Enterohemorrhagic Escherichia coli and Other Shiga Toxin-Producing E. coli

The term enteropathogenic Escherichia coli was originally used to refer to strains belonging to a limited number of O groups epidemiologically associated with infantile diarrhea ( 1 ). Subsequently, E. coli strains isolated from intestinal diseases have been grouped into at least six main categories on the basis of epidemiological evidence, phenotypic traits, clinical features of the disease they produce, and specific virulence factors. The well-described intestinal pathotypes or categories of diarrheagenic E. coli groups are enteropathogenic E. coli (EPEC), Shiga toxin-producing E. coli (STEC) or verocytotoxin-producing E. coli (VTEC) (including enterohemorrhagic E. coli [EHEC]), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli, and diffusely adherent E. coli. The general definition of an E. coli pathotype as “a group of strains of a single species that cause a common disease using a common set of virulence factors” ( 2 ) has been further refined for STEC to help assess the clinical and public health risks associated with different STEC strains ( 3 ). An empirical classification scheme was used to classify STEC serotypes into five “seropathotypes” (A through E) according to the reported association of serotypes with human intestinal disease, outbreaks, and hemolytic-uremic syndrome (HUS) ( 3 ). This classification system uses a gradient ranging from seropathotype A (high risk) to seropathotypes D and E (minimal risk). This definition has been of considerable value in cases of human infection but is also problematic because the majority of isolates from STEC infections are not fully characterized and coupled to reliable clinical information. Although the definition of HUS is distinct, the spectrum of diarrheal disease varies considerably and may include a range of symptoms from nonbloody to scanty blood to true hemorrhagic colitis. Additionally, the use of A through E adds confusion because Shiga toxin subtypes are also named alphabetically. Most importantly, the concept of (sero)-pathotypes collides with the requirements of a good taxonomy, which separates elements of each group into subgroups that are mutually exclusive, unambiguous, and, together, include all possibilities. In practice, a good taxonomy should be simple to apply, easy to remember, and easy to use. The need to define human pathogenic STEC and to identify factors of STEC that absolutely predict the potential to cause human disease is obvious in terms of clinical management, supportive or antibiotic treatment, quarantine measurements, risk assessment, surveillance, and outbreak investigations and management. This chapter presents a brief history of the concept of pathotypes and describes the possible alternatives for categorizing STEC based on phenotypic or molecular typing.

Shiga toxin (Stx) is one of the most potent biological poisons known. Stx causes fluid accumulation in rabbit ileal loops; causes renal damage in mice, rabbits, greyhounds, and baboons; and is lethal to animals upon injection. However, humans encounter Stx as a consequence of infection with Shigella dysenteriae type 1 or certain serogroups of Escherichia coli such as O157:H7. There are two immunologically distinct groups of Stxs, and this review discusses toxin classification, structure, and function and the virulence associated with Stx-producing E. coli (STEC).

O157:H7 is the most common enterohemorrhagic Escherichia coli (EHEC) serotype in North America, and it has been the principal causative agent of numerous food-poisoning outbreaks worldwide ( 1 , 2 ). Initially E. coli O157:H7 was recognized as a human pathogen in 1982 when it was isolated from 47 persons in two states who had developed bloody diarrhea after consuming hamburgers contaminated with this organism ( 3 ). Since then, E. coli O157:H7 has emerged as a major enteric pathogen, capable of causing localized infections and large outbreaks of gastrointestinal disease ( 4 ). Data accumulated from 1982 to 1996 showed that approximately two-thirds of the 3,000 cases of E. coli infections from 139 recognized outbreaks were associated with the ingestion of contaminated food products, whereas 22% of the reported cases were from direct person-to-person transmission and 10% were from drinking water ( 5 ). Surveillance data have demonstrated a high prevalence of E. coli O157:H7 among cattle and their environment, but a relatively low incidence of human infection. This supports the potential hypothesis that a subset of E. coli O157:H7 harbored in cattle may be responsible for the majority of human disease ( 6 ). To minimize or eradicate adverse effects on public health, the E. coli O157:H7 lineage has been the focus of numerous epidemiological, microbiological, genomic, forensic, and diagnostic studies. Overall, it is estimated that E. coli O157:H7 alone causes more than 76,000 infections and 61 deaths in humans due to severe complications annually in the United States ( 7 ). Symptoms include a range of gastrointestinal morbidities, such as severe abdominal cramping accompanied with little or no associated fever and a watery diarrhea that leads to severe bloody diarrhea ( 8 ). Although many infected individuals remain asymptomatic, approximately 15 to 20% of people infected with EHEC present severe enough symptoms to require hospitalization. In such severe cases, patients display renal dysfunction known as hemolytic-uremic syndrome (HUS), hemorrhagic colitis, and central nervous system failure with potentially lethal outcomes ( 9 – 11 ).

It is generally accepted that all actions of Shiga toxin (Stx) depend on its interaction with the receptor, globotriaosylceramide (Gb3), on eukaryotic cells. Although alternative receptors for Stx have been postulated, no definitive data have been forthcoming in support. Stx holotoxin is internalized by receptor-mediated endocytosis, retrograde transported via the Golgi apparatus and processed through in the endoplasmic reticulum, and released into the cytoplasm where it enzymatically inactivates ribosomes and inhibits protein synthesis ( Fig. 1 ). However, it is important to note that, in addition to Stx holotoxin, the B-subunit alone can interact with Gb3 in a physiologically meaningful manner where it activates signal transduction pathways in target cells ( Fig. 1 ) ( 1 ). An additional but unexplained anomaly is the interaction of Stx with eukaryotic cells in a Gb3-independent manner that leads to induction of cytokines by these cells ( 2 ). As shown in Fig. 1 , intracellular responses to Stx are diverse, including inhibition of protein synthesis, activation of cellular stress responses, and induction of cytokines and chemokines. It is likely that these different schemes take place in cell-specific activities during Shiga toxin-producing Escherichia coli (STEC) infections in humans, culminating in typical hemolytic-uremic syndrome (HUS). As depicted, it is clear that in some cases Stx can result in activation of p38 mitogen-activated protein kinase as well as apoptotic and necrotic cell death ( Fig. 1 ). The topic of HUS renal disease has been reviewed recently ( 3 – 5 ).

Enterohemorrhagic Escherichia coli (EHEC) was first recognized as a cause of human disease in 1983 and is associated with diarrhea and hemorrhagic colitis, which may be complicated by life-threatening renal and neurological sequelae (reviewed in reference 242 ). EHEC strains are defined by their ability to produce one or more Shiga toxins (Stx), which mediate the systemic complications of EHEC infections (reviewed in reference 243 ), and to induce attaching and effacing (A/E) lesions on intestinal epithelia. The ability of EHEC to induce such lesions is shared by enteropathogenic E. coli (EPEC), Escherichia albertii (formerly classified as eae-positive Hafnia alvei), and the murine pathogen Citrobacter rodentium. The A/E histopathology was first described in gnotobiotic piglets challenged with a strain of EHEC serotype O157:H7 ( 1 ) but has subsequently been observed in ruminant reservoirs and diverse animal models (reviewed in reference 244 ).

Among the thousands of bacterial species contained within the intestinal gut flora, it is accepted that each species requires the use of adhesin proteins, or some combination thereof, that bring the bacteria closer to the epithelia and allow them to colonize the intestine. In a similar way, enteric pathogens also require surface-localized adhesins for colonization of the host intestine and eventual establishment of disease. Enterohemorrhagic Escherichia coli (EHEC) and, in general, Shiga toxin-producing E. coli (STEC) strains are known to contain a large number of proteins responsible for adhesion and contribute to establishment, persistence, and tissue tropism observed during infection with these pathogens. Understanding how these adhesins work is critical to having a full picture of the pathogenic and pathophysiological process associated with EHEC. Further, because adhesins play such an important role in virulence, they are targets for therapeutic intervention. Thus, this review summarizes the current knowledge on the adhesive proteins in EHEC, emphasizing up-to-date information and discussing gaps in knowledge and future directions in the study of these virulence factors.

Since the first recognized Escherichia coli O157:H7 outbreak over 3 decades ago ( 1 ), investigators have sought to identify suitable animal hosts that allow study of enterohemorrhagic E. coli (EHEC)-mediated disease. The value of any animal infection model ultimately relies on its ability to reproduce the human disease and enable the mechanistic processes that lead to clinical disease, pathogen carriage, and transmission to be examined. As yet, no single animal model mimics the full spectrum of disease caused by EHEC infection. However, since Moxley and Francis's review in 1998 ( 2 ), several advances have been made in the field, including the generation of a Shiga toxin (Stx)-producing Citrobacter rodentium-murine model, a human intestine xenograft murine model, and a renewed interest in the use of rabbit models. This article reviews what is known about EHEC-mediated disease from human outbreaks and biopsy studies, and within a historical context, describes the features and limitations of EHEC infection models that are based on the three most commonly used species (pigs, rabbits, and mice). Recent new advances are highlighted and discussed in light of mounting evidence for the need to study the biology and virulence strategies of EHEC in the context of its niche within the intestine. The reader is directed elsewhere for excellent reviews on the environmental sources of EHEC infection ( 3 ), EHEC interactions with the intestinal epithelium ( 4 ), the molecular basis of pathogenicity ( 5 – 8 ), and the current status of treatment options ( 9 ).

As a species, Escherichia coli is highly successful, adapting to inhabit the lower intestine of warm-blooded animals. Commensal E. coli, part of the normal biota, resides harmlessly in the gut, producing vitamin K. However, E. coli also causes three types of disease in humans: urinary tract infections, sepsis in newborns, and diarrheal disease. Enterohemorrhagic E. coli (EHEC) plays a prominent role in the third type of illness. It has been estimated that, for the pan genome of E. coli, the nonpathogenic and pathogenic strains only contain a core set of genes comprising approximately 20% of any one genome ( 1 , 2 ). Much of the horizontally acquired genetic information is clustered within genomic islands in pathogens. As for EHEC, this has allowed the organism to not only attach and colonize the large intestine of humans and other animals, to outcompete commensal E. coli and other bacteria at the site of infection, but also to cause serious disease.

A series of outbreaks of infection with Shiga toxin (or verotoxin [VT])-producing Escherichia coli or enterohemorrhagic E. coli (EHEC) O157:H7 occurred in Japan in 1996, the largest outbreak occurring in primary schools in Sakai City, Osaka Prefecture, where more than 7,500 cases were reported ( 1 ). Although the reason for the sudden increase in the number of reports of EHEC isolates in 1996 is not known, the number of reports has grown to more than 3,000 cases per year since 1996 from an average of 105 cases reported each year during the previous 5-year period (1991–1995) ( 2 ). Despite control measures instituted since 1996, including designating EHEC infection as a notifiable disease, and the disease being monitored effectively through nationwide surveillance, the number of reports remains high, around 3,800 cases per year ( Fig. 1 ). Serogroup O157 predominates over other EHEC serogroups, but isolation frequency of non-O157 EHEC has gone up slightly over the past few years. Non-O157 EHEC has caused outbreaks where consumption of a raw beef dish was the source of the infection and some fatal cases were occurred. Laboratory surveillance consisting of prefectural and municipal public health institutes (PHIs) and the National Institute of Infectious Diseases has contributed to finding not only multiprefectural outbreaks but also recognizing sporadic cases that could have been missed as an outbreak without the aid of molecular subtyping of EHEC isolates. This short overview presents recent information on the surveillance of EHEC infections in Japan.

Escherichia coli strains that carry Shiga toxin genes are commonly isolated from the gastrointestinal tract of a wide variety of animal species ( Table 1 ). Intestinal carriage of most Shiga toxin-producing E. coli (STEC) strains by domestic and wild animals has little clinical relevance to either the animal hosts or humans. Most animals lack receptors for Shiga toxin, and in humans, the presence of additional virulence factors, in addition to the stx gene, is associated with disease outcomes ( 1 – 3 ). However, animals may harbor STEC strains that are pathogenic to humans. This article focuses on the role of animals as reservoirs for infection or as spillover hosts. Within the animal, these bacteria may be resident or transient in the gastrointestinal tract. Determining whether STEC is resident in flora or transient is not possible during cross-sectional observational epidemiological studies when only one sample is collected from an animal and there is no serial testing. Even under experimental conditions it is difficult to determine if repeated isolation from the feces over time is a result of replication of the organism in the animal or repeated exposure.

The worldwide trends for healthier lifestyles to reduce obesity and other complications arising from unhealthy diets have greatly increased the consumption of fresh fruits and vegetables. This increased demand, coupled with ever busier consumer lifestyles, also stimulated the growth of a “convenience” food industry and popularized the concept of bagged salad vegetables and fruits. It has been estimated that several millions of bags of fresh produce are sold daily in the United States. Bagged produce, also referred to as “fresh cut” or “precut,” is often regarded as ready-to-eat (RTE) and consumed without further intervention steps. However, because produce is predominantly cultivated in soil in open fields, it is susceptible to contamination and can contain high levels of complex microbial populations, occasionally including bacterial pathogens. As a result, increases in fresh produce demand and consumption coupled with changes in production practices have also contributed to increases in incidents of food-borne illness. In the United States, about 0.7% of the infections in the 1970s were attributed to fresh produce, but this increased to 6% in the 1990s ( 1 ). Since “fresh cut” products are often mass produced, broadly distributed, and marketed worldwide, a single pathogen contamination event can have broadly impacting consequences, and several large, produce-related outbreaks have occurred in many countries ( 2 , 3 ). In 2006, a large multistate outbreak in the United States that infected more than 200 persons was traced to bagged spinach contaminated with Escherichia coli O157:H7 ( 4 ). Several months later, another O157:H7 outbreak in a fast-food restaurant chain had initially implicated green onions but appeared to have been due to bagged lettuce. At about the same time, bagged lettuce was implicated in another O157:H7 outbreak that affected three states ( 5 ). Likewise, increased consumption of sprouts caused several outbreaks of Salmonella sp., E. coli O157:H7, and other Shigatoxin-producing E. coli (STEC) strains. STEC serotype O26:H11 strains caused an outbreak with alfalfa sprouts and, more recently, with clover sprouts, and the large outbreak of O104:H4 in 2011 in the European Union also implicated the consumption of sprouts ( 6 ). These large produce-related outbreak incidents worldwide have greatly raised concerns about the safety of fresh produce and about the microbiological and sanitary quality of fresh produce.

Shiga toxin-producing Escherichia coli (STEC) represents a major issue for public health because of the capability to cause large outbreaks and the severity of the associated illnesses ( 1 ). STEC strains are the only pathogenic group of E. coli that has a definite zoonotic origin, with ruminants and, in particular, cattle being recognized as the major reservoir for human infections ( 2 ). Most human infections are food borne, but the routes of transmission include direct contact with animals and a wide variety of environment-related exposures ( 3 ). Therefore, STEC public health microbiology spans the fields of medical, veterinary, food, water, and environmental microbiology, requiring a “One Health” perspective ( 4 ) and laboratory scientists with the ability to work effectively across disciplines. Public health microbiology laboratories play a central role in the surveillance of STEC infections, as well as in the preparedness for responding to outbreaks and in providing scientific evidence for the implementation of prevention and control measures. This article reviews in depth (i) how the integration of surveillance of STEC infections and monitoring of these pathogens in animal reservoirs and potential food vehicles may contribute to their control; (ii) the role of reference laboratories; and (iii) the public health perspectives, including those related to regulatory issues in both the European Union and the United States.

Shiga toxin (verotoxin)-producing Escherichia coli (STEC) strains were first described in 1977 by their ability to cause cytotoxic effects on Vero cells ( 1 ). In the early days, production of Shiga toxin (Stx) by E. coli was thought to be associated with certain human enteropathogenic E. coli (EPEC) strains ( 1 – 3 ). STEC was recognized as a zoonotic agent when the first known outbreaks occurred in 1982. STEC O157:H7, a rare serotype of E. coli, was isolated from patients developing hemorrhagic colitis (HC) after they ingested undercooked beef in restaurant chains ( 4 ). STEC O157 was isolated from the incriminated beef, indicating a possible transmission from a bovine reservoir. In the following years cattle were identified as a worldwide natural reservoir for STEC O157 and non-O157 strains and as an important source of food contamination ( 5 – 8 ). Repeated sampling of cattle revealed that the agent was occasionally present in the majority of cattle farms in Europe and America ( 9 ). However, recent findings on the epidemiology of enteroaggregative Shiga toxin-producing E. coli (EAEC-STEC) O104:H4 indicate that not all STEC strains have a zoonotic origin ( 10 , 11 ).

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause illness with a spectrum of severity ranging from mild (even asymptomatic) carriage to life-threatening disease ( 1 – 3 ). STEC infections are relatively uncommon; in the United States, extrapolation of data from FoodNet ( 4 ) to a nationwide population that exceeds 300,000,000 indicates there are fewer than 4,000 diagnosed cases of E. coli O157:H7 infection per annum. E. coli O157:H7 remains the near-exclusive cause of hemolytic-uremic syndrome (HUS) throughout most of the world, and the single serotype on which most data have been generated. Therefore, we emphasize this particular pathogen in this article. The European Food Safety Authority and the European Centre for Disease Prevention and Control report similar epidemiology: 4,000 confirmed infections caused by Stx-producing E. coli strains (mostly belonging to the O157 serogroup) in 27 European Union member states. The number of reported infections attributed to E. coli strains that produce Shiga toxins has increased since 2008 ( 5 ).

The mammalian host is equipped with two major types of immune response, innate and adaptive, that are essential for effective control and elimination of infectious agents. The innate immune system is the first line of host defense against invading microbial pathogens and is promptly activated by the recognition of pathogen-associated molecular patterns, such as lipopolysaccharide (LPS), flagellin, peptidoglycan, and CpG DNA ( 1 ). Pathogen-associated molecular patterns are recognized by specialized germline-encoded pattern recognition receptors (PRRs) expressed by immune cells. To date, a number of PRR families have been described, including the Toll-like receptors (TLRs), retinoic acid-inducible gene-I-like receptors, and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) ( 2 , 3 ). The first and most significant consequence of PRR-mediated pathogen recognition in the host is the rapid production of proinflammatory cytokines that stimulates the innate immune response ( 2 , 3 ). In addition, the innate immune response directs the development of the more specific and long-term adaptive response to a particular pathogen, mediated by B and T cells.

Shiga toxin (Stx)-producing Escherichia coli (STEC) colonizes the intestine and causes hemorrhagic colitis. STEC encodes a variety of colonization factors, but a significant subset of STEC, the enterohemorrhagic E. coli (EHEC) strains, have the locus of enterocyte effacement (LEE), the products of which allow the bacteria to intimately adhere to and form attaching and effacing lesions on intestinal tissue. The O157:H7 strains, which are responsible for the majority of large outbreaks due to STEC infection, are members of the EHEC group. All STEC strains make one or more Stxs; these pathogens may produce two immunologically distinct but highly similar Stxs, Stx1 and Stx2. These toxins are briefly described in the section on therapeutics targeted to the Stxs.

Shiga toxin-producing Escherichia coli (STEC) strains emerged in the late 1970s or early 1980s as highly significant zoonotic threats to public health. In 1982, two outbreaks of severe bloody diarrhea, related to a previously rare serotype of E. coli, O157:H7, were reported in the United States ( 1 ).

Bacterial exotoxins may cause damage to host cells by defined mechanisms. Depending on the presence of the globotriaosylceramide (Gb3) receptor, Shiga toxin may bind to cells and induce the ribotoxic stress response and apoptosis ( 1 , 2 ). The toxin can also induce a proinflammatory response in cells, an effect that may be dissociated from ribosome inactivation and can even occur in cells lacking protein synthesis machinery. Bacterial lipopolysaccharide (LPS) induces a host response by binding to Toll-like receptor 4 (TLR4) and activating specific intracellular pathways. The activation of proinflammatory pathways, if excessive, promotes damage to the host. This article addresses enterohemorrhagic Escherichia coli (EHEC) pathogenesis and the host response, examining the innate and adaptive immune responses to the bacteria and virulence factors and how they affect the process of colonization, transfer and transport of virulence factors in the circulation, activation of thrombosis and inflammation, and specific end-organ damage to the kidney and the brain.

The human gastrointestinal (GI) tract harbors trillions of bacterial cells belonging to more than 1,000 species ( 1 ), and the number of bacterial cells within the GI tract is 10 times higher than the number of human cells within our bodies ( 2 ). The GI microbiota plays essential roles in human nutrition, physiology, development, immunity, and behavior, with disruption of the structure and balance of this community leading to dysbiosis and disease ( 3 – 5 ). This fundamental association between host and bacteria relies on chemical signaling and nutrient availability and exchange. It is also clear that this important balance between host and microbiota can be severely disrupted by environmental stimuli. Two of the most common insults on the microbiota that induce dysbiosis are antibiotic treatment and infectious diseases. Both insults can lead to several disease states ranging from autism, to inflammatory bowel disease, to inflammatory bowel syndrome (IBS). It is also noteworthy that stress exacerbates these syndromes ( 3 ).

Upton Sinclair's novel, The Jungle, which described horrific conditions in historical Chicago meat packing plants, engendered numerous reforms and regulations of the industry, including the Pure Food and Drug Act and the Meat Inspection Act of 1906, which in turn led to vast improvements in the sanitary conditions under which meat and meat products were handled. The massive and highly publicized 1993 outbreak of Escherichia coli O157 associated with Jack in the Box had a similar broad impact for the microbiological safety of food, including the classification of this pathogen as an “adulterant” in ground beef, and led to the implementation of the formal Pathogen Reduction and Hazard Analysis and Critical Control Point Program for this bacterium and other food-borne agents in meat processing plants. These changes were credited with significant reduction in the incidence of human infection with E. coli O157 in the United States over the subsequent several years; however, this trend did not continue, and in recent years the incidence of disease due to E. coli O157 has remained stubbornly stable. Incidence of disease caused by non-O157 Shiga toxin-producing E. coli (STEC) has paradoxically steadily increased, although this trend is undoubtedly due in part to increased use of more efficient diagnostic procedures.

Based on outbreak data acquired from 1998 to 2008 by the U.S. Centers for Disease Control and Prevention (CDC), known food-borne etiological agents were estimated to have caused 4,589 outbreaks, 9,638,301 cases, 57,462 hospitalizations, and 1,451 deaths per year in the United States ( 1 ). From these data, it was estimated that 482,199 cases (5.0%), 2,650 hospitalizations (0.03%), and 51 deaths (0.0005%) were attributable to bacteria consumed from beef. Of the 4,589 outbreaks, 103 (2.2%) and 3 (0.065%) were further attributable to beef-acquired Shiga toxin-producing Escherichia coli (STEC) O157 and non-O157 strains. Of the outbreaks caused by STEC O157 and non-O157 STEC strains for all food commodities combined, 103 of 186 (55.3%) and 3 of 6 (50%), respectively, were attributable to beef. On the basis of data acquired from the CDC from 2000 to 2008, of 9,388,075 cases of domestically acquired food-borne illness in the United States caused by 31 major pathogens, 63,153 (0.67%) were due to STEC O157 and 112,752 (1.20%) were due to non-O157 STEC infection ( 2 ). In the same study, of 35,796 hospitalizations, 2,138 (5.97%) were due to STEC O157 and 271 (0.76%) were due to non-O157 STEC; of 861 deaths, 20 (2.32%) were due to STEC O157 and 0 were due to non-O157 STEC infection.

It is well documented that animals and, in particular, ruminants can carry a range of potentially harmful pathogens, including verocytoxigenic Escherichia coli (VTEC), in their gastrointestinal tract. VTEC can reportedly survive for several months in the environment, in feces, and in soil, which allows for the recycling of VTEC among food animals and wildlife and prolonged environmental contamination. VTEC contamination of fresh produce may arise from irrigation water, manure or compost applied to soil as a fertilizer, and feces of wildlife or farmed animals. Table 1 summarizes some of the diverse VTEC outbreaks over the past few years (2006–2013). Of significant interest is that apart from the newly emerging vehicles of infection, serogroups other than O157 are increasingly causing outbreaks, many of which have severe outcomes with cases of hemolytic-uremic syndrome (HUS) and fatalities. While outbreaks are important and gain notoriety, the contribution of sporadic cases of human VTEC infection cannot be ignored. The data show that although foods of animal origin such as meat and dairy products and fresh produce such as salads and vegetables are well recognized important vectors of infection, there have also been VTEC outbreaks related to direct contact with fecal matter through recreational activities including visiting petting zoos, attending agricultural fairs, and swimming in contaminated water ( 1 ). With so many routes of potential transmission, it is clear that the management of this pathogen requires a multidisciplinary approach with cooperation among the disciplines of human medicine, animal and veterinary sciences, and environmental and food scientists. A veterinary public health approach to managing VTEC focuses on collation of data on prevalence of VTEC in different sources, the importance of each source as a reservoir of human pathogen VTEC, and risk factors for transmission to humans.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a food-borne pathogen that can lead to complications such as hemorrhagic colitis and hemolytic-uremic syndrome (HUS), serious sequelae. In the United States, the most common E. coli serotype causing outbreaks is O157:H7, although non-O157 serotypes also cause the same disease, but in much fewer cases. The highest incidence rate is among children of preschool age ( 1 , 2 ).

Human infection with Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) is relatively rare, but the consequences can be serious, especially in the very young and the elderly. Outcomes associated with STEC O157 infection include hemorrhagic colitis, renal failure, and death ( 1 – 5 ). In 2012, the overall laboratory-confirmed annual incidence of STEC O157 in the United States was 1.1 cases per 100,000 population ( 6 ). However, the incidence in children less than 5 years of age was 4.7 cases per 100,000 population ( 6 ).

In May 2011, an outbreak caused by Escherichia coli of serotype O104:H4 spread throughout Germany ( 1 ). The next month, France also reported a cluster of E. coli O104:H4 infections ( 2 ). A total of 46 deaths, 782 cases of hemolytic-uremic syndrome (HUS), and 3,128 cases of acute gastroenteritis were officially attributed to this new clone of enterohemorrhagic E. coli (EHEC) (last update from European Centre for Disease Prevention and Control, 27 July 2011). Most or all victims (although diagnosed in different countries in Europe) became infected in Germany or France. The phenotypic and genotypic characterization of the E. coli O104:H4 indicated that the isolates from the French and German outbreaks were common to both incidents. Fenugreek seeds imported from Egypt, from which sprouts were grown, were implicated as a common source. However, there is still much uncertainty about whether this is truly the common cause of the infections, as tests on the seeds did not allow the detection of any E. coli isolate of serotype O104:H4.

We have observed a vertical leap into our understanding of EHEC’s virulence. In the past edition of this book, the locus of enterocyte effacement (LEE) and its encoded type 3 secretion system (T3SS) had been recently discovered ( 1 , 2 ). However, few effectors were known at the time, with Tir ( 3 ) and intimin ( 4 ) dominating research on the molecular mechanisms involved in the formation of attaching and effacing (AE) lesions. Structural insights into T3SS came later, with the description of the EscF needle ( 5 ) and the EspA filament ( 6 ) forming the unique translocon of the EHEC and EPEC T3SSs. The number of effectors quickly expanded from the six LEE-encoded effectors, to the first hints that effectors encoded outside of the LEE existed ( 7 ), to the large expansion of their repertoire ( 8 ). Next-generation sequencing of many EHEC genomes also highlighted the fact that different strains of EHEC and enteropathogenic E. coli (EPEC) carry different combination of these effectors ( 9 ). Vigorous research was initially devoted to understanding the mechanism through which EHEC engaged the actin cytoskeleton to form AE lesions. These studies involved Tir and intimin interactions, but also extensive studies in the EspFu/TccP effectors ( 3 , 10 – 16 ). More recently, studies of non-AE-related effectors and their role in more discrete actin rearrangements, as well in modulation of the host immune response, have taken the front seat ( 17 – 24 ). Looking forward, we need to understand how different combinations of T3SS effectors affect the virulence potential of EHEC strains. We are also starting to study the hierarchy of secretion of these effectors ( 25 , 26 ) and how they work in concert. Knowledge of which effectors are acting within a mammalian cell at any given time, and how their functions amplify or antagonize their phenotypes, is the next frontier in understanding the role of these proteins in the bacterial/host interplay. Another still unresolved issue is how the T3SS is regulated to shift from secreting the translocon proteins (EspA, EspB, and EspD) to secreting bona fide effectors within epithelial cells. There is also the question of how the EspA filament is disassembled during the infection process to allow the close contact between the bacteria and the host. Finally, the big question that remains open is, how does EHEC cause diarrhea in the human intestine, and which are the main players in this disease process?