Serotyping, stx2 Subtyping, and Characterization of the Locus of Enterocyte Effacement Island of Shiga Toxin-Producing Escherichia coli and E. coli O157:H7 Strains Isolated from the Environment in France

Twenty-seven Shiga toxin-producing Escherichia coli (STEC) strains were isolated from 207 stx-positive French environmental samples. Ten of these strains were positive for stx₁, and 24 were positive for stx₂ (10 were positive for stx_2vh-a or stx_2vh-b, 19 were positive for stx_2d, and 15 were positive for stx_2e). One strain belonged to serotype O157:H7, and the others belonged to serogroups O2, O8, O11, O26, O76, O103, O113, O121, O141, O166, and O174. The environment is a reservoir in which new clones of STEC that are pathogenic for humans can emerge.     

Phenotypic and Genotypic Characterization of Biofilm Forming Capabilities in Non-O157 Shiga Toxin-Producing Escherichia coli Strains

The biofilm life style helps bacteria resist oxidative stress, desiccation, antibiotic treatment, and starvation. Biofilm formation involves a complex regulatory gene network controlled by various environmental signals. It was previously shown that prophage insertions in mlrA and heterogeneous mutations in rpoS constituted major obstacles limiting biofilm formation and the expression of extracellular curli fibers in strains of Escherichia coli serotype O157:H7. The purpose of this study was to test strains from other important serotypes of Shiga toxin-producing E. coli (STEC) (O26, O45, O103, O111, O113, O121, and O145) for similar regulatory restrictions. In a small but diverse collection of biofilm-forming and non-forming strains, mlrA prophage insertions were identified in only 4 of the 19 strains (serotypes O103, O113, and O145). Only the STEC O103 and O113 strains could be complemented by a trans-copy of mlrA to restore curli production and Congo red (CR) dye affinity. RpoS mutations were found in 5 strains (4 serotypes), each with low CR affinity, and the defects were moderately restored by a wild-type copy of rpoS in 2 of the 3 strains attempted. Fourteen strains in this study showed no or weak biofilm formation, of which 9 could be explained by prophage insertions or rpoS mutations. However, each of the remaining five biofilm-deficient strains, as well as the two O145 strains that could not be complemented by mlrA, showed complete or nearly complete lack of motility. This study indicates that mlrA prophage insertions and rpoS mutations do limit biofilm and curli expression in the non-serotype O157:H7 STEC but prophage insertions may not be as common as in serotype O157:H7 strains. The results also suggest that lack of motility provides a third major factor limiting biofilm formation in the non-O157:H7 STEC. Understanding biofilm regulatory mechanisms will prove beneficial in reducing pathogen survival and enhancing food safety.     

Presence and Characterization of Shiga Toxin-Producing Escherichia coli and Other Potentially Diarrheagenic E. coli Strains in Retail Meats

To determine the presence of Shiga toxin-producing Escherichia coli (STEC) and other potentially diarrheagenic E. coli strains in retail meats, 7,258 E. coli isolates collected by the U.S. National Antimicrobial Resistance Monitoring System (NARMS) retail meat program from 2002 to 2007 were screened for Shiga toxin genes. In addition, 1,275 of the E. coli isolates recovered in 2006 were examined for virulence genes specific for other diarrheagenic E. coli strains. Seventeen isolates (16 from ground beef and 1 from a pork chop) were positive for stx genes, including 5 positive for both stx₁ and stx₂, 2 positive for stx₁, and 10 positive for stx₂. The 17 STEC strains belonged to 10 serotypes: O83:H8, O8:H16, O15:H16, O15:H17, O88:H38, ONT:H51, ONT:H2, ONT:H10, ONT:H7, and ONT:H46. None of the STEC isolates contained eae, whereas seven carried enterohemorrhagic E. coli (EHEC) hlyA. All except one STEC isolate exhibited toxic effects on Vero cells. DNA sequence analysis showed that the stx₂ genes from five STEC isolates encoded mucus-activatable Stx2d. Subtyping of the 17 STEC isolates by pulsed-field gel electrophoresis (PFGE) yielded 14 distinct restriction patterns. Among the 1,275 isolates from 2006, 11 atypical enteropathogenic E. coli (EPEC) isolates were identified in addition to 3 STEC isolates. This study demonstrated that retail meats, mainly ground beef, were contaminated with diverse STEC strains. The presence of atypical EPEC strains in retail meat is also of concern due to their potential to cause human infections.Escherichia coli is an important component of the intestinal microflora of humans and warm-blooded mammals. While E. coli typically harmlessly colonizes the intestinal tract, several E. coli clones have evolved the ability to cause a variety of diseases within the intestinal tract and elsewhere in the host. Those strains that cause enteric infections are generally called diarrheagenic E. coli strains, and their pathogenesis is associated with a number of virulence attributes, which vary according to pathotype (54). Currently, diarrheagenic E. coli strains are classified into six main pathotypes based on their distinct virulence determinants and pathogenic features, including enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC)/Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and diffusively adherent E. coli (DAEC) (37).Among diarrheagenic E. coli strains, STEC strains are distinguished by the ability to cause severe life-threatening complications, such as hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) (30). Other symptoms of STEC infection include watery diarrhea, bloody diarrhea, and hemorrhagic colitis (HC). STEC strains that cause HC and HUS are also called EHEC. Although individuals of all ages are at risk of STEC infection, children younger than 5 years of age and the elderly are more likely to suffer from severe complications (51). Outbreaks and sporadic cases of STEC infections have been reported frequently worldwide.The pathogenesis of STEC infection in humans is not fully understood. The major virulence factors implicated in STEC infection are potent Shiga toxins, which are classified into two groups: Stx1 and Stx2 (23). Additional factors that contribute to virulence have also been described, including intimin (encoded by the eae gene), an outer membrane protein involved in the attachment of E. coli to the enterocyte, and EHEC hemolysin (encoded by EHEC hlyA), which acts as a pore-forming cytolysin and causes damage to cells (41).The first STEC O157 infections were reported in 1982, when E. coli O157:H7 was involved in outbreaks associated with two fast food chain restaurants in the United States (44). Since then, ever-increasing numbers of cases and outbreaks due to STEC O157 have been reported worldwide. Although non-O157 STEC strains have also been associated with human cases and outbreaks, few laboratories have been looking for them, and their potential in causing human infections may be underestimated (2). Recently, though, the significance of non-O157 STEC strains as human pathogens has become more recognized. In the United States alone, there were 23 reported outbreaks of non-O157 STEC infection between 1990 and 2007 (10).Shiga toxin-producing E. coli can be transmitted through different routes, including food and water, person-to-person contact, and animal-to-person contact (9). Most human infections are caused by consumption of contaminated foods (16). Domestic and wild ruminant animals, in particular cattle, are considered the main reservoir of STEC and the main source for contamination of the food supply. Retail meats derived from animals could potentially act as transmission vehicles for STEC and other diarrheagenic E. coli strains. However, there is limited information about STEC contamination in retail meats, and fewer data exist about the presence of other diarrheagenic E. coli strains in retail meats. In the present study, we investigated 7,258 E. coli isolates from four types of meat samples (beef, chicken, pork, and turkey) collected during 2002 to 2007 to assess STEC contamination of retail meats. In addition, the presence of other potentially diarrheagenic E. coli strains was examined by detecting specific virulence determinants among E. coli isolates collected in 2006.     

Novel Microarray Design for Molecular Serotyping of Shiga Toxin-Producing Escherichia coli Strains Isolated from Fresh Produce

Serotyping Escherichia coli is a cumbersome and complex procedure due to the existence of large numbers of O- and H-antigen types. It can also be unreliable, as many Shiga toxin-producing E. coli (STEC) strains isolated from fresh produce cannot be typed by serology or have only partial serotypes. The FDA E. coli identification (FDA-ECID) microarray, designed for characterizing pathogenic E. coli, contains a molecular serotyping component, which was evaluated here for its efficacy. Analysis of a panel of 75 reference E. coli strains showed that the array correctly identified the O and H types in 97% and 98% of the strains, respectively. Comparative analysis of 73 produce STEC strains showed that serology and the array identified 37% and 50% of the O types, respectively, and that the array was able to identify 16 strains that could not be O serotyped. Furthermore, the array identified the H types of 97% of the produce STEC strains compared to 65% by serology, including six strains that were mistyped by serology. These results show that the array is an effective alternative to serology in serotyping environmental E. coli isolates.     

Comparison of Shiga Toxin Production by Hemolytic-Uremic Syndrome-Associated and Bovine-Associated Shiga Toxin-Producing Escherichia coli Isolates

There is considerable diversity among Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria, and only a subset of these organisms are thought to be human pathogens. The characteristics that distinguish STEC bacteria that give rise to human disease are not well understood. Stxs, the principal virulence determinants of STEC, are thought to account for hemolytic-uremic syndrome (HUS), a severe clinical consequence of STEC infection. Stxs are typically bacteriophage encoded, and their production has been shown to be enhanced by prophage-inducing agents such as mitomycin C in a limited number of clinical STEC isolates. Low iron concentrations also enhance Stx production by some clinical isolates; however, little is known regarding whether and to what extent these stimuli regulate Stx production by STEC associated with cattle, the principal environmental reservoir of STEC. In this study, we investigated whether toxin production differed between HUS- and bovine-associated STEC strains. Basal production of Stx by HUS-associated STEC exceeded that of bovine-associated STEC. In addition, following mitomycin C treatment, Stx2 production by HUS-associated STEC was significantly greater than that by bovine-associated STEC. Unexpectedly, mitomycin C treatment had a minimal effect on Stx1 production by both HUS- and bovine-associated STEC. However, Stx1 production was induced by growth in low-iron medium, and induction was more marked for HUS-associated STEC than for bovine-associated STEC. These observations reveal that disease-associated and bovine-associated STEC bacteria differ in their basal and inducible Stx production characteristics.     

Serotypes and Virulence Gene Profiles of Shiga Toxin-Producing Escherichia coli Strains Isolated from Feces of Pasture-Fed and Lot-Fed Sheep

Shiga toxin-producing Escherichia coli (STEC) strains possessing genes for enterohemolysin (ehxA) and/or intimin (eae), referred to here as complex STEC (cSTEC), are more commonly recovered from the feces of humans with hemolytic uremic syndrome and hemorrhagic colitis than STEC strains that do not possess these accessory virulence genes. Ruminants, particularly cattle and sheep, are recognized reservoirs of STEC populations that may contaminate foods destined for human consumption. We isolated cSTEC strains from the feces of longitudinally sampled pasture-fed sheep, lot-fed sheep maintained on diets comprising various combinations of silage and grain, and sheep simultaneously grazing pastures with cattle to explore the diversity of cSTEC serotypes capable of colonizing healthy sheep. A total of 67 cSTEC serotypes were isolated, of which 21 (31.3%), mainly isolated from lambs, have not been reported. Of the total isolations, 58 (86.6%) were different from cSTEC serotypes isolated from a recent study of longitudinally sampled healthy Australian cattle (M. Hornitzky, B. A. Vanselow, K. Walker, K. A. Bettelheim, B. Corney, P. Gill, G. Bailey, and S. P. Djordjevic, Appl. Environ. Microbiol. 68:6439-6445, 2002). Our data suggest that cSTEC serotypes O5:H⁻, O75:H8, O91:H⁻, O123:H⁻, and O128:H2 are well adapted to colonizing the ovine gastrointestinal tract, since they were the most prevalent serotypes isolated from both pasture-fed and lot-fed sheep. Collectively, our data show that Australian sheep are colonized by diverse cSTEC serotypes that are rarely isolated from healthy Australian cattle.     

Diversity of Shiga Toxin-Producing Escherichia coli (STEC) O26:H11 Strains Examined via stx Subtypes and Insertion Sites of Stx and EspK Bacteriophages

Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen that may be responsible for severe human infections. Only a limited number of serotypes, including O26:H11, are involved in the majority of serious cases and outbreaks. The main virulence factors, Shiga toxins (Stx), are encoded by bacteriophages. Seventy-four STEC O26:H11 strains of various origins (including human, dairy, and cattle) were characterized for their stx subtypes and Stx phage chromosomal insertion sites. The majority of food and cattle strains possessed the stx_1a subtype, while human strains carried mainly stx_1a or stx_2a. The wrbA and yehV genes were the main Stx phage insertion sites in STEC O26:H11, followed distantly by yecE and sbcB. Interestingly, the occurrence of Stx phages inserted in the yecE gene was low in dairy strains. In most of the 29 stx-negative E. coli O26:H11 strains also studied here, these bacterial insertion sites were vacant. Multilocus sequence typing of 20 stx-positive or stx-negative E. coli O26:H11 strains showed that they were distributed into two phylogenetic groups defined by sequence type 21 (ST21) and ST29. Finally, an EspK-carrying phage was found inserted in the ssrA gene in the majority of the STEC O26:H11 strains but in only a minority of the stx-negative E. coli O26:H11 strains. The differences in the stx subtypes and Stx phage insertion sites observed in STEC O26:H11 according to their origin might reflect that strains circulating in cattle and foods are clonally distinct from those isolated from human patients.     

Use of Antibody Responses against Locus of Enterocyte Effacement (LEE)-Encoded Antigens To Monitor Enterohemorrhagic Escherichia coli Infections on Cattle Farms

Enterohemorrhagic Escherichia coli (EHEC) is a significant zoonotic pathogen causing severe disease associated with watery and bloody diarrhea, hemorrhagic colitis, and the hemolytic-uremic syndrome (HUS) in humans. Infections are frequently associated with contact with EHEC-contaminated ruminant feces. Both natural and experimental infection of cattle induces serum antibodies against the LEE-encoded proteins intimin, EspA, EspB, and Tir and the Shiga toxins Stx1 and Stx2, although the latter are poorly immunogenic in cattle. We determined whether antibodies and/or the kinetics of antibody responses against intimin, Tir, EspA, and/or EspB can be used for monitoring EHEC infections in beef cattle herds in order to reduce carcass contamination at slaughter. We examined the presence of serum antibodies against recombinant O157:H7 E. coli intimin EspA, EspB, and Tir during a cross-sectional study on 12 cattle farms and during a longitudinal time course study on two EHEC-positive cattle farms. We searched for a possible correlation between intimin, Tir, EspA, and/or EspB antibodies and fecal excretion of EHEC O157, O145, O111, O103, or O26 seropathotypes. The results indicated that serum antibody responses to EspB and EspA might be useful for first-line screening at the herd level for EHEC O157, O26, and most likely also for EHEC O103 infections. However, antibody responses against EspB are of less use for monitoring individual animals, since some EHEC-shedding animals did not show antibody responses and since serum antibody responses against EspB could persist for several months even when shedding had ceased.     

Low-Density Macroarray Targeting Non-Locus of Enterocyte Effacement Effectors (nle Genes) and Major Virulence Factors of Shiga Toxin-Producing Escherichia coli (STEC): a New Approach for Molecular Risk Assessment of STEC Isolates

Rapid and specific detection of Shiga toxin-producing Escherichia coli (STEC) strains with a high level of virulence for humans has become a priority for public health authorities. This study reports on the development of a low-density macroarray for simultaneously testing the genes stx₁, stx₂, eae, and ehxA and six different nle genes issued from genomic islands OI-122 (ent, nleB, and nleE) and OI-71 (nleF, nleH1-2, and nleA). Various strains of E. coli isolated from the environment, food, animals, and healthy children have been compared with clinical isolates of various seropathotypes. The eae gene was detected in all enteropathogenic E. coli (EPEC) strains as well as in enterohemorrhagic E. coli (EHEC) strains, except in EHEC O91:H21 and EHEC O113:H21. The gene ehxA was more prevalent in EHEC (90%) than in STEC (42.66%) strains, in which it was unequally distributed. The nle genes were detected only in some EPEC and EHEC strains but with various distributions, showing that nle genes are strain and/or serotype specific, probably reflecting adaptation of the strains to different hosts or environmental niches. One characteristic nle gene distribution in EHEC O157:[H7], O111:[H8], O26:[H11], O103:H25, O118:[H16], O121:[H19], O5:H−, O55:H7, O123:H11, O172:H25, and O165:H25 was ent/espL2, nleB, nleE, nleF, nleH1-2, nleA. (Brackets indicate genotyping of the flic or rfb genes.) A second nle pattern (ent/espL2, nleB, nleE, nleH1-2) was characteristic of EHEC O103:H2, O145:[H28], O45:H2, and O15:H2. The presence of eae, ent/espL2, nleB, nleE, and nleH1-2 genes is a clear signature of STEC strains with high virulence for humans.Since the early 1980s, Shiga toxin-producing Escherichia coli (STEC) has emerged as a major cause of food-borne infections (17, 30). STEC can cause diarrhea in humans, and some STEC strains may cause life-threatening diseases, such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). On the basis of its human pathogenicity, this subset of STEC strains was also designated enterohemorrhagic E. coli (EHEC) (22, 25). Numerous cases of HC and HUS have been attributed to EHEC serotype O157:H7 strains, but it has now been recognized that other serotypes of STEC belong to the EHEC group. The STEC seropathotype classification is based upon the serotype association with human epidemics, HUS, and diarrhea and has been developed as a tool to assess the clinical and public health risks associated with non-O157 EHEC and STEC strains (18). Only a few serotypes of STEC have been reported as most frequently associated with severe disease in humans. Besides E. coli O157:[H7], five other serotypes, namely O26:[H11], O103:H2, O111:[H8], O121:[H19], and O145:[H28], account for the group of typical EHEC (25). (Brackets indicate genotyping of the flic or rfb genes; the absence of brackets indicates data obtained with the conventional serotyping approach using specific antisera, as described in Materials and Methods.) Atypical EHEC group strains of serotypes O91:[H21], O113:H21, and O104:H21 are less frequently involved in hemorrhagic diseases than typical EHEC but are a frequent cause of diarrhea (8, 12, 25). Recent data from Enter-Net, a global surveillance consortium of 35 countries that tracks enteric infectious diseases, showed that the number of human cases of illness caused by non-O157 EHEC increased globally by 60.5% between 2000 and 2005, while at the same time the number of cases linked to EHEC O157 increased by only 13% (1). In the past few years, new serotypes of EHEC that differ from those previously known as typical and atypical EHEC have emerged (6, 8, 23, 24, 31). These EHEC strains were identified as important causes of food-borne infections in humans and were described as “new emerging EHEC.”The production of Shiga toxin (Stx) by EHEC is the primary virulence trait responsible for HUS, but many E. coli non-O157:H7 strains that produce Stx do not cause HUS. Identification of human-virulent STEC by detection of unique stx genes may be misleading, since not all STEC strains are clinically significant for humans (11). Besides the ability to produce one or more types of Shiga toxins, typical EHEC strains harbor a genomic island called the “locus of enterocyte effacement” (LEE). Atypical EHEC strains are negative for the LEE but may carry other factors for colonization of the human intestine (6, 25). The LEE carries genes encoding functions for bacterial colonization of the gut and for destruction of the intestinal mucosa, thus contributing to the disease process (25). The LEE eae gene product intimin is directly involved in the attaching and effacing (A/E) process (37). The LEE includes regulatory elements, a type III secretion system (TTSS), secreted effector proteins, and their cognate chaperon (13, 29). In addition to the intimin, most of the typical EHEC strains harbor the plasmid-borne enterohemolysin (ehxA), which is considered an associated virulence factor (6, 25).A number of other pathogenicity island (PAI) candidates, including O island 122 (OI-122) and O island 71 (OI-71), have been found in EHEC and EPEC strains, but their role in disease is not fully clear. Within the EHEC group, both O157:H7 strains (19, 34) and non-O157 strains (18, 35) present a variable repertoire of virulence determinants, including a collection of non-LEE-encoded effector (nle) genes that encode translocated substrates of the type III secretion system (9, 20). Our objective was to identify type III secreted virulence factors that distinguish EHEC O157 and non-O157 strains constituting a severe risk for human health from STEC strains that are not associated with severe and epidemic disease, a concept called “molecular risk assessment” (MRA) by Coombes et al. (9). Supporting the MRA approach requires the development of diagnostic tests based on multiplex nucleic acid amplification and microfluidics-based detection using standardized platforms applicable in hospital service or public health laboratories. It is now feasible to develop low-density DNA arrays that can be used to examine the gene inventory from isolated strains, offering a genetic bar coding strategy. A recent innovation in this field is the introduction of the GeneSystems PCR technology (5, 36). In this study, we have developed a GeneDisc array designed for simultaneous detection of genes encoding Shiga toxins 1 and 2 (stx₁ and stx₂), intimins (eae), enterohemolysin (ehxA), and six different nle genes derived from genomic islands OI-71 and OI-122. We focused our efforts on the detection of the OI-122 genes, ent/espL2 (Z4326), nleB (Z4328), and nleE (Z4329), and the OI-71 genes, nleF (Z6020), nleH1-2 (Z6021), and nleA (Z6024). The macroarray presented here was evaluated for its specificity and ability to discriminate between STEC causing serious illness in humans and other E. coli strains.     

Fates of Acid-Resistant and Non-Acid-Resistant Shiga Toxin-Producing Escherichia coli Strains in Ruminant Digestive Contents in the Absence and Presence of Probiotics

Healthy ruminants are the main reservoir of Shiga toxin-producing Escherichia coli (STEC). During their transit through the ruminant gastrointestinal tract, STEC encounters a number of acidic environments. As all STEC strains are not equally resistant to acidic conditions, the purpose of this study was to investigate whether acid resistance confers an ecological advantage to STEC strains in ruminant digestive contents and whether acid resistance mechanisms are induced in the rumen compartment. We found that acid-resistant STEC survived at higher rates during prolonged incubation in rumen fluid than acid-sensitive STEC and that they resisted the highly acidic conditions of the abomasum fluid, whereas acid-sensitive strains were killed. However, transit through the rumen contents allowed acid-sensitive strains to survive in the abomasum fluid at levels similar to those of acid-resistant STEC. The acid resistance status of the strains had little influence on STEC growth in jejunal and cecal contents. Supplementation with the probiotic Saccharomyces cerevisiae CNCM I-1077 or Lactobacillus acidophilus BT-1386 led to killing of all of the strains tested during prolonged incubation in the rumen contents, but it did not have any influence in the other digestive compartments. In addition, S. cerevisiae did not limit the induction of acid resistance in the rumen fluid. Our results indicate that the rumen compartment could be a relevant target for intervention strategies that could both limit STEC survival and eliminate induction of acid resistance mechanisms in order to decrease the number of viable STEC cells reaching the hindgut and thus STEC shedding and food contamination.Shiga toxin-producing Escherichia coli (STEC) strains are food-borne pathogens that cause human diseases ranging from uncomplicated diarrhea to hemorrhagic colitis (HC), as well as life-threatening complications, such as hemolytic-uremic syndrome (HUS). Most outbreaks and sporadic cases of HC and HUS have been attributed to O157:H7 STEC (http://www.cdc.gov/ecoli/outbreaks.html; http://www.euro.who.int). However, in some geographic areas, non-O157:H7 STEC infections are considered to be at least as important as E. coli O157:H7 infections, but they are often underdiagnosed (21, 46). In spite of diverse virulence characteristics, one common trait of pathogenic STEC strains could be resistance to the gastric acidity in humans. Indeed, it has been suggested that acid resistance of E. coli O157:H7 is negatively correlated with the infectious dose required for this organism to cause disease in humans (17).Healthy cattle and other ruminants appear to be the main reservoir of STEC strains. However, colonization of the cattle gastrointestinal tract (GIT) by STEC seems to be a transient event, with a mean duration of 14 days to 1 month (4, 8, 38). The site of STEC persistence and proliferation in the GIT depends on the STEC strain and seems to vary from one individual to another. Some previous studies identified the rumen as the primary site of colonization (8), whereas other studies referred to the cecum, the colon, or the rectum (10, 18, 23, 32, 42). Although STEC strains adhere in vitro to bovine colonic mucosa, forming the characteristic attaching and effacing lesions (35), they are very rarely associated with tissues in animal carriers and are generally isolated from the digesta (8). STEC does not, therefore, seem to colonize the gut mucosa, except for the anorectal mucosa, which has been described as the preferred colonization site for O157:H7 strains but not for non-O157:H7 strains (24, 32). During their transit through the ruminant GIT, STEC strains encounter various acidic conditions. Volatile fatty acid (VFA) concentrations are high in the rumen of grain-fed animals, and the pH may vary from 5.0 to 6.5. In these conditions, VFAs are in the undissociated form and can freely enter the bacterial cells, dissociate, and acidify the cytosol. In hay-fed animals, less fermentation occurs in the rumen, and the pH remains between 6.5 and 7. In the abomasum, STEC encounters strongly acidic conditions, regardless of the diet, due to the presence of mineral acids, resulting in a pH below 3. Then the pH increases from the proximal part to the distal part of the small intestine, and in the cecum and the colon STEC encounters more neutral pH conditions.All STEC strains are not equally resistant to acidic conditions (2, 9, 30, 45). Therefore, it could be hypothesized that acid-resistant (AR) STEC survives and persists better in the GIT of ruminants than acid-sensitive (AS) STEC. Acid resistance mechanisms can be induced during exposure to a moderately acidic environment (12, 26, 41). The rumen contents of a grain-fed animal could be such an environment favorable for the induction of acid resistance in STEC. While the diet does not seem to affect the acid resistance of an E. coli O157:H7 strain (19), grain feeding increases the number of acid-resistant generic coliforms (15, 19), either by inducing acid resistance mechanisms in the rumen or by selecting acid-resistant E. coli strains during passage through the abomasum. Hence, generic coliforms behave differently than E. coli O157:H7 in ruminants (19), and the potential ecological advantage conferred by acid resistance to non-O157:H7 STEC strains for persistence in the ruminant GIT has never been investigated.Inhibition of STEC proliferation in the ruminant gut may be mediated through probiotic supplementation. Several studies have demonstrated the capacity of certain lactic acid bacteria or yeast to reduce E. coli O157:H7 counts in vitro (1, 34) or in vivo (5, 40). The mechanisms of action of probiotics are not well characterized but could involve competition for nutrients and adhesion sites in the GIT, an increase in the VFA concentration and a decrease in the pH, production of antimicrobial molecules, or interference with quorum-sensing signaling (27-29). However, the impact of probiotics on non-O157:H7 STEC has been poorly investigated (36). Although not all non-O157:H7 STEC strains are pathogenic, limiting their carriage by ruminants should decrease the risk of food-borne illness. The impact of probiotics and of the physicochemical conditions of the rumen digesta on the survival of non-O157:H7 STEC strains or on induction of acid resistance mechanisms could have significant implications for farm management practices and food safety.The purpose of this work was to investigate whether the level of acid resistance, determined using an in vitro assay, confers an ecological advantage to STEC strains in ruminant digestive contents and whether acid resistance mechanisms are induced in the rumen compartment. Moreover, we evaluated the potential of probiotics to limit STEC survival and induction of acid resistance in the ruminant GIT.     

Horizontal Transmission of Shiga Toxin-Producing Escherichia coli within Groups of Dairy Calves

To examine the dissemination of Shiga-toxigenic Escherichia coli (STEC) within cattle groups, dairy calves on two farms utilizing different calf-rearing practices were exposed to a traceable STEC strain. Test strain dissemination differed significantly between farms, with a higher prevalence being associated with group penning. Pen floors and calf hides may be the main environmental mechanisms of transmission. Dairy calf husbandry represents a control point for reducing on-farm STEC prevalence.     

Biofilm-Forming Abilities of Shiga Toxin-Producing Escherichia coli Isolates Associated with Human Infections

Forming biofilms may be a survival strategy of Shiga toxin-producing Escherichia coli to enable it to persist in the environment and the food industry. Here, we evaluate and characterize the biofilm-forming ability of 39 isolates of Shiga toxin-producing Escherichia coli isolates recovered from human infection and belonging to seropathotypes A, B, or C. The presence and/or production of biofilm factors such as curli, cellulose, autotransporter, and fimbriae were investigated. The polymeric matrix of these biofilms was analyzed by confocal microscopy and by enzymatic digestion. Cell viability and matrix integrity were examined after sanitizer treatments. Isolates of the seropathotype A (O157:H7 and O157:NM), which have the highest relative incidence of human infection, had a greater ability to form biofilms than isolates of seropathotype B or C. Seropathotype A isolates were unique in their ability to produce cellulose and poly-N-acetylglucosamine. The integrity of the biofilms was dependent on proteins. Two autotransporter genes, ehaB and espP, and two fimbrial genes, z1538 and lpf2, were identified as potential genetic determinants for biofilm formation. Interestingly, the ability of several isolates from seropathotype A to form biofilms was associated with their ability to agglutinate yeast in a mannose-independent manner. We consider this an unidentified biofilm-associated factor produced by those isolates. Treatment with sanitizers reduced the viability of Shiga toxin-producing Escherichia coli but did not completely remove the biofilm matrix. Overall, our data indicate that biofilm formation could contribute to the persistence of Shiga toxin-producing Escherichia coli and specifically seropathotype A isolates in the environment.     

Diverse Virulence Gene Content of Shiga Toxin-Producing Escherichia coli from Finishing Swine

Shiga toxin-producing Escherichia coli (STEC) infections are a critical public health concern because they can cause severe clinical outcomes, such as hemolytic uremic syndrome, in humans. Determining the presence or absence of virulence genes is essential in assessing the potential pathogenicity of STEC strains. Currently, there is limited information about the virulence genes carried by swine STEC strains; therefore, this study was conducted to examine the presence and absence of 69 virulence genes in STEC strains recovered previously from finishing swine in a longitudinal study. A subset of STEC strains was analyzed by pulsed-field gel electrophoresis (PFGE) to examine their genetic relatedness. Swine STEC strains (n = 150) were analyzed by the use of a high-throughput real-time PCR array system, which included 69 virulence gene targets. Three major pathotypes consisted of 16 different combinations of virulence gene profiles, and serotypes were determined in the swine STEC strains. The majority of the swine STEC strains (n = 120) belonged to serotype O59:H21 and carried the same virulence gene profile, which consisted of 9 virulence genes: stx_2e, iha, ecs1763, lpfA_O113, estIa (STa), ehaA, paa, terE, and ureD. The eae, nleF, and nleH1-2 genes were detected in one swine STEC strain (O49:H21). Other genes encoding adhesins, including iha, were identified (n = 149). The PFGE results demonstrated that swine STEC strains from pigs raised in the same finishing barn were closely related. Our results revealed diverse virulence gene contents among the members of the swine STEC population and enhance understanding of the dynamics of transmission of STEC strains among pigs housed in the same barn.     

Wild Ungulates as Disseminators of Shiga Toxin-Producing Escherichia coli in Urban Areas

Background

In 2008, children playing on a soccer field in Colorado were sickened with a strain of Shiga toxin-producing Escherichia coli (STEC) O157:H7, which was ultimately linked to feces from wild Rocky Mountain elk. We addressed whether wild cervids were a potential source of STEC infections in humans and whether STEC was ubiquitous throughout wild cervid populations in Colorado.

Methodology/Principal Findings

We collected 483 fecal samples from Rocky Mountain elk and mule deer in urban and non-urban areas. Samples testing positive for STEC were higher in urban (11.0%) than non-urban (1.6%) areas. Elk fecal samples in urban areas had a much higher probability of containing STEC, which increased in both urban and non-urban areas as maximum daily temperature increased. Of the STEC-positive samples, 25% contained stx1 strains, 34.3% contained stx2, and 13% contained both stx1 and stx2. Additionally, eaeA genes were detected in 54.1% of the positive samples. Serotypes O103, and O146 were found in elk and deer feces, which also have the potential to cause human illness.

Conclusions/Significance

The high incidence of stx2 strains combined with eaeA and E-hyl genes that we found in wild cervid feces is associated with severe human disease, such as hemolytic uremic syndrome. This is of concern because there is a very close physical interface between elk and humans in urban areas that we sampled. In addition, we found a strong relationship between ambient temperature and incidence of STEC in elk feces, suggesting a higher incidence of STEC in elk feces in public areas on warmer days, which in turn may increase the likelihood that people will come in contact with infected feces. These concerns also have implications to other urban areas where high densities of coexisting wild cervids and humans interact on a regular basis.     

Variation in Acid Resistance among Shiga Toxin-Producing Clones of Pathogenic Escherichia coli

Pathogenic strains of Escherichia coli, such as E. coli O157:H7, have a low infectious dose and an ability to survive in acidic foods. These bacteria have evolved at least three distinct mechanisms of acid resistance (AR), including two amino acid decarboxylase-dependent systems (arginine and glutamate) and a glucose catabolite-repressed system. We quantified the survival rates for each AR mechanism separately in clinical isolates representing three groups of Shiga toxin-producing E. coli (STEC) clones (O157:H7, O26:H11/O111:H8, and O121:H19) and six commensal strains from ECOR group A. Members of the STEC clones were not significantly more acid resistant than the commensal strains when analyzed using any individual AR mechanism. The glutamate system provided the best protection in a highly acidic environment for all groups of isolates (<0.1 log reduction in CFU/ml per hour at pH 2.0). Under these conditions, there was notable variation in survival rates among the 30 O157:H7 strains, which depended in part on Mg²⁺ concentration. The arginine system provided better protection at pH 2.5, with a range of 0.03 to 0.41 log reduction per hour, compared to the oxidative system, with a range of 0.13 to 0.64 log reduction per hour. The average survival rate for the O157:H7 clonal group was significantly less than that of the other STEC clones in the glutamate and arginine systems and significantly less than that of the O26/O111 clone in the oxidative system, indicating that this clonal group is not exceptionally acid resistant with these specific mechanisms.     

Dual-Serotype Biofilm Formation by Shiga Toxin-Producing Escherichia coli O157:H7 and O26:H11 Strains

Escherichia coli O26:H11 strains were able to outgrow O157:H7 companion strains in planktonic and biofilm phases and also to effectively compete with precolonized O157:H7 cells to establish themselves in mixed biofilms. E. coli O157:H7 strains were unable to displace preformed O26:H11 biofilms. Therefore, E. coli O26:H11 remains a potential risk in food safety.     

The Sinorhizobium meliloti Insertion Sequence (IS) Element ISRm14 Is Related to a Previously Unrecognized IS Element Located Adjacent to the Escherichia coli Locus of Enterocyte Effacement (LEE) Pathogenicity Island

ISRm14 is 2695 basepairs (bp) in size and bordered by 22 bp imperfect inverted repeats (IRs). A 9-bp target sequence is duplicated upon ISRm14 transposition. The DNA strand that putatively encodes the transposase enzyme carries three open reading frames (ORFs) designated ORFs1 to 3, which specify putative proteins of 15.9 kDa, 13.1 kDa, and 61.1 kDa, respectively. According to its structural characteristics, ISRm14 belongs to the recently proposed IS66 family of IS elements. The ORFs1 to 3 encoded putative proteins displayed significant similarities to ORFs of the previously unrecognized IS element ISEc8, which is inserted adjacent to the locus of enterocyte effacement (LEE) pathogenicity island of Escherichia coli EDL933. Analyses of the distribution of ISRm14 in a natural S. meliloti population showed its widespread occurrence in 66% of the strains tested with a copy number ranging from 1 to 6. Received: 13 May 1999 / Accepted: 14 June 1999     

Heterogeneity of Shiga Toxin-Producing Escherichia coli Strains Isolated from Hemolytic-Uremic Syndrome Patients, Cattle, and Food Samples in Central France

A detailed analysis of the molecular epidemiology of non-O157:H7 Shiga toxin-producing Escherichia coli (STEC) was performed by using isolates from sporadic cases of hemolytic-uremic syndrome (HUS), animal reservoirs, and food products. The isolates belonged to the O91 and OX3 serogroups and were collected in the same geographical area over a short period of time. Five typing methods were used; some of these were used to explore potentially mobile elements like the stx genes or the plasmids (stx₂-restriction fragment length polymorphism [RFLP], stx₂ gene variant, and plasmid analyses), and others were used to study the whole genome (ribotyping and pulsed-field gel electrophoresis [PFGE]). The techniques revealed that there was great diversity among the O91 and OX3 STEC strains isolated in central France. A close relationship between strains of the same serotype having the same virulence factor pattern was first suggested by ribotyping. However, stx₂-RFLP and stx₂ variant analyses differentiated all but 5 of 21 isolates, and plasmid analysis revealed further heterogeneity; a unique combination of characteristics was obtained for all strains except two O91:H21 isolates from beef. The latter strains were shown by PFGE to be the most closely related isolates, with >96% homology, and hence may be subtypes of the same strain. Overall, our results indicate that the combination of stx₂-RFLP, stx₂ variant, and plasmid profile analyses is as powerful as PFGE for molecular investigation of STEC diversity. Finally, the non-O157:H7 STEC strains isolated from HUS patients were related to but not identical to those isolated from cattle and food samples in the same geographical area. The possibility that there are distinct lineages of non-O157:H7 STEC, some of which are more virulent for humans, should be investigated further.