coli strains, multilocus enzyme electrophoresis (MLEE) was used at 10 enzyme loci to determine the genetic diversity of E. coli and the relationship of pathogenic clones to commensal clones. Results showed that pathogenic E. coli strains do not have a single evolutionary origin but have actually arisen several times [6], likely due to the lateral transfer of specific virulence factors which are subject to strong natural selection. E. coli is a diverse species with both commensal and pathogenic strains. E. coli strains may not have always been pathogenic; the one common ancestor evolved into pathogenic strains due to the acquisition of mobile genetic elements such as plasmids and pathogenicity islands (PAIs), as well as due to integration of bacteriophages and transposons. Changes in microbial populations can lead to the evolution of entirely new pathogens, development of new virulent strains in old pathogens, adaptation to new niches, the development of antibiotic resistance, or to changes in the ability to survive in adverse environmental conditions. The virulence determinants encoded on these mobile genetic elements are supposed to be highly interchangeable among bacterial species, and though initially mobile may become ‘locked’ into the genome [7]. Each pathotype of E. coli has unique virulence mechanisms, with the exception of enterohemorrhagic E. coli (EHEC), which is a clonal group derived from enteropathogenic E. coli (EPEC) [8]. Physiologically, E. coli is versatile and well adapted to its characteristic habitats, and can respond to environmental signals such as pH, temperature, osmolarity, as well as a multitude of other stimulants. There are several highly adapted clones that have acquired specific virulence elements which confer an increased ability to adapt to new niches. Diseases caused or effects of infection depend on the distribution and expression of the specific array of pathogenic (virulence) determinants possessed by the organism, including adhesins, secretion systems, and toxins, and the ability to withstand host defenses. The very diversity of E. coli and its pathogenic clones is due to the continued arrival of different virulence determinants into the population, from other E. coli species or other enteric pathogens such as Salmonella, Shigella, and Yersinia. This again emphasizes the idea that pathogenic E. coli do not originate from a single ancestor, but instead have arisen several times from several ancestors [9]. Virulence Determinants, How They Are Acquired How do bacteria adapt to the life-style of a pathogen? Ecological niches that non-pathogenic bacteria might inhabit, such as soil, are very different from the niches encountered upon infecting a vertebrate host [5]. These hosts have defenses that have evolved through co-evolution with microbes – physical barriers, such as the skin and the mucuscovered epithelia, up to the more elaborate antimicrobial peptides and immune responses enacted by the host. The factors and mechanisms that pathogens have evolved to circumvent these defenses are termed virulence factors. Several highly adapted E. coli clones have acquired specific virulence attributes, which has conferred upon them the ability to adapt to new niches and thus cause a spectrum of diseases [10]. The virulence determinants of each E. coli pathotype are distinct, but can generally be categorized as either colonization factors or secreted proteins. The colonization factors, such as adhesins, enable the bacteria to bind closely to the intestinal mucosa and resist clearance. Most frequently, these adhesins form distinct structures on the bacterial cell surface termed fimbriae or pili, though they also include outer-membrane proteins such as intimin in EPEC and EHEC, and other non-fimbrial proteins. Secreted proteins, including toxins and other effector proteins, interfere with the normal physiological processes of host cells such as protein synthesis and the regulation of intracellular messengers such as cAMP and cGMP. By one means or another, pathogenic strains of E. coli have perfect mechanisms to acclimate to new environmental pressures and to survive in novel niches which they previously did not inhabit, consequently causing damage to host tissues and leading to disease. Genes can be taken up as naked DNA or transferred in the form of

 

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plasmids, conjugative transposons, or bacteriophages, and the transferred DNA can range in size from less than 1 to more than 100 kb, and encode entire metabolic pathways [5]. Virulence is often conferred to bacteria by pathogenicity islands, which refer to clusters of virulence-associated genes that are found on the chromosomes of pathogenic bacteria but are absent from non-pathogenic strains. These islands often show evidence of having been acquired from other bacteria [11], including a nucleotide base composition different from the native chromosome in general and possibly the presence of mobile genetic elements at the termini. These fragments of genetic material can lead to increased virulence or even change a non-pathogenic organism to a pathogen. Bacteriophages, the viruses that infect bacteria, are important vehicles for horizontal gene exchange between different bacterial species and account for a good share of the strain-to-strain differences within bacterial species, such as E. coli. Studies have indicated that some pro-phages carry additional genes (termed lysogenic conversion genes) that are not required for the phage life cycle. Instead, many lysogenic conversion genes from prophages in pathogenic bacteria encode proven or suspected virulence factors. They are postulated to change the phenotype or fitness of the lysogen [5]. Phages have thus emerged as prime suspects in the adaptation of pathogens to new hosts and the emergence of new pathogens or epidemic clones. Phages can also serve as anchor points for genome rearrangements, and protect a bacterium from lytic phage infection, and, most importantly, have the ability to introduce new virulence factors. Transposable elements are discrete DNA segments that have the ability to move from site to site in a genome, independent of extensive DNA sequence homology [12]. These transposable elements often cause spontaneous mutations, regulate the expression of genes near their insertion sites, and induce cycles of chromosome breakage and rearrangement. They play a special role in bacterial evolution because of their ability to move between the chromosome and the various plasmid and phage DNAs resident in a bacterial cell and, when piggybacked on these molecules, to move between unrelated bacteria in a population. Virtually, any gene can become associated with a transposable element, and elements called transposons containing genes whose functions are unrelated to movement are now common. It has been suggested that, especially during periods of drastic environmental change, transposable elements make great contributions to the adaptability and evolution of bacterial populations [12]. Besides pathogenicity islands, bacteriophages, and transposons, plasmids play an important role in the transfer of genetic information between clones. Bacterial plasmids are self-replicating, extrachromosomal replicons and are key agents of change in microbial populations. Naturally occurring plasmids are able to promote the dissemination of a variety of traits, from antibiotic resistance to the ability to metabolize certain substances, and recombinant plasmids based off these wild type plasmids have been essential to the field of molecular biology. E. coli strains have been found to possess a wide variety of plasmid types, including those associated with virulence. Some of these are essential for virulence in the various pathotypes of E. coli, and it has been shown that the majority of these E. coli virulence plasmids have evolved from a single plasmid backbone type through the acquisition of traits that are essential for and specific to the particular pathotypes [13]. E. coli Pathotypes Though E. coli are historically classified based on the serology of the O (lipopolysaccharide, LPS) and H (Hauch, flagellar) antigens, more recently the terms virotype and/or pathotype have been used, to refer to a group of strains of a single species that cause a common disease using a common set of virulence factors. Only the most successful combination of virulence factors have persisted to become specific pathotypes, and each pathotype represents a family of E. coli clones that share virulence determinants, which were acquired by horizontal gene transfer between E. coli and other bacterial species [10-11]. Individual strains of each pathotype possess a distinct set of virulenceassociated characteristics that determine the pathological and clinical features of the diseases they cause; only the most successful combinations of virulence factors persist to become specific pathotypes. There are more than 180 O serogroups of E. coli, each of which is further subdivided into more than 60 H serotypes, to give more than 10,000 possible combinations [11]. The clonal nature of these pathogenic bacteria is seen in the fact that they generally belong to distinctive O serogroups and O:H serotypes and has recently been inferred by sequencing studies and multilocus enzyme electrophoresis (MLEE) of different E. coli clones [6].

 

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Eight distinct pathotypic categories (also known as pathovars) of E. coli have been described, which are broadly classified into diarrheagenic E. coli (DEC) or extraintestinal E. coli (ExPEC). Two pathovars are extraintestinal, the uropathogenic E. coli (UPEC), and the neonatal meningitis E. coli (NMEC). Six pathovars are diarrheagenic: enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), and diffusely-adhering E. coli (DAEC) (Fig. 1) [10]. Several evolutionary events have permitted the differentiation of the different pathovars. For example, EPEC produce a characteristic intestinal histopathology known as the attaching and effacing (A/E) lesion, and the ability to form this lesion is conferred by the chromosomally-located Locus of Enterocyte Effacement (LEE) Pathogenicity Island. The LEE is in fact present in a family of pathogens, including EHEC, all able to confer the attaching and effacing lesion [10, 14]. EPEC also produce a type IV pilus, known as the bundle-forming pilus (BFP), expressed from 14 genes on a virulence-associated plasmid (pEAF) carried by certain EPEC strains [15-16]. Bundle-forming pili are thought to mediate both initial binding to host cells and interbacterial interactions leading to the formation of three-dimensional microcolonies of attached bacteria [17]. In the case of EHEC, this pathovar is distinguished from other strains on the ability to produce Shiga toxins (Stx), the key virulence determinant for these strains, which is transmitted among Shigella and E. coli strains by toxinencoding bacteriophages [18]. For EPEC and EHEC, the majority of virulence determinants are encoded on ‘O’ islands or plasmids and so these were the focus for comparison. Analysis of the originally described 177 ‘Ospecific’ islands provides insights into the evolution of the two strains[19]. Homologous sequences can be demonstrated for nearly all the ‘O157’ islands in EPEC E2348/69, with only 14 showing little nucleotide homology (below 55%). Sixty-nine of the islands have 49% nucleotide homology [17]. This divergence offers considerable range for differences in the carriage and expression of virulence determinants. Therefore, variation in these Oislands impacts host adaptation, tissue tropism and virulence and this assumption is a simplification that belies the evolution adapting the strains to different hosts and the complex interactions on the host mucosa that lead to an asymptomatic or pathogenic outcome. In addition, not all strains of Stx-producing E. coli are able to cause the more serious clinical syndromes associated with EHEC infection. Those that can usually carry other virulence determinants in addition to Stx, such as the LEE pathogenicity island or a distinctive hemolysin known as enterohemolysin or EHEC hemolysin [10-11]. In fact, the only E. coli pathotypes that share virulence determinants are EPEC and EHEC, likely because EHEC strains have evolved from EPEC ancestors [20-22]. This demonstrates the compound effect that multiple virulence determinants can have on a pathogen. For ETEC strains to cause disease, they must attach to the epithelia of the small intestine, colonize, secrete either of both of two varieties of enterotoxin, the heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST), and evade host defenses while causing damage to the host. ETEC adhesins, known as colonization factors (CFs), allow binding to the small intestinal mucosa – a region where E. coli normally does not display tropism. Human ETEC CFs can be either plasmid or chromosomally encoded; however the majority are plasmid-encoded and appear to have been horizontally-acquired due to the presence of flanking insertion sequences and transposons [13]. The colonization factors themselves have undergone extensive evolution, resulting in at least 22 human ETEC CF genetic variants. For example, the pCOO plasmid was the first ETEC CF-encoding plasmid to be sequenced. Isolated from strain C921b-1, this plasmid encodes the CS1 and CS3 variants of CFs [13]. Genome sequencing of ETEC strain E24377A showed this strain has six plasmids, ranging in size from 5 to 80Kb. The CS1 antigen of this strain is encoded on the pETEC_73 plasmid, which is similar to the pCOO plasmid in its possession of CS1 and in the RepI1 backbone of the plasmid. This indicates that the CS1 operon was introduced into an ancestral plasmid and maintained by certain strains, prior to the integration of RepFIIA components into the pCOO plasmid [13] and its maintenance in other ETEC strains. Indeed, the presence of other CF-encoding plasmids with the same Rep backbones, such as the CFA/I on the pH10407_95 plasmid from strain H10407, further suggests that the ETEC CF operons have been acquired on multiple occasions on multiple plasmid backbones. EAEC is another pathovar which is a very heterogeneous pathogen and a complicating factor is that some EAEC strains are pathogenic while others are not. These strains are often recovered from apparently healthy individuals and there was a failure of some studies to show a correlation between EAEC and disease [23]. Although three major EAEC phylogenetic groups, EAEC1, EAEC2, and AA/DA (aggregative adherence/diffuse adherence) have been

 

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identified on the basis of multilocus enzyme electrophoretic (MLEE) patterns, EAEC phylogeny overlaps with the also heterogeneous DAEC group [24]. However, members of each of the three clusters show conserved plasmid and chromosomal loci, suggesting the most EAEC, like other pathotypes of diarrheagenic E. coli, show a conserved linkage of virulence genes [23]. The primary virulence factor of EAEC is the aggregative adherence phenotype, which is associated with aggregative adherent fimbriae (AAF) and localized to a 55-65MDa plasmid, the pAA plasmid [13]. Similar to the ETEC colonization factors (CFs), EAEC adhesins are multiple and diverse and allelic variants of AAF have been identified. A study by Jenkins et al. differentiated two groups of EAEC on the basis of the presence or absence of genes on the pAA plasmid, and thus designates “typical” and “atypical” EAEC with typical strains possessing pAA-associated genes, including aggR, as well as certain chromosomal islands that are apparently co-inherited[25]. Another EAEC virulence factor identified as a putative cause of diarrhea was the enteroaggregative heat-stable toxin EAST-1[26]. This toxin activates guanylate cyclase and causes ion secretion; however, no association has been identified between EAST-1 and diarrheal illness, and EAST1 has been detected in other diarrheagenic E. coli pathotypes. Three plasmids from EAEC strains have been completely sequenced: pO42, which belongs to the AAF/II+ strain O42; 55989p, which belongs to AAF/III+ strain 55989; and pO86A1, which has a novel AAF-like operon [13]. Finally, there are no known pathogenicity islands in EAEC; however, islands associated with other members of the family Enterobacteriaceae have been found in EAEC. Examples include a hemolysin-pyleonephritis-associated pili island associated with ExPEC and the high pathogenicity island originally described in Yersinia, which has genes for the synthesis of the siderophore yersiniabactin and its uptake protein [23]. Commensal E. coli LEE PAI (Intimin) Lpf fimbriae

CF As PAI

Atypical EPEC

LT/ST Enterotoxins

EHEC plasmid

EAF plasmid (Bfp)

Shi PAI

pAA plasmid

Inv plasmid

Afa/Dr fimbriae

Enterotoxins

Stx genes CFA

EIEC ETEC Bfp Lpf

Typical EPEC

EHEC STEC

Afa/Dr

EAEC

DAEC

Figure 1: Escherichia coli encompass a continuously evolving group that includes both commensal and pathogenic strains. The pathogenic diversity of E. coli is a result of deletion or acquisition of genes, which confer virulence properties to different bacterial isolates. Only the most successful combinations of virulence factors, commonly encoded on mobile genetic elements, have persisted to become part of specific E. coli pathotypes. Virulence determinants encoded by these elements include the EPEC adherence factor (EAF), EHEC virulence, and EIEC invasion plasmids; the Locus of Enterocyte Effacement pathogenicity island (LEE PAI) of EHEC and EPEC; the plasmid-encoded heat stable and heat labile enterotoxins of ETEC, and the bacteriophageencoded Shiga toxin (Stx) of EHEC. Other categories of pathogenic E. coli, such as EAEC, EIEC and DAEC possess unique combinations of virulence determinants. Abbreviations: EPEC, enteropathogenic E. coli; EHEC, enterohemorrhagic E. coli; STEC, Shiga toxin-producing E. coli; ETEC, enterotoxigenic E. coli; EAEC, enteroaggregative E. coli; EIEC, enteroinvasive E. coli; DAEC, diffusely adherent E. coli.

EPIDEMIOLOGY OF INTESTINAL Escherichia coli PATHOTYPES Intestinal E. coli pathotypes (or diarrheagenic E. coli, DEC) cause significant morbidity and mortality worldwide in children under 5 years of age, especially in the developing world. ETEC strains alone are responsible for millions of diarrheal episodes and an estimated 380,000 deaths each year [27-28]. The majority of diarrhea cases due to DEC are caused by ETEC; however other DEC pathotypes also cause significant disease, such as EPEC and, increasingly, EAEC. Shiga toxin-producing E. coli (STEC) infections are relatively rare in both developed and developing countries; however, STEC, especially EHEC of the serotype O157:H7, are considered important pathogens due to

 

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the potential for life-threatening sequelae, such as hemorrhagic colitis and hemolytic uremic syndrome. Widespread use of oral rehydration therapy since the 1980’s has reduced the severity of disease and mortalities due to acute diarrheal episodes. Since the reduction of serious disease due to acute diarrhea, the incidence of persistent diarrhea (defined as diarrhea lasting >14 days) has increased, and approximately 50% of those who die as a result of diarrheal disease have persistent diarrhea [29]. In conjunction, detection of EPEC and EAEC strains that are associated with persistent diarrhea has increased. All intestinal E. coli pathotypes are transmitted via the fecal-oral route and most infections occur as a result of contaminated food or water. Humans are the major reservoir of EPEC, EAEC and ETEC, while cattle are the main reservoir for STEC. EPEC, EAEC and ETEC have all been isolated from various animals, but the role of these animals in transmission is unclear. Epidemiology of EPEC EPEC is a major cause of persistent diarrhea in children less than 2 years of age throughout the developing world where it is endemic [30]. It is estimated that 5-10% of all diarrheal cases in children are caused by EPEC when identification is based on molecular methods, or 10 - 20% when based on serotyping or adherence to cultured human epithelial cells [30]. All EPEC carry the eae gene, encoding the receptor intimin, which is contained within the LEE pathogenicity island. EPEC strains are classified as either typical or atypical EPEC based on the presence (typical) or absence (atypical) of bundle-forming pili (BFP) [31]. Although detection of BFP is considered the best criteria for accurate classification of EPEC, many investigators distinguish EPEC strains by the presence or absence of the gene encoding the BFP (bfpA) or of EPEC adherence factor plasmid (pEAF) which carries bfpA [32]. Typical EPEC have been a leading cause of persistent watery diarrhea in children of developing countries, but have been supplanted over the last decade by emerging atypical EPEC strains in both developed and developing countries [30]. EAEC, followed by typical EPEC, were the predominant diarrheagenic E. coli isolated from children under 5 years of age that were hospitalized for acute or persistent diarrhea in Dar es Salaam, Tanzania [33]. Results of a study in Brazil showed a similar trend, although EAEC and, in this case, atypical EPEC, were the predominant diarrheagenic E. coli pathogens in children less than 5 years of age presenting with diarrhea to hospitals or clinics in two large urban centers in Sao Paulo State, Brazil [34]. Typical EPEC was also recovered in this study, but mostly from a population at high risk of typical EPEC infection in the past. This observation supports the hypothesis that the shift in prevalence from typical to atypical EPEC in Brazil and other parts of South America may be due to improved sanitation and/or other living conditions or factors that allowed for the emergence of atypical EPEC strains [32, 35]. Other studies conducted in Mexico, Peru and Uruguay also found atypical EPEC (typical EPEC was also detected in diarrhea cases but at a lower prevalence) as one of the most frequently isolated diarrheagenic E. coli pathotypes in young children with acute or persistent diarrhea [36-43]. Gomes et al. (2004) reported atypical EPEC in children, adults and AIDS patients with diarrhea, in three urban centers in Brazil, indicating that atypical EPEC is an important cause of diarrhea in adults and the immunocompromised as well as in children. EPEC outbreaks involving adults have occurred [44-45] and presumably a high dose is ingested because in volunteer studies diarrhea can be induced with doses of 108 – 1010 CFU/ml and after neutralization of gastric acid with sodium bicarbonate [46]. Atypical EPEC is also an important cause of sporadic and epidemic diarrhea in developed countries. Sporadic cases are mainly detected in children, while epidemics affect both children and adults. Atypical EPEC made up 71% (30/42) of diarrheagenic E. coli isolated from children less than 14 years old with persistent diarrhea in Australia [47]. Similarly, a study in Norway found EPEC more frequently than any other enteric pathogen in the stools of children less than 2 years of age. All but one of the 44 EPEC isolates were atypical EPEC, and one-third of the patients from whom atypical EPEC was recovered had persistent diarrhea [48]. A subsequent study by Afset et al. (2004) was unable to show a significant association between atypical EPEC and diarrhea in children less than 5 years old in Norway; however, an association was detected between atypical EPEC and persistent diarrhea [49]. While typical EPEC are very rarely isolated in developed countries, outbreaks of diarrhea due to atypical EPEC have occurred in the United States, Finland and Japan [44-45, 50]. An outbreak in Minnesota affected >100 patrons and workers of a restaurant. Foodborne transmission of EPEC was suspected, but no single vehicle was implicated, and contamination of various foods by an infected restaurant worker could not be ruled out [44]. No source of infection was identified in a diarrheal outbreak in Finland that affected both children and adults [45]. A waterborne

 

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diarrheal outbreak occurred in Japan that affected children from 12-15 years old [50], while another Japanese outbreak involved infants in a daycare facility [51]. Humans are considered to be the major reservoir of typical EPEC, although there are increasing reports of isolation from dogs, cats, monkeys and deer [52-56]. EPEC that carried eae, bfpA and/or the EAF plasmid, but belonged to the non-classical EPEC serotype O157:H45 were isolated from cattle in Switzerland [57]. EPEC strains of this serotype have been isolated from humans, and one such strain caused an outbreak in Japan [58-59]. Atypical EPEC are shed by domesticated animals (cats, dogs, cattle, sheep, pigs, rabbits, chickens, duck, geese, and pigeons) and wildlife (monkeys, deer) [53-55, 60-62]. These animals may serve as reservoirs of atypical EPEC infection in humans, as several studies have identified aEPEC from animals that carried virulence genes or displayed phenotypes associated with human infection. These findings led to the suggestion that colonization by these isolates is not restricted to a particular species [53, 55, 62]. Despite isolation of typical and atypical EPEC from multiple animal sources and animal products, currently there is no evidence of zoonotic transmission. Typical EPEC generally belong to the serogroups O26, O55, O86, O111,O114, O119, O125, O126, O127, O128, O142, O158 [35] which are referred to as classic EPEC serotypes. Eighty-one percent of atypical EPEC do not belong to these classic EPEC serogroups and 26.6% are untypeable. The most frequent serogroups of atypical EPEC are O26, O51, O55, O111, O119, and O145, some of which are also classic EPEC serotypes. Initially, typical and atypical EPEC were classified based on serotype, however, new methods to distinguish between atypical and typical EPEC became necessary due to shared serotypes between these groups. These methods include detection of virulence genes such as eae and bfpA and virulence traits such as formation of attaching and effacing lesions and pattern of adherence on epithelial cells. Typical EPEC exhibit a localized adherence (LA) pattern, whereas atypical EPEC can exhibit localized-like (LAL), diffuse (DA), or aggregative adherence (AA) patterns. Epidemiology of EAEC EAEC is an important cause of diarrhea in children and adults in both developing and developed countries. EAEC strains can cause acute and persistent (>14 days) diarrhea along with inflammation. EAEC was first described in 1987 in a child from Chile with persistent diarrhea [63]. Several studies have since shown an association between EAEC and persistent diarrhea in children throughout the developing world [64-67]. More recently, a meta-analysis of 41 case-control studies involving populations from developing and industrialized countries demonstrated more frequent isolation of EAEC from children with acute diarrheal illness compared to controls [68]. A significant association was also detected between EAEC and acute diarrhea in children of industrialized countries, HIV-infected adults from developing countries, adults from developing countries, and adult travelers to developing regions [68]. EAEC is second only to ETEC as a cause of diarrhea in travelers to developing countries, being responsible for 24.1% and 16% of cases in Latin America and South Asia, respectively [69]. There is also evidence that EAEC causes sporadic diarrhea in adults of industrialized countries [70-71]. In studies conducted at sites in Brazil and Peru, EAEC was one of the most frequently isolated pathotype of diarrheagenic E. coli recovered from children with diarrhea [38, 72]. Most EAEC cases are sporadic, but several food and waterborne outbreaks of diarrhea affecting both children and adults in Europe, the UK, the US and Japan have been described [73-74]. In 1993, 40.6% of 2,697 Japanese children who ate school lunches developed diarrhea, which was persistent in 10% of cases [73]. Two outbreaks of diarrhea due to EAEC occurred within 10 days of each other at a holiday farm restaurant in Italy [74]. The infection sources in both the Japanese and Italian outbreaks were identified epidemiologically; EAEC was not recovered from the implicated food items. EAEC has been recovered from both asymptomatic individuals and those with diarrhea, which suggests that humans are likely the reservoir of EAEC infection. Fecally-contaminated food and water may serve as source of infection, and vehicles include produce, unpasteurized dairy products, sauces and baby bottles [75]. Other risk factors include travel to developing countries, poor hygiene, host susceptibility and possibly immunosuppression due to HIV infection [68, 75-76].

 

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EAEC has been recovered from animals such as calves, piglets and horses, which may indicate that these animals are potential reservoirs for human EAEC infection [77-78]. However, Uber et al. (2006) found that clinical isolates of EAEC from calves, piglets and horses with diarrhea were not typical EAEC, in that they lacked the plasmidencoded aggR gene. These atypical EAEC animal isolates were also found to carry few or none of the other EAEC virulence markers which leads to the suggestion that animals may not be a reservoir for human pathogenic EAEC. Another study that examined clinical samples from horses included isolates from both diarrheal feces and extraintestinal sites [77]. One isolate was found to carry several putative virulence genes, including aggR, that are associated with EAEC diarrhea in humans. The risk of infection from animals appears low; therefore, humans remain the most likely reservoir of EAEC infection. Common EAEC serotypes include O44:H18 and O111:H12 as well as the serogroups O125 and O126, although much serotype diversity has been reported [75]. The aggregative nature of the bacterium results in many strains being untypeable for the O antigen. Further evidence of heterogeneity is found in volunteer studies that demonstrate virulent and avirulent strains of EAEC (some strains elicit diarrhea in volunteers, while others do not) [79]. These observations may explain the high frequency of EAEC recovery from asymptomatic individuals. Efforts to identify markers associated with EAEC pathogenicity are ongoing. The CVD432 probe can be useful in identifying EAEC strains, although sensitivity can range from 15–90%, depending on the geographic region [75]. The presence of this probe was associated with persistent diarrhea in children compared to healthy controls or to children with acute diarrhea in a case-control study conducted in Brazil [80]. Diarrhea was positively associated with heat stable enterotoxin (EAST1)-positive and EAST1/CVD432-positive EAEC strains in this study [80], although few studies have found an association between the virulence of EAEC strains and any one or group of these markers. The heterogeneity of EAEC strains, as well as host factors, likely contribute to the role of EAEC in both acute and persistent diarrhea with a range of accompanying symptoms, including intestinal inflammation, abdominal pain, nausea, vomiting, low-grade fever and blood or mucus in stools. Epidemiology of STEC Shiga-toxigenic E. coli (STEC) strains are not a major cause of diarrhea, although STEC infection can lead to severe and life-threatening disease. Many STEC are able to form attaching and effacing lesions in the intestine through the expression of the proteins encoded on the LEE, but it is the production of Shiga toxins (Stx) that defines this E. coli pathotype. Toxin production is also thought to be responsible for the damage leading to serious sequelae such as hemolytic uremic syndrome (HUS). Some strains of STEC that cause bloody diarrhea, a risk factor for HUS, are also known as enterohemorrhagic E. coli (EHEC), which is a subset of STEC. Unlike other DEC pathotypes, cattle are the main animal reservoir of STEC and are the major source of direct and indirect transmission. Since the emergence of E. coli O157:H7, other STEC serotypes have also been recognized as causes of serious disease [81]. Both O157 and non-O157 STEC infections are reported mainly in developed countries such as the United States, Canada, UK, Japan, Australia, Europe and Argentina. Outbreaks are common and receive much attention in the press but most STEC cases are sporadic. Only 25.7% of STEC infections in the US in 2008 were outbreak-associated [82]. As, with other DEC pathotypes, children are most at risk of infection and are also more likely to develop severe disease, although HUS can occur at all ages. The elderly are also vulnerable to developing severe manifestations of STEC infection and they are most likely to succumb to STEC-associated disease even in the absence of severe complications [83]. STEC infections are rare in developing countries and few outbreaks are reported. In a meta-analysis of 19 studies which examined the pathogens isolated from cases of persistent diarrhea in children under 6 years of age residing in low to middle income countries, there were only two studies which tested for EHEC, and neither detected it in cases or controls [84]. Several studies in Brazil have identified STEC in human clinical isolates, including the first report in that country of O111:NM infection [85] and possibly the first outbreak in Brazil caused by E. coli O157:H7 [8687]. A study in the Amazon found 0.63% of cases carried STEC. STEC infection was found to be significantly associated with EPEC diarrhea cases, although only 3 cases were positive for STEC [41]. E. coli O157:H7 or nonmotile strains of the serogroup O157 (O157:H- or O157:NM) have been detected in a collection of food, animal and human clinical isolates from South America [88]. These O157 isolates carried several virulence markers associated

 

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with human disease. In children over 2 but less than 12 years-old in Mexico City, 8.6% of diarrheal pathogens were STEC [42]. Most of the STEC isolates carried the stx2 gene which is associated with development of HUS. Converse to the trend of low prevalence of STEC-associated disease in Mexico and many South American countries, Argentina has the highest rates of HUS in the world at 12.2 cases per 100,000 inhabitants in 2002. Elsewhere, most HUS cases are associated with the O157 serogroup although, in Argentina, STEC of non-O157 serogroups were isolated from 40% of cases with diarrhea or HUS [89]. Chile and Uruguay also have high rates of HUS [90-91]. STEC was detected in a small number of controls in several case-control studies conducted throughout the developing world. Often, these isolates carried virulence genes or were of serotypes associated with human disease [36, 42, 72]. A study in Nigeria showed STEC were frequently isolated from children with and without diarrhea [92]. Humans may serve as an important reservoir of STEC infection in areas where asymptomatic STEC carriage is coupled with inadequate sanitation. One of the largest outbreaks in a developing country occurred in South Africa and Swaziland where contaminated water sickened thousands of people; fatalities and cases of renal failure were also reported. E. coli O157:H7 was isolated from 22.5% of 89 stools from patients [93]. Many developed countries report multiple STEC outbreaks each year; however, these are usually small, affecting tens of people or less. Large outbreaks, sickening hundreds of people, are less frequent. In 2000, an estimated 2,300 people were infected with E. coli O157:H7 due to a contaminated municipal water supply in the town of Walkerton, Ontario, Canada. Twenty-seven cases developed HUS and 7 died of STEC infection [94]. In the same year as the Walkerton outbreak, Canada reported 45 other outbreaks of E. coli O157:H7 infection. In 2006, E. coli O157:H7contaminated spinach caused 205 cases in 26 US states and in Canada, and 3 deaths [95]. In the 20-year period from 1982 to 2002, 350 outbreaks were reported in the US [96]. Although higher incidence occurs in the northern states, outbreaks were reported in 49 states affecting 8,598 people [96]. Since the 2006 spinach outbreak, several other outbreaks have been reported. A cluster of E. coli O157:H7 cases was linked to a Taco Bell fast-food outlet in the northeastern US in 2006. This outbreak had an 11% HUS rate and lettuce, cheddar cheese and beef were linked to cases [97]. Other outbreaks in the US from 2007 to 2010 have been linked to frozen beef patties, ground beef, bladetenderized steaks, pepperoni on frozen pizzas and raw cookie dough [98]. Most STEC infections in industrialized countries are sporadic. Analysis of data from FoodNet shows that outbreaks accounted for only 25.7% of E. coli O157 cases in 2008[82]. Overall, 513 E. coli O157 infections were reported resulting in an incidence of 2.12 cases per 100,000 people. Non-O157 STEC infections in 2008 totaled 205 with an incidence of 0.45 cases per 100,000 people. In children (< 18 years old), 77 cases of HUS were reported (incidence of 0.73 cases per 100,000 children). There was no significant change in the incidence of E. coli O157 infections or HUS compared to the previous three years; however, E. coli O157 infections have decreased 25% (95% confidence interval: 8%-39%) compared to the first three years of surveillance (1996-1998)[82]. The decrease in incidence since the mid-late 1990’s plateaued by the mid-2000’s, and incidence has not yet reached the target of 1.0 E. coli O157 infections per 100,000 people proposed in the CDC Healthy People by 2010 Program suggesting unresolved and/or unrecognized issues in food safety still exist[82]. Transmission of STEC is via the fecal-oral route. The infectious dose of E. coli O157:H7 is estimated to be approximately 100 organisms and is presumed to be low for non-O157 STEC [99]. This low dose and the ability of E. coli to survive a variety of conditions may contribute to the diversity of vehicles and transmission routes reported. Transmission of STEC has been reported via contaminated water, food items, person-to-person contact and animal contact. Airborne particles contaminated with E. coli O157 were implicated in one outbreak [100] and airborne transmission of E. coli O157 was demonstrated between pigs in a controlled setting [101]. Among food items implicated in STEC infections, meat, produce, milk and milk products have been reported [96, 102-103]. Produce items including sprouts, lettuce and spinach have been linked to outbreaks [96, 102-103]. Although there are many potential sources of STEC infection, cattle remain the most important reservoir. At the beef and dairy herd level, O157 is ubiquitous [104]. Small ruminants such as sheep and goats are also carriers of STEC and indirect transmission from sheep to humans was demonstrated following a Norway sausage outbreak [105-106]. Pigs were thought to only serve as a mechanical vector, but recent studies have demonstrated that pigs are competent biological reservoirs of STEC [95]. The 2006 spinach-associated outbreak of E. coli O157:H7 was likely the result of feral pigs shedding the outbreak strain onto spinach fields. The outbreak strain was also identified in cattle about

 

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one mile away from the implicated fields [95]. Interestingly, STEC have a wide distribution in both food items and production animals in countries that have low STEC prevalence in humans, such as India and Brazil [87-88, 107110]. Some of the STEC from food and animals carried several genes and/or serotypes associated with human disease; however, many isolates lacked important virulence genes such as the eae gene [87, 108]. Runoff water from fecally-contaminated fields is likely responsible for contamination of drinking and recreational water [103]. Little is known about the behavior of STEC in soil and/or water but this will likely prove important to the ecology of STEC and STEC-related disease. Risk factors for sporadic STEC infection vary geographically, but in general, eating outside the home and eating undercooked beef are the most common exposures. Risk may differ for exposures depending on age. For example, Werber et al. (2007) found that transmission through food items was less of a risk in children less than 3 years of age, whereas in children over 10 years of age, only food items, such as lamb meat and raw spreadable sausage were found to be significant risk factors [111]. In general, risk factors are similar for E. coli O157 and non-O157 STEC infection; however, a study in Australia found that O157 was associated with eating hamburgers, eating out of the home and living on or visiting a farm while non-O157 infections were associated with eating chicken deli meat and working with animals [112]. A study of sporadic STEC infection in New Mexico found that those infected with nonO157 were more likely to be nonwhite, < 5 years old and urban residents compared to those infected with O157 [113]. Seasonality has been observed in both STEC infection and prevalence in cattle, both of which increase during summer months [103, 114]. E. coli O157:H7 is a highly virulent serotype and the most recognized and characterized of the STEC; however, there are 250 recognized serogroups of non-O157 STEC and over 100 of these serogroups have been associated with disease in humans, and along with flagellar antigen types, the variety of STEC serotypes is vast [115]. As the use of methods to identify non-O157 STEC becomes more widespread, non-O157 STEC serotypes are increasingly detected and approximately 19-100% of STEC-associated disease is due to non-O157 serotypes [115]. The nonO157 serotypes most associated with severe disease in humans are motile and non-motile (NM) strains of O26:H11/NM, O103:H2, O111:H8/NM, O145:H28/NM. Other emerging serotypes include O118:H16/NM, O121:H19/NM [102]. STEC that produce Stx2 are associated with severe human disease, while strains producing only Stx1 are not, although these strains are isolated from some cases of HUS and bloody diarrhea [116]. While the Stx1 gene is quite conserved, several variants of Stx2 have been identified that are associated with differences in disease severity. Stx2 and Stx2c are variants that are more frequently isolated from HUS patients, whereas patients with STEC producing Stx2d are more likely to have uncomplicated diarrhea [103]. Some Stx2 variants are mainly detected in animal strains such as Stx2e, which contributes to the pathogenesis of edema disease in pigs [117-118] and Stx2f which is found in STEC of avian origin [103]. Variants of intimin have also been identified in attaching and effacing E. coli. Although 17 intimin types have been described, the main intimin types are α, β, γ, ε [103, 119]. In general, EPEC carry α-intimin, while γ-intimin is found in STEC serogroups O157, O55 and O145. Other important STEC serogroups, O103 and O121 have the ε-intimin type, and STEC O26:H11 carry β-intimin [103]. LEE-negative (intimin-negative) serotypes, such as O113:H21, have been associated with severe disease and produce an alternate adhesin and cytotoxin [115, 120]. Epidemiology of ETEC ETEC is the most commonly isolated pathotype of DECs. It is a major cause of pediatric diarrhea which causes an estimated 1.4 million deaths per year [28, 121]. ETEC can cause cholera-like diarrhea in groups of all ages, including travelers to the developing world where ETEC is endemic. Asymptomatic carriage of ETEC in individuals of all ages is common, which provides a reservoir for cycles of infection and re-infection [28]. ETEC infection is a major cause of diarrhea in children under 2 years of age in the developing world [122]. A survey by Wenneras et al. (2004) of studies conducted between 1970 and 1999 showed high incidence of ETEC in children less than one year old (69 million diarrheal episodes per year) with 210 million episodes per year for children 1-4 years old (average 52 million episodes). Diarrhea due to ETEC infection in children less than 2 years of

 

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age may exacerbate or result in malnourishment, potentially leading to reduced physical and/or cognitive development. ETEC is associated with diarrhea in children under 5 years, although it is estimated almost 50 million children in this age group are asymptomatic carriers [28]. Compared to younger children, 5-15 year-olds have reduced incidence (114 million cases per year for the entire age group, average of approximately 10 million cases per year) and ETEC is no longer associated with diarrhea in this older age group [28]. Although ETEC-associated diarrhea is mainly considered to affect young children, approximately 25% of ETEC cases are in adults who can experience severe dehydration compared to children [122]. ETEC is endemic in almost all developing countries with peak incidence occurring during warm and wet seasons [122]. Outbreaks of ETEC can also occur in developing countries, although clusters of ETEC-associated diarrhea have been mistaken for cholera outbreaks. Two unrelated ETEC outbreaks in the Brazilian Amazon Rainforest were originally thought to be cholera [123]. Travelers to countries where ETEC is endemic are susceptible to diarrhea due to ETEC infection. ETEC is a major cause of traveler’s diarrhea (TD) and may be responsible for 20-40% of cases [122, 124]. In a meta-analysis, Shah et al. (2009) reported that 30.4% of global TD cases are due to ETEC. The prevalence of ETEC-associated TD in different regions were similar to this global prevalence: 33.6% in Latin America, 31.2% in Africa, 30.6% in South Asia, but only 7.2% in Southeast Asia [69]. ETEC-associated diarrhea also impacts soldiers deployed to countries where ETEC is endemic; 70% of US troops experienced at least one bout of diarrhea during deployment to Iraq and ETEC was the most commonly isolated enteropathogen from these cases at a prevalence of 32% [125]. Sporadic endemic cases of ETEC diarrhea are rare in developed countries, except in communities lacking adequate water quality and sanitation; however, several food and waterborne outbreaks have been reported in the US, Japan and Europe [122]. ETEC was identified as the etiologic agent of a large food borne outbreak at a sushi restaurant in Nevada in 2004. Poor food-handling practices and infected food handlers likely contributed to this outbreak since the butterfly shrimp implicated in these outbreaks was distributed to other restaurants that were not involved in the outbreak [126]. Another ETEC outbreak in 2004 occurred at a corporate lunch in Illinois [127]. Cucumber salad and Asian crispy noodle salad were associated with diarrhea through epidemiological methods, although no food was available for testing. In general, the ETEC strain responsible for this outbreak resulted in diarrhea of longer duration (median of 7 days) compared to other reported ETEC outbreaks (median of 4 days) [127]. As with other DEC pathotypes, transmission of ETEC occurs via the fecal-oral route. Contaminated food and water are the most common sources of infection. Humans are the major reservoir of ETEC, so improved personal hygiene and sanitation capabilities (clean water and latrines) should reduce incidence of ETEC infection. Infants in low socio-economic households in the developing world are most susceptible to ETEC-associated diarrhea [122]. Breastfeeding is thought to be protective since exposure to ETEC through contaminated food and water is reduced. However, protection is limited since children are often weaned or started on solid foods at a very young age, and contaminated weaning food has been suggested as a likely source of ETEC infection in infants [122]. Risk of ETEC diarrhea in children in Brazil was associated with preparation of food (beans, rice and soup) in the morning that was fed to children in the afternoon [128]. This association suggests that improper food storage is a risk factor for ETEC diarrhea. ETEC is also a cause of serious diarrheal disease in young animals, particularly swine, cattle and rabbits, but these strains carry different toxin and colonization factors than human ETEC strains and, therefore, do not appear to have zoonotic potential [122]. ETEC strains isolated from humans are highly variable. There are 78 different serotypes and many isolates are untypeable. The most common ETEC serotypes detected in a collection of isolates representing the global diversity of ETEC were O6, O78, O8, O128 and O153 [129]. Only 34 flagellar H-antigen types were identified in this collection, five of which accounted for half the ETEC isolates [129]. A subsequent study in Egypt showed that the serotypes O43 and O159 were most prevalent and five H types also accounted for half of the Egyptian isolates, but these serotypes differed from the previous study [130]. Analysis of ETEC outbreaks occurring in the US between 1996 and 2003 found that ETEC of the serotype O169:H41 were isolated in 10/16 outbreaks and were the sole

 

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serotype identified in 6 of the outbreaks [131]. Prior to 1996 only 1/21 outbreaks were due to O169:H41 ETEC. These serotypes appear to be emerging in both Japan and the US [132]. The finding of multiple lineages within a serotype goes against other studies which demonstrated that each serotype belonged to its own lineage despite some intra-serotype variation [133-134]. ETEC strains produce colonization factor antigens (CFAs) that mediate binding to the small bowel. Over 22 CFA types have been recognized and more are thought to exist. The most common types are CFA/I, coli surface antigen (CS) 1, CS2, CS3, CS4,CS5, CS6, CS7, CS14, CS17 and CS21 [10, 122]. Approximately 75% of ETEC express CFA/I, CFA/II or CFA/IV, although 30-50% of ETEC express no typeable CFA [10, 122]. This may be due to the absence of CFA or our inability to detect all types of CFA. Colonization factors, such as K88 and K99, that are expressed by ETEC of animal-origin are very different from those of ETEC isolates from humans and likely contribute to species-specificity [10, 122]. ETEC can produce each or both of the plasmid-encoded toxins, heat-labile toxin (LT) and heat-stable toxin (ST). ETEC strains producing LT only are thought to be less pathogenic than strains producing ST only or LT and ST, since LT-producing ETEC are often isolated from asymptomatic individuals [122]. Indeed, Qadri et al. (2005) reported a possible increasing trend in LT-producing ETEC strains in Bangladesh and Latin America. There are two variants of LT, LT1 and LT2. LT2 is associated with animals, while LT1 are mainly isolated from human ETEC cases. Genotyping techniques found 16 different LT1 types among 51 ETEC strains isolated from children with and without diarrhea in Brazil [135]. Considering that 16 LT types were identified in only 51 samples from one country, it is likely that significant diversity of ETEC LT1 exists. Functional differences in these LT types may account for the variations in incidence and disease severity observed among LT-producing ETEC strains. Similar to LT, ST has two major variants, STa (or STI), which is mainly associated with human disease, and STb (or STII) which is predominant in animals. STa can be further split into two subtypes, STh (or STIb) and STp (or STIa). STh were thought to mainly be found in humans, while STp were considered of porcine origin, although both STh and STp have been isolated from humans. Bölin et al. (2006) found that the distribution of the two STa subtypes varies by geographic region with equal prevalence of STh and STp among pediatric diarrhea cases in Egypt and Guatemala, but few STp-producing ETEC detected in Bangladesh. CONCLUSION Diarrhea is the second leading cause of death in children under 5 years old in the developing world. DEC strains are responsible for millions of episodes of diarrhea each year that affect children and adults worldwide. ETEC is endemic in developing countries and is the most frequently isolated bacterial pathogen from children with diarrhea, as well as from adult travelers to endemic areas. Increasing outbreaks of food and waterborne ETEC infection have been reported in the United States, many of them associated with the emerging serotype O69:H41. EPEC strains are also widespread, particularly in the developing regions. Atypical EPEC strains, lacking the bundleforming pilus, are emerging in both developing and industrialized countries and are associated with persistent diarrhea. Unlike typical EPEC, atypical EPEC strains have been isolated from a variety of animal species. The role of animals in atypical EPEC transmission is unknown, as are the factors underlying the emergence of atypical EPEC. EAEC strains have emerged as the second leading cause of travelers’ diarrhea following ETEC, and as one of the most frequently isolated DEC pathotypes in children with diarrhea. Persistent diarrhea in children has been associated with EAEC, in both developing and industrialized countries, and outbreaks affecting children and adults have been reported. EAEC are identified phenotypically by a “stacked brick” adherence pattern on cultured epithelial cells; however, EAEC strains are very diverse and a limited factor for epidemiological studies is that no genetic markers have been detected that identify all EAEC. STEC continue to be an important cause of sporadic and epidemic diarrhea and the more serious HUS in industrialized countries. STEC are present in cattle and other animal species worldwide, but are detected at low levels, if at all, in children with or without diarrhea in developing countries. The threat posed to human health by the

 

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great diversity of E. coli strains carrying stx genes is unknown. In addition, the growing number and variety of transmission routes warrants continued surveillance of STEC in humans, animals and the environment. DEC natural history is complex and the underlying factors responsible for shifts in DEC pathotypes or strains, including loss and acquisition of mobile elements as well as anthropogenic changes, must be understood to inform effective interventions that reduce exposure to DEC and decrease the burden of disease. REFERENCES [1] [2] [3] [4] [5] [6]

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[109] Oliveira MG, Brito JR, Gomes TA, et al. Diversity of virulence profiles of Shiga toxin-producing Escherichia coli serotypes in food-producing animals in Brazil. Int J Food Microbiol. 2008;127:139-46. [110] Sehgal R, Kumar Y, Kumar S. Prevalence and geographical distribution of Escherichia coli O157 in India: a 10-year survey. Trans R Soc Trop Med Hyg. 2008;102:380-3. [111] Werber D, Behnke SC, Fruth A, et al. Shiga toxin-producing Escherichia coli infection in Germany: different risk factors for different age groups. Am J Epidemiol. 2007;165:425-34. [112] McPherson M, Lalor K, Combs B, et al. Serogroup-specific risk factors for Shiga toxin-producing Escherichia coli infection in Australia. Clin Infect Dis. 2009;49:249-56. [113] Lathrop S, Edge K, Bareta J. Shiga toxin-producing Escherichia coli, New Mexico, USA, 2004-2007. Emerg Infect Dis. 2009;15:1289-91. [114] Brooks JT, Sowers EG, Wells JG, et al. Non-O157 Shiga toxin-producing Escherichia coli infections in the United States, 1983-2002. J Infect Dis. 2005;192:1422-9. [115] Johnson KE, Thorpe CM, Sears CL. The emerging clinical importance of non-O157 Shiga toxin-producing Escherichia coli. Clin Infect Dis. 2006;43:1587-95. [116] Ostroff SM, Tarr PI, Neill MA, et al. Toxin genotypes and plasmid profiles as determinants of systemic sequelae in Escherichia coli O157:H7 infections. J Infect Dis. 1989;160:994-8. [117] MacLeod DL, Gyles CL, Valdivieso-Garcia A, et al. Physicochemical and biological properties of purified Escherichia coli Shiga-like toxin II variant. Infect Immun. 1991;59:1300-6. [118] Makino S, Watarai M, Tabuchi H, et al. Genetically modified Shiga toxin 2e (Stx2e) producing Escherichia coli is a vaccine candidate for porcine edema disease. Microb Pathog. 2001;31:1-8. [119] Blanco M, Schumacher S, Tasara T, et al. Serotypes, intimin variants and other virulence factors of eae positive Escherichia coli strains isolated from healthy cattle in Switzerland. Identification of a new intimin variant gene (eae-eta2). BMC Microbiol. 2005;5:23. [120] Bettelheim KA. The non-O157 shiga-toxigenic (verocytotoxigenic) Escherichia coli; under-rated pathogens. Crit Rev Microbiol. 2007;33:67-87. [121] Bryce J, Boschi-Pinto C, Shibuya K, et al. WHO estimates of the causes of death in children. Lancet. 2005;365:1147-52. [122] Qadri F, Svennerholm AM, Faruque AS, et al. Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention. Clin Microbiol Rev. 2005;18:465-83. [123] Vicente AC, Teixeira LF, Iniguez-Rojas L, et al. Outbreaks of cholera-like diarrhoea caused by enterotoxigenic Escherichia coli in the Brazilian Amazon Rainforest. Trans R Soc Trop Med Hyg. 2005;99:669-74. [124] Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11:142-201. [125] Monteville MR, Riddle MS, Baht U, et al. Incidence, etiology, and impact of diarrhea among deployed US military personnel in support of Operation Iraqi Freedom and Operation Enduring Freedom. Am J Trop Med Hyg. 2006;75:762-7. [126] Jain S, Chen L, Dechet A, et al. An outbreak of enterotoxigenic Escherichia coli associated with sushi restaurants in Nevada, 2004. Clin Infect Dis. 2008;47:1-7. [127] Yoder JS, Cesario S, Plotkin V, et al. Outbreak of enterotoxigenic Escherichia coli infection with an unusually long duration of illness. Clin Infect Dis. 2006;42:1513-7. [128] Sobel J, Gomes TA, Ramos RT, et al. Pathogen-specific risk factors and protective factors for acute diarrheal illness in children aged 12-59 months in Sao Paulo, Brazil. Clin Infect Dis. 2004;38:1545-51. [129] Wolf MK. Occurrence, distribution, and associations of O and H serogroups, colonization factor antigens, and toxins of enterotoxigenic Escherichia coli. Clin Microbiol Rev. 1997;10:569-84. [130] Peruski LF, Jr., Kay BA, El-Yazeed RA, et al. Phenotypic diversity of enterotoxigenic Escherichia coli strains from a community-based study of pediatric diarrhea in periurban Egypt. J Clin Microbiol. 1999;37:2974-8.

 

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[131] Beatty ME, Bopp CA, Wells JG, et al. Enterotoxin-producing Escherichia coli O169:H41, United States. Emerg Infect Dis. 2004;10:518-21. [132] Nishikawa Y, Helander A, Ogasawara J, et al. Epidemiology and properties of heat-stable enterotoxin-producing Escherichia coli serotype O169:H41. Epidemiol Infect. 1998;121:31-42. [133] Pacheco AB, Guth BE, Soares KC, et al. Random amplification of polymorphic DNA reveals serotype-specific clonal clusters among enterotoxigenic Escherichia coli strains isolated from humans. J Clin Microbiol. 1997;35:1521-5. [134] Pacheco AB, Soares KC, de Almeida DF, et al. Clonal nature of enterotoxigenic Escherichia coli serotype O6:H16 revealed by randomly amplified polymorphic DNA analysis. J Clin Microbiol. 1998;36:2099-102. [135] Lasaro MA, Rodrigues JF, Mathias-Santos C, et al. Genetic diversity of heat-labile toxin expressed by enterotoxigenic Escherichia coli strains isolated from humans. J Bacteriol. 2008;190:2400-10.

 

Pathogenic Escherichia coli in Latin America, 2010, 25-47

25

CHAPTER 3 Enteropathogenic Escherichia coli (EPEC) Tânia AT Gomes1* and Bertha González-Pedrajo2 1

Departmento de Microbiologia, Imunologia, e Parasitologia, Universidade Federal de São Paulo, São Paulo, Brazil; 2Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México, D.F., Mexico Abstract: Enteropathogenic Escherichia coli (EPEC) comprise two groups of distinct organisms classified as typical EPEC (tEPEC) and atypical EPEC (aEPEC). tEPEC were leading infantile diarrheal agents in developing countries, whereas aEPEC prevailed in developed countries. Nowadays, tEPEC are less frequent while aEPEC are emerging enteropathogens of children and adults (including HIV-infected patients) in developing countries. EPEC infections can lead to severe secretory acute and persistent diarrheal diseases. Both EPEC groups contain the locus of enterocyte effacement (LEE), which encodes a Type Three Secretion System and various effector proteins that alter several signaling mechanisms of intestinal cells, leading to the development of attaching and effacing (A/E) lesions. The distinction between tEPEC and aEPEC strains is based on the expression of the bundle-forming pilus (BFP) adhesive-structure, which is restricted to tEPEC. Both EPEC groups lack the Shiga toxin genes of another A/E lesion-producing pathogen, enterohemorrhagic E. coli. aEPEC are much more heterogeneous than tEPEC in terms of phenotypic characteristics and virulence determinants. Humans are the only reservoir of tEPEC, whereas aEPEC strains may be found in humans and diverse animal species. Diagnosis is currently performed in research laboratories that use molecular methods to detect specific virulence properties that distinguish tEPEC from aEPEC strains. Antibiotics are indicated to treat more severe or persistent diarrheal cases, but resistance has been detected worldwide. Prophylactic measures are common to other diarrheal infections and vaccines based on surface or secreted proteins that were shown to induce antibodies (IgG and SIgA) responses in endemic areas are under development.

INTRODUCTION The first epidemiological studies suggesting that certain Escherichia coli strains were agents of severe childhood diarrhea were published at the end of the 19th century [reviewed in [1]]. In subsequent years, various studies suggested the involvement of certain E. coli strains of specific serogroups as agents of infantile diarrhea in Europe and the United States [reviewed in [2] and [1]]. Notwithstanding these various studies, general recognition of E. coli as agent of human diarrhea was attained by John Bray [3], who described the association of antigenically homogeneous E. coli strains with outbreaks of infantile diarrhea (“summer diarrhea”) in England. At the same period, Varela et al. [4] described the involvement of an E. coli strain (E. coli-gomez) that caused fatal diarrhea in an infant in Mexico. Subsequently, various experimental infections were published that corroborated the potential etiologic role of certain E. coli strains in diarrheal diseases [reviewed in [5]]. In 1955, Neter [6] created the term enteropathogenic E. coli (EPEC) to designate those E. coli strains epidemiologically associated with childhood diarrhea and to differentiate these strains from E. coli strains of the normal flora. However, although EPEC was the first diarrheagenic E. coli pathotype identified, their pathogenic potential was only confirmed and widely accepted when their ingestion by volunteers promoted evident symptoms of diarrhea [7]. Due to the epidemiological association of E. coli strains of certain serogroups and serotypes with diarrhea, until the 1970s, detection of specific serogroups (classical O groups) and serotypes was the only method available for EPEC identification and to distinguish pathogenic from non-pathogenic E. coli strains [5]. The development of molecular *Address correspondence to: Dr. Tânia A. Tardelli Gomes, Departmento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, Rua Botucatu, 862, 3º. Andar, Vila Clementino, São Paulo, S. Paulo, 04023-062, Brazil. Tel: 55-11-5083.2980; E-mail: [email protected]. Alfredo G. Torres (Ed) All rights reserved - © 2010 Bentham Science Publishers Ltd.

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and cellular biology techniques and of tissue culture assays has contributed a great deal of information about the virulence factors of EPEC thus allowing the use of such techniques to identify EPEC and to study their virulence mechanisms [8]. Currently, the EPEC pathotype is subdivided into typical EPEC (tEPEC) and atypical EPEC (aEPEC). This classification is based on the occurrence of the virulence-associated EAF (EPEC adherence factor) plasmid (pEAF) in tEPEC and its absence in aEPEC [9]. Both EPEC groups produce a characteristic lesion in the intestinal cells known as attaching and effacing (A/E) lesions, which result from the cooperative action of proteins encoded in a pathogenicity island named locus of enterocyte effacement (LEE). In addition, tEPEC and aEPEC strains lack the genes encoding Shiga toxins (Stx), heat-labile and heat-stable toxins, and are non-invasive [9]. Although tEPEC strains were major causative agents of acute diarrhea in very young children in developing countries (including Latin American countries) until the 1990s, there is currently a clear decrease in their frequency in many of these countries [10, 11]. In contrast, aEPEC strains, which are important agents of diarrhea in developed countries since the 1960s, are emerging agents of acute and persistent diarrhea affecting children and adults worldwide [8, 11, 12]. The main characteristics of tEPEC and aEPEC are summarized in Table 1. Table 1: Main features of typical and atypical EPEC Features

Typical EPEC

Atypical EPEC

Most common serotypes

O55:H6, O55:NM, O86:H34, O111ab:H2, O111ab:NM, O119:H6, O127:H6, O127:H40, O142:H6, O142:H34

O26:H11, O55:H7, O55:H34, O86:H8, O111ac:H9, O111:H25, O119:H2, O125ac:H6, O128ab:H2

Attaching-effacing lesion

Yes

Yes

Present

Absent

EAF plasmid (BFP expression) Stx genes

No

No

Adherence patterns a

LA

LA-like, AA, DA, LAc

LEE Region

Present

Present

Regulation

per, ler, quorum sensing

ler, quorum sensing

Reservoir

Humans

Humans, animals

a

Adherence pattern in HeLa/HEp-2 cell: LA, localized adherence; LAL, localized adherence-like; DA, diffuse adherence; AA, aggregative adherence.

b

NM, non-motile.

c

The LA phenotype in aEPEC strains is independent of BFP expression and usually is detected in prolonged assays (6 h).

SEROTYPES In 1987, the World Health Organization [13] defined EPEC as E. coli strains belonging to 12 different O groups also known as classic serogroups: O26, O55, O86, O111, O114, O119, O125, O126, O127, O128, O142, and O158. It is currently known that some serotypes within these serogroups may comprise both typical and atypical EPEC strains, as well as other diarrheagenic pathotypes [14, 15]. The most frequent serotypes among tEPEC strains of the classic serogroups are O55:H6, O55:NM (non-motile), O86:H34, O111ab:H2, O111ab:NM, O119:H6, O127:H6, O127:H40, O142:H6, and O142:H34. Most of these serotypes correspond to genetically related clones, when studied by Multilocus Enzyme Electrophoresis (MLEE) and other molecular methods [11, 14]. However, the frequency of these serotypes has changed over the years and some tEPEC serotypes belonging to non-classical EPEC serogroups have now been identified, e.g. O88:H25, and O145:H45 [16]. Regarding aEPEC strains, various epidemiological studies conducted in different geographic areas have reported a large antigenic diversity with at least 109 different serogroups (mostly non-EPEC serogroups) and more than 200 different H types [reviewed in [12]]. The most frequent aEPEC serogroups are O26, O51, O55, O111, O145, and O119, whereas the most frequent serotypes are O26:H11, O55:H7, O55:H34, O86:H8, O111ac:H9, O111:H25, O119:H2, O125ac:H6, and O128ab:H2 [12, 17]. A considerable number of aEPEC strains are O and/or H nontypable and many are non-motile.

Enteropathogenic Escherichia coli (EPEC)

Pathogenic Escherichia coli in Latin America 27

PATHOGENESIS After passing through the gastric barrier, EPEC adhere to the mucosa of the small and large intestines, determining complex alterations that lead to diarrhea. The colonization process is proposed to occur in three phases [8]. The first phase is superficial and non-intimate and the factors that mediate initial adherence have not been definitively characterized, but some studies report the possible involvement of a type IV fimbriae named bundle forming pilus (BFP), other less characterized fimbrial and afimbrial structures, as well as the flagella, thus indicating that this phenomenon is multifactorial [18, 19]. After initial adherence, a type III secretion system (T3SS) is mounted and various effector proteins are injected, whose signaling effects promote diverse alterations in the host epithelium. Finally, there is an intimate adherence that culminates with the A/E lesion. Morphologically, the A/E lesion includes the effacing of the intestinal microvilli and the formation of actin-rich pedestal-like structures on which EPEC bacteria rest (Fig.1). In severe infections, there is complete destruction of the intestinal absorptive epithelium, with marked villous atrophy and thinning of the mucosal layer. This lesion could explain the diarrhea presented by infants due to the extensive destruction of intestinal microvilli, but there are currently many evidences that other factors participate in the process of diarrhea like alterations in the transport of ions and water, opening of the tight junctions and mucosal inflammation [19].  

 

mv PY

Figure 1: Attaching-effacing lesions in rabbit ileum infected with atypical enteropathogenic E. coli showing effacement of microvilli (mv) and pedestals (arrows) (Bar 26 μm).

MECHANISMS AND VIRULENCE FACTORS INVOLVED IN THE INTERACTION OF EPEC WITH HOST CELLS Adherence The tEPEC strains produce the so called localized adherence (LA) pattern to HeLa/HEp-2 cell surfaces after 3 h of contact [20], which reflects the formation of compact microcolonies on cell surfaces mediated by BFP [21]. These fimbriae also promote and stabilize bacterial interconnection within the microcolonies (Fig. 2A) [21]. Microcolony formation is also observed in natural infected children, and in ex vivo human biopsies [22], reviewed in [23]]. In contrast, the majority of aEPEC strains produce a modified LA pattern termed LA-like (LAL) [24] or poor LA [25], in which loosen clusters of bacteria are observed in fewer cells. Usually, the establishment of LAL is slower requiring prolonged incubation periods (6 h assays) (Fig. 2B). While the LAL pattern is characteristic of the strains of most aEPEC serotypes [26-32], some aEPEC strains express alternative adherence patterns in vitro, such as diffuse adherence (DA) (Fig. 2C) or aggregative adherence (AA) (Fig. 2D) [26, 27, 32-34]. Some tEPEC strains were shown to produce biofilms on a flow through continuous culture system, and a model of EPEC biofilm formation has been proposed [35]. Using several EPEC isogenic mutants to form biofilms, it was shown that adhesins such as BFP and the EspA filament of the T3SS were involved in bacterial aggregation during biofilm formation on abiotic surfaces. Whether biofilms are involved in the virulence of EPEC remains to be established.

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A

C

B

D

Figure 2: Patterns of adherence to HeLa cells of typical and atypical enteropathogenic E. coli. A. Localized adherence, B. Localized adherence-like; C. Aggregative adherence; D. Diffuse adherence. Microscopic magnitude: 1,000 x.

Adhesins BFP

BFP, the first virulence factor to be identified in pEAF [21], is encoded by the bfp operon which comprises 14 genes (mostly related to its biogenesis), of which the first, bfpA, encodes the pilin protein named bundlin [[36, 37] reviewed in [17]]. The rope-like filaments of BFP interconnect EPEC bacteria into microcolonies to promote non-intimate bacterial adhesion of EPEC to enterocytes in the small bowel and are also involved in dispersion of the bacteria through the intestinal mucosa [38-41]. BFP probably mediates the initial attachment by binding to N-acetyl-lactosamine-containing or to similar receptors on host cell surfaces [42]. The contribution of BFP to the virulence of EPEC has been established by studies in volunteers who ingested EPEC strains carrying mutations in genes of the bfp operon and had much less severe diarrhea than the individuals that received the wild type strain [43]. Intimin Intimin is required for intimate bacterial adhesion to epithelial cells and cytoskeletal reshuffling [[44], reviewed in [17]]. It is an outer membrane protein of 94 kDa with a high variability in amino acid composition at its C-terminal domain (280-amino acid C-terminal sequence Int280). The highly conserved intimin N-terminal domain is inserted in the bacterial outer membrane, whereas the extracellular C-terminal adhesive domain is exposed to the environment [45]. Based on subtle differences at the nucleotide sequence of the C-terminal portion of the molecule, more than 27 intimin subtypes have been described [46-51]. These subtypes were named with Greek letters, being the alpha and beta types more common among tEPEC, whereas intimin subtypes alpha, beta, gamma, zeta, delta, and epsilon appear to be the most frequent among aEPEC strains of different serotypes worldwide [28, 48, 52]. Usually most tEPEC strains of a certain serotype carry the same intimin subtype, [11] whereas not all aEPEC serotypes have the same intimin subtype. For instance, while aEPEC strains of serotype O51:H40 isolated in Brazil and in Spain possess intimin subtype theta [48, 53], certain aEPEC serotypes carry different intimin sub-types, e.g., O80:H26 carrying either intimin subtype beta or epsilon [48, 52]. The exposed variable portion of the intimin molecule connects to its receptor protein Tir (translocated intimin receptor), which is translocated into the cytosol of the targeted eukaryotic cell through a T3SS. After its translocation, Tir is inserted in the plasma membrane exposing its middle portion at the cell surface as a loop. Intimin interacts with this loop region, inducing clustering of adjacent Tir molecules whereas the amino- and carboxy- portions of Tir are exposed to the cytosol [54].

Enteropathogenic Escherichia coli (EPEC)

Pathogenic Escherichia coli in Latin America 29

The Tir C-terminal domain is phosphorylated on its Y474 residue and triggers actin polymerization in tEPEC strains while Tir of enterohemorrhagic E. coli (EHEC) strains are non-phosphorylated and employ other effector proteins for the same function. In addition, it has been demonstrated that aEPEC strains may carry either phosphorylated or non-phosphorylated Tir molecules [reviewed in [55]]. Although the host tissue distribution of EPEC strains is probably multifactorial, some in vitro studies suggest that different intimin subtypes can determine tropism for different intestinal sites (sites of preferential adhesion) [56]. Thus, intimin subtyping may yield important information concerning tissue tropism [reviewed in [17]]. EFA/LIF Lymphocyte inhibitory factor (LifA) is a very large surface protein described in tEPEC strains, which inhibits proliferation of mitogen-activated lymphocytes and the synthesis of pro-inflammatory cytokines [57]. Efa1 (EHEC factor adhesin 1) was first described as a potential adhesin in some EHEC strains [58]. The lifA and efa1 genes are almost identical [58] and are located in a pathogenicity island named O island 122 (OI-122). Besides efa1/lifA, PAI O122 comprises other putative virulence genes: sen, pagC, nleB and nleE [59]. Efa1/LifA seems to contribute to EPEC adherence to epithelial cells in the absence of BFP, and is critical for intestinal colonization by Citrobacter rodentium, an A/E lesion-producing bacterial murine pathogen [60]. There is evidence indicating that efa1/lifA encodes a critical protein product that regulates bacterial colonization, crypt cell proliferation, and epithelial cell regeneration during in vivo colonization [60]. Although Efa1/LifA has been implicated in the attachment of aEPEC strains to host cells [61], its association with diarrheal diseases is controversial [33, 62]. In a recent study in Brazil, the efa1/lifA gene was found to be more frequent among tEPEC (62%) than among aEPEC (30%) strains [63]. However, although tEPEC and aEPEC strains may harbor complete and incomplete PAI O122, a strong association between the presence of a complete PAI O122 (with simultaneous occurrence of efa1/lifA, sen, pagC, nleB and nleE) and diarrhea was observed only in aEPEC. This observation led the authors to suggest that the detection of complete PAI O122 could help to identify potential more pathogenic aEPEC strains [63]. Other Adhesins The complete genomic sequence of tEPEC prototype strain E2348/69, which has been recently published, revealed the presence of eight intact and five incomplete fimbrial operons as well as ten regions encoding putative nonfimbrial adhesins [64]. However, among the intact operon products identified, thus far only BFP were confirmed to play a role in microcolony formation in vitro [21] and diarrhea in human volunteers [43]. Other fimbriae encoded by tEPEC strain E2348/69 include the type 1 fimbriae, but mutants in these fimbriae showed no interference with in vitro adherence [reviewed in [17]]. In addition, EPEC E2348/69 also has conserved fimbrial genes encoding homologs of the long polar fimbriae (LPF) [65]. LPF were originally identified in Salmonella enterica serovar Typhimurium and were shown to direct the attachment of this organism to murine Peyer's patches in vivo [66]. These fimbriae also mediate microcolony formation contributing to the colonization by EHEC O157:H7 in some animal models [67]. In tEPEC, the lpf region (lpfABCDE) encodes predicted proteins with about 60% homology to the Salmonella LPF, but initial studies have indicated that LPF is apparently not necessary for adherence and A/E lesion formation on human biopsies as these functions were unaltered in an EPEC strain E2348/69 lacking the lpf gene cluster [65]. In fact, mutations in one or both of the known lpf loci (lpfA1 and lpfA2) in EHEC O157:H7 were shown to diminish colonization in animal models and to display an altered human intestinal tissue tropism [68, 69]. A number of polymorphisms within the lpfA genes have been recently identified and were used to classify distinct variants based on these major fimbrial subunit genes of EPEC and Shiga toxin producing E. coli strains (STEC) [70]. Both tEPEC and aEPEC strains were found to carry different lpfA variants. Among the tEPEC strains, the majority possessed only one of the two lpfA genes whereas most of the aEPEC strains possessed the lpfA1-2 and lpfA2-1 genes in combination with specific intimin alleles [71]. Recently, it was demonstrated that the E. coli common pilus (ECP), which is present in most E. coli isolates, may act in concert with BFP to stabilize interactions between EPEC and host cells [72]. However, the prevalence and significance of ECP to aEPEC pathogenesis has yet to be determined. In addition, the EspA filament of T3SS has

30 Pathogenic Escherichia coli in Latin America

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been proposed to be the adhesin mediating initial adherence by EPEC strains that lack BFP [38] but the initial adherence of aEPEC strains may probably be multifactorial. As mentioned previously, some aEPEC strains may express the diffuse adherence (DA) or aggregative adherence (AA) patterns in vitro. In some aEPEC strains DA is a consequence of the expression of daa and afa operons, which encode adhesins of the Dr family [73, 74]. A non-fimbrial structure conferring the DA phenotype to tissue culture cells in some aEPEC strains belonging to serotype O26:H11 has been described, despite the fact that this strain exhibited a LA pattern similar to the BFP-mediated LA. This structure is encoded by the chromosomal region designated Locus for Diffuse Adherence (LDA) [75], and its expression is induced by bile salts [76]. The minor structural subunit gene of this adhesin, ldaH, was found in few aEPEC strains of serogroups O5, O26, O111 and O145 [75], but its role in virulence of these aEPEC strains remains to be evaluated. In addition, aEPEC strains of serotype O125ac:H6 express AA in HEp-2 cells but lack EAEC-virulence-associated adhesins; the AA shown by aEPEC strains of this serotype was shown to be mediated by an outer membrane protein [15, 77]. Flagella Flagella contribute to the virulence of various pathogenic bacteria through motility, chemotaxis, and stimulation of IL-8 production in eukaryotic cells. Furthermore, in some species flagella were shown to promote adhesion to and invasion of host surfaces [78]. Some flagellar antigen types, such as H2 and H6 have been consistently identified among EPEC strains isolated in various epidemiological studies worldwide. However, conflicting data exist in the literature regarding the involvement of flagella in EPEC virulence, especially as an adhesin. Girón et al. [79] demonstrated that H2 and H6 flagella purified from tEPEC but not H7 flagella purified from EHEC O157:H7 bound to HeLa cells. In addition, flagella mutants of tEPEC strains were shown to be impaired in adherence and microcolony formation thus corroborating that flagella may mediate adhesion on cultured enterocytes in vitro [79]. However, another study could not confirm a role of flagella in adherence [38]. Studying a selected aEPEC strain (1711-4) of serotype O51:H40, the most prevalent aEPEC serotype in Brazil, Sampaio et al. [80] demonstrated that flagella was involved in aEPEC 1711-4 adhesion to and invasion of polarized intestinal cells (Caco-2 and T84 cells) in vitro as an isogenic aEPEC mutant unable to produce flagellin (the protein subunit of the flagellar filament) had a marked decrease in the ability to adhere and invade those cell lineages. Signaling Events Type Three Secretion System As mentioned previously, EPEC virulence and A/E lesion development are conferred by the chromosomal LEE pathogenicity island, which encodes a T3SS [81, 82]. T3SSs are used by many Gram-negative pathogenic bacteria to deliver effector proteins straight into eukaryotic cells, subverting different host cellular processes [83, 84]. The virulence-associated T3SS also known as the ‘injectisome’, assembles into a complex macromolecular structure of more than 20 different proteins that traverses the bacterial cell envelope [84-86]. It is composed of a multi-ring base that spans both membranes, and extends a needle-like projection that protrudes out of the cell from the bacterial surface [87-89]. In addition, a hydrophilic protein forms a tip complex at the distal end of the needle, and serves as an assembly platform for two hydrophobic pore-forming translocator proteins that form a pore in the host cell membrane [90, 91]. Effectors are thought to be transported through the hollow needle directly into the cytoplasm of the target cell through the translocation pore [89, 90]. The injectisome is closely related to the bacterial flagellar export apparatus [83, 92]. The needle complex shares structural resemblance with the flagella basal body and a high degree of sequence similarity exists among eight proteins of their secretion apparatus [84, 93]. Moreover, phylogenetic studies indicate that both structures derived from a common evolutionary ancestor [94]. The T3SS in EPEC is composed of a cylindrical basal structure with two sets of membrane ring complexes joined by a periplasmic central rod (Fig. 3) [95]. The outer membrane ring is composed of the EscC protein, a member of the secretin family, which forms a channel for the delivery of large molecules through the outer membrane [96, 97]. Recently, the crystal structure of the periplasmic domain of EscC was solved and a homomultimeric ring-model of

Enteropathogenic Escherichia coli (EPEC)

Pathogenic Escherichia coli in Latin America 31

12 subunits was constructed [98]. The inner membrane ring predicted to be associated to the outer leaflet of the inner membrane, is formed by the lipoprotein EscJ that oligomerizes into a 24-subunit ring structure [99]. The EscD protein is also predicted to form a ring-like structure in the inner membrane and EscI is believed to form the inner rod [100]. In addition, several integral and associated inner membrane proteins form the export apparatus essential for protein secretion. Among these, five polytopic membrane proteins EscR, EscS, EscT, EscU and EscV, extensively conserved among different T3SSs and with the flagellar export apparatus, are proposed to be localized within a membrane patch in the center of the inner membrane ring [101, 102]. However, the precise function and localization of these proteins within the secretion apparatus is still unknown. Structural data is available for the Cterminal cytoplasmic domain of EscU, a member of the SpaS/YscU/FlhB family of proteins that undergo autocleavage and form part of a molecular switch that regulates a substrate secretion hierarchy [103]. HM EspD/B

EspA

EscC

EscF OM

Escl

EscJ

EscQ

EscD EscL

PG IM

EscU,R,S,T,V EscN

Chaperone-effector complex

Figure 3: Schematic representation of the injectisome from EPEC. Proteins are represented according to what is known for the EPEC T3SS (see the text), and for their orthologues in other virulence as well as the flagellar T3SSs [92, 98, 102, 104, 105]. As shown, the inner membrane component EscR interacts with EscU and EscS; EscD interacts with EscC (interaction not depicted in the figure) [106]. We have shown interactions between EscN-EscL and EscN-EscQ (González-Pedrajo B., et al. unpublished results). HM, host membrane; OM, outer membrane; PG, peptidoglycan; IM, inner membrane.

A fundamental component of all T3SSs is a highly conserved ATPase EscN/InvC/YscN/FliI that shares sequence similarity with the catalytic β subunit of the F0F1-ATPases and serves to energize the secretion process [107, 108]. It provides a docking interface for chaperone-effector complexes and induces chaperone release and unfolding of the secreted protein in an ATP-dependent manner [109-111]. EscN is the ATPase associated with the T3SS in EPEC and it is essential for the virulence of this bacterial pathogen [97, 112]. High resolution structural data were obtained for the catalytic domain of EscN and a hexameric ring model was built using the F1-ATPase coordinates [113]. The extracellular portion of the injectisome is formed by a needle-like extension which is a helical homopolymer of EscF subunits [87]. Furthermore, EPEC and other A/E pathogens possess a unique T3SS that has a filamentous extension, called the EspA filament, which extends from the needle and is thought to facilitate attachment to the host cells through the thick mucus layer [87, 114, 115]. A central channel within this structure appears to function as a conduit for the translocation of effector proteins into enterocyte cells [116, 117]. Finally, EspB and EspD are secreted by the T3SS in EPEC and form the translocation pore in the intestinal cell [118]. The assembly of the T3SS is a highly regulated process. It has been shown that protein secretion is induced in response to conditions similar to the ones found in the gastrointestinal tract [119]. The T3S proteins SepD and SepL constitute a molecular switch that controls the ordered secretion of translocators (EspA, EspB and EspD) and effector molecules, possibly in response to environmental cues such as low calcium concentrations [120]. This regulatory mechanism is used by A/E pathogens to ensure that translocators are secreted prior to effectors [120-122].

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LEE and Non-LEE Encoded Effectors Genome sequence analysis of the prototype typical strain E2348/69 revealed the existence of 21 T3SS effector genes carried on lambda-like prophages and integrative elements [64]. Seven of the effectors translocated through the T3SS are encoded within the LEE PAI (Tir, Map, EspB, EspF, EspH, EspZ and EspG), while the others are scattered throughout the chromosome and are referred to as non-LEE-encoded effectors (Nle) [[122] reviewed in [123]]. Translocated LEE effectors subvert normal host cell functions and are responsible for the formation of the A/E lesion (effacement of absorptive microvilli and induction of pedestals); and with the exception of EspZ [124], all have proven deleterious effects on the host cell [123]. The first LEE effector to be characterized was Tir; with Tirintimin interaction being essential for pedestal formation [54]. In addition to promoting intimate attachment, actin polimerization and cytoskeletal rearrangements, the Tir-intimin interaction also triggers phosphorylation of a host phospholipase [125], facilitates invasion of non-phagocytic cells [126] and downregulates the EPEC-mediated filopodia formation [127]. Tir is also involved in tight junction (TJ) disruption [123]. The contribution of the LEE effectors in the disease process has been studied in animal infection models using EHEC and Citrobacter, indicating that Tir is essential while the other effectors have a smaller contribution to virulence [123]. Many of the EPEC translocated effectors have multiple functions and the ability to cooperate with one another (reviewed by [128, 129]. Map (Mithochondrial-associated protein) is targeted to the mitochondria affecting its structure and function [130], it induces transient filopodia formation [127], and is essential for disruption of intestinal barrier function and alteration of TJ structure [131]. More recently, Map was shown to act as a guaninenucleotide exchange factor regulating actin dynamics [132]. EspF, another multifunctional effector is also targeted to the host mitochondria initiating the mitochondrial death pathway [133]. It has a role in disrupting the intestinal barrier function [134], remodeling of the brush border microvilli [135], and redistribution of TJ proteins [136]. Additionally, EspF has been implicated in cell death via apoptosis [137] and in inhibition of phagocytosis [138]. EspG and its Nle homolog EspG2 have been shown to trigger actin stress fiber formation and the destruction of the microtubule networks beneath adherent bacteria [70, 139, 140]. More recently, it was demonstrated that both EspG and EspG2, play a role in the inhibition of intestinal membrane chloride transport [141], and that they activate the host cysteine protease calpain during EPEC infection, leading to host cell loss and necrosis [142]. The effector EspH localizes to the host cell membrane and is a modulator of the host actin cytoskeleton structure [143]. EspH, Tir, and Map collaborate to organize the assembly and disassembly of actin filopodia and pedestals [127]. Recently, it has been shown that EspH counteracts macrophage phagocytosis by binding to RhoGEFs, inactivating the host Rho GTPase signalling pathway [144]. EspZ (previously SepZ) is the translocated effector most recently identified that can be detected beneath the site of bacterial attachment [124]. A function has recently been identified for this effector, it was demonstrated that it interacts with the host protein CD98, enhancing phosphorylation of focal adhesion kinase (FAK), and promoting host cell survival mechanisms during infection [145]. EspB, which is also a translocator protein essential for the delivery of effectors, acts as an effector modulating the host cell cytoskeleton [146]. It also participates in microvilli effacement and in preventing phagocytosis [147]. Non-LEE effectors also have roles in EPEC virulence, although relatively little is known about their cellular function [148]. Since the major EPEC virulence properties have been attributed to the LEE effectors, the non-LEE effectors are proposed to function as accessory factors for an efficient infection [reviewed in [123]]. In contrast to the LEE-encoded effectors that are conserved among all the A/E pathogens, there is a considerable variation in the repertoire of non-LEE effector proteins between strains [64]. To state some of their functions, NleA is reported to inhibit protein trafficking [149] and to disrupt TJs [150]. NleB has been associated with diarrheal disease due to aEPEC [62]. NleE participates in the induction of the signaling pathways required for polymorphonuclear leukocytes transepithelial migration, and it has been demonstrated that it is capable of inhibiting NF-kappaB activation [151, 152]. NleH has anti apoptotic activity during EPEC infection [153]. As previously mentioned, EspG2 has functional redundancy with EspG, indicating that LEE and non-LEE effectors can function together to alter specific cellular processes [140, 154]. EspJ is involved in inhibition of receptor-mediated phagocytosis [155], and the cyclomodulin Cif induces apoptosis [156]. Several other Nle proteins have been identified but their function is still unknown. Most effectors require chaperone proteins for efficient translocation into host cells [157]. T3S chaperones typically form homodimers that interact with their cognate effector through a chaperone-binding domain located within the

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first 100 amino acids of the effector protein. It has been proposed that chaperones promote translocation by stabilizing the effectors in the cytoplasm, maintaining them in a secretion-competent conformation, masking their cellular localization or ‘toxic’ domains, regulating their synthesis, and by targeting them to the secretion apparatus [84, 157]. In EPEC two effector chaperones have been identified. CesF, which binds to EspF [158], and CesT, which was initially shown to bind and stabilize Tir and Map [159-161]; however, additional studies have demonstrated interactions with multiple LEE and non-LEE effectors [162, 163]. The participation of these chaperones in establishing a hierarchal translocation of effectors has also been demonstrated [162, 164]. Autotransporters EPEC also encodes virulence-associated proteins that are secreted via a type V secretion mechanism [reviewed in [165]]. EspC is the most studied autotransporter protein in this bacterial pathogen. It has a conserved serine protease motif similar to the IgA protease and has been shown to have enterotoxic activity [166, 167]. In addition, it has been demonstrated that EspC produces epithelial damage on HEp-2 cells [168] and that it proteolyses hemoglobin [169]. Recently, it was shown that EspC internalization into host cells is also dependent on the T3SS, suggesting cooperation between two secretion systems [170]. Two other putative autotransporter proteins have been identified in the genome sequence of the prototype strain E2348/69, but their function is still unknown [64]. Regulation The LEE contains 41 genes organized in five major polycistronic operons (LEE1 to LEE5) and several smaller transcriptional units, which are positively regulated by Ler, a key transcriptional regulator of EPEC virulence encoded by the first gene of the LEE1 operon [reviewed in [171]]. Ler regulates LEE gene expression by counteracting the repression imposed by the global regulator H-NS [172]. In addition, two other LEE-encoded transcriptional regulators have been identified, GrlA and GrlR, that have positive and negative roles in ler expression, respectively [122, 173, 174]. Moreover, in tEPEC strains, the EAF plasmid-encoded regulator PerC also plays a role in ler positive regulation, linking the expression of BFP with the expression of the LEE [172, 175, 176]. Additionally, in EPEC and EHEC, Ler also regulates non-LEE encoded virulence factors e.g., espC, nleA and lpf, so it is considered a global regulator of EPEC virulence [68, 177-179]. Invasion Some in vivo studies have shown the presence of EPEC cells inside human enterocytes [22, 180, 181] and different cell lines in vitro [182-185]. However, despite these evidences, invasiveness has not been considered a pathogenic characteristic of tEPEC strains in vivo and strains in this pathotype have been considered extracellular pathogens [8]. Studies conducted to evaluate the invasive ability of aEPEC strains are somewhat controversial. Former studies with some collections of aEPEC strains have shown that these strains invade HEp-2 cells less efficiently than tEPEC prototype E2348/69 [30] or rarely invade these cells [33]. In contrast other studies have shown that some aEPEC strains invade epithelial cells efficiently [50, 80, 186, 187]. Scaletsky et al. [188] reported a case of persistent diarrhea caused by an aEPEC (O18ab) strain that invaded HeLa and rabbit intestinal cells as observed by transmission electron microscopy. In addition, Rosa et al. [186] and Yamamoto et al. [50] have shown that a subset of aEPEC strains are able to invade undifferentiated intestinal Caco2 cells more efficiently than differentiated cells of the same lineage suggesting that undifferentiated cells express basolateral receptors necessary for aEPEC invasion. Hernandes et al. [187] showed that an aEPEC strain of serotype ONT:NM invaded HeLa cells as a result of intimin-Tir interaction with the subsequent cytoskeleton reorganization, as an eae mutant of this strain remained adherent but was no longer invasive. Furthermore, Bulgin et al. [189] showed that EspT, a T3SS-dependent effector protein belonging to the WXXXE family of effector proteins, promoted invasion of non-phagocytic cells by the trigger mechanism. The pathogenic role of the invasive ability of some aEPEC strains is presently unknown. As invasive organisms may be protected from destruction by the immune system and some antibiotics that do not penetrate eukaryotic cells, invasion could contribute to the permanence of certain aEPEC strains in the intestine, resulting in the persistent diarrhea reported in recent studies [190, 191].

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Induction of Mucus hypersecretion A putative new virulence phenomenon has been recently described with two aEPEC strains isolated from diarrheic children in Brazil, which consisted of induction of mucus hypersecretion in rabbit ligated ileal loops and in cultured human mucin-secreting intestinal HT29-MTX cells [192]. The same phenomenon could not be observed with tEPEC strain E2348/69 tested in the same conditions. Mucus hypersecretion was associated with increased production of secreted MUC2 and MUC5AC mucins and membrane-bound MUC3 and MUC4 mucins after infection of HT29MTX cells by an unidentified non-secreted effector molecule. Interestingly, adhering aEPEC cells grew in the presence of membrane-bound mucins, thus exploiting the mucins increased production for its own growth benefit. It is currently not known whether mucus hypersecretion is a virulence mechanism used by aEPEC strains to infect the human host. Furthermore, it remains to be investigated how frequent this property can be found among aEPEC strains. DIVERSITY OF VIRULENCE PROPERTIES The tEPEC strains are generally more homogeneous in their virulence characteristics, expressing the LEE- and the EAF plasmid-virulence genes [11]. Conversely, besides the genes on the LEE, many aEPEC strains carry genes encoding virulence factors of other E. coli pathotypes (even from extra-intestinal pathogenic E. coli) in different combinations [11, 26, 28, 32, 77], reflecting the heterogeneity of the group. However the role of these genes or different genes combinations in aEPEC pathogenesis is unknown. It is also recognized that some tEPEC and EHEC strains may loose pEAF or the stx-encoding phages during infection, respectively, thus generating E. coli isolates devoid of these genes, which would be diagnosed as an aEPEC isolate [193, 194]. Using DNA microarray analyses to search for genes associated with diarrhea, Afset et al. [62] found that the genes present in PAI O122 (efa1/lifA, set/ent, nleB and nleE) and certain genes located outside this PAI (lpfA, paa, ehxA and ureD) were associated with diarrhea but these associations may vary among different serotypes and in distinct geographic areas. It is apparent that aEPEC is more likely than tEPEC to receive virulence genes by horizontal transmission (i.e., from transmissible plasmids, PAIs, transposons or bacteriophages) in the intestine and/or environment. Lacher et al. [195] showed that EPEC strains are spread in four main clusters: EPEC 1 containing only tEPEC strains with H6 flagellar antigen, EPEC 2 containing tEPEC and aEPEC carrying H2 antigen, EPEC 3 including tEPEC and aEPEC with H34 antigen and EPEC 4 comprising tEPEC and aEPEC with H6 antigen. Bando et al. [196] have combined data generated by MLST and presence of pathogenic E. coli virulence factor-encoding genes to make a phylogenetic analysis of a collection of EPEC strains with other diarrheagenic E. coli pathotypes. With this approach, they showed that tEPEC and aEPEC of the classical EPEC serogroups were distributed on clusters that closely correlated with these clonal groups. However, they have also shown that aEPEC strains are distributed in all E. coli phylogenetic groups (A, B1, B2 and D) with at least two main distinct genomic backgrounds (named Clusters I and III). According to these authors, the acquisition and expression of virulence factors derived from non-EPEC pathotypes by various aEPEC clonal groups could be due to their particular genomic background, with Clusters I and III being associated with severe and mild diarrhea, respectively. EPIDEMIOLOGY AND IMPACT IN LATIN AMERICA For many decades, studies conducted worldwide have shown that tEPEC serotypes were strongly associated with diarrhea in children <1 year of age, mainly in poor children of urban centers in the first six months of life [reviewed in [8]]. In this age group, these serotypes were considered the main cause of endemic diarrhea in developing countries, including Latin America [197-200]. The frequency of tEPEC serotypes in children older than one year of age were reported to diminish with increasing age and adults rarely contracted EPEC infections, except under exceptional conditions [8]. The increased resistance in older children and adults was credited to the development of immunity or the loss of receptors for some specific adhesin [8]. In developed countries, there was a significant decrease in the frequency of tEPEC since the 1960s, and after that period, these bacteria were rarely detected in these countries, whereas aEPEC are frequently found in diarrheic patients [191, 201] reviewed in [1, 8]. In some developing countries, such decrease in the frequency of tEPEC strains has also been detected in recent years [11, 202-207]. The reasons for such decrease probably comprise better control of hospital infections, improvements in therapy, sanitation measures and in nutrition of infants [11].

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The epidemiology of aEPEC strains worldwide has been recently reviewed [12, 203]. It is noticeable that this pathotype has shown a more significant role in diarrhea in developing countries [205, 206, 208]. In many of these studies the frequency of aEPEC strains in both diarrheic and non-diarrheic individuals are similar, thus strong association of aEPEC serotypes with endemic diarrhea has yet to be demonstrated. Even so, aEPEC strains have been found in diarrheic patients of various ages and in adult patients with HIV-AIDS [28]. Moreover, some serotypes have been incriminated as agents of diarrheal outbreaks, but thus far no outbreaks due to aEPEC have been reported in Latin America. A large outbreak caused by serotype O111:H9 was described in Finland involving more than 600 people [209] while in the U.S.A. an outbreak due to an aEPEC of serotype O39:NM involved more than 100 adults [210]. Another outbreak associated with aEPEC was described in infants at a daycare center in Japan, in which four aEPEC isolates of serotype O55:NM with identical Pulsed Field Gel Electrophoresis patterns were detected in four patients [211]. In addition, a waterborne diarrheal outbreak affected students between 12 and 15 years old in Japan [211]; seven of 41 diarrheic individuals carried aEPEC isolates of serotype ONT:H45. RESERVOIRS AND TRANSMISSION Thus far, studies have shown that tEPEC are rarely found in animals and their reservoirs comprise only humans [11]. Many children acquire infection in public hospitals, usually by contact with other patients hospitalized with diarrhea. Although not completely characterized, it is apparent that tEPEC may be transmitted by ingestion of contaminated food and water [212]. The substitution of bottle feeding for breast-feeding enhances the risk of contracting diarrhea [212].

In contrast, various reports have described the isolation of aEPEC strains from various healthy and diarrheic animal species, such as cattle, sheep, goat, pig and poultry, as well as in domestic animals, deers and marmosets [reviewed in [12]]. Although there is no evidence of direct transmission from animals to humans, some aEPEC strains isolated from animals belong to serogroups implicated in human diseases, for example, O26, O103, O119, O128 and O142 [reviewed in [12]], suggesting that these animals may represent important reservoirs of aEPEC, which can be transmitted to humans. CLINICAL ASPECTS Diarrhea due to EPEC varies from a fatal to a subclinical infection, probably depending on host factors [8]. Various studies have shown that tEPEC strains can induce abundant secretory diarrhea with mucus (but without blood) with important losses of fluid and electrolyte in feces. Vomiting and low-grade fever are also observed in EPEC-infected patients. Furthermore, EPEC infection may lead to severe malabsorption of nutrients, and even evolve to food intolerance, resulting in nutritional aggravation and persistence of diarrhea [213]. The infective dose of EPEC necessary to cause disease in infants has not been established. Studies in volunteers indicate that a high infective dose is necessary to induce diarrhea (109 and 1010 bacteria), but in children it is presumed to be much lower [8]. Although adult volunteers ingesting large numbers (109 to 1010) of EPEC cells presented short incubation periods (12 to 24 h) to develop diarrhea [7, 193, 214, 215], the incubation periods in infants is unknown [216, 217]. The examination of intestinal biopsies of infected infants have shown that EPEC adhere to the mucosa of the small and large intestines, and intimate attachment to the small bowel mucosa has been demonstrated in many (but not all) cases of diarrhea by EPEC [218-221]. The presence of the A/E lesion appears to be related to fluid secretion, and disarrangement of the digestive-absorptive enzyme system, which leads to malabsorption of nutrients. Edema, neutrophil infiltrate, and reduced enzymatic activity in the intestinal mucosa have been found following EPEC infection [181, 218, 222]. IMMUNE RESPONSE Antibody response to the O antigen of certain O serogroups has been demonstrated in volunteers convalescing from experimental EPEC infection [193, 223] and in children older than 1 year of age [6]. In addition, most EPEC virulence factors, such as the major adhesins (intimin, and BFP) and cell-signaling proteins (EspA, EspB and Tir)

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were shown to induce an immune response in serum (IgG and IgM), and milk, colostrum and saliva (sIgA) of healthy and sick human hosts and in colostrum and milk of bovines [224-236]. In breast milk, IgA and other factors (i.e., oligosaccharide fractions) were shown to contribute to immunity by blocking bacterial adherence [232]. A strong antibody response in serum and saliva of children reactive to EPEC virulence factors was shown to develop early, since children around 12 months of age presented an antibody repertoire equivalent to that of an adult at the same geographical area [234]. Taken together, these findings suggest that the reduction in the frequency of tEPEC in children older than 2 years of age may be at least in part due to the development of a repertoire of anti-EPEC antibodies. It has been demonstrated that purified flagellin induces IL-8 production by Toll-like receptor 5 (TLR5) activation in enterocyte monolayers infected with tEPEC [79]. Zhou et al. [237] demonstrated that flagellins H6 and H34 of tEPEC stimulated IL-8 production in T84 monolayers. Using a flagellin deficient mutant, Sampaio et al. [80] have recently shown that the flagella of aEPEC strain 1711-4 (serotype O51:H40) induced IL-8 production in Caco-2 cells at an early stage of infection (3 hours). As a fliC derivative mutant of this strain still induced IL-8 levels at least 2 times higher than that of uninfected monolayers at 24 h post-infection, it was hypothesized that at a later stage of infection the wild-type aEPEC 1711-4 could stimulate IL-8 production in a flagellin-independent pathway (24 h) [80]. The significance of these findings in vivo remains to be confirmed. DIAGNOSIS Currently, molecular assays are more appropriate methods for EPEC identification. These methods include PCR and multiplex PCR, as well as genetic probes, to detect pEAF (EAF probe) [238], bfpA (encoding the major pilin subunit of BFP), eae genes (or other conserved LEE-genes) and the stx genes (encoding Shiga toxins) [18, 44, 239-241]. EPEC strains have been identified by the presence of the LEE region and absence of the stx genes; lack of stx distinguishes EPEC from enterohemorrhagic E. coli (EHEC). tEPEC and aEPEC strains are differentiated mainly by the presence of the EAF sequence and/or the bfpA gene in the former group. Although all tEPEC strains carry bfpA, some aEPEC strains contain a defective bfp operon, thus resulting in positive reactions to this gene. For this reason and to differentiate atypical from typical EPEC, search for BFP expression by immunological methods is recommended [11, 26, 242]. As aEPEC strains are very heterogeneous and may often be isolated from non-diarrheic children, it should be important to demonstrate the potential ability of E. coli strains diagnosed as aEPEC to promote A/E lesions on epithelial cells. This could be achieved in vitro by the Fluorescent Actin Staining Method described by Knutton et al. [243]. Nonetheless, the use of methods to identify BFP expression and ability to promote A/E lesions is so far restricted to research laboratories. TREATMENT AND PROPHYLAXIS Oral rehydration therapy noticeably reduces early mortality and is the most effective therapy against mild EPEC infections. However, it has been shown that children with EPEC may not respond to this therapeutic measure, and some children may present intolerance to cow’s milk, require hospitalization and develop persistent diarrhea [213]. This is probably due to the damage caused in the intestinal mucosa, which may require time to fully recover its functions (digestion, absorption, and defense) [8]. Antibiotic therapy is only recommended for more severe cases or cases of persistent diarrhea associated with aEPEC [203, 244]. However, the antimicrobial susceptibility patterns of tEPEC and aEPEC vary among the different geographic areas studied and antibiotic resistance are common in many areas of the world [26, 198, 216, 244-247]. Consequently, susceptibility testing must be performed before the establishment of therapy. A conserved conjugative plasmid carrying antibiotic resistance was identified among tEPEC strains of serotypes O111:H2 (and non-motile), and aEPEC strains of serotype O119:H2 of different geographical areas [248]. More recently, this plasmid was identified in tEPEC strains of other serotypes (O55:H6, O127:H6, and O119:H6) and in aEPEC strains of serogroups O55 and O119 [245]. As already mentioned, breast milk may contain antibodies against EPEC O antigens and outer membrane proteins, which inhibit EPEC adherence to tissue culture cells [232]. Breast-feeding has a protective effect [212, 224, 225,

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249, 250] probably due to the cooperative protective effect of IgA antibodies to EPEC components and the presence of oligosaccharides that inhibit adhesion [232], or a direct inhibitory effect of lactoferrin on EPEC [251]. Besides breast feeding, protection offered by appropriate sanitary conditions and corrected hygiene and alimentation practices could prevent the infection of poor children in developing countries [252]. An EPEC vaccine would benefit poor children as these conditions are difficult to be implemented in these countries. However, there is currently no licensed vaccine available against EPEC infections of humans [reviewed in [253]]. A plant based vaccine was constructed using BfpA (the structural subunit A of the bundle-forming pilus) as antigens to induce high levels of phytosecreted BfpA in BALB/c mice by the induction and detection of fecal anti-BfpA antibodies [254]. Since natural infections in rabbits with a REPEC (Rabbit EPEC) strain induce A/E lesions in a manner similar to human EPEC [255, 256], the development of vaccines against human EPEC could benefit from the elaboration of a vaccine against REPEC or other A/E lesion producing pathogen. Some laboratories are trying to develop vaccines by inactivating some virulence genes to generate attenuated vaccines lacking genes encoding important virulence factors, such as intimin or the Ler-encoding gene [223, 255]. A deletion mutation in the ler gene in a wild-type REPEC strain was constructed resulting in diminished T3SS-secreted effector proteins by the mutant strain and in preventing REPEC from adhering intimately to the rabbit intestinal mucosa in vivo with vaccinated animals, demonstrating no clinical evidence of disease. It remains to be determined whether this vaccine could protect against A/E producing E. coli strains in different hosts [257, 258]. Other groups have cloned specific EPEC or EHEC virulence genes to induce protective responses in the host, mainly the gene encoding intimin. In a recent study, two distinct fragments of intimin β (one of the most frequent EPEC intimin subtype) were expressed in Lactobacillus casei strains for nasal immunization of mice. This procedure induced specific antibodies in the sera (IgG) and nasal washes (sIgA), which were active against the intimin immunodominant region and inhibited EPEC adhesion to epithelial cells in vitro [259]. In another recent study, an attenuated Vibrio cholerae strain (CVD 103-HgR) expressing the C-terminal portion of intimin in fusion with ClyA (a secreted protein from Salmonella enterica serovar Typhi) has been reported [260]. This construction showed moderate protection efficacy in rabbits challenged with a virulent REPEC strain. In addition, EspA was lately expressed in Lactococcus lactis NZ9000 and used to immunize mice; antisera from immunized animals were shown to interrupt the interaction of E. coli O157:H7 with the host cell [261]. Despite the assays were designed to produce an EspA-oral vaccine against E. coli O157:H7 infections, they could also be an alternative candidate to prevent EPEC infections. ACKNOWLEDGEMENT Work in the laboratory of T.A.T.G. is supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Work in the laboratory of B.G.P. is supported by grants 81847 from CONACyT and IN224708 from DGAPA, UNAM. We thank Dr. Waldir P. Elias and B.G.P. lab students and academic technician Norma Espinosa Sánchez for useful discussion and contributions to this work. REFERENCES [1] [2] [3] [4] [5] [6]

Robins-Browne RM. Traditional enteropathogenic Escherichia coli of infantile diarrhea. Rev Infect Dis. 1987;9(1):28-53. Levine MM, Kaper JB, Black RE, et al. New knowledge on pathogenesis of bacterial enteric infections as applied to vaccine development. Microbiol Rev. 1983;47(4):510-50. Bray J. Isolation of antigenically homogeneous strains of Bacterium coli neopolitanum from summer diarrhoea of infants. J Pathol Bacteriol. 1945;57:239-47. Varela G, Aguirre A, Carrillo J. Escherichia coli-gomez nueva especie aislada de un caso mortal de diarrea. Bol Med Hosp Infant Mex. 1946;3(3). Levine MM. Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent. J Infect Dis. 1987;155(3):377-89. Neter E, Westphal O, Luderitz O, et al. Demonstration of antibodies against enteropathogenic Escherichia coli in sera of children of various ages. Pediatrics. 1955;16(6):801-8.

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CHAPTER 4 Enteroaggregative Escherichia coli Fernando Navarro-Garcia1*, Waldir P Elias2, Jose Flores3 and Pablo C Okhuysen3 1

Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), México DF, Mexico;  2Laboratory of Bacteriology, Instituto Butantan, São Paulo, SP, Brazil; 3Division of Infectious Diseases, The University of Texas at Houston Medical School, Houston, Texas, USA Abstract: Enteroaggregative Escherichia coli (EAEC) have been identified in children and adults living in developed and developing countries as well as in travelers returning from developing countries and in patients infected with HIV. In addition to the acute complications of diarrhea, such as dehydration and death, EAEC can also cause persisting diarrhea that can lead in turn to malnutrition and impaired growth and development in children. EAEC strains are defined by the ability to produce a “stacked brick” appearance when placed on HEp-2 epithelial cells in culture, and contaminated food appears to be the main source of EAEC infection. EAEC are genetically heterogeneous. EAEC strain 042 is the prototypical strain for the study of virulence factors and pathogenicity; however, emergence of atypical EAEC has been described. Three major features of EAEC pathogenesis have been defined; abundant adherence to the intestinal mucosa, elaboration of enterotoxins and cytotoxins, and induction of mucosal inflammation. Infection with strains that possess specific virulence factors correlate with elevated levels of fecal cytokines and inflammatory markers. Unfortunately, the diagnosis of EAEC infection remains limited to research laboratories and therefore, are of little use in guiding antibiotic therapy. EAEC induce short-term immunity in healthy individuals which suggests that immunoprophylaxis is a potential option for control of EAEC.

INTRODUCTION The pathogenesis and protective immunity to enteroaggregative Escherichia coli (EAEC) and diffusely adhering E. coli (DAEC) are the poorest understood compared to the other four diarrheagenic categories: enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shiga toxin-producing E. coli (STEC) and enteroinvasive E. coli (EIEC) [1]. Considerable advances have been made on the understanding of pathogenesis and prevention of these last four categories, even though diarrheagenic E. coli is still loosely classified into these six major categories based on clinical associations, serotyping, phenotypic assays and identification of virulence characteristics.

Figure 1: The adherence pattern of enteroaggregative E. coli (EAEC) to HEp-2 cells. EAEC adheres to HEp-2 cells in a pattern known as auto-aggregative, in which bacteria adhere to each other in a 'stacked-brick' configuration.

A common feature that defines EAEC strains is the ability to produce a “stacked brick” appearance (Fig. 1) when placed on HEp-2 epithelial cells in culture [1]. This phenotype is derived from the ability of EAEC to attach to other EAEC, to the epithelial cells, and to the surface of tissue culture plates. At the molecular level (described later in this *Address correspondence to: Dr. Fernando Navarro-Garcia. Department of Cell Biology, CINVESTAV-IPN, Ap. Postal 14-740, 07000 México DF, Mexico. Tel: (52-55) 5747-3990. Fax: (52-55) 5747-3393. E-mail: [email protected] Alfredo G. Torres (Ed) All rights reserved - © 2010 Bentham Science Publishers Ltd.

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chapter in detail) the aggregative phenotype determinants (also referred to as the AA phenotype) of EAEC are contained in a large plasmid (pAA) that carries a number of virulence genes that are under the control of the master aggR regulator. Shortly after the initial description of EAEC in 1987 by Nataro et al. [2] an empirically derived probe originally denominated CVD432 or AA was developed in the early 1990’s by Baudry et al. [3] and served as the basis for a PCR based assay a few years later [4]. This AA probe was later shown to hybridize with a region of in the pAA coding for an ABC transporter apparatus that is needed to translocate the antiaggregative protein dispersin across the bacterial cell membrane [5] The study of EAEC experienced a breakthrough in 1994, when the ability of this pathotype to cause diarrhea was left in no doubt when a volunteer study demonstrated that the prototypic EAEC strain 042, originally identified in a child with diarrhea in Peru, elicited diarrhea in the majority of volunteers challenged with 109 c.f.u. in a study at the Center for Vaccine Development, University of Maryland [6]. However, volunteers exposed to other EAEC strains in this challenge study did not develop diarrhea. This provided initial insights into the variable and complex pathogenesis of EAEC. EAEC strain 042 (serotype O44:H18), which caused diarrhea in the healthy volunteer challenge, became then the prototypical EAEC strain for the study of virulence factors and EAEC pathogenicity [6] and was the subject of a recent EAEC genome characterization [7]. Thus, the majority of the virulence factors described in EAEC have been characterized in the EAEC prototype strain 042. Epidemiologic studies showed that the majority of isolates hybridizing with the AA probe also contained the master aggR regulator and that EAEC isolates that hybridized with the AA probe were found to be associated with persisting diarrhea in case-controlled studies. However, it was also noted that not all EAEC strains causing diarrhea hybridized with primers specific for aggR. This led to the suggestion that EAEC be classified into “typical” EAEC when aggR is present and “atypical” EAEC when aggR is absent [8]. The literature thus, contains studies on EAEC that rely solely on the aggregative phenotypic HEp-2 assay, solely on the use of molecular probes and yet others that use a combination of these two approaches to identify EAEC. The heterogeneity among strains from geographically diverse regions not conforming to these characteristics further complicates the classification of EAEC into discrete groups [9] and will likely require of a reclassification in the near future. EPIDEMIOLOGY AND IMPACT IN LATIN AMERICA EAEC has been identified in all regions of the world where it has been studied. Children and adults living in developing countries [10-14], as well as travelers returning from developing countries are susceptible to infection with EAEC [15, 16]. EAEC can also result in persisting asymptomatic infection in adults and children [17, 18]. In the US, a recent study showed that gene probe positive EAEC but not EAEC defined by HEp-2 adherence, was more commonly identified in children under the age of five with acute diarrhea than in age matched healthy controls [19-21]. Although atypical EAEC has been identified in calves, piglets and horses [22], animals have not been implicated as a source or reservoir for human infection. Contaminated food appears to be the main source of EAEC infection and has been implicated in several food borne outbreaks of diarrhea [23, 24] in several parts of the world. In Mexico, EAEC is commonly found in food, including desserts and salsa [25, 26] and may explain the high rates of travelers’ diarrhea seen in US visitors to that country. Also, in Brazil EAEC was detected in the lacteal contents of feeding bottles of low socio-economic class infants brought to the outpatient clinic of a public hospital, demonstrating the possible transmission of EAEC by contaminated food [27]. In Latin America, EAEC is among the most common bacterial isolates recovered from children with diarrhea and has been identified in Colombia [28], Mexico [29, 30], Guatemala [31], Brazil [10, 11, 32-42], Chile [43], Peru [44], Argentina [45], Ecuador [46] and Jamaica [16]. In some areas of Brazil, EAEC surpasses ETEC as the predominant pathogen identified [47].

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In addition to the acute complications of diarrhea such as dehydration and death, EAEC can also cause persisting diarrhea [48] that can lead in turn to malnutrition and impaired growth and development. Steiner and colleagues demonstrated that in Brazilian slums, infections with EAEC were associated with intestinal inflammation as evidenced by elevated levels of fecal cytokines including IL-8 and that even in the absence of diarrhea, infection with EAEC led to impaired growth and development [49]. EAEC has also been associated with chronic diarrhea in patients infected with HIV [17, 50-55]. Although studies in Africa linked EAEC to persistent and bloody diarrhea in HIV associated infection [52], there is a paucity of data on the prevalence or complications associated with HIV-related EAEC infection in Latin America. In developed countries, diarrhea due to opportunistic pathogens in patients with HIV has decreased dramatically in response to the introduction of highly active antiretroviral therapy and this may also be the case in Latin America. EAEC can be identified in 19% to 33% of traveler’s diarrhea cases depending on the region of the world visited [16, 56-58]. In Latin America, the proportion of EAEC associated cases of travelers' diarrhea is seen in 25% of travelers, which is significantly higher to the rates observed in travelers visiting Africa, where the proportion is less than 2%, and south Asia (15%) [59]. Unlike ETEC, which causes diarrhea within the first two weeks of travel and is not associated with asymptomatic carriage in US visitors to Mexico, EAEC infection can persist for longer periods of time [60]. EAEC PATHOGENESIS Once ingested, the localization of infection in the gastrointestinal tract has not been well defined. EAEC infection has been associated with increased nitric oxide production and alterations in lactulose permeability in aboriginal children in Australia [61]. Studies done on endoscopic intestinal specimens demonstrate that EAEC can bind to jejunal, ileal, and colonic epithelium [62]. Studies in volunteers have demonstrated that EAEC 042 can be recovered from duodenal aspirates [6]. In a separate study, organ cultures of small and large intestinal mucosa from children were used to examine the interactions of EAEC with human intestine. In this study, five EAEC strains were evaluated for their ability to bind to jejunal, ileal, and colonic mucosa. The affinity for segments of the intestinal tract was heterogeneous among the EAEC isolates studied [63]. Although a great diversity of adhesins, toxins and proteins involved in EAEC pathogenesis have been described, the prevalence of these virulence factors-encoding genes is highly variable and none of these have been found in all EAEC strains [33, 40, 64-68]. In addition to the expression of adhesins and toxins, several other potential virulence factors have been described in EAEC which are encoded in plasmids or in the chromosome [69]. Nevertheless, pathogenesis studies have suggested three major features of EAEC pathogenesis: (i) abundant adherence to the intestinal mucosa, (ii) elaboration of enterotoxins and cytotoxins, and (iii) induction of mucosal inflammation (Fig. 2). Virulence Factors HyE EAST1 Pet Pic

Flagellin

ShET1 Dispersin Adhesins

EAEC

EA

EC AAFs

B

Vesicular traffic

Epithelial cell

A

B

IL-8 Inflammation

Targets Guanylyl Cyclase Adenylyl Cyclase Fodrin Membrane

A

Mucus

Figure 2: Schematic representation of enteroagregative E. coli virulence factors and their targets in the mucosal epithelium. Some of the targets of virulence factors are extrapolated from their known function in other pathogens o similarity to other factors (HylE, ShET1 and EAST1).

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Abundant Adherence to the Intestinal Mucosa Several authors demonstrated that the AA pattern is associated with the presence of fimbrial and afimbrial adhesins in EAEC strains. However, the genes encoding these adhesins are found in low prevalence, indicating a high diversity of adhesive structures responsible for the AA pattern [55, 70-76]. Afimbrial Adhesins Afimbrial adhesins, or outer membrane proteins (OMP) associated with the AA, have been observed in several EAEC strains of different serotypes [72, 74, 76, 77]. Debroy et al. [72] and Wai et al. [76], demonstrated that OMPs of 30 and 38 kDa, respectively, were responsible for the AA in some EAEC. A 18 kDa OMP present in EAEC strains belonging to serotypes O44:H18 and O126:H27, was associated with AA in HEp-2 cells and scum formation on broth cultures [77]. Spencer et al. [78] showed that the expression of that OMP also occurred in other serotypes that hybridized with the DAEC genetic probe described by Bilge et al. [79]. The mannose-resistant hemagglutination of human erythrocytes expressed by the prototype strain 042 in low temperature was associated with a 16 kDa OMP named cryohemagglutinin [80]. Monteiro-Neto et al. [74] described a 58 kDa OMP, named aggregative protein 58 (Ap58) responsible for the AA pattern of EAEC strains of the serotype O111:H12. The Ap58 adhesin is encoded by the ap58 gene located in the chromosome of the prototype strain 236. However, the distribution of Ap58 seems to be restricted to the O111:H12 serotype [74]. Recently, Bhargava et al. [81] reported the presence of an OMP adhesin (Hra1) in EAEC 042 that is an accessory factors involved in autoaggregation, biofilm formation and aggregative adherence, demonstrating the multifaceted adhesion and aggregation factors seen in this phenotype. Fimbrial Adhesins The presence of different fimbriae has been demonstrated in several EAEC strains employing electron microscopy studies [82-84]. Old et al. [85] described a fibrillar adhesin present in two strains of serogroup O78 able to mediate agglutination in rat and mice erythrocytes in the presence of mannose. Only a few of EAEC fimbriae have been genetically characterized, and certainly, the best studied are the Aggregative Adherence Fimbriae (AAFs) I, II and III [55, 71, 75]. AAF/I, II and III are encoded in high-molecular weight plasmids and are necessary for the expression of AA, in the prototype strains 17-2, 042 and 55989, respectively [55, 75, 86]. AAF/I is a flexible, bundle-forming fimbriae (2- to 3- nm diameter) responsible for the AA on HEp-2 cells and mannose-resistant hemagglutination of human erythrocytes [75]. In the prototype strain 17-2, the genes responsible for AAF/I biogenesis are located in two regions of the pAA1 plasmid, designated region 1 (containing the aggABCD operon that encodes the pilin, chaperonin and usher proteins) and region 2 (containing the AggR regulator-encoding gene) [75, 87, 88]. AAF/II was described in the prototype strain 042 as the adhesin responsible for the AA to HEp-2 cells and human colonic mucosa in tissue explants, as well as for biofilm formation in tissue cultures and polystyrene [71]. This fimbrial structure was observed as semi-rigid bundles of filaments (5-nm diameter) and detected by its ability to produce mannose-resistant hemagglutination of human erythrocyte [6]. The genes responsible for AAF/II biogenesis are also located in a high-molecular weight plasmid (pAA2) in two regions. However, these two regions show a unique fimbrial genetic organization, where the pilin-, chaperonin- and the regulator-encoding genes (aafA, aafD and aggR) are located in region 1, and the usher-encoding gene (aafC) takes place in region 2 [86]. These two regions are separated by a ~12-kb sequence harboring toxin-encoding genes [86]. The third AAF was identified in strain 55989 by Bernier et al. [55]. AAF/III is also involved in bacterial aggregation and AA to cultivated epithelial cells. Distinctly of AAF/I and II, AAF/III is long and individual flexible filaments. The genes responsible for AAF/III fimbria (agg-3ABCD and aggR) are organized in two regions similar to AAF/I genetic organization. Another fimbrial structure has been described in the atypical EAEC strain C1096. In this strain, the aggregative adherence to cultivated epithelial cells and to abiotic surfaces is mediated by a type IV pili, encoded by genes (operon pilLMNOPQRSTUV), located in a plasmid of the IncI1 incompatibility group. The gene pilS encodes the

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major pili subunit [73, 89]. Recently, a new adhesin responsible for the AA in EAEC strains isolated in Denmark named Had has been described by Boisen et al. [70]. Had is responsible for the AA in EAEC strains lacking the AAFs. The ultrastructure of this adhesin has not been showed, however, the had gene has been previously described as encoding a pilin related to the Dr family of adhesins. A large number of EAEC strains lack the genes encoding these fimbriae, indicating that they express uncharacterized adhesins mediating the AA pattern [22, 33, 40, 65, 73, 90]. The EAEC heterogeneity in terms of fimbrial repertoire is similar to the observed among ETEC colonization factor antigens (CFA) [1]. Dispersin, Shf and Irp-2 Czeczulin et al. [65] identified several putative virulence genes in the pAA2 plasmid (strain 042) in addition to aafABCD-aggR (AAF/II-encoding genes), pet (Pet) and astA (EAST1). Among them are aspU or aap and shf. Dispersin is a 10.2 kDa immunogenic secreted protein encoded by the aap (anti-aggregation protein) gene, formerly known as aspU [65, 91]. This protein is secreted to the extra cellular milieu being covalently attached to the bacterial surface, neutralizing the negative charge of the bacterial surface and allowing the AAF/II projection [5, 91]. A 042 mutant in aap shows that dispersin acts decreasing bacterial autoaggregation allowing dispersion along the intestinal mucosa [91]. Dispersin secretion across the bacteria outer membrane is dependent of an ABC protein transporter system [92]. The shf gene shows homology with two genes described in Shigella flexneri 2a (shf1 e shf2), and encodes a predicted protein similar to IcaB of Staphylococcus epidermidis, related to biofilm formation [65, 93]. Besides pic and setAB (see below), the irp-2 gene was also localized in the 042 chromosome [65]. This gene encodes the iron-repressible high-molecular weight protein 2 (Irp-2) and is involved in the biosynthesis of a yersiniabactin responsible for iron acquisition in Yersinia [94]. Some authors showed a high prevalence of this gene among EAEC strains [22, 94], although, the role of Irp-2 in EAEC pathogenesis is unclear. Biofilm Formation Another characteristic of EAEC is the ability to form biofilm on biotic and abiotic surfaces in vitro, as result of bacterial aggregation [62, 63, 95, 96]. Biofilm of EAEC is a multifactorial phenotype distinct of the non-pathogenic E. coli biofilms [97]. It has been shown the involvement of AAF/II, type I fimbriae, Fis and YafK in the formation of biofilm by the prototype strain 042 [96, 98]. Fis is a DNA-binding protein involved in growth phase-dependent regulation and YafK is a secreted protein required for transcription of the genes involved in AAF/II biogenesis [96]. EAEC adherence to intestinal tissues is characterized as a biofilm composed by aggregates of bacteria in association with a thick mucus layer [62, 63, 95]. This characteristic biofilm formed by EAEC in vivo has been associated with the persistence of some cases of EAEC diarrhea [96, 99]. Invasion EAEC is considered a non-invasive pathogen, although some authors have demonstrated the invasive phenotype of some strains [90, 100, 101] using cells in culture. Benjamin et al. [101], showed that EAEC strains were internalized by HeLa cells, which was mediated by the cellular cytoskeleton components. Abe et al. [100] demonstrated the invasive phenotype to T84 cells and human colonic biopsies by the EAEC strain 236 (serotype O111:H12). These observations, in addition to the fact that children with EAEC diarrhea in Mexico can present bloody stools [29], suggest that the invasion ability of EAEC may be a virulence trait of at least some strains. Elaboration of Enterotoxins and Cytotoxins EAEC produces cytotoxic effects such as microvillus vesiculation, enlarged crypt openings, and increased epithelial cell extrusion [8]. Numerous putative virulence factors such as the plasmid-encoded Pet and EAST-1 toxins [102, 103]; the chromosomally encoded ShET1 toxin [104, 105], HlyE [106], Pic, a mucinase widely associated with pathogenic E. coli and Shigella spp [105], and a novel type VI secretion system [107] have been associated to these cytotoxic effects in EAEC. In an effort to identify cytotoxins and enterotoxins, Navarro-Garcia et al. [108] analyzed culture supernatants from strains that caused outbreaks of EAEC diarrhea in Mexican hospitals. Two major proteins of 108 and 116 kDa (now

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known as Pet and Pic, respectively) were secreted. Interestingly, Pet and Pic are secreted by EAEC using the type V secretion system or also called autotransporter mechanism, and belong to the serine protease autotransporter of Enterobacteriaceae family. Thus, both proteins undergo two cleavage steps before a functional protein is released into the extracellular space. The passenger domains contain the protease motif (GDSGSP) characteristic of all proteins of the SPATE group [109]. Plasmid-encoded Toxin (Pet) It has been shown that a 104 kDa protein, termed Pet (plasmid-encoded toxin), is required for EAEC-induced damage to human intestinal mucosa [108]. Pet is a member of the autotransporter class of secreted proteins and together with Tsh, EspP, EspC, Pic, SigA, Hbp, Sat and SepA proteins comprises the serine protease autotransporter of Enterobacteriaceae (SPATE) subfamily. The defining feature of autotransporters is their self-contained secretion system [102]. Pet causes raises in Isc (short-circuit current) and decreases in electrical resistance of rat jejunum mounted in the Ussing chamber, an enterotoxic effect that is accompanied by mucosal damage, increased mucus release, exfoliation of cells and development of crypt abscesses [108]. Pet appears to be a cytoskeleton-altering toxin, since it induces contraction of the cytoskeleton, loss of actin stress fibers, and release of focal contacts in HEp-2 and HT29/C1 cell monolayers, followed by complete cell rounding and detachment. Interestingly, Pet cytotoxicity and enterotoxicity depend on Pet serine protease activity, since both effects are inhibited by phenylmethylsulfonyl fluoride (PMSF) and are not induced by Pet S260I, which is mutated in the catalytic serine and thereby lacks in vitro protease activity [110]. It has been shown that Pet enters the eukaryotic cell and that trafficking through the vesicular system appears to be required for the induction of cytopathic effects. Moreover, the Pet serine protease motif is the main requisite for the cytopathic effects, because the internalization assays have demonstrated that Pet and mutant S260I are found inside epithelial cells, but that only native Pet produces the cytopathic effects [111]. All these data suggest an intracellular target for Pet. Indeed, Pet produces degradation of erythroid spectrin [112]. Pet internalization by clathrin-coated vesicles was found to be the essential mechanism because two reagents for blocking this pathway totally inhibited Pet internalization (monodansylcadaverine and sucrose). Whereas drugs for blocking endocytosis (filipin and methyl--ciclodextrin), through caveolae mechanism, was unable to inhibit Pet effects on the cytoskeleton. Moreover, small interfering RNA (siRNA), designed to knock down clathrin gene expression in HEp-2 cells, prevented Pet internalization, and thereby the Pet-induced cytotoxic effect. However, the use of siRNA to knock down caveolin expression had no effect on Pet internalization, and the cytotoxic effect was clearly observed [113]. Pet endocytosis is a rapid event in HEp-2 cells, since it is possible to find Pet in early endosomes, as short as 8 min after the initial interaction. This rapid Pet endocytic process has also been observed in various bacterial toxins, in which, after endocytosis through clathrin-dependent or -independent mechanisms, toxins are delivered to early endosomes and a small fraction is subsequently delivered to lysosomes for its degradation. In the endocytic transport of Pet, it is possible to observe that a small fraction reached the lysosomes for its possible degradation. However, most of Pet followed a transport to other organelles [114]. Furthermore, brefeldin A (BFA) is able to inhibit the cytoskeletal effects caused by Pet. BFA disrupts the Golgi apparatus and causes multiple alterations in the vesicular transport. Inhibition of cell intoxication by BFA is classically associated with toxins, which undergo retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER), such as Cholera toxin, Shiga toxin and ricin [111]. These findings were confirmed by localizing Pet in the Golgi apparatus at sequential times and then in the ER. ER is an attractive compartment for translocation, since it contains factors that facilitate entry to the cytosol by reducing or displaying proteins before their departure from the ER membrane. Additionally, the ER contains a protein of machinery for translocation, named Sec61p, which is involved in the reverse translocation of misfolded proteins from the ER lumen to the cytosol. The Sec61p translocon complex appears to be responsible in this essential pathway for the intoxication by diverse toxins, since they have the ability to associate with the Sec61p complex, such as the A subunit of ricin, Pseudomonas exotoxin A, and Cholera toxin. Thus, Pet is able to colocalize with Sec61α, the major protein in the Sec61p complex, and this colocalization occurred immediately after Pet localization in the ER. Additionally, antibodies against Sec61α were able to precipitate Pet and antibodies against Pet immunoprecipite Sec61. Furthermore, Pet is unable to intoxicate

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mutant CHO cells (cholera toxin resistant), which have translocation defects [114]. Finally, Pet colocalizes with one of its intracellular targets, fodrin. An intracellular target, -fodrin (II spectrin), has been found for Pet. Pet binds and cleaves epithelial fodrin (between M1198 and V1199) in vitro and in vivo, causing fodrin redistribution within the cells, to form intracellular aggregates as membrane blebs. Interestingly, both enterotoxic and cytotoxic effects depended upon the serine protease motif, the active site used for α-fodrin degradation. Furthermore, Pet internalization is needed to produce the cytotoxic and cytoskeletal damage, since α-fodrin is an intracellular protein. Thus, this mechanism appears to be a new system of cellular damage identified in bacterial toxin, which includes the internalization of the protease to allow finally specific α-fodrin degradation to destroy the cell [115]. So far, it is clear that purified Pet follows a sequence of events in order to damage the epithelial cells. However, all these events have been deduced from research performed in in vitro systems using purified Pet toxin (37 µg/ml) and have not yet been shown to occur during the infection of epithelial cells by EAEC. Recently, it was reported that the secretion of Pet by EAEC during the infection is regulated at the transcriptional level, since secretion is inhibited in eukaryotic cell culture medium, although Pet is efficiently secreted in the same medium supplemented with tryptone. Efficient Pet secretion in DMEM/tryptone increases cell detachment in HEp-2 cell adherence assay. Pet toxin is efficiently delivered by EAEC as it occurs when 37 µg/ml of purified Pet protein are used. Thus, there is a correlation between Pet secretion by EAEC, the internalization of Pet into epithelial cells, cell detachment and cell death in EAEC-infected cells. All these data indicate that Pet is an important virulence factor in the pathogenesis of EAEC infection [116]. Protein Involved in Colonization (Pic) Pic was identified as a second SPATE member in EAEC. This protein was recognized by the sera from the same children who were infected during an outbreak of diarrhea in a pediatric ward of a Mexican hospital and which were used to identify Pet [108]. In contrast to Pet, Pic is localized in the EAEC chromosome [65, 105] and it was found that Pic was identical to a protein termed Shmu (Shigella mucinase), which is encoded on the Shigella she pathogenicity island (PAI) [117]. Interestingly, the pic gene has a unique characteristic among the autotransporter proteins since there are two oppositely oriented genes in tandem within the pic (she) gene, set1B and set1A [105, 117], which encode the 7- and 20-kDa subunits of the 55-kDa ShET1 toxin [104]. The sequences flanking pic are different in EAEC and S. flexneri, and in the case of S. flexneri the she PAI contains another SPATE, SigA, which is similar to Pet. Pic catalyzes gelatin degradation which can be abolished by disruption of the predicted proteolytic active site. Through functional analysis of Pic, this proteolytic site is involved in Pic’s mucinase activity, serum resistance, and hemagglutination. Phenotypes identified for Pic suggest that it is involved in the early stages of pathogenesis and most probably promotes intestinal colonization [105, 117]. Recently, it has been shown that Pic did not damage epithelial cells, cleave fodrin, or degrade host defense proteins embedded in the mucus layer (sIgA, lactoferrin and lysozyme). However, by using a solid-phase assay to evaluate the mucinolytic activity of EAEC Pic, it has been documented a specific, dose-dependent mucinolytic activity. A serine protease inhibitor and an enzymatically inactive variant of Pic were used to show that the Pic serine protease motif is required for mucinolytic activity. Additionally, Pic binds mucin, and this binding was blocked in competition assays using monosaccharide constituents of the oligosaccharide side chains of mucin. Moreover, Pic mucinolytic activity decreased when sialic acid was removed from mucin. Thus, Pic is a mucinase with lectin-like activity that can be related to its reported hemagglutinin activity [118]. A homologue protein in uropathogenic E. coli, PicU (96% identity at the amino acid level), has been characterized and shared similar functions [119]. Subsequent investigations by Heimer et al. (2004) revealed that picU was expressed during experimental infection in the mouse model of urinary tract infection. However, co-challenge of wild-type organisms and the picU mutant in the mouse model suggested that PicU encoding strains had no competitive advantage in colonization [120]. Nevertheless, since PicU is a secreted protease, it is possible that

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protein secreted by the wild-type strains may complement the picU deficiency of the mutant strains. Indeed, challenge of mice separately with wild-type and picU mutant strains revealed that the wild type strain colonized the bladder to a greater extent and displayed higher levels of neutrophil infiltration into the lumen, epithelium, and submucosa. Unfortunately, while these data showed a trend, they were not statistically significant [120]. In addition, it should not be discounted that loss of PicU function may be complemented by the homologous Vat and Sat serine protease autotransporters. Thus, the role of PicU in uropathogenic E. coli infection remains enigmatic. Shigella Enterotoxin 1 (ShET1) Culture filtrates of S. flexneri 2a strain M4243 cause significant fluid accumulation in rabbit ileal loops, when the bacteria is grown in iron-depleted medium. Also, when tested in Ussing chambers, a greater rise in potential difference and short circuit current was seen with such filtrates compared with the medium control. Ultrafiltration and gel exclusion size fractionation of M4243 filtrate revealed that the activity was approximately 60 kDa. Genes encoding this enterotoxin, named ShET1 (Shigella enterotoxin 1), were cloned. Thus, ShET1 is elaborated in vivo, where it elicits an immune response, may be important in the pathogenesis of diarrheal illness due to S. flexneri [104]. ShET1 toxin is a subunit toxin encoded by setA and setB, which are thought to form an oligomeric toxin consisting of a single 20-kDa SetA protein associated with a pentamer of five 7-kDa B subunits (SetB) [121]. ShET1 appears to induce intestinal secretion via cAMP and cGMP, however the precise mechanism of action and detailed biochemistry remains elusive. As mention above, unusually, the setAB genes are encoded within the pic gene but on the complementary strand and thus have the same prevalence characteristics and disease associations as pic [105]. A study of the genetic regulation of these overlapping genes suggests that pic and the setBA loci are transcribed as complementary 4-kb mRNA species. The major pic promoter is maximally activated at 37°C in exponential growth phase. It was also suggested that the setB gene is transcribed from a promoter which lies more than 1.5 kb upstream of the setB structural gene; setA may be transcribed via read-through of the setB transcript and possibly by its own promoter [122]. Hemolysins Hemolysin E (HlyE) of Escherichia coli is a pore-forming hemolytic protein encoded by the hlyE (clyA, sheA) gene that was first identified in E. coli K-12. A study done to determine if hlyE was present in other E. coli pathotypes demonstrated that 19 of 23 (83%) of STEC, 7 of 7 (100%) of EIEC, 6 of 8 (80%) EAEC strains, and 4 of 7 (58%) ETEC strains tested possess a complete hlyE. The remaining STEC, EAEC, and ETEC strains and 9 of the 17 tested EPEC strains were shown to harbor mutant hylE derivatives, containing 1-bp frameshift mutations that cause premature termination of the coding sequence [123]. However, the role of HlyE in EAEC has not been determined. HlyE is a 34-kDa, predominantly α-helical protein, which oligomerizes into higher order structures to form a poreforming toxin mediating cytolytic and cytopathic effects on cultured human cells [106]. Several lines of evidence, including a complex regulatory circuit and recognition by convalescent antisera, indicate a role in disease [124, 125]. However, the occurrence of hlyE amongst non-pathogenic bacteria [123, 126] suggests that if HlyE plays a role in mediating disease then it is a minimal role. A 120-kDa heat-labile toxin that cross-reacts with antibodies against part of the tandem repeat region of E. coli alpha-hemolysin, and is capable to increase intracellular calcium levels and to stimulate protein phosphorylation via calcium-dependent kinases in HEp-2 cells, was identified in EAEC strains by Baldwin et al. [127]. These authors suggested that this toxin could play an important role in the development of EAEC-caused diarrhea, promoting cellular changes that lead to death of intestinal epithelial cells. The production of alpha-hemolysin was detected in 13% of EAEC strains and strongly associated with HeLa cells detachment of the glass coverslip during the adherence assay [128]. Fernandez-Prada et al. [129] demonstrated that the infection of human and murine macrophages with alpha-hemolysin-producing EAEC cause macrophage cell death accompanied by release of lactate dehydrogenase activity and interleukin 1. Such macrophage destruction accompanied by proinflammatory cytokines release may contribute to the clinical manifestation of EAEC-caused diarrhea. The production of plasmidmediated and thermoregulated contact hemolysin, detected by close contact between bacteria and erythrocytes

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achieved by centrifugation, was observed in EAEC strains isolated from cases of infantile diarrhea [130]. The role of  and contact hemolysins in the pathogenesis of EAEC-caused diarrhea has not been fully determined. Enteroaggregative Heat-stable Toxin 1 (EAST1) EAST-1, encoded by the astA gene adjacent to pet, is present in a wide variety of strains and different pathovars [65]. EAST1 is a 38 amino acids peptide (4.1 kDa). The sequence of EAST1 contains four cysteines that are involved in the formation of two disulfide bridges in a C1–C2 and C3–C4 conformation [131]. Using a synthetic peptide spanning residues 8–29, Savarino et al. [103] observed enterotoxic activity in an Ussing chamber assay using freshly isolated rabbit ileum. However, the presence of EAST-1 in EAEC 17-2, a strain which did not cause diarrhea in volunteers, suggests that EAST-1 is not the sole mediator of diarrhea. This observation is confirmed by studies which demonstrated EAST-1 from EAEC 17-2 had identical activities to EAST-1 derived from EAEC 042 during in vitro models of toxicity [6, 131]. Furthermore, the astA gene is detected in commensal and diarrheagenic E. coli strains [99]. EAST1 is often compared to E. coli STa (heat-Stable Toxin a) enterotoxin [103]. Like EAST1, STa toxin is known to cause secretory diarrhea [132]. The interaction of STa toxin with guanylyl cyclase C (GC-C), its enteric receptor, induces an increase of the concentration of intracellular cGMP (cyclic Guanosine MonoPhosphate) in the intestinal cells, leading to anion secretion and diarrhea. Structural similarities to STa have led to speculation that EAST1 is a ligand for GC-C [121]. However, EAST1 and STa are immunologically different since no cross-neutralization of EAST1 was observed using polyclonal anti-STa antibodies [103]. Induction of Mucosal Inflammation by EAEC The initial host inflammatory response to EAEC infection is dependent on the host innate immune system and the type of EAEC strain causing infection. The role of putative virulence genes and clinical outcomes is not well understood; however, the presence of several EAEC virulence factors correlate with finding elevated levels of fecal cytokines and inflammatory markers in stools of adults and children with diarrhea, including interleukin (IL)-1ra, IL-1β, IL-8, interferon (INF)-γ, lactoferrin, fecal leukocytes, and occult blood [58, 133]. In a quest to identify factors that may be responsible for the inflammatory presentation of EAEC-infected patients, it was reported that the flagellin of EAEC strains induced IL-8 secretion from Caco-2 cells in culture [134]. IL-8 release was subsequently linked to binding to the Toll-like receptor 5 (TLR-5) on the target epithelium [135]. TLR5 signals through P38 mitogen-activating protein kinase (MAPK) and nuclear factor kappa B (NF-κB) to induce transcription of pro-inflammatory cytokines from epithelial and monocytic cells [136]. MAPK is a member of a family of stress-related kinases that influences a diverse range of cellular functions, including host inflammatory responses to microbial products [136]. It has also suggested a role for plasmid-encoded factors in IL-8 induction [49, 58]. IL-8 is an important pro-inflammatory chemokine involved in EAEC pathogenesis. IL-8 is responsible for recruiting neutrophils to the epithelial mucosa without mucosal injury, and facilitates intestinal fluid secretion [137139]. Travelers to Mexico who developed symptomatic illness due to EAEC infection excreted high concentrations of fecal IL-8, compared to travelers who did not develop diarrhea due to EAEC infection [140]. Interestingly, IL-8 levels are higher in feces of patients infected with aggR- or aafA-harboring strains compared with those infected with strains negative for these factors. Recently, it was also shown that EAEC strains containing aggR, aggA and aap were more likely to cause increased IL-8 induction from non-polarized HCT-8 IECs than EAEC negative for those genes [133]. In a search to identify additional factors that could account for the IL-8 release from epithelial cells infected with EAEC strain 042, it was found that polarized T84 intestinal cells release IL-8, even when infected with 042 mutated in the major flagellar subunit FliC. IL-8 release from polarized T84 cells was found to require the AggR activator and the AAF fimbriae, and IL-8 release was significantly less when cells were infected with mutants in AafB, a cryptic protein encoded in the region 2 of the AAF/II fimbriae biogenesis gene cluster [141]. In addition to IL-8, intestinal epithelial cells infected with EAEC 042, have been shown to upregulate the following genes: IL-6, tumor necrosis factor (TNF)-α, growth-related gene product (GRO)-α, GRO-, intercellular adhesion molecule (ICAM)-1,

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granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-1α. These cellular responses are primarily mediated by flagellin (fliC) of EAEC [141]. It is clear that multiple factors contribute to EAEC-induced inflammation, and further characterization of the nature of EAEC pro-inflammatory factors will greatly advance the understanding of this emerging pathogen. HOST GENETIC SUSCEPTIBILITY TO EAEC IL-8 is a potent chemokine that is released into the human respiratory and intestinal mucosa by epithelial cells, macrophages and polymorphonuclear cells in response to infection. In the case of EAEC, IL-8 is produced in vitro and in vivo in response to flagellin, the monomeric component of EAEC flagella via contact with the Toll-like receptor 5 in the intestinal mucosa. Single Nucleotide Polymorphisms (SNP) in the IL-8 gene promoter region are associated with severe pulmonary infections and increased levels of secreted IL-8 [142]. Based on this observation, Jiang et al. conducted a study of 69 Caucasian healthy adult students from North America visiting Mexico for at least 3 weeks who were at risk for travelers’ diarrhea due to EAEC to investigate if host IL-8 gene SNPs were associated with diarrheal disease. This study demonstrated that a SNP that resulted in a transition from C to T in the –251 position of the IL-8 gene promoter was associated with increased levels of IL-8 and symptomatic EAEC infection. Interestingly, in this study EAEC was identified by HEp-2 adherence, and the association of symptomatic EAEC diarrhea with the high IL-8 producing alleles was similar in individuals infected with typical and atypical EAEC [140]. Additional studies in different geographical locations are needed to validate this observation. Similarly, studies in Latin American children and adults repeatedly exposed to EAEC are needed to dissect differences in susceptibility to infection in individuals with sporadic and endemic EAEC infection. TREATMENT AND PROPHYLAXIS The diagnosis of EAEC infection remains limited to research laboratories and results are available in most cases a long time after infection has resolved and therefore are of little use in guiding antibiotic therapy. As with all other enteropathogens causing acute pediatric diarrhea, the mainstay for the treatment of EAEC infection is rehydration, preferably with oral rehydration therapy followed by the reintroduction of food as tolerated. As with other self-limiting infections, the decision to use antibiotic therapy should be done on an individual basis. The selection of the antibiotic to be used should take into account the age of the person, region of the world where infection was acquired and the local antimicrobial susceptibility patterns. In general, travelers from developed countries visiting Latin America should be reminded of basic principles of hygiene, advised to avoid tap water, to drink bottle water instead and to stick to the saying “cook it, peel it, boil it or forget it”. It should be kept in mind that even cooked food, served in restaurants may become contaminated due to unhygienic conditions. In studies of travelers’ diarrhea, infection with EAEC responds to antibiotic therapy [15]. Most strains are susceptible to fluoroquinolones, azithromycin and rifaximin. However, the prevalence of antibiotic resistance in EAEC isolated from children of developing nations is high [143], including reports of emerging resistance to fluoroquinolones [100]. In addition, fluoroquinolones can rarely cause tendon rupture. Nevertheless, for most parts of the world, fluoroquinolones remain a choice for the treatment of travelers’ diarrhea due to EAEC [13, 144] and have been recommended by the Infectious Diseases Society of America for the treatment of EAEC infection [144]. Antibiotics with and without antimotility agents have been shown to be safe and effective therapy for EAEC in US adult travelers [145, 146]. Although antibiotics are highly effective in preventing travelers’ diarrhea, their cost, potential side effects and the contribution to the development of resistance [147] limits routine use. Rifaximin, a non absorbable antibiotic with low side effect profile [148] is effective in preventing travelers’ diarrhea but not US Food Drug Administration approved for this indication. Studies of cost benefit are needed to compare chemoprophylaxis and early initiation of therapy of travelers' diarrhea. The cost of generic ciprofloxacin for 1- to 3-day treatment is certainly less than the cost of 14 days of rifaximin used

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for chemoprophylaxis [148]. In patients with HIV and EAEC infection antimicrobial efficacy data is scarce. In a small, randomized, double-blind, placebo-controlled, cross-over study done with 24 cases of EAEC treated with ciprofloxacin 500 mg orally twice daily for 7 days or a placebo demonstrated that ciprofloxacin decreased the number of unformed stools by 50% and improved gastrointestinal symptoms and was associated with the eradication EAEC [53]. Of interest, nitazoxanide a broad spectrum anti-infective agent currently approved for the treatment of cryptosporidiosis and frequently used in HIV infection associated diarrhea was shown in vitro to decrease the formation of EAEC biofilm, block hemagglutination and prevent AAF/II filament formation[149]. THE PROSPECTS OF AN EAEC VACCINE Although EAEC infection responds to antibiotic therapy, the widespread geographic distribution of this infection and increasing rates of antibiotic resistance limit pharmacological approaches for control. Several lines of evidence suggest that immune based control of EAEC infection is possible. Antibodies raised against membrane components can effectively block EAEC infection in vitro [72] and this has also been demonstrated using secretory immunoglobulin A (IgA) derived from human colostrums [150]. Studies of natural history of EAEC suggest that short-term immunity develops in healthy individuals and protects against symptomatic disease [60]. Thus, immunoprophylaxis is a potential option for control of EAEC. Initial studies in volunteer challenge studies suggested that individuals with serum antibodies to dispersin would be less likely to develop symptomatic infection after exposure to EAEC [75]. This raised the possibility that dispersin, being a protein unique to EAEC [91] could represent a valuable vaccine target. Recently, however, dispersin has been identified in commensal E. coli and DAEC strains [151] and the fact that seroconversion to dispersin is seen in travelers without evidence of EAEC infection [152]. The development of an effective EAEC vaccine will require identifying pathogen-specific antigens across an enormously heterogeneous group of strains. In addition to identification of host immune responses that correlate with protection in the context of host-immunogenetic factors. ACKNOWLEDGEMENT We thank Paul S. Ugalde for the artistic work in the Fig. 2. F.N-G. was supported by Conacyt grants (60714-M and 128490) and W.P.E. was supported by FAPESP and CNPq grants. P.C.O. was supported by National Institutes of Health grant R01 AI54948, grant UL1RR024148 to the Center for Clinical and Translational Sciences of the University of Texas Medical School at Houston, and grant DK56338 to fund the Texas Gulf Coast Digestive Diseases Center. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8]

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[117] Rajakumar K, Sasakawa C, Adler B. Use of a novel approach, termed island probing, identifies the Shigella flexneri she pathogenicity island which encodes a homolog of the immunoglobulin A protease-like family of proteins. Infect Immun. 1997;65:4606-14. [118] Gutierrez-Jimenez J, Arciniega I, Navarro-Garcia F. The serine protease motif of Pic mediates a dose-dependent mucolytic activity after binding to sugar constituents of the mucin substrate. Microb Pathog. 2008;45:115-23. [119] Parham NJ, Srinivasan U, Desvaux M, et al. PicU, a second serine protease autotransporter of uropathogenic Escherichia coli. FEMS Microbiol Lett. 2004;230:73-83. [120] Heimer SR, Rasko DA, Lockatell CV, et al. Autotransporter genes pic and tsh are associated with Escherichia coli strains that cause acute pyelonephritis and are expressed during urinary tract infection. Infect Immun. 2004;72:593-7. [121] Fasano A, Noriega FR, Liao FM, et al. Effect of shigella enterotoxin 1 (ShET1) on rabbit intestine in vitro and in vivo. Gut. 1997;40:505-11. [122] Behrens M, Sheikh J, Nataro JP. Regulation of the overlapping pic/set locus in Shigella flexneri and enteroaggregative Escherichia coli. Infect Immun. 2002;70:2915-25. [123] Ludwig A, von Rhein C, Bauer S, et al. Molecular analysis of cytolysin A (ClyA) in pathogenic Escherichia coli strains. J Bacteriol 2004;186:5311-20. [124] Lithgow JK, Haider F, Roberts IS, et al. Alternate SlyA and H-NS nucleoprotein complexes control hlyE expression in Escherichia coli K-12. Mol Microbiol. 2007;66:685-98. [125] von Rhein C, Hunfeld KP, Ludwig A. Serologic evidence for effective production of cytolysin A in Salmonella enterica serovars typhi and paratyphi A during human infection. Infect Immun. 2006;74:6505-8. [126] von Rhein C, Bauer S, Simon V, et al. Occurrence and characteristics of the cytolysin A gene in Shigella strains and other members of the family Enterobacteriaceae. FEMS Microbiol Lett. 2008;287:143-8. [127] Baldwin TJ, Knutton S, Sellers L, et al. Enteroaggregative Escherichia coli strains secrete a heat-labile toxin antigenically related to E. coli hemolysin. Infect Immun. 1992;60:2092-5. [128] Gomes TA, Abe CM, Marques LR. Detection of HeLa cell-detaching activity and alpha-hemolysin production in enteroaggregative Escherichia coli strains isolated from feces of Brazilian children. J Clin Microbiol. 1995;33:3364. [129] Fernandez-Prada C, Tall BD, Elliott SE, et al. Hemolysin-positive enteroaggregative and cell-detaching Escherichia coli strains cause oncosis of human monocyte-derived macrophages and apoptosis of murine J774 cells. Infect Immun. 1998;66:3918-24. [130] Haque MA, Ohki K, Kikuchi M, et al. Contact hemolysin production by strains of enteroaggregative Escherichia coli isolated from children with diarrhea. J Clin Microbiol. 1994;32:1109-11. [131] Menard LP, Lussier JG, Lepine F, et al. Expression, purification, and biochemical characterization of enteroaggregative Escherichia coli heat-stable enterotoxin 1. Protein Expr Purif. 2004;33:223-31. [132] Greenberg RN, Hill M, Crytzer J, et al. Comparison of effects of uroguanylin, guanylin, and Escherichia coli heat-stable enterotoxin STa in mouse intestine and kidney: evidence that uroguanylin is an intestinal natriuretic hormone. J Investig Med. 1997;45:276-82. [133] Huang DB, DuPont HL, Jiang ZD, et al. Interleukin-8 response in an intestinal HCT-8 cell line infected with enteroaggregative and enterotoxigenic Escherichia coli. Clin Diagn Lab Immunol. 2004;11:548-51. [134] Steiner TS, Nataro JP, Poteet-Smith CE, et al. Enteroaggregative Escherichia coli expresses a novel flagellin that causes IL-8 release from intestinal epithelial cells. J Clin Invest. 2000;105:1769-77. [135] Hayashi F, Smith KD, Ozinsky A, et al. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature. 2001;410:1099-103. [136] Khan MA, Kang J, Steiner TS. Enteroaggregative Escherichia coli flagellin-induced interleukin-8 secretion requires Tolllike receptor 5-dependent p38 MAP kinase activation. Immunology. 2004;112:651-60. [137] Kucharzik T, Hudson JT, 3rd, Lugering A, et al. Acute induction of human IL-8 production by intestinal epithelium triggers neutrophil infiltration without mucosal injury. Gut. 2005;54:1565-72. [138] Sansonetti PJ, Arondel J, Huerre M, et al. Interleukin-8 controls bacterial transepithelial translocation at the cost of epithelial destruction in experimental shigellosis. Infect Immun. 1999;67:1471-80. [139] Madara JL, Patapoff TW, Gillece-Castro B, et al. 5'-adenosine monophosphate is the neutrophil-derived paracrine factor that elicits chloride secretion from T84 intestinal epithelial cell monolayers. J Clin Invest. 1993;91:2320-5. [140] Jiang ZD, Okhuysen PC, Guo DC, et al. Genetic susceptibility to enteroaggregative Escherichia coli diarrhea: polymorphism in the interleukin-8 promotor region. J Infect Dis. 2003;188:506-11. [141] Harrington SM, Strauman MC, Abe CM, et al. Aggregative adherence fimbriae contribute to the inflammatory response of epithelial cells infected with enteroaggregative Escherichia coli. Cell Microbiol. 2005;7:1565-78. [142] Hull J, Ackerman H, Isles K, et al. Unusual haplotypic structure of IL8, a susceptibility locus for a common respiratory virus. Am J Hum Gene.t 2001;69:413-9.

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[143] Haider K, Faruque SM, Shahid NS, et al. Enteroaggregative Escherichia coli infections in Bangladeshi children: clinical and microbiological features. J Diarrhoeal Dis Res. 1991;9:318-22. [144] Guerrant RL, Van Gilder T, Steiner TS, et al. Practice guidelines for the management of infectious diarrhea. Clin Infect Dis. 2001;32:331-51. [145] Geyid A, Olsvik O, Ljungh A. Virulence properties of Escherichia coli isolated from Ethiopian patients with acute or persistent diarrhoea. Ethiop Med J. 1998;36:123-39. [146] Kawano K, Yamada T, Yagi T, et al. [Detection of enteroaggregative Escherichia coli from sporadic diarrhea patients]. Kansenshogaku Zasshi. 1998;72:1275-82. [147] Wagner A, Wiedermann U. Travellers' diarrhoea - pros and cons of different prophylactic measures. Wien Klin Wochenschr. 2009;121Suppl3:13-8. [148] DuPont HL. Systematic review: the epidemiology and clinical features of travellers' diarrhoea. Aliment Pharmacol Ther. 2009;30:187-96. [149] Shamir ER, Warthan M, Brown SP, et al. Nitazoxanide Inhibits Biofilm Production and Hemagglutination by Enteroaggregative Escherichia coli Strains by Blocking Assembly of AafA Fimbriae. Antimicrob Agents Chemother. [150] Fernandes RM, Carbonare SB, Carneiro-Sampaio MM, et al. Inhibition of enteroaggregative Escherichia coli adhesion to HEp-2 cells by secretory immunoglobulin A from human colostrum. Pediatr Infect Dis J. 2001;20:672-8. [151] Monteiro BT, Campos LC, Sircili MP, et al. The dispersin-encoding gene (aap) is not restricted to enteroaggregative Escherichia coli. Diagn Microbiol Infect Dis. 2009;65:81-4. [152] Huang DB, Brown EL, DuPont HL, et al. Seroprevalence of the enteroaggregative Escherichia coli virulence factor dispersin among USA travellers to Cuernavaca, Mexico: a pilot study. J Med Microbiol. 2008;57:476-9.

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CHAPTER 5 Shiga Toxin-Producing Escherichia coli Beatriz EC Guth1*, Valeria Prado2 and Marta Rivas3 1

Department of Microbiology, Immunology, and Parasitology, Universidade Federal de São Paulo, São Paulo, Brazil; 2Microbiology Program, Institute of Biomedical Sciences, Faculty of Medicine, Universidad de Chile, Santiago, Chile; 3Branch of Physiopathogenesis, Department of Bacteriology, Instituto Nacional de Enfermedades Infecciosas-ANLIS "Dr. Carlos G. Malbrán", Buenos Aires, Argentina. Abstract: Shiga toxin-producing Escherichia coli (STEC) can produce a wide spectrum of human diseases, being an important cause of both outbreaks and sporadic cases of bloody and non-bloody diarrhea, hemorrhagic colitis, the diarrhea-associated form of hemolytic uremic syndrome (HUS) worldwide. HUS is a major cause of acute renal failure in children. Albeit O157:H7 is by far the most important serotype in human infections, several different O:H serotypes of E. coli can harbor Shiga toxin (stx) genes, and actually some non-O157 strains cause illnesses that are comparable in severity to O157-induced diseases, posing a substantial concern to public health. Infections due to STEC have a proven zoonotic character as these bacteria are largely distributed among both domestic and wildlife animal species. STEC infections are transmitted to humans through contaminated food, water, and contact with infected animals or people. The cascade leading from gastrointestinal infection to renal impairment is complex, being the production of Stx the major pathogenicity determinant of STEC. However, a mosaic of different virulence traits comprising several adhesins and other toxins that may play a role in pathogenesis has also been described. There is no specific treatment to reduce the progression of HUS. Research is still necessary to improve our knowledge on the mechanisms of Stx infections and the pathophysiology of cell injury in HUS to lead to better therapeutic strategies to prevent the acute mortality and the long-term morbidity of HUS.

INTRODUCTION Shiga toxin-producing E. coli (STEC) is characterized by its ability to produce at least one member of a class of potent cytotoxins that have the ability to inhibit protein synthesis in eukaryotic cells. These toxins were named verotoxin (VT) for their activity in cultured Vero cells [1], as well as Shiga toxin (Stx) due to similarities to the toxin produced by Shigella dysenteriae 1 [2]. Thus, the names VTEC (Verotoxigenic E. coli) and STEC (Shiga toxin-producing E. coli) are equivalent and define all E. coli strains that produce one or more toxins of the Stx (VT) family [3]. Although association between certain E. coli strains isolated from children with diarrhea and production of cytotoxins have been reported since the 70’s [1], the importance of STEC as human pathogens gained notoriety only in the 80’s. In 1982, two outbreaks of severe bloody diarrhea, called hemorrhagic colitis (HC), related to a previously rare serotype of E. coli, the O157:H7 were reported in the United States [4]. Subsequent studies demonstrated that the O157 isolates that had caused these outbreaks produced Stx [5], and that this toxin was identical to the verotoxin produced by an O157:H7 strain isolated from HC in Canada [6]. The important events in the linkage of HUS and STEC were the reports by Karmali et al. [7, 8] describing the association of HUS with Stxproducing E. coli strains. Studies in several other countries subsequently confirmed the linkage between STEC infection and the development of HUS, and also with another post-diarrheal syndrome, thrombotic thrombocytopenic purpura (TPP), more commonly found in adults. The term enterohemorrhagic E. coli (EHEC) was originally used to classify a subset of STEC strains, being E. coli O157:H7 the prototype, considered to be highly pathogenic and responsible to cause severe human diseases, including HC and HUS, which besides expressing Stx, present other virulence attributes as the eae gene for bacterial intimate adherence and a plasmid of approximately 60 MDa known as “EHEC plasmid” [9]. However, as it was shown later the absence of virulence markers such as eae does not necessarily indicate that an isolate is less virulent for humans [10], thereby leading to the use of more general term STEC to refer to all types of Stx-producing E. coli isolates. In contrast to infections caused by other pathogenic E. coli, those associated with STEC have a proven zoonotic *Address correspondence to: Dr. Beatriz E C Guth, Department of Microbiology, Immunology, and Parasitology, Universidade Federal de São Paulo, São Paulo, 04023-062, Brazil; Tel:55-11-50832980; e-mail:[email protected] Alfredo G. Torres (Ed) All rights reserved - © 2010 Bentham Science Publishers Ltd.

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character. Although most of the animals are asymptomatic carriers of STEC, diarrheal disease have been reported in calves, dogs and cats [11, 12,]. The piglet edema disease, a serious STEC-related illness is caused by particular STEC strains [14]. Ruminant animals, especially cattle, have long been recognized as the main natural reservoir of STEC strains [15], however several different surveys have demonstrated that STEC strains occurred in the gastrointestinal tracts of many other domestic animals such as sheep, goats, water buffalos, pigs, dogs, and cats [16, 17, 18, 19]. STEC can also be found in wildlife animals and birds, and the possible role of these animals as reservoirs for domestic animals sharing the same environment has been suggested [17, 20]. As a consequence, direct and indirect animal contact was identified as important transmission route for STEC in the environment followed by person to person spread of STEC as second most important way of transmission [21, 22]. E. coli O157:H7 has become one of the most significant food-borne pathogens, exhibiting some characteristic features such as its low infectious dose and acid tolerance that certainly favors its transmission to humans by the food chain. Since the first reported O157:H7 outbreak, many others have been linked to the consumption of raw or undercooked foods of bovine origin, which is one of the most common sources of human STEC infection, but raw or inadequately pasteurized dairy products, fermented or dried meat, unpasteurized apple cider, and several types of produce had also been sources of infection [23]. The spectrum of vehicles implicated in transmission of STEC is expanding far beyond the initial meat-related outbreaks, and the environmental contamination of farmland and irrigation water with STEC explains their finding in vegetables, fruits and sprouts [17]. Over the years, it has been recognized that more than 400 different O:H serotypes of E. coli can harbor stx genes, and a considerable proportion of them have already been recovered from human hosts (http://www.lugo.usc.es/ecoli/SEROTIPOSHUM.htm). Apart from O157:H7, the serogroups and serotypes of STEC implicated in human diseases vary from country to country, however certain serotypes such as motile and non-motile strains of O26:[H11], O103:H2, O111:[H8] and O145:[H28] tend to be found frequently associated with HC and HUS in most countries [24]. Indeed, in some geographic regions the frequency of non-O157 STEC illness overlaps that of O157 [25, 26, 27, 28, 29]. However, the role of non-O157 STEC is certainly underestimated in several countries because, in contrast to O157:H7, most laboratories do not routinely screen for them. Therefore, considerable concern regarding the awareness of the clinical importance of non-O157 STEC and the need of rapid, cost-effective diagnostic methods to detect them has been recently discussed [31, 30]. Within Latin-American countries, STEC infections seem to be more common in the South, reports of sporadic cases of diarrhea and HUS come from Argentina, Chile, Uruguay, Paraguay and Brazil, and it is probable that the problem exists but it is underestimated in other countries. Large outbreaks have not been notified in Latin America, perhaps due to lacking of surveillance programs. STEC INFECTIONS In Latin America, STEC infections are endemic and contribute to the burden of acute diarrheal syndrome in children less than 5 years of age being responsible for 2% of total cases of acute diarrhea, and in a few studies correspond to 20-30% of bloody diarrhea [32, 33]. More recent surveys have shown that in Argentina STEC accounted for approximately 2% of diarrheal diseases. In a study carried out in 2007 in Neuquén City, the rate of STEC detection in fecal samples of adults and pediatric patients with bloody and non-bloody diarrhea was low in comparison with the high number of HUS cases reported in the same period. These results suggest that the strains of this region may have a higher pathogenic potential, which allows a rapid evolution to HUS [34]. The clinical spectrum of STEC infections is broad and varies from asymptomatic cases, watery diarrhea, bloody diarrhea (or HC) and severe systemic complications as HUS. HUS is characterized by acute renal failure, microangiopathic hemolytic anemia, and thrombocytopenia [35], and is the most common indication for renal transplant in children [36]. Although the kidneys are the main targets, other organs, including the central nervous system, lungs, pancreas, and heart, may be affected. Five to 10% of children with STEC infection develop HUS, and the mortality rate among these children in the acute phase is 5% [37, 38].Infection and disease occurs after ingestion of a low dose of bacteria (as low as 50 colony forming units [CFU] and the incubation period is about 3-4 days. After that period, a high proportion of patients present watery diarrhea, and about 30% (80% in Argentina) of them evolve on day 5-6 to bloody diarrhea, presenting stools with mucus and spots of blood and a minority present HC. Mild or no fever is observed. Abdominal pain and scramps are frequent and more relevant in older children, but

 

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vomiting and irritability are also observed. In some patients, the symptoms of HUS appear on day 6-8 from the onset of diarrhea and include: microangiopathic hemolytic anemia (hematocrit <30%, schistocytes), thrombocytopenia (<150,000 platelets/mm3) and acute renal failure (creatinine >1mg/dl). In some STEC infected patients a silent period between diarrhea and HUS is observed, in other HUS patients appears during the diarrhea episode, and in a low proportion of HUS cases, there is no prodromic diarrhea [39]. In some cases, the first symptoms of HUS are neurologic manifestations such as seizures and coma. During the course of HUS is common that patients present hypertension as a consequence of overload of intravascular volume and activation of renine-angiotensin-aldosterone system due to renal ischemia [39, 40, 41]. But congestive cardiac failure, pulmonar edema, coagulopathies, electrolytic disorders, like hyperkalemia, and neurologic involvement are also registered [42]. Appearance of HUS as a complication of STEC infection is the consequence of complex interactions between the bacteria and the host factors. The STEC serotype seems to be an important associated factor, and the risk for HUS has been reported to be 10-15% for O157:H7 STEC. On the other hand, the risk for HUS development is lower, around 2%, when the strain belongs to a non-O157 serotype. It is important to consider that a proportion of children (about 10%) can present an incomplete HUS with two out of three classical features [43, 32]. The severity of initial disease is also important, being bloody diarrhea a risk factor present in 60% of Chilean children with HUS [40, 44]. The most affected age group are children less than 5 years of age, but the mean age of children with HUS varies in different countries, and range between 16 months in Chile, 29 months in Argentina, 31 months in Canada, and 3.8 years in Minessota [33, 44, 45, 46]. These differences may be associated with differences in mechanisms of transmission. Sequelae Arterial hypertension, central nervous system dysfunction, (seizures, occasionally coma), chronic renal failure and involvement of other organs are the most serious complications in HUS patients. Mortality ranges from 2-10% and up to 30% of patients will develop sequelae due to severe hypertension, and/or increased proteinuria and decreased glomerular filtration [47]. Among the complications described are: myocardial ischemia, colonic necrosis, rectal stenosis and/or prolapsed. Almost 20% of patients have evidence of pancreatic involvement with increase of serum amylase and lipase, and diabetes mellitus is seen in a low proportion of cases [48]. Neurologic sequelae as hemiparesis, learning disorders and epilepsy have also been described. STEC PATHOGENESIS As with other enteric E. coli infections, the disease process is considered to involve colonization of the intestine and damage due to toxins. The production of Stx is essential for many of the pathological features and sequelae of STEC infection. However, pathogenesis is a multistep process, involving a complex interaction between a range of bacterial and host factors. During colonization, STEC overcome host defense mechanisms to establish them in the intestine. Gastric acidity is an important host defense mechanism in the gastrointestinal tract, and acid resistance is a general feature of STEC O157 and other serotypes, which facilitate their survival through the low pH of the stomach [49]. The bacteria pass through the small intestine, and virulence genes are turned on by environmental signals in the colon [50]. Adherence to intestinal epithelial cells is an early event on STEC infection, and markedly different between strains that contain the LEE pathogenicity island and those strains that are LEE-negative. In LEE-positive strains, adherence to enterocytes of the colon is produced by a characteristic mechanism called attaching and effacing (A/E) lesion, which involves ultraestructural changes, localized destruction of enterocyte microvilli, and intimate attachment of the bacterium to the apical cell surface. Beneath the adherent bacteria, there is a rearrangement of the cytoskeletal actin components and the formation of actin-rich pedestals. The bacterial genes required for A/E lesion formation are located on the LEE and are conserved in EPEC and STEC strains capable of inducing A/E lesions, but absent in other E. coli pathotypes and non-pathogenic E. coli [51]. On the other hand, the adherence mechanisms of LEEnegative STEC strains are less understood, and several proteins were proposed to be novel adhesive factors [reviewed in 52]. As mentioned before, production of Stx is the hallmark factor in STEC diseases. If sufficient Stx is produced, local damage in blood vessels in the colon results in bloody diarrhea. If sufficient Stx is absorbed into circulation, vascular endothelial sites rich in the toxin receptor are damaged, leading to impaired function. Following binding of

 

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toxin to its globotriaosylceramide (Gb3) receptor, Stx appears to induce its transport to clathrin-coated pits, from which the toxin molecule is taken up into the cell by receptor-mediated endocytosis [53, 54]. In this process, a fragment of cell membrane pinches off to produce a coated vesicle with toxin molecules on the internal surface of the membrane. The vesicles may fuse with lysosomal vesicles, resulting in destruction of the protein toxin, leading to protection of the cell. In cells that are susceptible, however, Stx in the vesicle is transported using the retrograde pathway to the Golgi apparatus and the endoplasmic reticulum, after which the A1 fragment enters the cytosol, where interaction of the A1 subunit with the 28S rRNA results in inhibition of protein synthesis [55]. The fatty acid content of the Gb3 receptor may influence the interaction of Stx with the cell [56]. Binding of Stx induced signaling that resulted in an increase in Stx entry into the cell. Expression of the Stx receptor is a primary determinant of susceptibility to tissue injury. Both quantity and type of Gb3 present on epithelial or endothelial cells may therefore influence susceptibility to Stx. The pro-inflammatory cytokines IL-1β and tumor necrosis factor-α may also up regulate expression of Gb3 on endothelial cells [57]. Shiga toxin has been shown to induce production of IL-8 by human colonic epithelial cells and the inflammation that develops likely impairs the intestinal epithelial barrier function, thereby facilitating passage of Stx from the lumen into the submucosal area [58, 59, 60]. Most aspects of bloody diarrhea and HUS appear to be attributable to the action of Stx on vascular endothelial cells and thrombotic microangiopathy is a central feature of the disease [61]. Bloody diarrhea is associated with lesions in small blood vessels in the colon. Gianantonio et al. [62] described the pathologic renal findings in the acute stage of HUS and showed that most of the lesions are expressions of intravascular clotting. The HUS is associated with renal glomerular lesions that are due to damage to endothelial cells, which become swollen and detach from the basement membrane. Fibrin thrombi develop, and there is narrowing or occlusion of the capillary lumen [63]. The compromise in the blood supply to the glomeruli is the major contributor to loss of kidney function, but damage to glomerular epithelial and proximal tubular epithelium may also contribute to kidney damage. It has been demonstrated that Stx directly affect renal tubular cells [64, 65], and it seems plausible that injured tubular cells trigger the activation of the coagulation and inflammatory systems, which in turn, sensitize endothelial glomerular cells to Stx toxic effects. Independent of which cell type is the first target of Stx, the extent of glomerular injury will be determinant in long-term outcome, as tubular damage can be repair, whereas affected glomeruli will become fibrotic reducing the number of functional nephrons. In addition to Stx, several other factors from the bacteria and from the host that may have importance on the inflammatory and thrombotic responses in the development of HUS have been described and will be presented below. STEC VIRULENCE FACTORS Stx is the major virulence determinant of STEC, but a repertoire of different virulence factors comprising several adhesins and other toxins that may play a role in STEC pathogenesis has also been documented. Shiga Toxin The members of the Stx family share a conserved AB5 subunit structure, consisting of a single catalytic A subunit of approximately 32 kDa and a pentameric B subunit (7,7 kDa monomers) that is responsible for the binding of the toxin to specific glycolipid receptors on the surface of target cells [66]. The existence of two major Stx groups, Stx1 and Stx2, were described based on sequence analysis of stx genes and neutralization assays. Each group contains the major Stx type and an increasing number of variants. The Stx1 group presently consists of Stx1, which is nearly identical to Stx of S. dysenteriae type 1, and two variants Stx1c [67, 68] and Stx1d [69]. Stx2, which shares less than 60% amino acid sequence with Stx1, comprise a more heterogeneous group of toxins and an expanding number of variants that include Stx2c [70], Stx2d [71, 72], Stx2e [73], Stx2f [74, 75], and Stx2g [76], which has not yet found in humans. Stx2dactivatable differs from the other Stx types as its biological activity can be activated by elastase, a constituent of the intestinal mucus [77]. The genetic information for the production of Stx1, Stx2 and certain Stx variants are located in the genome of temperate lambdoid bacteriophages integrated in the STEC chromosome [reviewed in 78]. A single STEC strain can possess one or more different stx genes, and although most of the Stx variants have been isolated from patients, not all of them are associated with severe disease. In this respect, it has been reported that STEC producing only Stx2 are more pathogenic than those producing Stx1 alone and those producing both Stx1 and Stx2. Moreover, STEC strains producing Stx2, Stx2c and Stx2dactivatable have been more

 

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frequently associated with HC and HUS when compared to the other members of Stx1 and Stx2 groups that were most commonly isolated from uncomplicated diarrhea and asymptomatic infections [79]. Therefore, information about the stx2 allele has considerable predictive value to assess the risk of HUS development in a patient that presents STEC infection [80]. A/E Lesion Certain STEC strains colonize the intestinal mucosa, inducing a characteristic histophatological lesion defined as “attaching and effacing” (A/E), which is genetically governed by a large pathogenicity island named the Locus of Enterocyte Effacement (LEE). The LEE pathogenicity island is organized into 5 major polycistronic operons called LEE1 to LEE5. The products of LEE are a type III secretion apparatus (LEE1 to LEE3), a protein translocation system (LEE4), and an adherence system consisting of an outer membrane protein called intimin and its translocated intimin receptor (Tir), both encoded by LEE5, and other effector proteins that are translocated by the secretion system. The secretion apparatus is a molecular syringe structure that begins inside the bacterial cytoplasm extends through the inner and outer membranes and passes through the host cell membrane. Secreted proteins are transferred from the bacterial cytoplasm to the host cell through this structure. The Tir protein becomes inserted into the host cell membrane and acts as the receptor for intimin on the bacterial surface. Tir and other secreted proteins (EspA, EspP, EspI, StcE) activate a number of signaling cascades that result in rearrangement of the intestinal epithelial cell architecture and in changes in the cell physiology, causing diarrhea and intestinal inflammation [81, 82]. Interestingly, EspJ, a non LEE-encoded secreted protein, has been identified as an anti-virulence factor. The intimin-coding genes eae present considerable heterogeneity in their 3’ end that encodes the C-terminal 280 amino acids involved in binding to the enterocytes and Tir [83, 84], and the corresponding changes in the amino acid sequence also represent antigenic variations. Based on the sequence and antigenic differences in this C-terminal cell binding domain, at least 17 distinct intimin types have been identified and classified with a nomenclature system based on the Greek alphabet (α1, α2, β1, β2,γ1, γ2/θ, δ/κ, ε, ζ, η, η2, ι, λ, , ν, ξ, o) [85, 86, 87]. These intimin variants are distributed among STEC and EPEC of human and animal origins. The γ1 intimin is associated with highly pathogenic STEC serotypes such as O157:H7 and O145:H− [88, 89]. It has been hypothesized that the wide variability in the polypeptide cell binding domain of intimin could play a role in the tissue tropism of the different intimin producing E. coli. EPEC, which produce β intimin, can colonise almost all regions of the small bowel, while binding of γ intimin-positive STEC strains is restricted to the follicle-associated epithelium of the Peyer’s patches [90]. Although the A/E lesion is not essential for bloody diarrhea and HUS in humans, most of the strains implicated in these syndromes are LEE-positive and the eae gene has been identified as a risk factor for HUS [91]. HC and HUS patients develop antibody responses to several LEE-encoded proteins. Furthermore, immunization with LEEencoded proteins can partially protect reservoir hosts against STEC infection [92, 93, 94]. It is important to point out that several LEE-positive STEC strains are not associated with HUS, and that the type III secretion system also secretes many other effector molecules encoded outside the LEE, that are referred to as non-LEE-encoded effectors (nle) [95]. At least three of these nle have been linked to non-O157 STEC that cause HUS [96]. Other Adhesion Mechanisms and Putative Adhesins Apart from the pivotal role of intimin in the adherence of LEE-positive STEC to human intestine, considerable research has been undertaken to discover other fimbrial and non-fimbrial adherence factors that may be involved in binding of these organisms to eukaryotic cells [reviewed in 97]. The involvement of a 60 MDa STEC plasmid referred to as pO157, in the adherence of STEC O157:H7 was initially suggested by Karch et al. [98]. These investigators found that the presence of the plasmid correlated with expression of fimbriae and adherence to Henle 407 but not HEp-2 cells. However, subsequent studies have produced conflicting results and there is no consistent in vitro evidence for a role for pO157 in STEC adherence [99, 100, 101]. Experimental infection studies indicate that pO157 is not required for EHEC O157:H7 virulence in gnotobiotic piglets [102], but it was more recently reported that pO157 influences colonization of the bovine terminal rectum [103]. In addition, it has been described that the plasmid-encoded serine protease EspP from O157:H7 STEC, which belongs to the family of serine protease autotransporters of Enterobacteriaceae (SPATES), contributes to adherence to bovine primary rectal cells and colonization of the bovine intestines [104]. However, the mechanism by which EspP affects intestinal colonization and adherence to cultured cells is unknown.

 

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A chromosomal genetic locus named efa-1 (EHEC factor for adherence) was characterized in a clinical O111:NM STEC strain, and this locus was shown to mediates adherence to cultured Chinese hamster ovary (CHO) cells [105]. Mutations on the efa-1 gene dramatically reduced the number of bacteria associated with the bovine intestinal epithelium, although the mutants still retain their ability to form both microcolonies and A/E lesions [106]. A truncated version of efa-1 was identified in the chromosomes of the two fully sequenced O157:H7 prototype EDL933 and Sakai strains [107, 108]. It was suggested that this gene influences bacterial adhesion and intestinal colonization [109]. Another gene significantly homologous to efa-1 and also located in pO157 known as toxB was described [110]. Although strains lacking toxB presented reduced adherence to cultured epithelial cells [109], studies using single and double O157:H7 toxB and efa1’ deletion mutants indicate that neither ToxB, nor the truncated version of Efa1, are required for efficient intestinal colonization of calves [106]. On the other hand, it has been suggested that these genes may indirectly influences adherence by modulating the production and secretion of LEE-encoded effector proteins that are required for the formation of A/E lesions [106]. The distribution of efa-1 and toxB has been described in different STEC serotypes and the close association on the presence of these genes with eae has been confirmed [111, 112, 113, 114]. Some other proteins have been implicated as mediators of adherence in O157 STEC including Iha (Vibrio cholerae IrgA homologue adhesin), and Cah (calcium-binding antigen 43 homologue), but with no obvious role in pathogenesis. Iha, a 67 kDa outer membrane protein, was associated with adherence to HeLa cells only when it is expressed in a non-fimbriated E. coli stain [115]. The iha gene, which is located adjacent to an O-island encoding urease and tellurite resistance in O157:H7 strain, is commonly distributed in many STEC serotypes isolated from humans and cattle, independently of the presence of eae [112, 113, 116]. Cah is a surface-expressed and heat-extractable protein that causes autoaggregation and changes in bacterial shape when it is expressed as a recombinant protein and also participates in the formation of biofilms [117]. Two chromosomal gene clusters closely related to the long polar fimbrial (lpf) operon of Salmonella enterica serovar Typhimurium were identified in O157:H7 STEC [118, 119]. Expression of E. coli O157:H7 lpf operon 1 (lpf1) in E. coli K-12 was shown to increase adherence to HeLa cells, and is associated with the appearance of long fimbriae [118]. The lpf2 operon has also been linked to adherence to epithelial cells [119]. In addition, E. coli O157:H7 mutants in one or both of the lpf loci showed diminished colonization abilities in animal models [120], and the role of Lpf as a colonization factor associated with persistence in the intestine was elucidated using a lamb model of infection [121]. The adhesion mechanisms of non-O157 LEE-negative serotypes have been the subject of intense research, since some of these bacteria have been associated with sporadic cases and small outbreaks of HC and HUS [10]. The occurrence of fimbrial and non-fimbrial proteins with adhesive functions, beyond some of those already known among LEE-positive strains, has been reported. Investigations on the attachment of O113:H21 STEC to HEp-2 cultured cells and to rabbit intestine in vivo showed that there were areas of microvillus effacement beneath the organism, but that the cytoskeletal rearrangement characteristic of the AE lesion did not develop [122]. An autoagglutinating adhesin named Saa (STEC autoagglutinating adhesin) was initially characterized in an O113:H21 STEC strain isolated during an outbreak of HUS in Australia [123]. Saa is a plasmid encoded outer membrane protein of approximately 56 kDa, with little homology to other known bacterial adhesins. Saa was associated with both an increase in adherence to Hep-2 cells when it was expressed as a recombinant protein in E. coli and with a reduction in adherence when its gene was mutagenized in the wild-type strain [123]. Because saa was detected in several HUS associated LEE-negative STEC isolates but in any of the LEE-positive strains, it was suggested that saa may be a marker for a subset of LEE-negative STEC strains capable of causing severe gastrointestinal and systemic illness in humans [123]. However, subsequent surveys showed that saa is also widely found in bovine STEC isolates [112, 113, 114, 124, 125], sometimes at greater frequencies than in human STEC strains, suggesting that Saa may have a more important role in attachment to the bovine gut than the human intestine [124]. The participation of Saa in the adherence process of an O125 STEC strain isolated from HC was also described [126]. Moreover, a Saa-independent mechanism of adherence has also been reported by Toma et al. [127]. Homologs of lpf genes have also been detected in non-O157 STEC strains. A similar region to the O157 lpf2 operon, also involved in adherence to epithelial cells, was characterized in a LEE-negative O113:H21 STEC strain [128]. Subsequent studies showed that the lpfA gene from STEC O113 (lpfAO113) is highly frequent among LEE-negative E. coli strains isolated from humans and animals [112, 113, 125, 129, 130], and thus may represent an important

 

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adherence factor in this group of pathogens. Recently, Galli et al. [131] using a collection of Argentinean LEEnegative STEC strains, determined that the different lpfA types were present in a wide variety of serotypes and no apparent association was observed between the types of lpfA1 or lpfA2 genes and the severity of human disease. The lpfA2-1 was the most prevalent variant identified, which was present in 95.8% of the isolates, and the lpfA1-3 and lpfA2-2, proposed as specific biomarkers of E. coli O157:H7, were not found in any of the serotypes studied. Other Toxins The plasmid-encoded hemolysin (Ehly) is a pore-forming cytolysin [132] belonging to the repeats-in-toxin (RTX) family [133] and responsible for the so called enterohemolytic phenotype observed among O157 and non-O157 STEC strains [134]. Production of Ehly has been identified at varied frequencies both in eae-positive and eaenegative STEC strains. Humoral immune response to this protein, detected in sera of patients recovering from HUS, demonstrates its expression during infection, and was the first evidence of an association of Ehly with the virulence of STEC [133]. It has been suggested that hemolysin could contribute to disease through lysis of erythrocytes and release of hemoglobin as a potential source of iron for the bacteria. A direct effect of Ehly on endothelial cells has not been investigated, but E. coli expressing the cloned enterohemolysin operon induced IL-1β [ 57], one of the cytokines that upregulates Stx receptor Gb3. Therefore, enterohemolysin might augment the effects of Stx on endothelial cells. Although this is an attractive hypothesis, there is no available data to support a role of enterohemolysin in the hemolytic anemia of HUS patients. A new member of the cytolethal distending toxin (CDT) family was identified in O157 and some particular nonO157 STEC serotypes associated with HUS, and designated CDT-V [135, 136]. Investigations on the biological effects of recombinant CDT-V from an O157 strain on human endothelial cells showed that CDT-V damages eukaryotic DNA, leading to G2/M cell cycle arrest and cell death [137]. These data demonstrate that CDT-V directly injures endothelial cells resulting in their dead, and may thus contribute in the pathogenesis of HUS. Subtilase cytotoxin, a novel member of the AB5 family of toxins, was identified for the first time in an O113:H21 STEC strain associated with an outbreak of HUS in Australia [138]. The presence of subAB genes was further identified in STEC strains belonging to different serotypes and isolated in other countries [113, 125, 139, 140, 141, 142]. This new toxin is composed of an enzymatically active A subunit, which is a subtilase-like serine protease, and a binding B subunit, which is related to a putative exported protein from Yersinia pestis. Intraperitoneal injection of purified SubAB into mice causes extensive microvascular tromboses, and necrosis in the kidneys, brain and liver [138], resembling the lesions observed in patients with HUS, and thus suggesting a contribution of this toxin to the pathogenesis of STEC disease. The simultaneous presence of cdt-V and subAB genes was recently reported in several LEE-negative STEC serotypes, some of which related to severe human diseases [113, 125, 142]. Additional Factors from the Bacteria and From the Host Several authors have reported the importance of the inflammatory and thrombotic responses in the development of HUS. In the STEC O157 infection, H7 flagellin appears to be a major contributor to colonic inflammation. Berin et al. [143] proposed that H7 flagellin played a major part in the activation of proinflammatory signals in human colon epithelial cells. Miyamoto et al. [144] reported that upregulation of proinflammatory cytokines and attraction of subepithelial neutrophils was observed following addition of H7 flagellin, which binds to toll-like receptor 5 leading to activation of NF-B and secretion of IL-8. Besides its putative role in STEC adherence, the plasmid-encoded serine protease EspP was shown to cleave coagulation factor V in human plasma [145], and this effect has been proposed to contribute to the mucosal hemorrhages observed in patients with HC. Moreover, the presence of proteolytically active EspP in other serogroups most frequently associated with HC and HUS suggests its role as a significant STEC virulence factor [146]. Other proteases (EspA, EspJ, EspI, StcE) are also produced by STEC and their role in the pathogenicity has been discussed elsewhere [15, 81]. The lipopolysaccharide (LPS) is a major product of the Gram-negative bacteria that induce the clinical syndrome of septic shock and renal cortical necrosis. Both Stx and LPS may be absorbed from the inflamed gastrointestinal tract and induce synergistic effects in Stx-induced HUS. Louise et al. [147] suggested that Stx may enhance the pro-coagulant

 

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effect of LPS. In vivo studies have demonstrated that toxicity of Stx is potentiated by LPS, and renal thrombotic microangiopathic lesions, mimicking those seen in human HUS, can be reproduced by infusion of Stx together with LPS [148, 149, 150]. Many substances, including prostaglandins, cytokines, vasoactive and procoagulant factors, are stimulated by LPS [151]. Stx-induced endothelial injury is the primary pathogenic event but LPS, chemokines and cytokines released by inflammatory cells [58] or injured cells [152] may contribute with this process. Stahl et al. [153] suggested that absorption of LPS in the early stages of STEC O157 infection may lead to direct activation of platelets or to binding to endothelium followed by binding to platelets, resulting in platelet consumption. The authors suggested that other factors, such as activation of platelets by Stx and/or other compounds [154 ], or endothelial cell injury, may also contribute to the thrombocytopenia and the thrombotic state observed in HUS. Since acute inflammatory infiltration of the gut and the presence of leukocytes in feces are seen in many STECinfected patients, several studies were conducted to analyze the role of PMN in the intestine. According with Wagner et al. [155], PMN recruitment in the intestine may also increase the risk of HUS by inducing the Stx2 prophage in vivo and augmenting Stx2 production, mainly through the production of H2O2. Either Stx directly or the combined effects of LPS and cytokines will activate the coagulation system, causing a prothrombotic state with platelet and PMN activation that will amplify endothelial injury. Patients show markers of endothelial injury, activation of the coagulation cascade and inhibition of fibrinolysis [156]. It has been suggested that the degree of the prothrombotic activation early in infection could be decisive in the course of the disease [157]. The activation of PMN is evidenced by a high peripheral blood PMN count at presentation, which has been correlated with a poor prognosis, and increased levels of serum elastase and IL-8. EPIDEMIOLOGY OF STEC IN LATIN AMERICA STEC O157:H7 has been responsible for numerous outbreaks and sporadic cases of infections in different parts of the world [14, 43, 158, 159, 160]. The relative ease of isolation of this serotype on the basis of its inability to ferment sorbitol may be contributing to an overestimation of its prevalence with respect to other STEC serotypes. At present, there is growing concern over the emergence of highly virulent non-O157 STEC serotypes that become globally distributed and associated with outbreaks and/or severe human illness [96]. These serotypes differ in their frequency of association with human disease and the severity of disease, suggesting differences in virulence characteristics [161] Important differences exist in the incidence of STEC infections and HUS in South America. A Regional Network for surveillance purposes is still inexistent and data are only restricted to few countries. The National Reference Laboratories participate in PulseNet Latin America and Caribbean, a regional network that performs pulsed-field gel electrophoresis (PFGE) on STEC strains. National Databases have been created in some countries. HUS is endemic in some countries of the Southern cone region and reporting is only mandatory in Argentina, Chile and Paraguay. If we compare the HUS situation, the annual incidence in Argentina is five times higher than in Chile, but the magnitude of the problem is still unknown in other countries. In Brazil, STEC infections are important public health issues at least in some regions. Infections due to non-O157 STEC have been reported since the early 70’s, mainly in cases of uncomplicated diarrhea in young children [28, 162]. In later years, O157 and non-O157 STEC strains have been recovered from severe human diseases such as bloody diarrhea, hemolytic anemia and HUS cases, and also from food and animals. The STEC prevalence among children with acute diarrhea varies depending on the studied region, but in general the incidence is relatively low [reviewed in Chapter: E. coli situation in Brazil]. From 1976 to 1999, STEC strains were isolated from patients with diarrhea in São Paulo City. Serogroups O26 and O111 accounted for most of the cases [28]. At present, different non-O157 serotypes, including important zoonotic serotypes, have been reported associated with diarrhea and HUS [29, 163, 164, 166, 165, 167]. Since the first description in the 1990’s, O157 STEC strains associated with bloody diarrhea and HUS have been identified in the southeastern region [28, 29, 168, 169]. In the last five years, STEC strains were isolated in São Paulo from 2.9% HIV adult patients and from 1.5% children with non-bloody diarrhea. Different serotypes and genotypes were identified among the isolates. The HIV patient with O157 infection was the only one with bloody diarrhea [168]. Although a nationwide surveillance system for HUS has been recently established, data are still inexistent or scarce. In a prospective study carried out in São Paulo City from 2001 to

 

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2005, thirteen children with clinical and laboratory diagnosis of HUS were enrolled in the survey, and O26, O157 and O165 STEC strains were isolated from three patients [164, 167, 169]. In addition, high levels of O111 and O157 LPS antibodies were detected in sera of two and seven HUS patients, respectively. Both microbiologic and serologic techniques provided evidences of STEC infection in more than 90% of the patients, and association with O157 infections accounted for almost 62% of the HUS cases. Ten patients were followed up from 2 to 5 years, one patient developed chronic renal disease [170]. Isolation of STEC strains from healthy dairy and beef cattle, water buffaloes, calves and goats at farm level, and also from hides and carcasses at abattoir level has been described in different Brazilian regions [reviewed in Chapter: E. coli situation in Brazil]. Non-O157 STEC strains prevailed in the animal reservoir, mainly stx2 producers, although E. coli O157 has also been isolated in some of them. The STEC frequency in cattle was higher in southern estates. In Chile, a National Surveillance System was established in 1999 and all clinical laboratories must report and send isolates to the Reference Laboratory. In the last two years, 96 strains were characterized and O157:H7 was the predominant serotype with more than 60% of the isolates and the stx2 genotype prevailed. A National E. coli O157 Database has been created and three restriction patterns are prevalent [171, 172]. Regarding the animal reservoirs, STEC strains were detected in 13.5% of bovine feces at slaughter level. Non-O157 serotypes and stx2-genotype were predominant. In pigs, the isolation rate was 6.2%, a value significantly lower than those reported several years before of 69%. The Stx2e variant was prevalent [173]. In Uruguay, reports of HUS are not mandatory and only a few cases are recognized each year. In 2002, the first HUS case associated with STEC O157 infection was recognized [174]. Moreover, Varela et al. [175] performed the comparison of STEC strains isolated from human diseases and food using typing techniques. Grazing cattle and sheep were recognized as main reservoirs of STEC, and non-O157 STEC strains were recovered from 42% of the sampled animals. Stx2 was the predominant genotype detected [176]. In Paraguay, reporting of HUS has been mandatory since 2005. In the last two years, fifteen HUS cases were reported by the laboratory-based surveillance system. The annual incidence was 0.6 cases per 100,000 children under 5 years old. In Asunción City, three sentinel sites have been established. National Databases have been created for molecular surveillance purposes but only few O157 and non-O157 strains have been isolated from HUS and diarrhea cases. In Argentina, data on human STEC infections are gathered through different strategies: 1) the National Health Surveillance System collects data of HUS cases and since the year 2000, the report is mandatory, and must be immediate and individualized; 2) the Sentinel Surveillance System through 25 HUS Sentinel Units; 3) the Laboratory-based Surveillance System through the National Diarrheal and Foodborne Pathogens Network; and 4) the Molecular Surveillance through the PulseNet of Latin America and Caribbean. Post-diarrheal HUS is endemic. Over the last 10 years, approximately 500 HUS cases were reported annually. The incidence has ranged between 7.8 and 17 cases per 100,000 children less than 5 year of age and the lethality was between 2 and 5% [46, Ministry of Health, Argentina, 2008, unpublished data). This rate is 10-fold higher than in other industrialized countries. In some studies, evidence of STEC infection was found in around 60% of Argentinean HUS cases, and E. coli O157 was the predominant serogroup isolated [46, 89, 177, 178]. Each year, the National Reference Laboratory receives samples from approximately 60% of the HUS cases reported. As part of the PulseNet Latin America and Caribbean, Argentinean databases for O157 and non-O157 STEC strains were created by the National Reference Laboratory. In the period 1988-2009, a total of 710 XbaI-PFGE patterns corresponding to 1775 STEC O157 strains were included. Two XbaI-PFGE patterns are prevalent, AREXHX01.0011 (n=176) and AREXHX01.0022 (n=100), representing 9.9% and 5.6% of the database, respectively. Among the non-O157 STEC strains, 891 XbaI-PFGE patterns, corresponding to 1190 isolates were established. Up to date, the patterns are classified and codified according to the following O-groups: O8, O22, O26, O91, O103, O111, O113, O121, O130, O145, O174 and O178. Different studies have demonstrated the role of ruminants, mainly cattle, as STEC reservoir [179, 180, 181, 182, 183], and foods as vehicle of transmission [184, 185, 186, 187, 188, 189]. In conclusion, STEC strains are

 

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widespread in South America, infect humans and animals, and contaminate food products, but the magnitude of the problem is different in each country. Different serotypes and genotypes were detected and the severity of clinical symptoms caused by STEC has been associated with the stx variants produced. In each country, a National Surveillance System and enhanced laboratory-based surveillance are needed to assess the real burden of diseases due to STEC. A regional network should be created to monitor the incidence of STEC in humans, animals, food and environment. STEC epidemiology is the interest in South America from a human health perspective and an economic perspective being these countries major exporters of primary products, particularly meat. TREATMENT AND CONTROL Currently, there is no specific treatment for STEC infections and HUS. For that reason these diseases can be considered within the category of “orphan diseases” and thus the main focus must be in prevention. Use of antibiotics in the initial period of diarrhea is not recommended. Some evidences have indicated that antimicrobials of different families could be a risk factor for developing HUS, by enhancing phage induction and subsequent stx gene expression, and by increasing Stx release after bacterial lysis induction [190]. Taking this into account, a rapid identification of patients infected by STEC using adequate diagnostic tools is very important to avoid the use of antimicrobials; diagnostic efforts must be concentrated in bloody diarrhea patients [190, 191]. At hospital level, ideally in intensive care units, it is recommended to observe patients with diagnosis of bloody diarrhea caused by STEC to measure variations in parameters as: weight, water and electrolytes balance and diuresis, hematocrit, appearance of signs of hemolysis like schistocytes and serum creatinine. Medical efforts must be oriented to correct anemia and renal damage. If HUS is suspected or confirmed it is important to have an adequate vein access for the eventually administration of parenteral solutions or transfusions. Ake et al. [41] recommended parenteral rehydration with isotonic crystalloids to protect the kidney and to diminish the need for dialysis. If the patient present edema, oligoanuria, cardiac overload or hypertension, a diuretic like furosemide it is initially recommended, but if no response occurred in few hours, peritoneal dialysis must be indicated. There is a consensus that the opportunity of dialysis is critical and influences the patient’s outcome (prognosis). Transfusions with blood red cells (filtered and irradiated) must be administrated when hemoglobin is under 7gr/l or with upper levels if there is cardio-respiratory compromise. Platelets transfusion should be avoided unless the level of platelets is less than 20.000 /mm3 with high bleeding risk. The diet recommended for these patients is hyper caloric and low in proteins, sodium and calcium [192]. Some new strategies to prevent systemic and renal damage in STEC infections have been investigated, and are focused on the neutralization of the Shiga toxin deleterious activity. The following strategies can be pointed out: a) Synthetic molecules analogues of Gb3 receptor, like Synsorb-Pk, have been evaluated in clinical trials. A multicenter controlled trial enrolled 150 children with HUS and Synsorb was administered orally, but no efficacy in diminishing mortality, need or duration of dialysis, or indication for transfusions was observed. Conclusions of this study indicated that this kind of strategy would be more efficient in the early period of STEC infection, but with no benefits when HUS is already present [193]; b) Other molecules with greater affinity for Gb3 than Synsorb have been developed, and evaluated in preclinical studies. In rats, these molecules showed to be protective against a lethal dose of STEC O157, nevertheless evaluation in humans is still pending. [194]; c) A group of investigators have explored genetic recombinant techniques in bacterial strains to create non-pathogenic E. coli expressing Gb3 sugars in the LPS. Studies in animal models have shown good results with these molecules for binding Stx1 and Stx2 and protecting rats from a lethal dose of O157 STEC [195]; d) Other interesting therapeutic strategy proposed to reduce binding and uptake of Stx was based on short-term inhibition of host Gb3 synthesis with C-9, a specific inhibitor of glucosylceramide synthase. In vitro studies have shown that pre-treatment of human renal tubular epithelial cells for 48 h with C9 produced a significant reduction in cellular Gb3 levels [196]. This molecule if administered in the early course of STEC infection could be useful to prevent Stx damages; e) A recent therapeutic strategy was based in the use of intravenous chimeric human monoclonal antibodies against Stx1 and Stx2. The ability of antibodies to specifically neutralize Stx was well established. In vitro studies and pharmacological evaluations in mice revealed that antibodies protected animals from lethal dose of Stxs [197]. Safety and pharmacological properties of these monoclonal antibodies were then evaluated in phase I studies in healthy volunteers, and the results suggested that they are safe and well tolerated [198]. Future clinical studies in infected children are necessary.

 

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Prevention with Vaccines Considering that STEC infections are zoonotic, one strategy has been the development of vaccines to prevent or diminish shedding of the bacteria in the animal reservoir. Cattle are the main reservoir and vaccines based on antigens involved in intestinal colonization have been tested in this host with promising results. One vaccine using as antigens Type III secreted proteins was administered to cattle and a decrease in shedding from 23 to 9% was observed [94]. Another vaccine based on a combination of antigens involved in STEC adherence, such as EspA, intimin, and Tir significantly reduced shedding of EHEC O157 from experimentally infected cattle and the protection correlated with immune response [199]. A new technology based on iron requirement of pathogenic bacteria has also been explored. An anti-E.coli O157 vaccine based on siderophore receptor and porin proteins was evaluated in cattle naturally shedding the pathogen, and a significant reduction in prevalence and duration of shedding was observed [200]. Other vaccine candidates are based on attenuated strains with deletions on ler and stx genes, and immunized mice with this vaccine were protected against infection with wild-type O157:H7 STEC [201]. A DNA vaccine has been developed with a non-toxic Stx2 [202]. This vaccine was tested in mice and was able to elicit systemic Stx-specific antibody response for native Stx2 and conferred partial protection to Stx2 challenge in vivo. On the other hand, there is scarce experience related to vaccines against STEC for humans. One evaluated vaccine is based on the concept that IgG antibodies against the O-specific polysaccharide of E. coli O157:H7 may confer immunity by lysing the inoculum in the intestine. A phase 1 trial was conducted in adult volunteers with a parenteral vaccine containing O157 polysaccharide conjugated to a recombinant exotoxin A of Pseudomonas aeruginosa (O157-rPA) showing that the vaccine was safe and immunogenic [203]. Afterwards, the same vaccine was evaluated in a phase 2 trial in children, and safety was confirmed, no significant adverse reactions were seen and high specific antibodies response were observed [204]. Albeit promising, it is clear that this vaccine does not represent a solution in preventing human STEC infections as it has considered only O157 serogroup. Currently, new approaches including common antigens for STEC serogroups and serotypes are needed to protect humans. REFERENCES [1] [2] [3]

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[150] Sauter KA, Melton-Celsa AR, Larkin K, et al. Mouse model of hemolytic-uremic syndrome caused by endotoxin-free Shiga toxin 2 (Stx2) and protection from lethal outcome by anti-Stx2 antibody. Infect Immun. 2008;76:4469-78. [151] Shultz PJ, Raij L. Endogenously synthesized nitric oxide prevents endotoxin induced glomerular thrombosis. J Clin Invest. 1992;90:1718-25. [152] Ray P, Acheson D, Chitrakar R, et al. Basic fibroblast growth factor among children with diarrhea-associated hemolytic uremic syndrome. J Am Soc Nephrol. 2002;13:699-707. [153] Stahl AL, Svensson M, Morgelin M, et al. Lipopolysaccharide from enterohemorrhagic Escherichia coli binds to platelets through TLR4 and CD62 and is detected on circulating latelets in patients with hemolytic uremic syndrome. Blood. 2006;108:167-76. [154] Karpman D, Papadopoulou D, Nilsson K, et al. Platelet activation by Shiga toxin and circulatory factors as a pathogenetic mechanism in the hemolytic uremic syndrome. Blood. 2001;97: 3100-8. [155] Wagner PL, Acheson DW, Waldor MK. Human neutrophils and their products induce Shiga toxin production by enterohemorrhagic Escherichia coli. Infect Immun. 2001;69:1934-7. [156] Bielaszewska M, Karch H. Consequences of enterohaemorrhagic Escherichia coli infection for the vascular endothelium. Thromb Haemost 2005; 94: 312-8. [157] Chandler WL, Jelacic S, Boster DR, et al. Prothrombotic coagulation abnormalities preceding the hemolytic-uremic syndrome. N Engl J Med. 2002;346:23-32. [158] Rangel JM, Sparling PH, Crowe C, et al. Epidemiology of Escherichia coli O157:H7 outbreaks, United States, 1982-2002. Emerg Infect Dis. 2004;11:603-9. [159] Sartz L, De Jong B, Hjertqvist M, et al. An outbreak of Escherichia coli O157:H7 infection in southern Sweden associated with consumption of fermented sausage; aspects of sausage production that increase the risk of contamination. Epidemiol Infect. 2008;136:370-80. [160] Greenland K, de Jager C, Heuvelink A, et al. Nationwide outbreak of STEC O157 infection in the Netherlands, December 2008-January 2009: continuous risk of consuming raw beef products. Euro Surveill. 2008;14:19129. [161] Karmali MA, Mascarenhas M, Shen S, et al. Association of genomic O island 122 of Escherichia coli EDL 933 with verocytotoxin-producing Escherichia coli seropathotypes that are linked to epidemic and/or serious disease. J Clin Microbiol. 2003;41:4930-40. [162] Giraldi R, Guth BEC, Trabulsi LR. Production of Shiga-like toxin among Escherichia.coli strains and other bacteria isolated from diarrhea in São Paulo, Brazil. J Clin Microbiol. 1990;28:1460-1462. [163] Cantarelli V, Nagayama K, Takahashi A, et al. Isolation of Shiga toxin-producing Escherichia coli (STEC) serotype O91:H21 from a child with diarrhea in Porto Alegre city, RS, Brazil. Braz J Microbiol. 2000;31:266-70. [164] Guth BEC, Souza RL, Vaz TMI, et al. First Shiga Toxin-producing Escherichia coli isolate from a patient with hemolytic uremic syndrome, Brazil. Emerg Infect Dis. 2002;8:535-536. [165] Guth BEC, Vaz TMI, Gomes TAT, et al. Re-emergence of O103:H2 Shiga toxin-producing Escherichia coli infections in São Paulo, Brazil. J Med Microbiol. 2005;54:805-806. [166] De Toni F, Souza EM, Pedrosa FO, et al. A prospective study on Shiga toxin-producing Escherichia coli in children with diarrhoea in Paraná State, Brazil. Lett Appl Microbiol. 2009;48:645-647. [167] Souza RL, Nishimura LS, Guth BEC. Uncommon Shiga toxin-producing Escherichia coli serotype O165:HNM as cause of hemolytic uremic syndrome in São Paulo, Brazil. Diag Microbiol Infect Dis. 2007;59:223-225. [168] Irino K, Vaz TMI, Kato MAMF, et al. O157:H7 Shiga toxin-producing Escherichia coli strains associated with sporadic cases of diarrhea in São Paulo, Brazil. Emerg Infect Dis. 2002;8:446-447. [169] Bastos FC, Vaz TMI, Irino K, et al. Phenotypic characteristics, virulence profile and genetic relatedness of O157 Shiga toxin-producing Escherichia coli isolated in Brazil and other Latin American countries. FEMS Microbiol Lett. 2006;265:89-97. [170] Guth BEC, Souza RL, Andrade MC, et al. Hemolytic Uremic Syndrome in pediatrics care units in São Paulo, Brazil, from 2001 to 2005. 7th International Symposium on Shiga Toxin (Verocytotoxin) - Producing Escherichia coli Infections. Buenos Aires: Argentina. 2009;May 10-13,p54-55. [171] Olivares B, Araya P, Ibáñez D, et al. Genetic characterization of Chilean isolates of O157:H7 Shiga toxin producer Escherichia coli. 2003-2008. Public Health Institute of Chile. 7th International Symposium on Shiga Toxin (Verocytotoxin) - Producing Escherichia coli Infections. Buenos Aires: Argentina. 2009;May 10-13,p61. [172] Olivares B, Araya P, Prat S, et al. Laboratory surveillance system for STEC, Public Health Institute, ISP-Chile, 2007September 2008. 7th International Symposium on Shiga Toxin (Verocytotoxin) - Producing Escherichia coli Infections. Buenos Aires: Argentina. 2009;May 10-13,p61. [173] Vidal RM, Valenzuela P, Corvalán L, et al. Serotype and virulence genes identification in Shiga toxin - producing Escherichia coli (STEC) isolated from pig and bovine at slaughter in Chile. 7th International Symposium on Shiga Toxin (Verocytotoxin) - Producing Escherichia coli Infections. Buenos Aires: Argentina. 2009;May 10-13,p76.

 

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[174] Gadea MDP, Varela G, Bernadá M, et al. Primer aislamiento en Uruguay de Escherichia coli productora de toxina Shiga del serotipo O157:H7 en una niña con síndrome urémico hemolítico. Rev Med Uruguay. 2004;20:79-81. [175] Varela G, Chinen I, Gadea P, et al. Detección y caracterización de Escherichia coli productor de toxina Shiga a partir de casos clínicos y de alimentos en Uruguay. Rev Arg Microbiol. 2008;40:93-100. [176] Gadea MDP, Balseiro V, González S, et al. Phenotypic and genotypic characterization of Shiga toxin - producing Escherichia coli strains isolated from animal in Uruguay. 7th International Symposium on Shiga Toxin (Verocytotoxin) Producing Escherichia coli Infections. Buenos Aires: Argentina 2009; May 10-13,p.79. [177] Rivas M, Balbi L, Miliwebsky E, et al. Síndrome Urémico Hemolítico en niños de Mendoza, Argentina: su asociación con la infección por Escherichia coli productor de toxina Shiga. Medicina (Buenos Aires). 58:1-7,1998 [178] Miliwebsky E, Balbi L, Gómez D, et al. Síndrome Urémico Hemolítico en niños de Argentina. Asociación con la infección por Escherichia coli productor de toxina Shiga. Bioquímica y Patología Clínica. 63:113-121,1999. [179] Gioffre A, Meichtri L, Miliwebsky E, et al. Detection of Shiga toxin-producing Escherichia coli in cattle in Argentina. Evaluation of two procedures. Vet Microbiol. 2002;87:301-3. [180] Meichtri L, Miliwebsky E, Gioffré A, et al. Shiga toxin-producing Escherichia coli in healthy young beef steers from Argentina: prevalence and virulence properties. I J Food Microbiol. 2004;96:189-198. [181] Leotta GA, Deza N, Origlia J, et al. Detection and characterization of Shiga toxin-producing Escherichia coli in captive wild mammals. Vet Microbiol. 2006;118:151-7. [182] Masana MO, Leotta GA, Del Castillo LL, et al. Prevalence, characterization, and genotypic analysis of Escherichia coli O157:H7/NM from selected beef exporting abattoirs of Argentina. J Food Protect. 2010;73:649-56. [183] Tanaro JD, Leotta GA, Lound LH, et al. Escherichia coli O157 in bovine feces and surface water streams in a beef cattle farm of Argentina.. Foodborne Path Dis. 2010;7:475-7. [184] Chinen I, Tanaro JD, Miliwebsky E, et al. Isolation and characterization of Escherichia coli O157:H7 from retails meats in Argentina. J Food Protect. 2001;64:1346-51. [185] Gómez D, Miliwebsky E, Fernández Pascua C, et al. Aislamiento y caracterización de Escherichia coli productor de toxina Shiga en hamburguesas supercongeladas y quesos de pasta blanda. Rev Arg Microbiol. 2002;34:66-71. [186] Rivas M, Caletti MG, Chinen I, et al. Home-prepared hamburger as the source for a sporadic case of hemolytic uremic syndrome, Argentina. Emerg Infect Dis. 2003;9:1184-6. [187] Oteiza JM, Chinen I, Miliwebsky E, et al. Isolation and characterization of Shiga toxin-producing Escherichia coli from precooked sausages (Morcillas). Food Microbiol. 2006;23:283-8. [188] Chinen I, Epsztein S, Melamed CL, et al. Shiga toxin-producing Escherichia coli O157 in beef and chicken burgers, and chicken carcasses in Buenos Aires, Argentina. I J Food Microbiol. 2009;132:167-71. [189] Almada G, Estrella P, Ottavianoni L, et al. Relación clonal de Escherichia coli O157:H7 aislada en un caso de SUH, un portador asintomático y muestras de alimentos. La Pampa, Argentina, 2008. Ind Cárn Latinoam. 2009;158:8-11. [190] Wong CS, Jelacic S, Habeeb RL, et al. The risk of the hemolytic-uremic syndrome after antibiotic treatment of Escherichia coli O157:H7 infections. N Engl J Med. 2000;342:1930-6. [191] Saldar N, Adnan S, Gangnon RE, et al. Risk of hemolytic uremic syndrome after antibiotic treatment of Escherichia coli O157:H7 enteritis. JAMA. 2002;288:996-1001. [192] Oakes R, Siegler R, McReynolds M, et al. Predictors of fatality in postdiarrheal hemolytic uremic syndrome. Pediatrics. 2006;117:1656-62. [193] Trachtman H, Cnaan A, Christen E, et al. Effect of an oral Shiga toxin-binding agent on diarrhea-associated haemolytic uraemic syndrome in children. JAMA. 2003;290:1337-44. [194] Watanabe M, Matsuoka K, Kita E, et al. Oral therapeutic agents with highly clustered globotriose for treatment of Shiga toxigenic Escherichia coli infections. J Infect Dis. 2004;189:360-8. [195] Pinyon RA, Paton JC, Paton AW, et al. Refinement of a therapeutic Shiga toxin-binding probiotic for human trials. J Infect Dis. 2004;189:1547-55. [196] Silberstein C, Copeland DP, Chiang W-L, et al. A glucosylceramide synthase inhibitor prevents the cytotoxic effects of Shiga toxin-2 on human renal tubular epithelial cells. J Epith Biol Pharmacol. 2008;1:71-75. [197] Mukherjee J, Chios K, Fishwild D, et al. Human Stx2-specfic monoclonal antibodies prevent systemic complications of Escherichia coli O157:H7 infection. Infect Immun. 2002;70:612-9. [198] Dowling TC, Chavaillaz PA, Young DG, et al. Phase I safety and pharmacological study of chimeric murine-human monoclonal antibody alpha Stx2 administered intravenously to healthy adult volunteers. Ant Agents and Chem. 2005;49:1808–1812. [199] McNelly TN, Mitchell MC, Roser T, et al. Immunization of cattle with a combination of purified intimin-531, EspA and Tir significantly reduces shedding of Escherichia coli O157:H7 following oral challenge.Vaccine. 2010;3:1422-1428.

 

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[200] Fox JT, Thomson DU, Drouillard JS, et al. Efficacy of Escherichia coli O157:H7 siderophore receptor/porin proteinsbased vaccine in feedlot cattle naturally shedding E. coli O157. Foodborne Pathog Dis. 2009;6:893-899. [201] Liu J, Sun Y, Zhu L, et al. Towards an attenuated enterohemorhagic Escherichia coli O157:H7 vaccine characterized by a deleted ler gene and containing apathogenic Shiga toxins. Vaccine. 2009;27:5929-5935. [202] Bentancor LV, Bilen M, Brando RJ, et al. A DNA vaccine encoding the enterohemorrhagic Escherichia coli Shiga-like toxin 2 A2 and B subunits confers protective immunity to Shiga toxin challenge in the murine model. Clin Vaccine Immunol. 2009;16:712-718. [203] Konadu EY, Parke JC, Jr, Trant TH, et al. Investigational vaccine for Escherichia coli O157: phase 1 study of O157 Ospecific polysaccharide-Pseudomonas aerugionsa recombinant exoprotein A conjugates in adults. J Infect Dis.1998;177:383-387. [204] Ahmed A, Li J, Shiloach Y, et al. Safety and immunogenicity of Escherichia coli O157 O-specific polysaccharide conjugate vaccine in 2-5 year-old children. J Infect Dis. 2006;193:512-21.

 

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CHAPTER 6 Enterotoxigenic Escherichia coli Jose Flores and Pablo C. Okhuysen* Division of Infectious Diseases, The University of Texas Health Science Center, 6431 Fannin Street, MSB 2.112, Houston, Texas, USA, 77030.. Abstract: Enterotoxigenic Escherichia coli (ETEC) have been identified as a major bacterial pathogen responsible for infantile diarrhea in developing nations. ETEC are also the most common bacterial pathogen responsible of acute infectious diarrhea in adults traveling from industrialized nations to less developed countries. After ingestion, ETEC first attaches to epithelial cells lining the small intestine mucosa through an interaction mediated by adhesins known as colonization factor antigens (CFA), ETEC then produce either one or both of the well identified heat-labile (LT) and heat-stable (ST) enterotoxins. The LT of ETEC is structurally and immunologically related to the pentameric V. cholerae enterotoxin while ST is a small, non immunogenic peptide. Polymorphisms in host blood group antigens and the IL-10 gene promoter have been associated with diarrheal disease due to ETEC. The diagnosis of ETEC depends on the identification of the LT and ST toxin by molecular probes or immune based assays. Clinically, diarrhea due to ETEC cannot be differentiated from diarrhea due to other enteropathogens. The correction and maintenance of hydration is essential to prevent dehydration due to ETEC. . Several antimicrobials have demonstrated efficacy to treat ETEC associated travelers’ diarrhea, including Rifaximin, various fluoroquinolones and Azithromycin. Strategies for ETEC vaccines have focused on targeting CFAs of ETEC (CFA/I, CFA/II, and CFA/IV), the immunogenic LT or the use of whole cell killed organisms expressing CFAs.

INTRODUCTION Escherichia coli, initially named Bacterium coli, was initially incriminated as the cause of infantile diarrhea outbreaks during the 1940's and 1950's and was first referred to as enteropathogenic E. coli [1-5]. Further studies done on ligated rabbit ileal loops demonstrated that these E. coli strains elicited the accumulation of fluid in a manner that was similar to what was observed with Vibrio cholerae animal models [6, 7]. Enterotoxigenic E. coli (ETEC) are now defined by their ability to produce at least one of two well characterized enterotoxins known as LT (heat labile) and ST (heat stable) toxins. ETEC were first implicated in adult diarrhea in 1971 [8]. Subsequent human challenge studies determined that a dose of 1x108 to 1x1010 colony forming units was necessary to cause diarrhea [9]. ETEC ASSOCIATED PEDIATRIC DIARRHEA In developing countries, ETEC are the most common bacterial pathogen identified in children with acute diarrhea less than 5 years of age. It is estimated that annually, ETEC are responsible for 200 million cases of diarrhea worldwide resulting in approximately 150,000 deaths [10]. ETEC can cause repeated infections in children, particularly those under the age of 24 months. A study in the Nile Delta in Egypt showed that children experience 48 bouts of diarrhea annually, with more than half of the recurring attacks of diarrhea being due to ETEC [11]. Older children and adults are less susceptible but can also experience dehydrating diarrhea due to ETEC. Worldwide, it is estimated that 66% of all ETEC identified produce LT either alone or in combination with ST (LT/ST) and therefore, LT producing strains are responsible for most of the ETEC related disease burden. PEDIATRIC ETEC INFECTIONS IN MEXICO AND LATIN AMERICA In the 1970’s, ETEC LT was identified in 23% of Mexican children presenting with acute diarrhea [12]. A community based prospective study done a decade later, in 1986-87, in children living in Guadalajara, Mexico, investigated the relationship between infant feeding patterns and ETEC LT diarrheal disease. In this study, a cohort *Address correspondence to: Dr Pablo C. Okhuysen, Division of Infectious Diseases, The University of Texas Health Science Center, 6431 Fannin Street, MSB 2.112, Houston, Texas, USA, 77030; Phone: +1-713-500-6736, email: [email protected] Alfredo G. Torres (Ed) All rights reserved - © 2010 Bentham Science Publishers Ltd.

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of 98 Mexican women-infant pairs was followed for up to 50 weeks. Strictly formula-fed children had an incidence of diarrhea over three times that of strictly breast-fed infants and twice that of breast-fed and supplementally fed children. This hazard was inversely impacted by the overall amount of LT-ETEC-specific, immunoglobulin A antibodies the infant received via the mother's breast milk [13]. A more recent study published in 2006 done in central Mexico to assess the impact of vitamin A on infant diarrhea, showed that during a 15 month period of observation 41 of 92 (42%) participants in the placebo arm experienced ETEC infection with 23% of these infections being associated with diarrhea [14]. This suggests that in peri-urban areas, ETEC associated diarrhea remains a significant problem in central Mexico. In contrast, a prospective study conducted in the peri-urban areas of Lima, Peru showed that although diarrheagenic E. coli were the most common enteropathogen identified, only 3% of diarrheal episodes were due to ETEC with a majority of the cases being due to enteroaggregative, enteropathogenic and diffusely adherent E. coli [15]. ETEC AS A CAUSE OF TRAVELERS’ DIARRHEA ETEC also affects adults from developed nations traveling to developing countries and this E. coli pathotype remains the leading bacterial cause of travelers’ diarrhea in Mexico [16]. ETEC is more commonly identified among travelers to Latin America and the Caribbean compared to travelers visiting Africa or Asia; ETEC is identified in about 30.4% of travelers’ diarrhea cases overall, with rates in Latin America/Caribbean of 33.6%, Africa 31.2%, south Asia 30.6%, and Southeast Asia of only 7.2% [17]. ETEC is also the leading cause of bacterial diarrhea among military personnel conducting operations in developing nations [18]. After short stays in Mexico, US visitors develop ETEC diarrhea and seroconversion to LT [19]. With extended stays, the colonization and ETEC associated diarrhea rates decrease in travelers and the frequency in visitors resemble the rates of ETEC associated colonization and infection seen in inhabitants of endemic areas. However, immunity in visitors to developing countries is short lived in the absence of repeated exposure. ETEC VIRULENCE FACTORS ETEC Colonization Factor Antigens To cause diarrheal disease, ETEC must first attach to epithelial cells lining the small intestine mucosa through an interaction mediated by proteinaceous surface adhesins, also known as Colonization Factor Antigens (CFA), which can be non-fimbrial, fimbrial, helical or fibrillar. More than 25 distinct colonization factors (CF) have been identified; many of them are fimbriae or pili (Table 1). The types of CF are subdivided by type, antigenicity, molecular weight, N-terminal amino acid sequence of the major subunit, and also according to structural morphology. Human ETEC CFs are designated as coli surface antigens (CS) followed by a number that corresponds to the chronological order of identification, with the exception of CFA/I (Table1). ETEC can also infect animals and are distinguished by CFs that are distinct from those seen in human derived isolates. CFs in animal derived ETEC are referred to as K, F, or P. The most common animal ETEC LT CFs are F4, F5 and F6, also referred to as K88, K99 and 987P fimbriae. Table 1: Nomenclature of some human ETEC colonization factors. CF designation CFA/I CFA/II CFA/II CFA/II CFA/IV CFA/IV CFA/IV CS7 CFA/III 2230 PCF0148

CS number PCF0159 PCF09 PCF0166 8786 CS17 PCF020 CS19 CS20 Longus CS22

CFA/I CS1 CS2 CS3 CS4 CS5 CS6 CS7 CS8 CS10 CS11

CS12 CS13 CS14 CS15 CS17 CS18 CS19 CS20 CS21 CS22

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The predominant CFAs expressed by ETEC strains affecting humans include CFA/I, and the CFA families CFA/II and CFA/IV family [20]. However, the distribution of particular CFs among ETEC strains varies geographically and changes over time. As an example, while CFA/I is the most commonly identified CF phenotype in Chile [21], CFA/II and CFA/IV are the most commonly identified CF phenotypes in Egypt [22]. Importantly, it is estimated that 50% of all ETEC strains have no identifiable CF. CFA/I represent the prototype of class 5 fimbriae, the largest class of human-specific ETEC CFs. The CFA/I fimbriae is a polymer typically consisting of more than 1,000 copies of the major pilin subunit CfaB, and 1 or a few copies of the tip-residing adhesive minor subunit CfaE. The CfaB structure has a 7-stranded β-sandwich fold and displays an Ig-like fold adopted by all known class 1 pilins, Caf1 subunits, and the Dr adhesins [23]. CfaB binds to host cell membrane glycosphingolipids, including the Lewis blood group antigens. This is of interest since it was recently shown that individuals with the Le(a+b–) blood type are significantly at higher risk of acquiring diarrhea caused by CFA/I group ETEC than Le(a–b+) individuals [24]. The CFA/II strains express CS antigen 3, either alone or in conjunction with CS1 or CS2. CFA/IV strains express CS6, either alone or in conjunction with CS4 or CS5. The CFA/IV CS6 has a non-fimbrial short oligomeric assembly, has two non-covalent associated subunits (CssA and CssB) and is recognized for its ability to bind different extracellular matrix proteins, including fibronectin [25]. The majority of the CFs except for longus (also known as CS21) and CFA/III are synthesized and transported via a chaperone-usher-dependent pathway. In general, CFs are encoded in a four-gene operon consisting of a periplasmic chaperone, a major fimbrial subunit, an outer membrane usher protein and a minor subunit. The minor subunit is located at the tip of the fimbriae, with the N-terminal half of the protein responsible for binding to the host cell receptor [26]. Longus and CFA/III are both synthesized as Type IV pili in a process analogous to the Type II secretion pathway [27]. Both CFs and flagella anchor ETEC for initial long-range attachment to host cells, however for some strains including the prototypic strain H10407 a closer and more intimate attachment is facilitated by the outer-membrane proteins Tia and TiaA [20]. ETEC Heat Labile Toxin (LT) The heat labile (LT) enterotoxin of ETEC not only induces secretory diarrhea and neutralizing antibodies but is also an important immune adjuvant. LT is closely related to cholera toxin and shares 82% amino acid homology. The toxin is a pentameric holotoxin composed of 5 B subunits that mediate attachment to the intestinal epithelial cell and an A unit that is responsible for activating cellular adenyl cyclase by virtue of its ADP ribosylating activity. The 86 kDa LT binds preferentially to the gangliosides GM1 and GD1b present on the surface of the intestinal epithelium. In addition to the GM1 Ganglioside, the LTB subunits can also bind to polylactosaminoglycan-containing receptors [28], brush borders galactoproteins [29] and two other glycoproteins present in the human intestine [30]. The LT toxin can be further classified into 2 major families known as LT I and LTII (Fig. 1). The LT I and LT II toxins differ in host affinity and also in genetic sequences. ETEC LT containing LTI are more common in humans and can be sub classified into LTh (derived from humans) and LTp (derived from pigs). It has recently been recognized that LT I strains are genetically diverse with over 16 distinct types reported to date [31]. A retrospective study done in a collection of 54 ETEC strains in Brazil demonstrated that toxin types from asymptomatic individuals were more diverse genetically than those associated with diarrheal disease. The prototypic ETEC strain H10407 (Bangladesh) produces LT1. Recently an LT2 producing strain has been identified. At the molecular level, LT2 has 5 polymorphic sites. Although LT1 and LT2 are similar functionally in vitro, they differ in their adjuvant activity resulting in the generation of higher neutralizing titers in animal models [32]. LT is encoded on a 60 kb virulence plasmid termed Ent, which contains three distinct major regions: (i) a pathogenicity island containing enterotoxin genes, (ii) a region involved in plasmid replication and maintenance, and (iii) a region including tra genes that cause the self-transmissibility [33]. The eltA and eltB genes encoding the A and B subunits form a single transcriptional unit. A histone-like nucleoid structuring (H-NS) repressor protein appears to

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be the main gene expression modulator. The plasmid encoded eltAB operon is silenced by H-NS, which blocks the binding of RNA polymerase to the gene promoter [34]. LT secretion by ETEC is dependent on the chromosomally located gene leoA. It has recently been shown that up to 95% of the LT secretion is vesicle-bound [35].

Figure 1: Structure of the heat-labile toxin. (Protein Data Bank entry 1LTS)

Subsequently to the LTB binding to the target cell, LT is endocytosed by the cell either through caveolin-coated vesicles, clathrin-coated vesicles, or the Arf6 endocytic pathway [36, 37]. After endocytosis, LT travels to the endoplasmic reticulum (ER) via a retrograde transport pathway where as is the case with V. cholera CT; LTA undergoes post-translational processing to generate A1 and A2 peptides. The LTA1 is then translocated to the cytosol by the ER-associated degradation pathway, or degradasome [38]. LTA1 catalyzes the ADP ribosylation of the GTPbinding regulatory component of adenylate cyclase in the presence of NAD, resulting in elevation of intracellular cyclic AMP levels [39]. Gene sequences encoding LT have been rarely identified in other Enterobacteriaceae such as Citrobacter and Enterobacter. However, the clinical relevance of these non-E. coli isolates containing LT is unknown. As mentioned above, in addition to being an enterotoxin, LT is a potent mucosal immune adjuvant. After internalization of the toxin A unit, the B subunit has been shown to bind to Toll-like receptors (TLR) 1 and 2 [40] in dendritic cells and this may in part explain the immune-modulatory and adjuvant properties of LT. Heat Stable Toxin (ST) The second major toxin, ST, is only 18 amino acids long and binds to guanylate cyclase (GC-C), a member of receptor cyclases that includes the atrial natruretic peptide. ST increases intracellular levels of cGMP. As the case with LT, there are several ST toxin types (Figs. 2 and 3). ST are classified into 2 major types by virtue of distinct phenotypes: The STa (ST I; estA encoded) is identified mostly in human stools while Stb (ST II) is identified mostly in animal stools. In addition, there are two distinct forms of STa, namely STh (or ST Ib) and Stp (or ST Ia). There are several allelic forms of the estA gene [41]. Various other enteric bacteria have been shown to elaborate identical or highly homologous ST's. Some of these bacteria include Yersinia enterocolitica, Citrobacter freundii, and Klebsiella pneumoniae [42]. However, the clinical significance of ST in other Enterobacteriaceae remains unknown.

Figure 2: Structure of heat-stable enterotoxin B. (Protein Data Bank entry 1ETL)

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Figure 3: Structure of heat-stable enterotoxin B. (Protein Data Bank entry 1 EHS)

STs are synthesized as precursor proteins and are then converted to the active forms with intramolecular disulfide bonds after being released into the bacterial periplasm. The active STs are finally translocated across the outer membrane through a tunnel made by TolC [43]. Once released into the small intestine lumen, ST binds reversibly to the extracellular domain of GC-C on the villous and crypt epithelial cells. This interaction activates the intracellular catalytic domain of GC-C leading to intracellular accumulation of cGMP [44-46]. The activation of the cGMPdependent protein kinase II leads to phosphorylation of the cystic fibrosis transmembrane regulator (CFTR), driving Cl- secretion and inhibition of NaCl absorption [47]. It has been proposed that humans with cystic fibrosis do not respond to ST as well as carriers of wild type CFTR [48]. Activation of GC-C is regulated by the intestinal and kidney-enriched PDZ protein (IKEPP), which inhibits ST activation of GC-C [49]. Although GC-C is the primary receptor mediating STa-stimulated intestinal secretion, there are alternate receptors not associated with GC-C activity [50, 51], some of them can stimulate duodenal HCO3- secretion independent from the CFTR pathway [52]. The Serine Protease Autotransporter EatA In addition to the enterotoxins LT and ST, several other putative toxins have been described, including EatA, which is a serine protease autotransporter of ETEC. EatA is homologous to the wider group of Enterobacteriaceae serine protease autotransporters, which act as extracellular proteases. EatA possesses specificity for p-nitroanilideconjugated oligopeptides; these cleaved oligopeptides are substrates for cathepsin G, which in turn modulates or cleaves a diverse array of other extracellular products, including proteoglycans and cell surface protease-activated receptors, damaging the epithelial cell surface [53]. HOST GENETIC FACTORS PREDISPOSING ETEC INFECTION Several factors in the host impact the occurrence, duration, severity and resolution of ETEC associated diarrhea in a particular individual. An association between the anti-inflammatory IL-10, also known as human cytokine synthesis inhibitory factor, and susceptibility to ETEC LT associated diarrhea has been identified. Children living in ETEC endemic Latin America countries, such as Mexico, with intermediate and high levels of IL-10 cytokines in their stool, clear symptomatic and asymptomatic ETEC infections less efficiently compared to other children with low or no detectable levels of IL-10 in their stools [54]. Adult travelers harboring high production associated IL-10 single nucleotide polymorphisms (SNP) have a increased risk for developing diarrhea when naturally exposed to ETEC compared to individuals with normal or low IL-10 associated SNPs [55]. There is evidence that several enteropathogens use human blood group antigens as their receptors. Children with the Le (a+b-) blood group have increased susceptibility to diarrhea caused by ETEC (24). The H antigen (O group) lacks the terminal sugar residue, the toxin is unable to use it as a receptor [56]. It is also plausible that individuals with blood group O are less likely to become infected by ETEC, as individuals possessing the blood group O are 50% less likely to become infected with V. cholerae than non-blood group O individuals [57].

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DIAGNOSIS ETEC were initially identified by their ability to cause fluid accumulation in ligated animal ileal loops. With the characterization of the LT and ST genes, molecular probes specific for these toxins were developed. Most laboratories use colony hybridization or polymerase chain reaction assays with probes based on the LT and ST sequences applied to E. coli isolated from stool cultures. GM1 binding ELISA can measure the production of LT. Until recently, ETEC testing could only be done in a few research laboratories; commercial laboratories have now begun offering ETEC specific diagnostic testing. Probe hybridization on stool culture plates or the use of PCR done directly with DNA purified from feces increases the sensitivity for the detection of ETEC [58]. However, the specificity of these methods of testing remains unclear since PCR or whole plate probe hybridization can detect a low number of colonies. Inexpensive, field ready diagnostic tests are needed for an accurate assessment of ETEC epidemiology in the Americas. TREATMENT Pediatric ETEC Infection ETEC associated diarrhea cannot be differentiated from diarrhea due to other pathogens based on clinical features. The treatment of diarrheal disease due to ETEC is the same as that for other acute secretory diarrheal diseases, including cholera. The correction and maintenance of hydration is always most important intervention. When unable to tolerate oral rehydration, rapid rehydration using intravenous fluids may be required initially for patients with severe dehydration. After restoration of blood pressure and major signs of dehydration, patients can be placed on oral rehydration solutions for the remainder of therapy. For all other patients with lesser degrees of dehydration, therapy with oral rehydration solutions alone can be used until the diarrhea ceases. Antimicrobial therapy for ETEC in children is not routinely recommended. Treatment of Adults with Travelers’ Diarrhea There are several antimicrobial agents available for the treatment of travelers’ diarrhea. The antimicrobial choice depends on the epidemiology of the visited region, the microbial resistance patterns, the antimicrobial safety profile and the possible expected interactions. Several antimicrobials have demonstrated efficacy in the treatment of ETEC associated diarrhea, including Rifaximin, fluoroquinolones and Azithromycin. The current recommendations for the treatment of travelers' diarrhea occurring in an ETEC endemic region include: a) azithromycin, 1gr [59] or 500mg as a single dose [60], either with or without loperamide, b) rifaximin for three days either 400mg twice a day [61] or 200mg three times a day [62], also either with or without loperamide [63], or c) a fluoroquinolone antibiotic, such as 500mg levofloxacin as a single dose [64], 750mg ciprofloxacin as a single dose [65] or 500mg twice daily for 3 days [66]. Rifaximin is a rifamycin derivative antimicrobial recently approved by the FDA for the use in travelers diarrhea. The mechanism of action involves inhibition of bacterial RNA synthesis by binding to the beta-subunit of the bacterial DNA-dependent RNA polymerase. Less than 0.4% of rifaximin is absorbed after oral administration [67] which limits the potential for drug interactions and side effects. Although in vitro studies have demonstrated that rifaximin is capable of inducing the cytochrome P450 3A4 isoenzyme, clinical drug-drug interaction studies using midazolam and ethinyl estradiol have not demostrated such an interaction [68]. Antimicrobial Prophylaxis In general, travelers should be reminded of basic principles of hygiene, advised to avoid drinking tap water, to drink bottled water instead and to adhere to the saying “cook it, peel it, boil it or forget it”. It should be keep in mind that even cooked food, served in restaurants may be contaminated due to unhygienic conditions. Although not routinely recommended, antibiotic prophylaxis is highly effective in preventing ETEC associated diarrhea [69] and should be limited to travelers at greatest risk of disease or its complications. A drawback for the routine use of chemoprophylaxis is the emergence of antibiotic resistance [70]. Studies of cost benefit are needed to

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compare chemoprophylaxis with early initiation of therapy of TD. The cost of generic ciprofloxacin for 1- to 3-day treatment is certainly less than the cost of 14 days of rifaximin used for chemoprophylaxis [69]. ETEC VACCINE DEVELOPMENT Natural history studies of ETEC infections in children in developing countries suggest that recurrent infections induce protective immunity, as reflected by declining rates of ETEC diarrhea with age, lower ratios of symptomatic to asymptomatic ETEC infections with increasing age, and the protective relationships between initial ETEC infections and subsequent infections that have similar toxin and/or colonization factor phenotypes [71]. These data suggest that immunization against ETEC may be an effective preventive strategy not only against ETEC but possibly diarrhea due to other enteropathogens. Strategies for ETEC vaccines have focused on targeting the three major CFAs of ETEC (CFA/I, CFA/II, and CFA/IV), the immunogenic LT or the use of whole cell killed organisms expressing CFAs. However, the heterogeneity in CF antigens poses a problem due to the regional differences and changes over time. LT Based ETEC Vaccines Since ETEC LT is immunogenic, confers protective immunity and is a potent mucosal adjuvant, several LT vaccines have been developed with and without CF components. Vaccines in development include whole cell killed vaccines, live attenuated Shigella/LT vaccines, live attenuated ETEC and plant and bacterial based systems that express either LT or other ETEC antigens. Interestingly, there is evidence that two LT based vaccines not only confer protection against ETEC LT but also other enteric pathogens. The oral killed whole cell/recombinant B subunit (WC/rBS) cholera vaccine (Dukoral TM) is currently the only vaccine available in the market (but not in the US) that has shown to prevent ETEC associated diarrhea in travelers. WC/rBS cholera vaccine has been shown to prevent up to 40% of all travelers' diarrhea episodes [72]. Furthermore, vaccinated travelers develop less severe forms of diarrhea compared to their unvaccinated counterparts [73]. The oral killed vaccine approach is being pursued by several investigators. E. coli K12 over-expressing CFA/I have recently been engineered and could be useful as an oral killed CF-ETEC vaccine [72]. A vaccine trial in Morocco done in Finnish travelers with done with the WC/rBS prevented 23% of all diarrhea episodes, 52% of ETEC related diarrhea and 82% of ETEC and Salmonella diarrhea [74]. A more recent study done in US travelers to developing countries with a transdermal LT patch based vaccine showed a 75% protective efficacy against moderate to severe diarrhea, less duration of diarrhea (0.5 vs 2.1 days; p=0.0006) and fewer loose stools (3.7 vs. 10.5) in diarrhea due to all causes [75]. The mechanisms responsible for this favorable effect against diarrhea due to other pathogens in unknown, it is plausible that antitoxin antibodies in the intestinal lumen block other enteropathogens in a non-specific manner or that by preventing ETEC LT and therefore the damage induced by subclinical infections of ETEC LT, the mucosa remains less susceptible to other organisms [76, 77]. It is also plausible that ETEC LT based vaccines induce tolerance in the GI tract to enteric pathogens. Live Attenuated Vaccines Several live attenuated oral vaccines have been tested, some of them have shown good immunogenicity in human volunteers, including the attenuated CFA/II expressing PTL003 ETEC strain [78] and the CFA/I expressing ACAM2010 ETEC strain [79]. Live attenuated Shigella [80]) and Salmonella [81] vectors for expression of ETEC fimbrial and LT antigens are also in development. Future Vaccines Vaccines consisting of transgenic plant-delivered antigens offer a new strategy for development of safe and inexpensive vaccines. Vaccine antigens can be eaten with the edible part of the plant or purified from plant material. Recently, transgenic corn expressing ETEC LT has shown to stimulate a specific antibody response in adults when fed with about 2g of plant material [82].

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Unlike fimbriae or LT, STa has a very poor immunogenicity. However, STa can become immunogenic after being coupled with a strongly immunogenic carrier protein, such as LT, An animal model of infection that used ST/LT immunized piglets demonstrated that this approach protected piglets when challenged with a STa-positive ETEC strain [83]. The geographical and temporal heterogeneity in colonization factors found on the surface of ETEC and the subtypes of LT and ST produced pose significant challenges for the development of a single component vaccine for ETEC. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21]

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