Pathogenic Escherichia coli in Latin America

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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


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


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



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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8


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].



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



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].



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



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



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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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.


Gomes and González-Pedrajo

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)


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.



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].



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



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



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].



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



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].



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 0.05) was seen in the animals carrying E. coli isolates harboring stx2 (STEC) sequences. Statistical analysis of 12 parameters revealed several risk-factors for the presence of STEC: increases in carriers were observed in puppies up to two years old and increases were found in association with springtime. Males showed more positive results than females. With respect to habitat, a high proportion of household dogs living outdoors were infected, and dogs that cohabited with other pets increased the STEC carrier risk. In cats, the prevalence of the STEC strains carrying the stx2 sequence was (4/149); all of them were eae− belonging to serotypes O22:H8 (H-), ONT:H8 and ONT:H19. The risk-factor analysis of transmission of this pathogen in this species was also investigated. In the same survey, out of 149 sampled, 113 were healthy cats, and 4 of them (2.7%) were stx+. Carriers were detected only in the Spring. Kittens of up to one year of age exhibited significant differences with high STEC carrier results, and the female was the most prevalent sex. Similar to household dogs, the household cats living outdoors had a high risk of infection, and cohabitation with other pets increased the number of STEC carriers [102]. According to the Fisher test, no significant differences were seen in the prevalence of STEC in dogs and cats. Using a previously published PCR multiplex assay [103] for screening for stx genes, and using bacterial growth from the confluence zone as a template, as few as 20 CFU were detected in rectal swabs of dogs. The efficiency of the isolation of strains from samples with a presumptive positive diagnosis was about one-third. Although the number of CFUs evaluated was up to 300 CFUs in positive samples, a diagnosis of certainty by isolation was not reached in two-thirds of the samples [98]. Thus, the efficiency of the isolation was smaller in this study than in the other groups observed in cattle [31, 34, 104]. When performing the screening on asymptomatic animals or healthy animals, the relative load can be reduced. In this case, the possibility of recovering positive colonies decreases. On the other hand, the microbiota of monogastrics is different than that in ruminants; probably monogastric microbiota is more competitive in relation to STEC. Both dogs and cats are domestic animals that have lived together with humans in a close relationship for thousands of years and, consequently, there is a high probability of transmission of microorganisms among these hosts [96]. The feeding of these pets in Argentina includes (always or sporadically) raw meat, and there are no nutritional


Pestana de Castro et al.

restrictions that could determine a feeding change. The presence or absence of intimin in strains from the same swab sample means that the ecological niche is shared by different types of E. coli. The findings tended to demonstrate that STEC strains circulated among pets with a low load. Interestingly, the prevalence in pets did not present higher values than those registered for European regions, where the incidence of HUS was much lower. There were household pets related to sporadic cases of HUS in Buenos Aires. Preliminary research of rectal swab samples from 36 dogs and 20 cats associated with 15 sporadic cases of HUS reported in Buenos Aires during the period 2005-2008, showed a deviation in relation with the prevalence estimated in the same geographical area. Screening for stx1/stx2 and rfb0157 was done by multiplex PCR. STEC isolates were further characterized by biochemical tests and serotyped by standard methods. Screening for STEC was positive in three dogs (8.3%) and two cats (10%). The two STEC strains isolated from dogs belonged to the serotype O178:H19, one of them exhibited the genetic profile stx2c (vh-a) and the other stx2c (vh-b), and both were eae− and ehxA−. One STEC strain recovered from a cat belonged to the serotype O145:H- with the profile stx2 (vh2) ehxA+ and eae+. The isolation of a STEC strain of the serotype O145:H- from a household cat, exhibiting the same serological and virulence profile of regional strains associated with HUS, showed the important role pets could play in the epidemiology of HUS [105, 106]. The existence of these strains of common circulation to humans and animals suggests the need for actions in the field of sanitation guidelines, to encourage pet owners to adopt hygienic measures that could contribute to the control of the disease. In Brazil, a total of 92 E. coli strains were isolated from the 25 diarrheic dogs, and all of them were investigated for the presence of Stx genes (stx 1 and stx2) by PCR. Twelve (13.0%) of the strains carried the stx gene; seven (7.6%) carried only the stx1 gene, five (5.4%) the stx2 gene, and none carried both genes. All isolated STEC strains were tested by using the O157 latex agglutination test kit; non-O157 was detected in the isolated strains [107]. The occurrence of stx genes (40.0%) among E. coli samples from diarrheic animals in this study are in accordance with the results on the same subject reported by Hammermuler et al. [108] (44.4%, with the presence of stx1 or stx2 genes alone, among STEC strains). However, it is noteworthy that Nakazato et al. [109], in Brazil, did not find STEC strains carrying stx 1 or stx 2 genes among 146 diarrheic and 36 healthy dogs examined. Paula and Marin [107] also could find among 12 STEC isolates, seven (58%) presenting a multi-drug resistant (MDR) phenotype to four or more antimicrobial drugs. The carrying of MDR E. coli by dogs represents a potential hazard for Brazilian people having contact with such animals, running the risk of spreading resistance genes. INTRODUCTION TO ENTEROTOXIGENIC ESCHERICHIA COLI Enterotoxigenic Escherichia coli (ETEC) are a group of pathogenic E. coli strains that produce watery diarrhea in humans and in some domestic animals. ETEC is a major agent of human diarrhea in children and in travelers to developing countries. In some regions of the world, i.e. Egypt and Bangladesh, ETEC strains are the first cause of diarrhea in young children. ETEC strains are also routinely isolated from cases of diarrheal disease occurring in neonatal and post-weaning pigs and neonatal calves. ETEC in Humans, Cattle, Pigs and Sea Foods Diarrheal diseases account for significant losses to producers worldwide. ETEC, first described by Sack in 1971 [110], is characterized by possessing genes encoding for one or both defined groups of enterotoxins: heat stable (ST) and heat labile (LT). There are two major types of non-related STs, STa (18- or 19-amino acid peptide) and STb (48-amino acid peptide) [111]. In addition, two variants of STa, designated STp (ST porcine or STIa) and STh (ST human or STIb), were initially identified in strains isolated from pigs or humans, respectively. Both variants can be found in human ETEC strains. STb is associated primarily with ETEC strains isolated from pigs. ETEC strains may also express any of the two major serogroups of LT toxins, LT-I or LT-II. LTs are oligomeric toxins highly related to cholera toxin [111], but are non-immunologically related. Only variants of LT-I have been associated with human or animal disease. ETEC strains comprise various types of O serogroups, and the preferred host is dictated by the presence of specific colonization factors. Both, the enterotoxins and the colonization factors, are plasmid-encoded. In spite of the existence of several O serogroups in the ETEC group, an association of colonization factors and serogroups has been

Escherichia coli Animal Reservoirs


observed. F4 (K88), F5 (K99), F6 (987P) and F18 adhesins bind specifically to intestinal cells of different animal species. This host tissue specific adhesion makes it difficult for the animal ETEC strains to cause infection in humans. In consequence, ETEC is not considered a zoonotic pathogen. Initial studies in Argentina failed to identify in diarrheic calves E. coli strains with F5, ST and LT virulence factors [112]. A later study in 1990 [113] did not report finding ETEC in any of the fecal samples from 452 diarrheic calves. In another survey published in 1992, ETEC was [114] found in 1.5% of calf fecal samples. The presence of ETEC in pigs was evaluated from 1992 to 1997 in another study [84], and the STIa gene was detected in E. coli strains isolated from 21% piglets with diarrhea and LTI in 3.1% of the pigs. No toxin gene was amplified from E. coli strains isolated from either healthy piglets or their dams. Serogroup O64 appears to be a prevalent ETEC for pigs in Argentina [84], while it is an uncommon serogroup associated with non-toxigenic E. coli in Spain [115]. Mercado et al. [116] also failed to detect ETEC strains in specimens from 100 diarrheic or septicemic calves and 27 older cattle from outbreaks or individual cases submitted for laboratory diagnosis in Buenos Aires province, Argentina. In Brazil, ETEC investigations started as early as 1987, when different tests were compared to detect LT [117]. An adhesion factor from porcine ETEC, F42 was identified and biochemically characterized by Leite et al. [118]. In another study, 300 pigs with diarrhea, housed in farms located in the State of Sao Paulo, were investigated for the colonization of pathogenic E. coli strains, and 24 E. coli strains were found to produce enterotoxin STb, 5, LT, and 3, STa [119]. That work also showed the possible existence of a new E. coli colonization factor other than F4, F5 and F6, participating in colibacillosis provoked by E. coli in pigs. Silva et al. [120] found that 7.1% of all diarrheagenic strains isolated from urban pigeons belonged to the ETEC pathotype. A Bolivian-Japanese [121] consortium identified ETEC strains among other enteropathogenic bacteria in the water of La Paz River, Bolivia. Surprisingly, this is the only publication about contaminated fresh water in the region, in contrast to the Asiatic region, where many surveys focused on this problem [122, 123]. Peruvian researchers, analyzing Brazilian sea food samples, found a very low incidence of ETEC strains [124]. In another work, 32 E. coli strains were isolated from sea food. A total of 14 strains produced exotoxins, of which seven were LT and the other seven ST [125]. Ingestion of parsley was associated with an ETEC outbreak in Baja California, Mexico [126]. In Chile, a group [127] demonstrated in 1995 that the oral inoculation of calves with ETEC carrying a K99 pilus generated specific antibodies, but these antibodies did not neutralize the binding of the pilus to the bovine intestinal mucosa. An experimental vaccine combining ETEC and STEC bacteria was prepared in Corrientes, Argentina in 1999 [128]. A protection rate of 85% was obtained in vaccinated animals in mouse protection assays; unfortunately, this work did not have a continuation. INTRODUCTION TO EXTRAINTESTINAL PATHOGENIC ESCHERICHIA COLI Extraintestinal pathogenic E. coli (ExPEC) is a category of E. coli strains capable of causing disease outside of the intestine. In addition to human illness, ExPEC strains also cause extraintestinal infections in domestic animals and pets. It has been shown that ExPEC strains isolated from humans and animals share virulence traits and phylogenetic similarities. According to the definition of Johnson et al. [129], ExPEC strains contain at least two of the following virulence factors: P and S/F1 fimbriae, Afa-family adhesins, aerobactin siderophore and group 2 capsular polysaccharide antigens, besides numerous other putative virulence traits [130]. The ExPEC category also includes previously recognized groups of human and animal pathogenic E. coli strains, such as necrotoxigenic E. coli (NTEC), newborn meningitis-associated E. coli (NMEC), septicemic E. coli, avian pathogenic E. coli (APEC) and uropathogenic E. coli (UPEC). ExPEC in Human and Animal Species APEC is associated mainly with extraintestinal infections, principally of the respiratory tract, and systemic infections, both of which are principal causes of morbidity and mortality in chickens and turkeys, and are responsible for high economic losses in the world´s poultry industry. Swollen head syndrome (SHS) is a disease of



domestic poultry caused by a combination of a pneumovirus and adventitious bacteria, usually E. coli. Clinically, the disease occurs in broiler breeders causing swelling of the periorbital and infraorbital sinuses and submandibular edema. Young birds also showed signs of severe respiratory disease. Several studies were conducted in Brazil to determine the presence of E. coli virulence factors recovered from SHS in chickens. Parreira et al. [131], studying 50 SHS E. coli strains found that none of them agglutinated with antisera to adhesins K88, K99, F42, 987P and 2134P, and only 14% of strains agglutinated with antiserum to F41. Colicin V was produced by 78% of the E. coli strains and 80% produced aerobactin. In the serum resistance test, 36 (72%) of the strains showed resistance to normal chicken serum. Only seven (14%) strains expressed K1 capsular antigen, while motility was found in 62% of the strains. Efforts to define the SHS E. coli pathotype have shown that most SHS E. coli isolates have cytotoxic and lethal activities. Parreira and Yano [132] described for the first time a novel verocytotoxin named VT2y (Stx2y), which belongs to the STx family, and is produced by E. coli isolated from domestic poultry with SHS. The cytotoxic effect was neutralized by antiserum against Stx2, but not by antiserum against Stx1. The Stx2y toxin induced apoptosis in Vero, HeLa, CHO, CEF (primary chicken embryo fibroblast) and PCK (primary chicken kidney) cell lines, but was not lethal to mice [133]. Salvadori et al. [134] also investigated whether E. coli isolated from chickens with SHS produces a factor that is lethal to mice. They detected a lethal toxin similar to Bacillus cereus lethal toxin in the culture supernatants of E. coli strains, which killed mice within 10 min, and were not cytotoxic to Vero cells. In turn, Parreira and Gyles [135] demonstrated that SHS isolates were positive for the stx1 gene but had low titers for cytotoxicity in the Vero cell assay. The stx1 gene from one SHS E. coli isolate was cloned and sequenced and shown to be identical to that of the stx gene of S. dysenteriae. Cytotoxicity was also observed with supernatants, from the 30 avian pathogenic E. coli strains isolated from cellulitis lesions in chickens, avian septicemia and SHS, on two primary chicken cell lines (CEF and PCK) [136]. The cytotoxic effect, which was observed as early as 2 h after exposure of the cells, was maximal at 6 h and was evident as vacuolation, morphologically indistinguishable from that previously reported for culture supernatants of Helicobacter pylori. Supernatants of two vacuolating cytotoxinpositive cultures of H. pylori failed to induce vacuolation of the CEF and PCK cells, but caused the characteristic vacuolation in HeLa and Vero cells. The observations suggest that avian pathogenic E. coli produce a cytotoxin that is similar to the cytotoxin of H. pylori but may be specific for avian cells. An APEC strain designated SHS4, isolated from a chicken with clinical signs of SHS, adhered to but did not invade HEp-2 and tracheal epithelial cells. The strain harbored a 60MDa plasmid encoding adhesion genes, which may be responsible for the initial colonization of the upper respiratory tract of chickens [137]. In other work, Dias da Silveira et al. [138] found that colicin was characteristically produced by SHS E. coli strains, but failed to demonstrate a correlation between serotype, adhesion and invasion of in vitro cultured cells and hemagglutination patterns. Early work with avian septicemic E. coli isolates characterized a reduced number of strains with respect to their pathogenic phenotype [139]. They concluded that the adherence to and the invasiveness of HeLa cells were not related to the pathogenicity of these strains for chickens. Toxin production was correlated with the highest levels of pathogenicity. Some of the strains had mannose-resistant fimbriae. On the contrary, Vidotto et al. [140] examined a total of 45 E. coli isolates from chickens with colisepticemia and concluded that the characteristics exhibited by virulent strains were invasion for HeLa and chicken fibroblast cells, serum resistance, colicin V, and aerobactin production. None of the isolates were toxigenic or positive in hemagglutination tests. In order to detect phenotypic characteristics associated with pathogenicity, 25 E. coli strains isolated from clinical cases of colisepticemia in broiler chickens were studied by Ramirez Santoyo et al. [141]. Colicinogenicity occurred in 72% of the strains, 56% of all strains produced colicin V, 84% were positive for type 1 fimbriae, and 80% were positive for motility. None of the strains had hemolytic activity; however, all of them expressed at least one of the other characteristics studied. Sixty-three E. coli strains isolated from broilers with respiratory problems were examined for virulence factors by da Rocha et al. [142]. Interestingly, colicin production was observed in 55 (87.3%) of the strains, and 41.8% presented colicin V production, and 88.9% presented serum resistance, where as the operon pap was detected in 84.5% of the samples. The diversity of phenotypes detected in these studies partially explains the multifactorial nature of avian colisepticemia. A study of virulence-associated genes in 200 E. coli isolates from poultry with colibacillosis [143] examined the presence of 16 putative virulence genes by PCR. The seven virulence genes iutA, iss, cvaC, tsh, papC, papG and



felA were detected significantly more often among colibacillosis isolates than in fecal isolates from healthy birds, thereby confirming their worldwide occurrence and possible pathogenic role in colibacillosis. However, several of those genes were not detected in many colibacillosis isolates, and none were detected in 27.5% of those isolates, which points up the need for a search for variants of those genes, as well as yet undetected virulence factors. By colony hybridization, Vidotto et al. [144] demonstrated that sfaDE and facA are present in 40% and 30% of those isolates, respectively. Delicato et al. [145] also demonstrated the high frequency of the gene encoding the hemagglutinin Tsh among 305 avian pathogenic E. coli isolates in Brazil. Many of these results were in accordance with a recent study of Johnson et al. [146]. A large collection of avian E. coli isolates of known pathogenicity and serogroup were subjected to virulence genotyping and phylogenetic typing. Five genes carried by plasmids were identified as being the most significantly associated with highly pathogenic APEC strains: iutA, hlyF, iss, iroN, and ompT, underscoring the close association between avian E. coli virulence and the possession of ColV plasmids. APEC must resist the attack of incoming macrophages in order to cause disease. Rodrigues et al. [147] showed that resident murine peritoneal macrophages infected in vitro with an avian strain of E. coli underwent apoptosis 4 h after infection. Bastiani et al. [148] suggested that APEC may escape destruction by triggering macrophage apoptotic death through induced caspase 3/7 activation, the central caspases in apoptosis. The adherence pilus of avian pathogenic E. coli strains was examined by Vidotto et al. in 1997 [149], and they concluded that mannose-sensitive adherence to chicken tracheal cells correlated with the expression of type 1 fimbriae and that mannose-resistant adherence to chicken tracheal cells cannot always be attributed to P pili. A plasmid of 88 MDa encoding afimbrial adhesin genes has been also associated with adhesion properties to HEp-2 and tracheal epithelial cells in an avian septicemic E. coli strain by Stehling et al. [150]. As avian colisepticemia frequently occurs after respiratory tract damage, the primary site for infection allows bacteria to encounter an exposed basement membrane, where laminin and fibronectin are important components. Ramírez et al. [151] described two potential bacterial adhesins, which reacted with the basement membrane proteins laminin and fibronectin in a dot-blot analysis. Amabile de Campos et al. attempted to explore adhesion to tracheal epithelial cells, fimbrial expression and hemagglutination capacity of APEC strains isolated from chickens suffering from septicemia, SHS and omphalitis in different regions of Brazil [152]. They showed that adhesion, whether D-mannose resistant or D-mannose sensitive, is a characteristic observed in both pathogenic and commensal strains. Several strains with positive adherence had no genetic sequences related to the studied adhesin genes, which indicated that APEC strains probably possess a genome with adhesin genes besides those described elsewhere, as well as some not yet described. In studies of yolk sac infections, Rosario et al. [153] identified the ipaH gene, typical of enteroinvasive E. coli (EIEC) strains with biochemical properties that do not correspond to those described to the EIEC group. Invasion of HEp-2 cells with the formation of intercellular bridges or filipoidal-like protrusions were seen in some isolates, whereas serotypes and the presence of ColV plasmids agreed with the classification as extraintestinal E. coli strains. The results suggested the existence of specific clone complexes derived from EIEC strains adapted to the avian host. In other work, Rosario et al. [154] determined the serotypes and the presence of some virulence genes of E. coli strains isolated from different samples in a vertically integrated poultry operation in Mexico. The serogroup of 85% of the strains was determined, and the most commonly found included O19 (12%), 084 (9%), 08 (6%), and 078 (5%). In addition, 41 percent of the strains hybridized with one or more of the probes used (st, eae, agg1, agg2, bfp, lt, cdt, slt, and ipaH). Of these, ipaH (72%), eae (30%), and cdt (27%) were the most common. Results show that some avian E. coli strains isolated in Mexico are included as avian pathogenic E. coli serotypes not previously reported, which may suggest they could be specific for this geographic area. The wide distribution of the ipaH gene among non-motile strains may indicate that this invasiveness trait could be important in yolk sac infection pathogenesis, and some other genes could contribute to E. coli virulence. E. coli strains isolated from cellulitis presented phenotypic and genotypic characteristics of greater virulence than did fecal isolates [155]. The iss locus, associated with serum resistance, the iutA gene responsible for the aerobactin receptor, and the pathogenicity for 1-day-old chickens, were only associated with the cellulitis isolates. Salpingitis is another extraintestinal infection that affects broiler breeders. Based on the serogroups involved, pathogenicity for 1day-old chicks and virulence indicators, the E. coli salpingitis isolates were similar to those from cases of chronic respiratory disease [156].



Genetic relationships between avian E. coli strains isolated from extraintestinal sources have been evaluated by different typing methods. Forty-nine avian E. coli isolates from different outbreak cases of septicemia, SHS and omphalitis, and 30 commensal isolates from poultry with no signs of illness were characterized by the enterobacterial repetitive intergenic consensus (ERIC)-PCR technique and their serotypes were determined [157]. The ERIC-PCR profile allowed the grouping of the isolates into four main clusters (A-D), with the omphalitis isolates being grouped with the commensals and separated from the septicemia and SHS strains. This reinforces previous observations that omphalitis isolates are just opportunistic agents and are consistent with many reports that specific genotypes are responsible for causing specific diseases. When these strains were typed by isoenzyme profile and ribotyping analysis by restriction fragment length polymorphism (RFLP), isoenzyme analysis discriminated better among strains than did ribotyping analysis [158]. The enzyme profiles of the E. coli isolates allowed the identification of 33 clones that were organized into six main clusters. Most of the pathogenic strains were grouped together in a cluster, while commensal strains were assigned to the other clusters. Taken as a whole, these results demonstrate that pathogenic clones are more similar to one another, when compared with commensal strains, and may suggest that there is a correlation between the genetic background and the pathogenic characteristics of APEC strains. However, Carvalho de Moura et al. [159] could not distinguish among E. coli strains with different degrees of pathogenicity when testing the capability of ERIC and repetitive extragenic palindromic (REP) PCR to detect genetic diversity among E. coli strains isolated from chickens with colibacillosis and compared the genotypes so obtained with the O:H serotypes and virulence of those strains. The ERIC and REP-PCR methods had good discriminating power, and the dendrograms based on the different patterns revealed extensive genetic diversity among the avian strains. Those strains were allocated into four major clonal clusters, and those clusters corresponded to strains with different degrees of pathogenicity. The 32 serotypes detected were distributed in all clusters; however, strains with the same serotypes tended to form clusters with similar coefficients greater than 80%. Common virulence factors have been demonstrated in APEC and UPEC, and these findings may suggest that APEC strains represent a potential food-borne source of human UPEC infections. Both subcategories of ExPEC strains share virulence-associated traits and have overlapping O serogroups and phylogenetic types. The putative link between human and animal disease caused by E. coli deserves further attention. A reduced number of studies provided information about the isolation of ExPEC strains from other food-producing animals or pets in Latin American countries. E. coli strains isolated from pigs with urinary tract infection were investigated for the presence of virulence factors and a plasmid DNA profile [160]. The most frequent virulence factors presented by these strains were mannose-resistant fimbriae, including P fimbriae (54.8%) and aerobactin production (45.2%). The pap operon, detected by PCR, was found in 54.8% of the strains, which is similar to its frequency in human ExPEC strains. Other characteristics, such as the presence of mannose-sensitive hemagglutinin (16.1%), indicative of type 1 pili, and production of hemolysin (25.8%), colicin (38.7%) and toxins (22.6% for LT and for VT) were less frequent. No strains were positive for STa production. Plasmid profiles were variable among isolates from either the same or different farms. In a recent study, Siqueira et al. [161] compared the prevalence of virulence genes in E. coli strains isolated from 51 clinical cases of urinary tract infections, 52 of pyometra and from 55 fecal samples from healthy dogs by PCR. ExPEC-associated virulence factor genes encoding haemolysin (hlyA), uropathogenic specific protein (usp) and aerobactin iron transport system (iucD) were significantly associatedwith E. coli strains isolated from UTIs and pyometra. These genes are frequently identified in human UPEC strains. Eight E. coli strains obtained in Brazil from ostriches with respiratory disease also showed a virulence profile characteristic of ExPEC [162]. Serogrouping demonstrated that four isolates belonged to serogroup O2, two to serogroup O78, one to serogroup O9, and one to serogroup O21. The virulence genes encoding type 1 fimbria (fim) was found in all eight isolates, curli (csgA) in seven, aerobactin system (aer) in six, and P fimbria (pap), crl regulator protein (crl) and temperature-sensitive hemagglutinin (tsh) in one isolate each. All isolates analyzed were positive for mannose-resistant hemagglutination, adhered in vitro to ciliated tracheal epithelium, grew on irondeficient medium, and showed serum resistance. Five isolates exhibited high-t- intermediate pathogenicity when they were tested on one-day-old chickens. These results demonstrated strong similarities between E. coli strains isolated from respiratory disease in ostriches and septicemic E. coli strains isolated from poultry.



E. coli is also one of the bacteria that have been associated with otitis externa, produced primarily by rhabditiform nematodes and mites of the genus Raillietia in cattle from tropical and subtropical regions [163]. However, studies about the virulence traits of these E. coli isolates are lacking. In a study characterizing the E. coli strains isolated from 100 calves with diarrhea or septicemia and 21 older cattle with different pathologies, Mercado et al. [116] found that 21% of calves expressed the CS31A adhesin, and three of them produced the F17c fimbriae. All of the CS31A-producing strains exhibited at least one property of septicemic strains (resistance to serum, production of aerobactin or colicins) but none of them demonstrated heat-stable enterotoxigenic activity or expressed F5, F41, F17a or F17b fimbriae. CS31A+ E. coli isolates belonged to 10 serogroups, more commonly O8, O7, O17 and O21. These results suggested the worldwide distribution of plasmids containing CS31A or CS31A/F17c-associated sequences with additional extraintestinal virulence factor genes in cattle. Mercado et al. [164] also characterized 24 cytotoxic necrotizing factor (CNF)-producing E. coli strains isolated from cattle with diarrhea or extraintestinal infections. They found that the cnf2 allele was present in most of the isolates, while the cnf1 allele was present in only a reduced number of strains. Additionally, the virulence factor genes encoding hemolysin (hlyA), aerobactin iron transport system (iucD) and the outer membrane protein TraT (traT) were detected in more than 75% of the isolates. Cytolethal distending toxin genes cdtB-III and cdtB-IV were significantly associated with the cnf2 and cnf1 genes, respectively. Adhesin-encoding genes f17A, papC, sfa/foc y afaE-VIII and K1 capsular antigen were also detected among the CNF-producing strains. Interestingly, a cluster of strains belonging to serogroup O2 and B2 phylogenetic group was identified by RAPD-PCR. Finally, there is a need to know the role of ExPEC strains as etiological agents of disease in domestic and wild animals in Latin American countries. The human health hazard of ExPEC strains of animal origin in a region with intensive farming is of especially great concern. INTRODUCTION TO ENTEROPATHOGENIC Escherichia coli Evolution The term enteropathogenic E. coli (EPEC) was coined to describe strains associated with infantile diarrhea [165]. Independent of the track to be followed in this description, there was a gap in knowledge, because the major virulence factors were not known. Accordingly, Trabulsi et al. [166] described several serogroups for which there was a very strong prevalence in cases of infantile diarrhea. Experimental tests carried out in the rabbit ileal loop assay demonstrated that in most cases only serogroups associated with infantile diarrhea showed an increase of fluid in the intraluminal region of the loops. Accordingly, it was proposed that some active substances were released in vivo and that these were probably responsible for this effect. The increase in fluid, for example, could mimic the conditions in the gut of children infected with these particular strains; however there was no scientific basis to explain the etiology of these EPEC infections. The term enteropathogenic was established to refer to certain E. coli O serogroups or serotypes (O:H types) associated with infantile diarrhea that usually do not produce heat-labile and heat-stable enterotoxins and are not invasive. Since the discovery of enteropathogenic E. coli in the late 1940s, these serogroups were accepted worldwide as important agents of epidemic and non-epidemic infantile diarrhea in the first months of life [167]. Further studies [168, 169] demonstrated an adhesive characteristic, which differed between E. coli strains associated with diarrhea, that could provide a clue as to why strains that display a localized adherence (LA) to HeLa cells could explain the high virulence of some EPEC serotypes. Further studies showed a diffuse adherence to HeLa cells that had low pathogenicity or none for children. Several studies followed these data until it was established that the LA was not a true parameter to distinguish pathogenic EPEC from other low pathogenic E. coli, in infected children from 6 -12 months of age. The above phenotype was, in fact, due to a cascade of genotypic and respective phenotypic phenomena, which were exhaustively studied to reveal that the LA adherence was mediated by a bundle-forming pilus encoded in the Enteropathogenic Adherence Factor (EAF The pilus exhibited a cluster of 14 contiguous genes, and this was deemed the bfpA responsible for the synthesis of the bundling. The EAF plasmid (pEAF) was considered for many years as essential to determine what was called typical EPEC [169, 170, 171].



The strains encoding BFP and intimin are classified as typical EPEC (tEPEC). The strains harboring only the eae gene, without the presence of bfpA and expression of BFP, were designated as atypical EPEC (aEPEC). The tEPEC was the predominant pathotype associated with diarrhea in children over 1 year of age. The main serotypes were O55:[H6], O86:H34, O111:[H2], O114:H2, O119:[H6], O127:H6, O142:H6 and O142:H34. [172]. Recently, infections with tEPEC are decreasing dramatically as the cause of diarrhea in Brazil and other countries. Conversely, the infections caused by aEPEC are increasing exponentially in many cities in Brazil. The most common serotypes of aEPEC are O26:[H11], O55:[H7], O55:H34, O86:H8, O111ac:[H8], O111:[H9], O111:H25, O119:H2, O125ac:H6 and O128:H2 [172]. Furthermore, some aEPEC serotypes that do not belong to these classic serotypes are increasing in the community and in other countries, and similar epidemiologic data have been reported by other authors [173]. These strains seem to be involved in cases of diarrhea in both children and adults. Initially, when there was much attention placed on tEPEC, because it was found much more frequently in patients than was aEPEC, it had been assumed that humans were its only carriers. By extension of this assumption, the fecaloral route was considered virtually the exclusive route of infection, and, thus, only humans would be carriers and therefore reservoirs of tEPEC. If this premise was correct, there were further facts to be considered, including how long an infected person recovered from the disease or whether an imperceptibly infected patient continued to shed E. coli. However, at the time these questions remained unanswered due to a dearth of information, but in recent years this concept was somewhat disproved. Conversely, the findings that increased human infections were caused by aEPEC changed several factors. Among the most important was the report that adults, and not only children over 5-10 years of age, could be infected by aEPEC. Actually, findings in studies carried out by several groups [172, 174, 175, 176, 177] demonstrated that many isolates belonged to serotypes commonly found among domestic and companion animals [83, 109, 178, 179, 180]. As described below, many of the animal strains isolated were shown to be capable of transmission to humans and vice-versa. Since the kinetics of aEPEC infections either in humans or animals has not been widely studied, it was not easy to conclude whether rabbits, dogs, marmosets, bovine, ovine, cats or other animals were carriers and possible reservoirs of aEPEC, but this transmission route cannot be overlooked. The following sections will describe which isolates of aEPEC, as well as some tEPEC, have been reported mainly in Brazil and their incidence in the abovecited animals. Cattle as Reservoir of EPEC Calves are usually severely affected by enterotoxigenic E. coli (ETEC), but the disease is self-limited and rarely found in calves above one month of age. Older bovines are known as typical carriers of STEC/EHEC pathotypes [42, 43]. Although there are some discrepancies in the literature, these animals do not show diarrhea symptoms. To determine the occurrence, serotypes and virulence markers of EPEC strains in São Paulo, Brazil, 546 fecal samples from 264 diarrheic calves and 282 healthy calves from beef farms were screened by PCR. EPEC were isolated in 2.7% of the 546 animals. Although the IMS test was used, the STEC serotype O157:H7 was not detected. The most frequent EPEC serotypes were O26:H11, O123:H11 and O177:H11. The eae gene was detected in 100% of the EPEC strains. The intimin type 1 was the most frequently found. To our knowledge, this is the first report of the occurrence of the new intimin μB in one strain of animal origin. This new intimin was detected in one aEPEC strain of serotype O123:H?, isolated from diarrheic cattle. The enterohemolysin (ehxA) gene was detected in 80% of the EPEC strains. All 15 bovine EPEC strains isolated in this study were negative for both EAF and the bfpA gene. Overall, this study showed that cattle are reservoirs of atypical EPEC in Brazil. Among the EPEC isolated from diarrheic cattle, five EPEC strains (two of serotype O26:H11, two of O123:H11, and one of O177:H11) harbored intimin type 1, one other strain possessed intimin type θ/γ2 (O18:H7), and yet another, the new intimin μB (serotype O123:H?). Fluorescent actin staining (FAS) test confirmed four eae+ strains, but the three eae+ strains were FAS-negative, and one of those found to be FAS-negative was also intimin-negative



by Western blot. Three (43%) strains showed a NC pattern and one (14%), a LAL pattern, while three (43%) were negative in the HEp-2 cells adherence test. Among seven strains isolated from diarrheic animals, 86% possessed the enterohemolysin gene and expressed enterohemolytic activity. Six EPEC strains (4 of serotype O177:H11, one of O123:H11, and one of O123:H-), isolated from healthy cattle, harbored intimin type 1, while another strain possessed intimin θ/γ2 (O127:H40), and another, the intimin γ1 (serotype O145:H-). All 15 bovine EPEC strains were negative for both EAF and the bfpA gene, and, therefore, could be classified as aEPEC strains. It is not easy to explain the significance of these 15 aEPEC isolates. With the exception of serotype O127:H40, which was already reported in humans, the remaining isolates have not been isolated from cases of diarrheal disease in humans. Thus, based on these studies, cattle cannot be considered a reservoir of aEPEC for humans. Ovine as Reservoir of EPEC Previous studies have demonstrated the occurrence of distinct strains in sheep from different farms [83]. Depending on the number of strains studied, the absolute number of isolates of EPEC increases. However with some differences, the percentage of isolation of EPEC strains from ovines is not high. For example, among 86 ovine isolates, only five (5.81%) were eae+ and stx− and could be classified as aEPEC. Two isolates belonged to serotype O128:H2/β intimin; and the remainder were classified as O145:H2/γ, O153:H7/β and O178:H7ε. Based upon these findings, it is difficult to assume that ovines could be a reservoir of EPEC for humans. Pigs as Reservoir of EPEC There are a few reports of pigs as reservoirs of ETEC, and some EPEC-like isolates have been isolated from swine, mainly in other countries [181]. The study examined fecal samples from 198 pigs and 279 sheep at slaughter. The proportion of eae+ samples was 89% for pigs and 55% for sheep. By colony dot-blot hybridization, AE-producing E. coli (AEEC) were isolated from 50 and 53 randomly selected porcine and ovine samples, respectively. Strains of the serotypes O2:H40, O3:H8 and O26:H11 were found in both pigs and sheep. In pigs, O2:H40, O2:H49, O108:H9, O145:H28, and in sheep, O2:H40, O26:H11, O70:H40, O146:H21 were the most prevalent serotypes among typeable strains. Eleven different intimin types were detected, whereas γ2/θ was the most frequent, followed by β1, ε and γ1. All but two ovine strains tested negative for the Stx genes. All strains tested negative for the bfpA gene and the EAF plasmid. EAST1 (astA) was present in 18 of the isolated strains. Studies showed that pigs and sheep are a source of serologically and genetically diverse, intimin-harboring E. coli strains. Most of the strains show characteristics of aEPEC. Nevertheless, there are stx-negative AEEC strains belonging to serotypes and intimin types associated with classical EHEC strains (O26:H11, β1; O145:H28, γ1) [173]. The results of studies in which several pathotypes were sought in pig stool samples, did not point to any isolate that could be classified as aEPEC and tEPEC. This result leads to the possibility that pigs are not important carriers and reservoirs of this pathotype (unpublished data). Autochthonous and Exotic Wild Animals as Reservoirs of EPEC: Marmosets Monkey EPEC (MEPEC) or EPEC-like strains were the only groups of diarrheagenic E. coli isolated from fecal samples in research carried out in Brazil [179]. All of the isolates adhered to HeLa cells, and these results are shown in Table 3. The 21 eae+ E. coli strains belonged to 11 serogroups and 13 serotypes. Among these, O167:H9, O127, and O49:H46 were the most prevalent, identified in three animals each (23%), followed by serotypes O142:H6 and O132:H31 (each identified in two animals, 15%). Further, serotypes O139:H4, O128: H2, O26:H7, O167:H6, O33:NM, OR:H34, and O8:H10, were identified in one animal each. It is worth mention that some isolates from marmosets, i.e., serotype O132:H31, have all the characteristics of tEPEC, including presence of the eae and bfpA genes, BFP expression, and production of intimin type β and FAS+. Conversely this serotype of tEPEC was not found among humans, which may suggest that some animals could have their own tEPEC isolates. Symbols d or d/h means diarrhea (d) or healthy (h); Greek letters, types of intimin; LA, LAL, NC, DA and EA mean localized adherence or localized adherence-like; NC: Non-characteristic adhesion; DA: Diffuse adherence; EA: Enteroaggregative adherence.



Table 3: Characteristics of the E. coli eae+ isolates recovered from monkeys (Marmosets) healthy or with diarrhea. São Paulo, Brazil, 2003. Nº of Isolates

bfpA gene

BFP Expression

Intimin Subtype

Adherence

FAS

Serotype

2d 2d 2 d/h 1h 2 d/h 1d 1d 1d 1d 1d

+ +

+ +

β Ι γ2/θ λ β1 β1 β2 ε α1 α1

NC LAL, EA DA NC LA LAL,EA NC NC LAL LA

+ + + + + + + + + +

O128:H2 O49:H6 O127:H40 O33:NM O132:H31 O167:H9 O139:H14 O26:H7 O142:H6 O142:H6

Rabbits as Reservoir of EPEC Diarrhea in these animals is usually a serious problem because management procedures may include closed contact of the suckling and/or weanling rabbits. Thus most animals are infected earlier, usually either while suckling or just after weaning. In the case of REPEC (rabbit EPEC), the determination, even before determining the serotypes, of the biotype, as defined in reference [182], is important to allow assessment of the virulence of the isolates.. An important phenotype is the non-fermentation of rhamnose as indicative of higher pathogenicity. The virulence factors for REPEC are usually the production of fimbriae, which can be of two types: AF/R1 and AF/R2 (Table 4). Interestingly, AF/R1 is not the most common fimbrial antigen among REPEC. One study [177] showed that the AF/R1 was not detected in 178 strains. Among these isolates, 90 had the eae gene. Seventy-four were from diarrheic animals and all of them but one encoded the β intimin, which is unique compared to E. coli isolated from other animals. The virulence factor allowed the strains to be classified as aEPEC. The most prevalent serotype was O132:H2, present in 63 isolates (70%) of the 90 eae+ isolates. The AF/R2 fimbriae was found in 75 (83.3%) of the 90 eae+ isolates. Table 4: Summary of the study of 178 E. coli strains isolated from rabbits in Brazil. Number of Positive/total Number Isolated

Number of positive (percentage of isolates)

Strains not serotyped ( NT)

62/178

62 (34.83%)

Strains O132:H2

63/178

63 (35.39%)

Strains O128:H2

6/178

6 (3.37%)

Discrimination

Strains O153:H7

6/178

6 (3.37%)

Strains O103:H19

10/178

10 (5.62%)

Strains belonging to further serotypes

31/178

31 (17.42%)

Strains eae+ and serotype O132:H2

63/90

63 (70.00%)

Strains eae+ serotype O128:H2

6/90

6 (6.67%)

Strains eae+ serorype O153:H7

6/90

6 (6.67%)

Strains eae+ serotype O103:H19

0/90

0 (0.00%)

Strains of the serobiotype O132:H2:B28 among the eae+

49/90

42 (46.67%)


9/90

9 (10.00%)


6/90

6 (6.67%)

Strains eae+ of the serobiotype O153:H7:B28

6/90

6 (6.67%)

Strains AF/R2+ (only the eae+ were tested)

75/90

75 (83.33%)



Dogs as Reservoirs of EPEC The EAF plasmid and the bfpA gene have not been detected in EPEC from pigs, cattle, and rabbits, but some isolates of dog EPEC (DEPEC) and most typical EPEC of human origin harbor both EAF and bfpA [171, 183, 184]. Among the E. coli recovered from 182 fecal specimens collected from 146 dogs with diarrhea and 36 dogs without diarrhea, there were 23 (12.6%) that had the eae gene identified by PCR. These isolates were referred to as DEPEC. Twenty (13.7%) of the 146 isolates from diarrheic dogs, and three (8.3%) of the 36 isolates from non-diarrheic dogs were DEPEC [109]. The EAF plasmid was not detected in any of the DEPEC isolates, but the bfpA gene was detected in the isolates from two diarrheic dogs (S6 and C27, Table 4). The FAS test was carried out only with those DEPEC that showed adherence to HEp-2 cells. All isolates tested were FAS+, regardless of the pattern of adherence. There was a wide variety of serotypes among the DEPEC and serotypes O119:H2 (isolates SPA14 and BIO12) and O142:H6 (isolate S6) are noteworthy, as they have been identified as atypical and typical human EPEC serotypes, respectively. The DEPEC isolates in this study belonged to a wide variety of serotypes (Table 4). The serotype is often not determined for DEPEC, and only the serogroups of a limited number of isolates have been reported [96, 174, 178, 185, 186, 187]. It is significant that two isolates were serotyped as O111:H25 and two as O119:H2, because they are human atypical EPEC serotypes [172]. An outstanding finding is that one isolate belonged to serotype O142:H6, a typical human EPEC serotype. This isolate (S6) harbored the gene bfpA, expressed BFP, and adhered to HEp-2 cells in a LA pattern (Table 5). In a study characterizing non-STEC O157 strains isolated from dogs in Argentina, Bentancor and co-workers identified typical EPEC serotype O157:H45 and atypical EPEC serotype O157:H16 (188). These findings suggest that dogs, with or without diarrhea, may be a source of infection of typical and atypical EPEC infection for humans [172]. This has been described for EPEC of serotype O111:H- [189] in a study which demonstrated cross-infection between a dog and a child in the same house of a city of the state of São Paulo, Brazil. Table 5: Characterization of the eae+ E. coli isolates from diarrheic and non-diarrheic dogs in São Paulo, Brazil, 2004. Isolate 008 HE4 HE8 HE9 HE10 HE13 SPS14 SPA16 B1 B2N B4 B41-7 B17 S1 S6 BIO2 BIO4 BIO12 C27-colony 1 C27-colony2 C32 QSF4 SB2

bfpA + + -

Subtyping of Intimin β β γ γ γ β β NT γ NT NT NT γ NT α ε ε β γ β β κ β

Adhesion (HEp-2) LAL NC NC LAL NC LAL LAL LAL LAL LA NC NC LAL LAL LAL -

Serotype O98:H28 O11:H16 O111:H25 O111:H25 ONT:H40 O15:HO119:H2 ONT:HO88:H19 ONT:HO156:HO174:HO142:H6 O157:H16 O142:H6 O157:H16 O157:H16 O119:H2 O25:H8 O167:H6 O4:H6 O88:HO15:H-

(-) Negative; LAL: Localized Adherence Like; LA: Localized adherence; NC: Non characteristic adherence; Not Typeable: No reaction with primers (only α,β, γ and δ were available at the time of the research was carried out).



Cats as Reservoirs of EPEC Additional diarrheagenic bacteria have been studied in pets, as well as their potential role as sources of enteric pathogens for human infection. In this study, cats were investigated as carriers and excretors of EPEC, EHEC/STEC, and ETEC as possible causes of intestinal infections in humans [3, 172]. We examined fecal samples from 300 cats for diarrheagenic E. coli types. None of these were positive for genes encoding Shiga-toxins and enterotoxins, indicating that STEC and ETEC were not excreted by the animals. Fifteen eae+ E. coli strains (CEPEC; cat EPEC) were isolated from 14 cats (4.7%; 13 non-diarrheic and one diarrheic). Although further research was not performed with the CEPEC, these results may imply that cats can be an important reservoir for aEPEC and able to cause diarrheal disease in humans. Fig. 1 summarizes the interactions and relationships between animals and humans, based upon the reports in this section. The figure depicts, at left, humans as the only reservoir of typical EPEC. In text, it was emphasized that although tEPEC is disappearing as a major cause of diarrhea among children, a few serotypes, mainly O142:H6 and O127:H40, have been found in some animals that live in close contact with humans, and this observation cannot be overlooked. Therefore it is plausible to question whether the reservoir of these serotypes was human, canine s and/or simian (marmosets). Thus Fig. 1 also depicts some domestic and companion animal carriers of aEPEC. Because the diarrheal diseases by aEPEC is increasing, the role of these animals in the cycle of infection cannot be overlooked, and it is possible that nowadays the target of research on EPEC will focus more on these animals as putative reservoir of aEPEC for humans.

Figure 1: The past concept showing the putative reservoir for tEPEC and aEPEC for humans. The importance of humans as carries of tEPEC had its relevance strongly reduced. Among the animals (right part of the picture), some of them can be reservoirs of aEPEC, except marmoset and dogs that also showed to be carriers of tEPEC (particularly serotype O142:H6).

Conclusions about EPEC As a result of exploring several possibilities about animals as reservoirs, it can be stated, that to some extent they are reservoirs of aEPEC and tEPEC for humans. Further verification of this concept has been attempted by Rodrigo de Assunção Moura (University of São Paulo, Brazil to provide some additional findings, when strains of the same serotype, isolated from humans and different animal species, were matched and compared by using Multilocus Sequencing Typing (MLST) and Pulsed Field Gel Electrophoresis (PFGE) [190]. The phylogeny of 49 typical and atypical EPEC strains was inferred by Bayesian analysis. The strains were rooted with EHEC strain EDL933 and EPEC strain E2348/69; Salmonella enterica strain Ty2 was included as outgroup standard (data not shown). The relationships among the strains studied, constitute an original contribution to the epidemiology of EPEC, and describe the role of the sources of infection as possible reservoirs, in cases of cross- infection (animals and humans). ACKNOWLEDGEMENTS The co-authors thank those who participated in these studies and the institutions that gave financial support: USP, UBA, UNCPBA, CONICET, ANPCYT, INTA, CNPq, FAPESP, CIC-PBA, Min. Salud Nación Argentina and PPUA-SPU.



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A cluster of fourteen genes from Enteropathogenic Escherichia coli to HEp-2 cells for biogenesis of type IV pilus Mol Microbiol. 1996:20:325-37. [172] Trabulsi LR, Keller R, Gomes TAT. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis. 2002;8:508-13. [173] Hernandes RT, Elias WP, Vieira MAM, et al. An overview of atypical enteropathogenic Escherichia coli FEMS Microbiol Lett. 2009;297:137-49. [174] Drolet R, Fairbrother, JM, Harel J, et al. Attaching and effacing and enterotoxigenic Escherichia coli associated with enteric colibacillosis in the dog. Can J Vet Res. 1994;58:87-92. [175] Robins-Browne RM, Tokhi AM, Adams LM, et al. Adherence characteristics of attaching and effacing strains of Escherichia coli from rabbits. Infect Immun. 1994;62:1584-92. [176] Carvalho V, Irino K, Onuma DL, et al. 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Examination for heat-stable and Shiga-like toxins and for eaeA gene in Escherichia coli isolates obtained from dogs dying with diarrhea: 122 cases. J Am Vet Med Assoc. 1998;212:1735-6. [187] Holland RE, Walker RD, Sriranganathan N, et al. Characterization of Escherichia coli isolated from healthy dogs. Vet Microbiol. 1999;70:261-8. [188] Bentancor A, Vilte DA, Rumi MV, et al. Characterization of non-Shiga toxin- producing Escherichia coli O157 strains isolated from dogs. Rev Argent Microbiol. 2010;42:46-48.



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CHAPTER 16 Host-pathogen Communication Marcelo P Sircili1*, Cristiano G Moreira2 and Vanessa Sperandio2 1

Laboratório de Bacteriologia, Instituto Butantan, São Paulo, SP, 05503-900, Brazil, 2University of Texas Southwestern Medical Center, Department of Microbiology, Dallas, TX 75390-9048, USA Abstract: Chemical communication between pathogens and host mucosal cells corresponds to a dynamic array of molecular interactions. The signature molecules unique to microbial pathogens allow the mammalian immune system to recognize them as a foreign element. This recognition is usually mediated by receptor proteins, which can be classified as toll-like receptors, and recently described as nod-like receptors. These interactions result in innate immune responses targeted against the invading organism. Pathogens also elaborate a variety of proteins that actively engage host signaling pathways and subvert them to facilitate their growth and dispersal. The host function alterations are mediated by microbial pathogens including inflammatory responses, secretory responses, alteration of host cytoskeleton, disruption of epithelial tight junctions and apoptosis. Important interactions between pathogens and host cell involves chemical signaling, that depends on cell density and signaling molecules identified as autoinducers that function as hormone-like molecules in a phenomenon also known as quorum sensing. Pathogens can use these systems to colonize and cause disease in the host, and we will further discuss these mechanisms in this chapter. Chemical signaling involved in these interactions are potential targets for therapeutic strategies against infectious microbes.

BACTERIAL INTERCELLULAR COMMUNICATION Before 1970 bacterial ability to recognize molecules produced by another bacteria as well as act in a multicellular behavior communicating with another cell was a dogma. This premise started to change after observations made at the early seventies involving the bioluminescence phenomena in Vibrio fisheri strains [1]. They noticed that V. fisheri bioluminescence was only expressed after a particular cell density had been achieved during the culture growth. This phenomenon was attributed to the production of signaling molecules that allowed cells within a population to communicate with each other, later named as Quorum sensing [2]. Quorum sensing is a cell-to-cell signaling mechanism that refers to the ability of bacteria to respond to chemical hormone-like molecules called autoinducers. When an autoinducer reaches a critical threshold, the bacteria detect and respond to this signal by altering their gene expression [3]. BACTERIAL COMMUNICATION AND HOST INTERACTIONS It is estimated that in humans, the total microbial population within the gastrointestinal (GI) tract (1014) exceeds the total number of mammalian cells (1013) by at least an order of magnitude [4]. The GI tract is the site with the largest and most complex environment in the mammalian host. The density of bacteria along the GI tract can vary greatly, with the majority of the flora residing in the colon (1011 to 1012 bacterial cells/ml). Given the enormous number and diversity of bacteria comprising the GI environment, it should not be surprising that the members of this community somehow communicate among themselves and with the host itself to coordinate various processes [5]. INTER-KINGDOM COMMUNICATION Recent studies on bacterial signaling have revealed an ongoing communication between microorganisms and their hosts. Several host-derived signals are sensed by bacteria. Some of these compounds include tumor necrosis factor alpha [6], interleukin 1 [7], adenosine [8], epinephrine [9], and antimicrobial peptides [10]. These reports have suggested that bacteria can sense the metabolic stress of the host to take advantage of a weakened immune state. *Address correspondence to: Marcelo P. Sircili. Laboratório de Bacteriologia, Instituto Butantan. Av. Vital Brazil 1500 São Paulo, SP, Brazil CEP 05503900. Phone/fax: 55 11 3726 7222 ext 2075. e-mail: [email protected] Alfredo G. Torres (Ed) All rights reserved - © 2010 Bentham Science Publishers Ltd.


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On the other side of the cell-to-cell communication process, the ability of host cells to sense bacterial signaling molecules, such as acyl-homoserine lactones, has also been well shown [11]. The effects of these molecules are diverse and often deleterious for the host, leading to increased bacterial pathogenesis. The first host receptor for these molecules, the nuclear receptors PPRgamma and PPRbeta/delta were recently identified [12]. The activity of these molecules has been shown to modulate host immune responses, promote apoptosis and elicit pro-inflammatory responses. Research on chemical signaling between prokaryotes and eukaryotes has made significant progress in the recent past. Chemical signaling has an obvious mechanistically importance and implication for bacteria-host interactions during the infectious process. SIGNALING SYSTEMS Several signaling systems have been well studied between host-cell pathogens interactions, among them there are at least four involved in chemical signaling described in the literature: 1) AI-1 System, 2) AI-2 System, 3) AI-3/ epinephrine system, and 4) indole. The signaling systems described in E. coli are summarized in Fig. 1. AI-1 system

HSL

SdiA

-Interspecies communication -Biof ilm Formation

AI-2 system

AI-3 Epenephrine/norepinephrine system

AI-2

Lsr

-Lsr operon -Metabolic pathways

AI-3

QseC

QseBHSL

-Motility -HUS

QseF

QseE

- Via ler and espfu -AE lesion and Actin polymerization respectively

Figure 1: Schematic representation of signaling systems in E. coli

AI-1 System and Indole AI-1 system was first described in V. fisheri as responsible for lux operon activation, which culminates in bioluminescence control. The luxR/luxI family and their genes homologues are responsible for the production this respective autoinducer [2]. E. coli does not harbour one luxI homologue and does not produce the autoinducer, although harbours one luxR homologue which is called sdiA. SdiA is responsible for sensing the environmental AI-1 signals produced by other microorganisms [13]. Studies with SdiA mutants have shown that this regulator may be involved in biofilm formation in E. coli. SdiA has been proposed to interact with indole [14], one of the extracellular signals produced by E. coli [15], in addition to acylhomoserine lactones (AHLs) from other bacteria and interacts with them. However, conclusive data demonstrating that SdiA senses indole is lacking. In E. coli, SdiA, named after its ability

Host-pathogen Communication


to suppress cell division inhibitors, is a 240-amino-acid protein that belongs to the LuxR family of transcriptional regulators. Over-expression of SdiA induces the expression of the ftsQAZ locus involved in cell division. Salmonella enterica and E. coli’s SdiA detects quorum sensing signal AHLs produced by other bacteria, although they do not synthesize AHLs [16]. AHLs control social behavior like biofilm formation and virulence. Furthermore, SdiA decreases early E. coli biofilm formation in approximately 50-fold, enhances acid resistance, and it is required to reduce E. coli biofilm formation in the presence of AHLs as well as in the presence of the stationary-phase signal indole [14]. Indole is an E. coli quorum-sensing signal that works primarily at temperatures found outside the human host and reduces biofilm formation [17]. Therefore, SdiA, via interaction with AHLs, is a key protein for intraspecies and interspecies cell communication as well as for biofilm formation [18]. AI-2/luxS System AI-2 was first described in V. harveyi strains and is involved in the control of bioluminescence, in a convergent pathway with AI-1, since these strains possess both signals. It was first described as a universal signal, responsible for interspecies communication [19]. The molecule was described as a furanosyl-borate-diester [20] and the gene responsible for its production was called luxS [21]. The receptor for this system in V. harveyi is called LuxP. The LuxP system is restricted to Vibrio species, moreover there are differences among AI-2 molecules produced by different strains [22]. The AI-2 receptor in E. coli and Salmonella is the LsrB periplasmic protein [23]. Cocrystallization of LsrB with AI-2 demonstrated that its ligand was not a furanosyl-borate-diester, but a furanone [2R, 4S-2-methyl-2,3,3,4-tetrahydrofuran (R-THMF)] [22]. LuxS functions in the pathway for metabolism of S-adenosyl methionine (SAM), the major cellular methyl donor. Transfer of the methyl moiety to various substrates produces the toxic byproduct S-adenosylhomocysteine (SAH). In non-LuxS containing bacteria and eukaryotes, the enzyme SAH hydrolase metabolizes SAH to adenosine and homocysteine. However, in bacteria containing LuxS, two enzymes, Pfs and LuxS, act sequentially to convert SAH to adenine, homocysteine, and the signaling molecule Dihidroxipentanedione (DPD) [24]. DPD is a highly reactive product that can rearrange and undergo additional reactions, which suggests that distinct but related molecules derived from DPD may be the signals that different bacterial species recognize as AI-2. AI-3/ Epinephrine/Norepinephrine System AI-3/epinephrine/norepinephrine system was first described in enterohemorrhagic E. coli O157:H7 (EHEC) strains [9]. It was initially thought that the gene responsible for production of the molecule was luxS, since luxS mutant has diminished AI-3 production. Later, it was shown that luxS mutants have metabolic deficiencies and that the luxS gene is not responsible for AI-3 production [25]. AI-3 is an aromatic molecule that can only be eluted from C18 columns with organic solvents. The host hormones epinephrine and norepinephrine can rescue AI-3-dependent phenotypes in EHEC. Based on that, it has been proposed that this system is involved in host cell-bacteria communication, and inter-kingdom signaling. The AI-3 receptor, QseC sensor kinase [26] has been extensively described in EHEC as well as enteropathogenic E. coli (EPEC) [27] and other species. Subsequently studies showed that AI-3/ epinephrine/norepinephrine system is not restricted to E. coli strains [28]. AI-3 Signaling in EHEC In EHEC, the mammalian hormones epinephrine and norepinephrine, which are released by the host during stress, are sensed by the QseC receptor to regulate bacterial virulence genes [26, 28]. The qseC mutant is attenuated for virulence, which underscores the importance of this inter-kingdom communication to the development of disease [26]. EHEC is part of a group of pathogens that includes EPEC, Citrobacter rodentium, and Hafnia alvei, all of which are able to cause a lesion on the intestinal epithelial cells named the attaching and effacing (AE) lesion. The AE lesion is characterized by the destruction of the microvilli and rearrangement of the cytoskeleton to form a pedestal-like structure, which cups the bacteria individually [29]. The genes involved in the formation of the AE lesion are encoded within a chromosomal pathogenicity island named the Locus of Enterocyte Effacement (LEE) [30]. The LEE region contains five major operons: LEE1, LEE2, LEE3, tir (LEE5), and LEE4 [31, 32], which encode a type III secretion system (TTSS) [33], an adhesin (intimin) [34], and the intimin adhesin receptor (Tir) [35], which is translocated to the epithelial cell through the bacterial TTSS. The EHEC luxS mutant, who presents diminished AI-3 production and does not express the LEE-encoded TTTS system at normal levels, nonetheless still forms AE lesions on epithelial cells that were indistinguishable from those seen with wild type. The luxS mutant was


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still responding to eukaryotic cell signals to activate expression of the LEE genes. These signals were identified as the hormones epinephrine and norepinephrine. Epinephrine and norepinephrine can substitute for AI-3 to activate transcription of the LEE genes, type III secretion, and AE lesions on epithelial cells. Norepinephrine has been previously reported to induce bacterial growth, and there are reports in the literature that imply that norepinephrine might function as a siderophore [36]. Norepinephrine has been implicated as inducing expression of enterobactin and iron uptake in E. coli, suggesting that this is the mechanism involved in growth induction [37]. However, the role of norepinephrine in bacterial pathogenesis seems to be more complex, since several reports suggested that this signal also activates virulence gene expression in E. coli, such as the EHEC Stx toxin, by an unknown mechanism of induction [38]. Both epinephrine and norepinephrine are present in the GI tract. Norepinephrine is synthesized within the adrenergic neurons present in the enteric nervous system (ENS) [39]. EHEC QS SIGNALING CASCADE Concerning the EHEC AI-3/epinephrine/norepinephrine signaling cascade, a transcriptional regulator from the LysR family, designated as QS E. coli regulator (QseA) [40] has been recently identified. QseA is transcriptionally activated through QS and, in turn, binds to and directly activates transcription of the LEE-encoded regulator (Ler). The qseA mutant in EHEC has a striking reduction in type III secretion, but has no defect in motility, suggesting that QseA only regulates the LEE genes and plays no role in the flagella regulation. Additionally, the QseBC twocomponent system is responsible for the transcriptional activation of the flagella regulon in response to QS. It is well known that many two-component systems act to positively regulate their own transcription. QseBC is no exception to this rule and has also been shown to autoactivate its own transcription [41]. Early studies indicated that an isogenic mutant in the qseC sensor kinase was unable to respond to AI-3 or epinephrine given exogenously [26]. Interestingly, the motility of a luxS mutant can be restored either by the addition of AI-3 or epinephrine, and the transcription of flhDC genes is also activated by both signals. Motility and flhDC transcription in the qseC mutant however is unable to respond to the presence of either AI-3 or epinephrine, indicating that QseC may be sensing the presence of these cross-signaling compounds. QseBC regulates both its own transcription and flhDC expression, and participates in the regulation of other QS phenotypes, such as the LEE genes and Stx production [28, 42]. Three other genes in this signaling cascade have also been identified recently: qseD (encoding another regulator of the LysR family) and qseE and qseF (encoding a second two-component system), which are involved in regulating AE [43]. Additionally, QseE has also been shown to sense epinephrine, sulphate and phosphate, but not AI-3 [44]. The interaction of epinephrine with more than one sensor kinase would also impart a “timing” mechanism to this system, which is a desirable feature given that it would be inefficient for EHEC to produce both the LEE TTSS and flagella simultaneously. The AI-3-dependent QS signaling cascade is present in all Enterobacteriaceae (E. coli, Salmonella spp., Shigella spp., and Yersinia spp.). The most striking feature is that the genes encoding the transcriptional factors of this cascade are always in the exact same context in the chromosome of all these strains and share high levels of identity among these different species, suggesting that this signaling cascade is functionally conserved in Enterobacteriaceae. DRUG DEVELOPMENT Treatment of EHEC infections with conventional antimicrobials is highly ineffective, as it is well documented that antimicrobials activate the Stx phage to enter the lytic cycle, thereby producing and releasing Stx [45, 46]. To circumvent this matter, the development of drugs acting in the pathogens chemical signaling systems without eradicating them and without a selective pressure could be useful against these infections. Based in the fact that EHEC senses AI-3 through QseC, one possible way to prevent the disease would be a drug to block QseC and consequently the downstream signaling cascade. These antimicrobials will not only be useful against diarrheagenic E. coli, as EHEC and EPEC, but also against other pathogens such as Salmonella enterica, Shigella, and Yersinia pestis, all of which harbor this signaling cascade. One recent study was performed and was carried out a high-throughput screening, which employed a library with 150,000 small organic compounds to identify a lead structure the N-phenyl-4-[[(phenylamino)thioxomethyl]amino]benzenesulfonamide or LED209, which selectively blocked the signals binding (AI-3/epinephrine/NE) to QseC, preventing QseC’s autophosphorylation, and consequently inhibiting QseC-mediated activation of virulence gene



expression. LED209 inhibited EHEC pathogenesis but did not affect EHEC growth in vitro, i.e. without eliminating EHEC cells. This compound was not toxic to host cells, but inhibited expression of key virulence traits of EHEC pathogenesis (AE lesions and Stx production). LED209 was also able to inhibit Salmonella enterica serovar Typhimurium and Francisella tularensis virulence in vitro and in vivo [28]. Furthermore, the QseC-dependent interkingdom signaling system does not directly affect bacterial growth, i.e. the inhibition of this signaling pathway does not exert a selective pressure towards development of drug resistance. CONCLUDING REMARKS Chemical signaling between cells underlies the basis of multicellularity. Although bacteria are unicellular, bacterial populations also utilize chemical signaling, through hormone-like compounds named autoinducers, to achieve cellcell communication and coordination of behavior. Chemical signaling is also essential for an organism to survive, successfully adapt to ever changing environments and protect themselves from insults, which can be collectively considered stress. Successful stress responses require energy input, and the coordination of many complex signaling pathways within the cell. Co-evolution of prokaryotic species and their respective eukaryotic host have exposed bacteria to hormones and eukaryotic cells to autoinducers. Research on signaling between prokaryotes and eukaryotes has made significant progress in the recent past and it is now obvious that such phenomenon has important implications for bacteria-host interactions. Future studies will not only reveal novel layers of interactions between bacteria and eukaryotes but also help further understand the molecular mechanisms involved in bacteria-host communication. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18]

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CHAPTER 17 Future of Escherichia coli Research in Latin America Tânia AT Gomes1, Cristina Ibarra2, Fernando Navarro-Garcia3, Marina Palermo4, Valeria Prado5, Marta Rivas6 and Alfredo G Torres7* 1

Departmento de Microbiologia, Imunologia, e Parasitologia, Universidade Federal de São Paulo, São Paulo, Brazil; 2Departamento de Fisiología, Facultad de Medicina, Universidad de Buenos Aires; 3Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), México DF, Mexico; 4 Departamento de Inmunología, Academia Nacional de Medicina and ILEX-CONICET, Buenos Aires, Argentina; 5 Programa de Microbiología, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile; 6Servicio Fisiopatogenia, Instituto Nacional de Enfermedades Infecciosas – ANLIS “Dr. C. G. Malbrán”, Buenos Aires, Argentina; 7Department of Microbiology and Immunology, Department of Pathology and the Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, Texas, U.S.A. Abstract: In the 21st century, diarrhea is still a leading cause of illness and death especially in children in Latin American countries. ETEC, EHEC, EPEC, and EAEC remain as the major categories of pathogenic E. coli associated with diarrheal disease; however, it is evident that a shift in the serotypes responsible for human disease is occurring in this region. Recent reports implicated atypical EPEC as an important emerging category of E. coli and the association of EHEC O157:H7 as a cause of hemolytic uremic syndrome in Latin America is becoming evident. Significant improvements are required in the area of early diagnosis to increase the likelihood of an effective treatment. In this region, very few studies have addressed the emergence of antimicrobial resistance and high asymptomatic carriage rates for diarrheagenic E. coli (DEC), as well as non-human reservoirs and vehicles of transmission, are largely unknown. It is evident that broadening the epidemiological surveys to include emerging and re-emerging categories of DECs while increasing the capabilities of detection and novel treatment is a priority for the future of the E. coli research in Latin America.

INTRODUCTION Diarrheal episodes due to DEC infections remain as an important public health issue in Latin America because their association with morbidity and mortality of children under 5 years of age. During the past 5 years, the prevalence of specific serotypes of E. coli have been replaced by new categories of pathogenic E. coli, making this a new challenge because no much is known about the virulence of these new serotypes [1]. Thus, it is imperative that effective diagnostic and epidemiological strategies be implemented in hospitals, clinics, etc., to identify, categorize, and evaluate the new pathogenic E. coli isolates. Further, new and effective preventive and treatment measures need to be implemented to control E. coli infections. It is plausible that vaccination may offer a safe and effective means of preventing infections with the main categories of E. coli; therefore, more efforts need to be placed in this area of research. HOW CAN WE IMPLEMENT RELIABLE DIAGNOSTIC TEST TO IDENTIFY DIARRHEAGENIC E. coli? The majority of all known categories of DECs have been detected in Latin America. However, the epidemiology of DECs in this region is poorly understood, and very unevenly studied, regardless of the significant contribution that these pathogens make to the burden of illness in Latin America. Despite published epidemiological studies, these are restricted to 4 major countries: Argentina, Brazil, Chile and Mexico. Epidemiological studies in different countries could contribute to identify risk factors associated with country-specific habits, thus leading to recommendations of simple, rapid, and effective risk-reduction procedures to decrease morbidity and mortality. The lack of or very limited information available from the other countries could be related to the inability of the investigators to publish studies in peer-reviewed journals due to high cost, language barrier, or lack of interest by them or the official *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.

Future of Escherichia coli Research in Latin America


authorities. Other reason could be the lack of capacity to detect and further differentiate the different categories of diarrheagenic E. coli, either for research or clinical purposes, ignoring the fact that these organisms are among the most common causes of disease in these countries. The majority of the studies performed in Latin America are country-specific and sometimes city-specific, and multinational studies have been rarely performed. In many countries performing epidemiological studies, biochemical characterization of the isolates is often performed in the local clinical laboratories while serotyping and further classification of the E. coli pathotypes is performed in references labs in other countries in the continent or Europe. While this approach has result effective for some countries to produce reliable information, it is real barrier for other countries than would benefit from having local detection methods that will accelerate the identification and report of outbreaks, which currently are detected several months or years after they occurred. An example of an effective method in the US for surveillance, tracking trends, guiding prevention strategies, and detecting outbreaks is PulseNet [2]. This network for DNA "fingerprinting" of bacteria that cause foodborne illness, links state public health laboratories, the Center for Disease Control and Prevention (CDC) and the food regulatory agencies together so that multistate outbreaks can be rapidly detected, investigated, and control measures implemented. PulseNet Latin America and the Caribbean has been in operation since 2004 and this network if expanded to all the countries in Latin America should go a long way towards producing further epidemiological knowledge regarding distribution and endemic areas of E. coli O157:H7/NM and non-O157 STEC strains. Whenever possible, future studies need to use tests that are sensitive and specific to detect multiple E. coli pathotypes, without bias towards subtypes that have epidemiological relevance in other parts of the world, for example E. coli O157:H7 or typical enteropathogenic E. coli strains. As such methods necessarily require molecular biology and/or tissue culture, there is need to increase capacity for both on the different Latin American countries. Alternatively, the development and implementation of an appropriate diagnostic methodology in the different countries is required, although it is clear that the big differences in infrastructure remain as a limiting factor for some countries. Because stool culture is expensive, laborious and time consuming, and any evaluation that includes diarrheagenic E. coli increase the costs much more, perhaps the development of a simplified molecular method that will provide information directly from the stool sample, for example, it may be beneficial to screen DNA isolated from stool by multiplex PCR, and in case of positive results, the samples can be further investigated for diarrheagenic E. coli at reference laboratories within the same country. Considering that diarrhea incidence is higher in the areas with fewer resources, it is important to have rapid techniques of screening that can be easily performed, ideally, at the bedside, where the sample can be applied directly, either using chromatographic or immunoassay techniques. IDENTIFICATION OF NOVEL PATHOGENIC E. coli ISOLATES WITH CLINICAL RELEVANCE TO LATIN AMERICA Asymptomatic carriage of enteric pathogens in general and diarrheagenic E. coli in particular has been reported in many Latin American studies and the incidence seems to be high in some regions. Carriage rates are often higher for EAEC, pointing to strain variability and the likelihood that some strains are not pathogens, or at least less virulent than others. Therefore, in order to understand better the epidemiology of these pathogens, an in-depth characterization of isolates from large case-control studies needs to be planned and several countries involved to clarify the pathogenicity index of each category of DEC. Larger studies are more likely to uncover different types of associations with disease when these exist and to identify micro-outbreaks. Even better, population-based studies need to be performed as a way to link isolation rates to disease burden. It is also evident that mixed infections are present in parts of Latin America, including co-infections with pathogenic E. coli and other pathogens, i.e., Shigella, Campylobacter, or co-infection with other categories of pathogenic E. coli. Interactions between different pathogens have not been commonly studied in Latin America and this area of investigation could produce information regarding the effects on the human host. Regarding reservoirs and transmission: certain food products and animals have been directly associated with E. coli infection in humans. Contaminated water and person-to-person transfer of these organisms are probably


Gomes et al.

predominant routes for spread. Water supplies, sanitation, frequency of hand-washing with soap have been instrumental to the reduction of diarrheal episodes in many Latin American countries and they have proven to prevent the acquisition and spread of diarrheagenic E. coli and other pathogens. Likewise, stimulation of breastfeeding has been very effective in reducing the incidence of infantile diarrhea. However, the animal reservoir and/or the way how several categories of pathogenic E. coli survive in the environment are still unknown. It is well documented that cattle and swine serve as reservoirs for EHEC, and contaminated water must make the largest contribution to diarrheal prevalence, but other reservoirs for diarrheagenic E. coli are not well known. Close contact with other species of animals and evidence of zoonotic transmission of enteric organisms means that pathotypes with an animal reservoir will have a high chance of being transmitted to humans. E. coli PATHOGENESIS It is clear that the biology of the different E. coli pathotypes is complex, since each pathotype has a distinct subset of genes involved in the subversion of host responses and hijacking of host cell machinery. In many pathotypes, the same host machinery or process is targeted but the mechanism and outcome is different. Bacterial pathogenesis is a quickly evolving and expanding field. Genetics, genomics and proteomics efforts continue to identify more potential virulence factors, but our understanding of the interactions between virulence factors (i.e., effectors and toxins) and host components, remain incomplete. It is a considerable challenge to integrate the numerous host-cell targets and to translate this knowledge into an accurate understanding of the mechanisms by which effector proteins cause disease [3]. Other interesting field of the bacterial pathogenesis is related with protein secretion. Protein secretion plays a central role in modulating the interactions of bacteria with their host organisms. In pathogenic E. coli, secretion requires translocation across the outer as well as the inner membrane and a diversity of molecular machines have been elaborated for this purpose; at least the known six secretion systems are present in E. coli. A number of secreted proteins are destined to enter the host cell (effectors and toxins), and thus several secretion systems include apparatus to translocate proteins across the plasma membrane of the host also [4]. Expanding the knowledge about how the secreted proteins or effectors are secreted will allow us to direct the quest of their action mechanisms as an addressed initial approach. Pathogenic microbes subvert normal host-cell processes to create a specialized niche, which enhances their survival. A common and recurring target of pathogens is the host cell’s cytoskeleton, which is utilized by these microbes for purposes that include attachment, entry into cells, movement within and between cells, vacuole formation and remodeling, and avoidance of phagocytosis. Our increased understanding of these processes in recent years as well as in the next years will continue contributing to a greater comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology [5]. Regarding to how to understand better the interaction between the different E. coli pathotypes with their cell targets imply new approaches, including the study of clinical isolates of the different pathotypes (as a second step after studies in prototype strains, recognizing the E. coli diversity) and the use of relevant animal diseases models. Switching cultured cells to relevant animal disease models is crucial for understanding disease, yet such studies are often neglected, because cell-culture-based systems are easier to manipulate. However, currently it is possible to genetically engineer animal host to be susceptible to infection [6], as well as the re-engineering o the bacterial effector to extend the host range of this bacterium to include the ideal host (i.e. mice) [7]. These two approaches present an opportunity to probe the host–pathogen interface during disease [8]. On the other hand, new studies have showed that the host microbiota has a crucial role in mediating the outcome of disease, which adds another layer of complexity [9]. Thus, the knowledge in these different issues must be translated into a true understanding of disease. This remains the crucial challenge to all who are involved in this field. PREVENTION AND TREATMENT Additional clinical trials with antibiotics are needed to determine the efficacy of newly developed drugs in preventing or treating diarrheal episodes caused by antibiotic resistant strains of pathogenic E. coli. Scant knowledge of the clinical relevance of more recently described categories of pathogenic E. coli means that, in the cases where



antimicrobials are required, treatment protocols may not be optimal. For example, the use of antimicrobials for bloody diarrhea cases is contra-indicated when is associated with Shiga-toxin-producing E. coli infections because some evidence suggests that antibiotics increase the risk for HUS [10, 11]. In the majority of diarrhea episodes for which antimicrobials are not indicated, they are often prescribed because of the difficulty in distinguishing self-resolving infections from drug-indicated ones. Physicians and self-medicators would use less antimicrobial agents in this way if we have access to an early diagnosis and can determine which infections might be life-threatening. When antimicrobials are required, studies have shown that common E. coli pathotypes such as EPEC, ETEC, and EAEC are frequently resistant to almost all drugs available and affordable to patients in this part of the world so patients have no access to optimal treatment [12]. In the case of travelers diarrhea caused by ETEC and due to increase resistance, quinolones have replaced doxycycline and trimethoprim-sulphamethoxazole as drugs of choice, but quinolone resistance has since emerged and is increasing [13, 14]. The only promising alternative is rifamixin, a non-absorbable antimicrobial that can be used to treat infections by non-invasive E. coli pathotypes [15]. Availability and costs mean that rifamixin is presently limited to adjusting recommendations for travelers from affluent countries to compensate for the emergence of resistance, a niche occupied until now by ciprofloxacin. However, in the case of Shigella (similar to EIEC infections), which cause invasive dysentery, prevention of these invasive organisms from causing enteric infection, is relatively easier challenge for a non-absorbed antibiotic, like rifamixin, to be effective early during infection than treating enteric disease once the organisms have penetrated the mucosa and caused an inflammatory host response [16]. Other interesting alternative is the use of azithromycin, which has been demonstrated to be effective in the clinic for the treatment of shigellosis and it has a good in vitro activity against DECs; however, additional controlled clinical studies are needed to evaluate their use [17]. Continued study of the occurrence of functional diarrhea and other chronic gastrointestinal diseases is needed with an emphasis on the beneficial effects of prophylactic drugs on reducing these complications. An interesting novel therapeutic strategy is based on short-term inhibition of host Gb3 synthesis to reduce binding and uptake of Stx by host cells. Silberstein et al. [18] have recently shown that pre-treatment of human renal tubular epithelial cells for 48 h with C9 (Genzyme Corp.), a specific inhibitor of glucosylceramide synthase (the rate limiting first step in glycosphingolipid biosynthesis), caused a marked reduction in cellular Gb3 levels, as well as conferring a significant degree of resistance to Stx2-mediated cytotoxicity. The protective efficacy of C-9 was also demonstrated in an experimental model of Hemolytic Uremic Syndrome (HUS) in rats [19]. Glycosphingolipid inhibitors such as C-9 have been developed for treatment of glycosphingolipidoses such as Fabry and Tay-Sachs disease, but the findings of this study indicate that they may have utility to limit the progression of disease and development of HUS. Another new alternative therapeutic treatment is the use of intravenous humanized monoclonal antibodies anti-Shiga toxins 1 and 2 [20, 21]. The early administration during the initial phase of diarrhea by Shiga toxin-producing E. coli could neutralize the toxin in circulation and to prevent the HUS. Phase 2 clinical studies will star in the near future in Latin America to evaluate their efficacy. It is important to emphasize that their use required of an early diagnosis, identifying the subtypes of EHEC which constitute the major risk factor for development of HUS. Nutritional supplementation, in particular Zinc, has been proposed as a means for promoting small-bowel repair after infection, leading to shorter episodes of persistent diarrhea and resistance to reinfection. Some of these strategies could reduce the burden of disease from diarrheagenic E. coli. Preventing diarrheal episodes in the first place must be a primary goal [22]. VACCINATION Although significant advances have been made for vaccine research and development in some pathogenic E. coli categories, there still remain several important issues and challenges that need special attention. For example, the immunogenicity and efficacy of licensed vaccines, such as whole cell/recombinant B subunit (WC/rBS) oral ETEC vaccine, remain to be elucidated in infants, and they have yet to be fully implemented in areas where the disease is endemic, including Latin America [23, 24]. Vaccine candidates against enterotoxigenic E. coli and other


Gomes et al.

diarrheagenic E. coli strains also face serious challenges. For those candidate vaccines already in clinical trials, there are initial indications that the immunogenicity might be lower in infants in less developed countries than in those from industrialized countries, and, therefore, the clinical efficacy of these vaccines needs to be evaluated in infant populations in developing countries [24, 25]. Further, the diarrheagenic E. coli candidate vaccines also have to cope with the multiplicity of protective antigens which need to be included in the vaccine and with the geographical diversity of circulating serotypes of the bacteria. One additional problem is that the immune correlates of protection are not clearly understood for any of these intestinal pathogenic E. coli vaccines. The current problem in developing effective vaccines is complex, because it is necessary to create a multivalent vaccine of universal utility to those endemic regions where DECs are prevalent. It is necessary to understand the epidemiological reality of different geographic areas, to define which DEC pathotypes of mayor importance are, i.e., those associated with severe diarrhea and death. Then, the conserved immunologic epitopes need to be determined and included in a vaccine that ideally, it is administered by the oral route. Although many of these challenges remain to be overcome, the involvement of the public health sector and the high priority given to vaccine research and development in this field bode well for the future. Such a combined public health effort coupled with the extensive and active investigations being conducted by the academic and research community should finally bring hope for the control of diarrheal diseases in infants and young children around the globe. HEALTH PROMOTION AND EDUCATION Although several Latin American countries, through their Ministries of Health, have performed successful campaigns to educate people regarding the actions that have to be taken to reduce diarrheal cases caused by enteric organisms, several countries are not investing enough money or resources. For example, the recent earthquake in Haiti exposes some of the discrepancies in health and education that still persist in the American continent in the 21st century. The Caribbean Island Hispaniola, where the Dominican Republic and Haiti are located, has the lowest investment in public education in Latin America and the Caribbean [26]. As a result of unhygienic and cramped living conditions, the lack of access to health services, and risk-taking behavior, rural populations suffer high rates of persistent health problems including diarrhea, respiratory problems, and other infectious diseases. Limited access to education and generally poor educational quality has further exacerbated the marginalization of poor Latin American individuals. The Ministries of Health in each country need to initiate or continue promoting campaigns in critical areas like nutrition, preventive healthcare, water and sanitation, and education. In the area of preventive health, it is necessary to educate the population about health risks and providing them with the necessary tools to avoid these risks, including training on prevention of diarrheal diseases and the promotion of closer relations between community and health care providers. Further, it is important to emphasize improvement of family nutrition, with a particular focus on child health. As the gap between rich and poor in Latin America and the Caribbean countries continues to grow wider, bringing the benefits of education to the most disadvantaged children becomes progressively more difficult. Education cannot be effective if children in the region do not have access to adequate health care, good nutrition and live in a stable home environment. Therefore, a priority in the different countries needs to be the promotion of health, nutrition and early childhood development schemes within their education projects. REFERENCES [1] [2] [3] [4] [5] [6]

Ochoa TJ, Barletta F, Contreras C, et al. New insights into the epidemiology of enteropathogenic Escherichia coli infection. Trans R Soc Trop Med Hyg. 2008;102:852-6. Jones TF, Scallan E, Angulo FJ. FoodNet: overview of a decade of achievement. Foodborne Pathog Dis. 2007;4:60-6. Croxen MA, Finlay BB. Molecular mechanisms of Escherichia coli pathogenicity. Nat Rev Microbiol. 2010;8:26-38. Tseng TT, Tyler BM, Setubal JC. Protein secretion systems in bacterial-host associations, and their description in the Gene Ontology. BMC Microbiol. 2009;9:S2. Gruenheid S, Finlay BB. Microbial pathogenesis and cytoskeletal function. Nature. 2003;422:775-81. Lecuit M, Vandormael-Pournin S, Lefort J, et al. A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier. Science. 2001;292:1722–5.


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Wollert T, Pasche B, Rochon M, et al. Extending the host range of Listeria monocytogenes by rational protein design. Cell. 2007;129:891-902. Bhavsar AP, Guttman JA, Finlay BB. Manipulation of host-cell pathways by bacterial pathogens. Nature. 2007;449:82734. Lupp C, Robertson ML, Wickham ME, et al. Host-mediated inflammation disrupts the intestinal microbiota and promotes the overgrowth of Enterobacteriaceae. Cell Host Microbe. 2007;2:119–29. 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. 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. Erb A, Stürmer T, Marre R, et al. Prevalence of antibiotic resistance in Escherichia coli: overview of geographical, temporal, and methodological variations. Eur J Clin Microbiol Infect Dis. 2007;26:83-90. Minarini LA, Poirel L, Cattoir V, D et al. Plasmid-mediated quinolone resistance determinants among enterobacterial isolates from outpatients in Brazil. J Antimicrob Chemother. 2008;62:474-8. Rocha-Gracia R, Ruiz E, Romero-Romero S, et al. Detection of the plasmid-borne quinolone resistance determinant qepA1 in a CTX-M-15-producing Escherichia coli strain from Mexico. J Antimicrob Chemother. 2010;65:169-71. Koo HL, DuPont HL. Rifaximin: a unique gastrointestinal-selective antibiotic for enteric diseases. Curr Opin Gastroenterol. 2010;26:17-25. DuPont HL. Travellers' diarrhoea: contemporary approaches to therapy and prevention. Drugs. 2006;66:303-14. Cabada MM, White ACJ. Travelers' diarrhea: an update on susceptibility, prevention, and treatment. Curr Gastroenterol Rep. 2008;10:473-9. 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;1:71-5. Silberstein C, Lucero MS, Zottz E, et al. Effects of a glucosylceramide synthase inhibitor on an experimental model of Hemolytic Uremic Syndrome in rats.. 7th International Symposium of Shiga toxin (Verocytotoxin)-producing Escherichia coli infections 2009 10-13 May 2009; Buenos Aires. Argentina; 2009. Mukherjee J, Chios K, Fishwild D, et al. Production of and characterization of protective human antibodies against shiga toxin 1. Infect Immun. 2002;70:5896-9. 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. Walker RI. Considerations for development of whole cell bacterial vaccines to prevent diarrheal diseases in children in developing countries. Vaccine. 2005;23:3369-85. Girard MP, Steele D, Chaignat CL, et al. A review of vaccine research and development: human enteric infections. Vaccine. 2006;24:2732-50. Svennerholm A-M, Savarino SJ. Oral inactivated whole cell B subunit combination vaccine against enterotoxigenic Escherichia coli. In: Levine MM, Kaper JB, Rappuoli R, Liu MA, Good MF, editors. New Generation Vaccines. 3rd edn ed. New York: Marcel Dekker; 2004. DuPont HL. What's new in enteric infectious diseases at home and abroad. Curr Opin Infect Dis. 2005;18:407-12. Unknown. Programa de Promoción de la Reforma Educativa de América Latina y el Caribe (PREAL). 2006.

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Pathogenic Escherichia Coli in Latin America, 2010, 262-264

INDEX A Adherence EPEC 27-30 Antibiotic resistance reservoir 3-4 Autotransporters EPEC 33 B Bacterial intercellular communication 249 Bacterial communication and host interactions 249 C Characterization of diarrheagenic E. coli categories 99-100 Clinical aspects EPEC 35 Clinical management of E. coli: Introduction 116 Coliforms 2 Commensals 2-3 Concept of reservoirs 223 Complementary methods for diarrheagenic E. coli characterization 104-107 D DEC in Colombia 214-217 DEC in food, environment and animals, Mexico 199-201 DEC in human disease, Argentina 144-145 DEC in Mexico, conclusions 203-204 DEC in Peru 217-220 DEC in Uruguay 209-214 DEC pathotypes, Mexico 192-194 DEC treatment and antibiotic resistance, Mexico 202-203 Detection and presumptive identification 95-99 Detection and subtyping methods: Introduction 95 Diagnosis EPEC 36 Diagnosis ETEC 89 Diffusely adherent E. coli, Brazil 170-171 Diarrheagenic E. coli (DEC), Argentina: Introduction 142-144 Diarrheal diseases, Mexico: Introduction 191 Differential diagnosis 119 Diversity of virulence factors EPEC 34 Drug development 252-253 E EAEC, Argentina 156-157 EAEC, Brazil 162-164 E. coli O157:H7, Mexico 201-202 EHEC QS signaling cascade 252 Enteric pathogen 4-5 Alfredo G. Torres (Ed) All rights reserved - © 2010 Bentham Science Publishers Ltd.

Index

Enteroaggregative E. coli (EAEC): Introduction 48-49 Enteroinvasive E. coli (EIEC), Argentina 156 Enteropathogenic E. coli (EPEC): Introduction 25-26 Enterotoxigenic E. coli (ETEC): Introduction 84 EPEC, Brazil 164-166 EPEC in animals and food 156 EPEC reservoirs 235-240 Epidemiology 12-19 Epidemiology, Chile 179 Epidemiology, Mexico 194-198 Epidemiology EAEC 49-50 Epidemiology EPEC 34-35 Epidemiology STEC 72-74 Epidemiology STEC, Chile 180-182 Escherichia coli: the organism 1-2 ETEC, Argentina 153-156 ETEC, Brazil 167-168 ETEC reservoirs 230-231 Extraintestinal pathogenic E. coli reservoirs 231-235 Evolution 8-9 F Future in health promotion and education 260 Future in identification of novel pathogenic E. coli isolates 257-258 Future in prevention and treatment 258-259 Future in vaccination 259-260 Future of E. coli pathogenesis 258 Future of E. coli research: Introduction 256 H Host genetic factors ETEC 88 Host genetic susceptibility EAEC 57 I Immune responses EPEC 35-36 Implementing diagnostic test 256-257 Interaction with host intestine EHEC/EPEC and ETEC 122-126 Inter-kingdom communication 249-250 Invasion EPEC 33 M Methods for confirmation of diarrheagenic E. coli 99-104 Mucus hypersecretion 34 N Nosological classification 117 P Pathogenesis EAEC 50-57

Pathogenic Escherichia Coli in Latin America 263

264 Pathogenic Escherichia Coli in Latin America

Pathogenesis EPEC 27 Pathogenesis STEC 67-68 Pathotypes 10-12 Pediatric diarrhea ETEC 84 Pediatric infections ETEC 84-85 Perspectives related to STEC, Chile 187 Prognosis and sequelae 119-120 R Regulation EPEC 33 Reservoirs and transmission EPEC 35 S Serodiagnosis of STEC infections 109 Serotypes EPEC 26 Shiga toxin-producing E. coli (STEC): Introduction 65-66 Signaling systems 250-252 STEC, Argentina 145-153 STEC, Brazil 168-170 STEC food transmitted disease, Chile 183-185 STEC, reservoirs 223-230 STEC HUS D+ 118-119 STEC infections 66-67 Strategies for STEC control, Chile 186-187 Subtyping methods 107-108 Systemic host response during HUS 126-133 T Travellers’ diarrhea ETEC 85 Travellers’ diarrhea, Mexico 198-199 Treatment and control STEC 74-75 Treatment and prophylaxis EAEC 57-58 Treatment and prophylaxis EPEC 36-37 Treatment ETEC 89-90 Type III secretion system EPEC 30-33 V Vaccine EAEC 58 Vaccine ETEC 90-91 Virulence determinants acquisition 9-10 Virulence factors STEC, Chile 182-183 Virulence factors STEC 68-72 Virulence factors ETEC 85-88

Alfredo G. Torres

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