Persistent Circulation of Enterohemorrhagic Escherichia coli (EHEC) O157:H7 in Cattle Farms: Characterization of Enterohemorrhagic Escherichia coli O157:H7 Strains and Fecal Microbial Communities of Bovine Shedders and Non-shedders.

Cattle are carriers, without clinical manifestations, of enterohemorrhagic Escherichia coli (EHEC) O157:H7 responsible for life-threatening infections in humans. A better identification of factors playing a role in maintaining persistence of such strains in cattle is required to develop more effective control measures. Hence, we conducted a study to identify farms with a persistent circulation of EHEC O157:H7. The EHEC O157:H7 herd status of 13 farms, which had previously provided bovine EHEC O157:H7 carriers at slaughter was investigated. Two farms were still housing positive young bulls, and this was true over a 1-year period. Only one fecal sample could be considered from a supershedder, and 60% of the carriers shed concentrations below 10 MPN/g. Moreover, EHEC O157:H7 represented minor subpopulations of E. coli. PFGE analysis of the EHEC O157:H7 strains showed that persistent circulation was due either to the persistence of a few predominant strains or to the repeated exposure of cattle to various strains. Finally, we compared fecal microbial communities of shedders (S) (n = 24) and non-shedders (NS) (n = 28), including 43 young bulls and nine cows, from one farm. Regarding alpha diversity, no significant difference between S vs. NS young bulls (n = 43) was observed. At the genus level, we identified 10 amplicon sequence variant (ASV) indicators of the S or NS groups. The bacterial indicators of S belonged to the family XIII UCG-001, Slackia, and Campylobacter genera, and Ruminococcaceae NK4A21A, Lachnospiraceae-UGC-010, and Lachnospiraceae-GCA-900066575 groups. The NS group indicator ASVs were affiliated to Pirellulaceae-1088-a5 gut group, Anaerovibrio, Victivallis, and Sellimonas genera. In conclusion, the characteristics enhancing the persistence of some predominant strains observed here should be explored further, and studies focused on mechanisms of competition among E. coli strains are also needed.

Role of the Nitric Oxide Reductase NorVW in the Survival and Virulence of Enterohaemorrhagic Escherichia coli during Infection.

Enterohaemorrhagic Escherichia coli (EHEC) are bacterial pathogens responsible for life-threatening diseases in humans, such as hemolytic and uremic syndrome. It has been previously demonstrated that the interplay between EHEC and nitric oxide (NO), a mediator of the host immune innate response, is critical for infection outcome, since NO affects both Shiga toxin (Stx) production and adhesion to enterocytes. In this study, we investigated the role of the NO reductase NorVW in the virulence and fitness of two EHEC strains in a murine model of infection. We determined that the deletion of norVW in the strain O91:H21 B2F1 has no impact on its virulence, whereas it reduces the ability of the strain O157:H7 620 to persist in the mouse gut and to produce Stx. We also revealed that the fitness defect of strain 620 ΔnorVW is strongly attenuated when mice are treated with an NO synthase inhibitor. Altogether, these results demonstrate that the NO reductase NorVW participates in EHEC resistance against NO produced by the host and promotes virulence through the modulation of Stx synthesis. The contribution of NorVW in the EHEC infectious process is, however, strain-dependent and suggests that the EHEC response to nitrosative stress is complex and multifactorial.

Interplay between enterohaemorrhagic Escherichia coli and nitric oxide during the infectious process.

Enterohaemorrhagic Escherichia coli (EHEC) are bacterial pathogens responsible for life-threatening diseases in humans such as bloody diarrhoea and the hemolytic and uremic syndrome. To date, no specific therapy is available and treatments remain essentially symptomatic. In recent years, we demonstrated in vitro that nitric oxide (NO), a major mediator of the intestinal immune response, strongly represses the synthesis of the two cardinal virulence factors in EHEC, namely Shiga toxins (Stx) and the type III secretion system, suggesting NO has a great potential to protect against EHEC infection. In this study, we investigated the interplay between NO and EHEC in vivo using mouse models of infection. Using a NO-sensing reporter strain, we determined that EHEC sense NO in the gut of infected mice. Treatment of infected mice with a specific NOS inhibitor increased EHEC adhesion to the colonic mucosa but unexpectedly decreased Stx activity in the gastrointestinal tract, protecting mice from renal failure. Taken together, our data indicate that NO can have both beneficial and detrimental consequences on the outcome of an EHEC infection, and underline the importance of in vivo studies to increase our knowledge in host-pathogen interactions.

Paediatric haemolytic uraemic syndrome related to Shiga toxin-producing Escherichia coli, an overview of 10 years of surveillance in France, 2007 to 2016.

IntroductionHaemolytic uraemic syndrome (HUS) related to Shiga toxin-producing Escherichia coli (STEC) is the leading cause of acute renal failure in young children. In France, HUS surveillance in children aged < 15 years was implemented starting from 1996.AimWe present the results of this surveillance between 2007 and 2016.MethodsA voluntary nationwide network of 32 paediatric departments notifies cases. Two national reference centres perform microbiological STEC confirmation.ResultsOver the study period, the paediatric HUS incidence rate (IR) was 1.0 per 100,000 children-years, with a median of 116 cases/year. In 2011, IR peaked at 1.3 per 100,000 children-years, and decreased to 1.0 per 100,000 children-years in 2016. STEC O157 associated HUS peaked at 37 cases in 2011 and decreased to seven cases in 2016. Cases of STEC O26-associated HUS have increased since 2010 and STEC O80 associated HUS has emerged since 2012, with 28 and 18 cases respectively reported in 2016. Four STEC-HUS food-borne outbreaks were detected (three STEC O157 linked to ground beef and raw-milk cheese and one STEC O104 linked to fenugreek sprouts). In addition, two outbreaks related to person-to-person transmission occurred in distinct kindergartens (STEC O111 and O26).ConclusionsNo major changes in HUS IRs were observed over the study period of 10 years. However, changes in the STEC serogroups over time and the outbreaks detected argue for continuing epidemiological and microbiological surveillance.

Identification and prevalence of in vivo-induced genes in enterohaemorrhagic Escherichia coli.

Enterohaemorrhagic Escherichia coli (EHEC) are food-borne pathogens responsible for bloody diarrhoea and renal failure in humans. While Shiga toxin (Stx) is the cardinal virulence factor of EHEC, its production by E. coli is not sufficient to cause disease and many Shiga-toxin producing E. coli (STEC) strains have never been implicated in human infection. So far, the pathophysiology of EHEC infection is not fully understood and more knowledge is needed to characterize the "auxiliary" factors that enable a STEC strain to cause disease in humans. In this study, we applied a recombinase-based in vivo expression technology (RIVET) to the EHEC reference strain EDL933 in order to identify genes specifically induced during the infectious process, using mouse as an infection model. We identified 31 in vivo-induced (ivi) genes having functions related to metabolism, stress adaptive response and bacterial virulence or fitness. Eight of the 31 ivi genes were found to be heterogeneously distributed in EHEC strains circulating in France these last years. In addition, they are more prevalent in strains from the TOP seven priority serotypes and particularly strains carrying significant virulence determinants such as Stx2 and intimin adhesin. This work sheds further light on bacterial determinants over-expressed in vivo during infection that may contribute to the potential of STEC strains to cause disease in humans.

Emerging Multidrug-Resistant Hybrid Pathotype Shiga Toxin-Producing Escherichia coli O80 and Related Strains of Clonal Complex 165, Europe.

Enterohemorrhagic Escherichia coli serogroup O80, involved in hemolytic uremic syndrome associated with extraintestinal infections, has emerged in France. We obtained circularized sequences of the O80 strain RDEx444, responsible for hemolytic uremic syndrome with bacteremia, and noncircularized sequences of 35 O80 E. coli isolated from humans and animals in Europe with or without Shiga toxin genes. RDEx444 harbored a mosaic plasmid, pR444_A, combining extraintestinal virulence determinants and a multidrug resistance-encoding island. All strains belonged to clonal complex 165, which is distantly related to other major enterohemorrhagic E. coli lineages. All stx-positive strains contained eae-ξ, ehxA, and genes characteristic of pR444_A. Among stx-negative strains, 1 produced extended-spectrum ß-lactamase, 1 harbored the colistin-resistance gene mcr1, and 2 possessed genes characteristic of enteropathogenic and pyelonephritis E. coli. Because O80-clonal complex 165 strains can integrate intestinal and extraintestinal virulence factors in combination with diverse drug-resistance genes, they constitute dangerous and versatile multidrug-resistant pathogens.

C-source metabolic profilings of foodborne Shiga-toxin producing E. coli match serogroup differentiations and highlight functional adaptations.

The tropism of pathogenic STEC for foodstuffs and cattle reservoir is related to functional specializations. An investigation of C-source utilization patterns among and between STEC serogroups was performed using omnilog phenotypic microarrays (OM). OM functional groupings were compared with STEC phylogroups, seropathotypes, EFSA's molecular risk assessment groups and serogroups. OM INT reduction activities of 37 STEC strains growing on 190 C-substrates were compared. Each strain had its own specific C-utilization profile but 23% of the substrates was used by all strains, 47% by none, and 30% was variably metabolized. Galactose, mannose, N-acetyl-glucosamine (GlcNAc), and N-acetyl neuraminic acid (Neu5Ac) found in the mucus layer of the bovine small intestine were metabolized by all strains. The 56 most informative substrates divided the C-utilization patterns (CP) into three clusters with: (A) harboring all O157 and O145 strains; (B) all O26 strains, and (C) strains of the other serogroups. Significant correlations between INT reduction values of pair of strains per CP group supported these differentiations. CP of group A and B strains were respectively defective in the use of galactonic acid-γ-lactone and rhamnose. Most CP group C strains grew with l-lyxose. Adjusted Wallace coefficients analyses of the datasets indicated high probabilities for the prediction of the use of glycolic acid, ß-hydroxybutyric acid, l-lyxose and d-galactonic acid-γ-lactone and 5-keto-d-gluconic acid by a serogroup. The use of a C-substrate could be predicted from the classification of a strain into a phylogroup or seropathotype. Significantly lower numbers of C-substrates were used by seropathotype A strains like O157 ones. Improvements of STEC identification keys were proposed using the most discriminant C-substrates found in this study.

Distribution of Escherichia coli O157:H7 in ground beef: Assessing the clustering intensity for an industrial-scale grinder and a low and localized initial contamination.

Undercooked ground beef is regularly implicated in food-borne outbreaks involving pathogenic Shiga toxin-producing Escherichia coli. The dispersion of bacteria during mixing processes is of major concern for quantitative microbiological risk assessment since clustering will influence the number of bacteria the consumers might get exposed to as well as the performance of sampling plans used to detect contaminated ground beef batches. In this study, batches of 25kg of ground beef were manufactured according to a process mimicking an industrial-scale grinding with three successive steps: primary grinding, mixing and final grinding. The ground beef batches were made with 100% of chilled trims or with 2/3 of chilled trims and 1/3 of frozen trims. Prior grinding, one beef trim was contaminated with approximately 106-107CFU of E. coli O157:H7 on a surface of 0.5cm2 to reach a concentration of 10-100cells/g in ground beef. The E. coli O157:H7 distribution in ground beef was characterized by enumerating 60 samples (20 samples of 5g, 20 samples of 25g and 20 samples of 100g) and fitting a Poisson-gamma model to describe the variability of bacterial counts. The shape parameter of the gamma distribution, also known as the dispersion parameter reflecting the amount of clustering, was estimated between 1.0 and 1.6. This k-value of approximately 1 expresses a moderate level of clustering of bacterial cells in the ground beef. The impact of this clustering on the performance of sampling strategies was relatively limited in comparison to the classical hypothesis of a random repartition of pathogenic cells in mixed materials (purely Poisson distribution instead of Poisson-gamma distribution).

Enterohemorrhagic Escherichia coli Hybrid Pathotype O80:H2 as a New Therapeutic Challenge.

We describe the epidemiology, clinical features, and molecular characterization of enterohemorrhagic Escherichia coli (EHEC) infections caused by the singular hybrid pathotype O80:H2, and we examine the influence of antibiotics on Shiga toxin production. In France, during 2005-2014, a total of 54 patients were infected with EHEC O80:H2; 91% had hemolytic uremic syndrome. Two patients had invasive infections, and 2 died. All strains carried stx2 (variants stx2a, 2c, or 2d); the rare intimin gene (eae-ξ); and at least 4 genes characteristic of pS88, a plasmid associated with extraintestinal virulence. Similar strains were found in Spain. All isolates belonged to the same clonal group. At subinhibitory concentrations, azithromycin decreased Shiga toxin production significantly, ciprofloxacin increased it substantially, and ceftriaxone had no major effect. Antibiotic combinations that included azithromycin also were tested. EHEC O80:H2, which can induce hemolytic uremic syndrome complicated by bacteremia, is emerging in France. However, azithromycin might effectively combat these infections.

Variable tellurite resistance profiles of clinically-relevant Shiga toxin-producing Escherichia coli (STEC) influence their recovery from foodstuffs.

Tellurite (Tel)-amended selective media and resistance (Tel-R) are widely used for detecting Shiga toxin-producing Escherichia coli (STEC) from foodstuffs. Tel-R of 81 O157 and non-O157 STEC strains isolated from animal, food and human was thus investigated. Variations of STEC tellurite minimal inhibitory concentration (MIC) values have been observed and suggest a multifactorial and variable tellurite resistome between strains. Some clinically-relevant STEC were found highly susceptible and could not be recovered using a tellurite-based detection scheme. The ter operon was highly prevalent among highly Tel-R STEC but was not always detected among intermediately-resistant strains. Many STEC serogroup strains were found to harbor sublines showing a gradient of MIC values. These Tel-R sublines showed statistically significant log negative correlations with increasing tellurite concentration. Whatever the tellurite concentration, the highest number of resistant sublines was observed for STEC belonging to the O26 serogroup. Variations in the number of these Tel-R sublines could explain the poor recovery of some STEC serogroups on tellurite-amended media especially from food products with low levels of contamination. Comparison of tellurite MIC values and distribution of virulence-related genes showed Tel-R and virulence to be related.

Heterogeneity in Induction Level, Infection Ability, and Morphology of Shiga Toxin-Encoding Phages (Stx Phages) from Dairy and Human Shiga Toxin-Producing Escherichia coli O26:H11 Isolates.

Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are foodborne pathogens responsible for diarrhea and hemolytic-uremic syndrome (HUS). Shiga toxin, the main STEC virulence factor, is encoded by the stx gene located in the genome of a bacteriophage inserted into the bacterial chromosome. The O26:H11 serotype is considered to be the second-most-significant HUS-causing serotype worldwide after O157:H7. STEC O26:H11 bacteria and their stx-negative counterparts have been detected in dairy products. They may convert from the one form to the other by loss or acquisition of Stx phages, potentially confounding food microbiological diagnostic methods based on stx gene detection. Here we investigated the diversity and mobility of Stx phages from human and dairy STEC O26:H11 strains. Evaluation of their rate of in vitro induction, occurring either spontaneously or in the presence of mitomycin C, showed that the Stx2 phages were more inducible overall than Stx1 phages. However, no correlation was found between the Stx phage levels produced and the origin of the strains tested or the phage insertion sites. Morphological analysis by electron microscopy showed that Stx phages from STEC O26:H11 displayed various shapes that were unrelated to Stx1 or Stx2 types. Finally, the levels of sensitivity of stx-negative E. coli O26:H11 to six Stx phages differed among the 17 strains tested and our attempts to convert them into STEC were unsuccessful, indicating that their lysogenization was a rare event.

Recurrent Hemolytic and Uremic Syndrome Induced by Escherichia Coli.

A widespread belief is that typical hemolytic and uremic syndrome (HUS) does not recur. We report the case of a patient infected twice with raw milk taken from his own cow and containing a Shiga toxin-producing Escherichia coli O174:H21 that induced recurrent HUS causing severe renal and cerebral disorders. A genomic comparison of the human and bovine Shiga toxin-producing Escherichia coli O174:H21 isolates revealed that they were identical. Typical HUS may recur. Since milk from this animal was occasionally distributed locally, thereby posing a serious threat for the whole village, this particular cow was destroyed.

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.

Genome Sequence and Annotation of a Human Infection Isolate of Escherichia coli O26:H11 Involved in a Raw Milk Cheese Outbreak.

The consumption of raw milk cheese can expose populations to Shiga toxin-producing Escherichia coli (STEC). We report here the genome sequence of an E. coli O26:H11 strain isolated from humans during the first raw milk cheese outbreak described in France (2005).

Detection of non-O157 Shiga toxin-producing Escherichia coli in 375 grams of beef trim enrichments across multiple commercial PCR detection platforms.

Although serotype O157:H7 remains the pathogenic Shiga toxin-producing Escherichia coli (STEC) of primary concern worldwide, some focus in the United States has shifted to six particular non-O157 STEC serogroups (O26, O45, O103, O111, O121, and O145). Some of these serogroups have also emerged as concerns elsewhere around the world, including Europe. The objective of this work was to compare commercial detection methods with the U.S. Department of Agriculture (USDA) reference method for detection of non-O157 STEC in 375 g of beef trim using a limit of detection study design. Overall, the commercial platforms performed well, showing similar levels of sensitivity for detection of presumptive positives for O45, O26, O103, and O121 (PCR screen results only). For O111, one method that utilizes an integrated immunomagnetic separation and PCR approach was more sensitive than a PCR-only screen approach. Additionally, one commercial method showed more presumptive and confirmed positives overall. Use of an immunomagnetic separation tool, such as antibody-coated beads, aided considerably with the confirmation procedures and is an important step when confirming suspect samples. A secondary goal of this study was to evaluate isolation and International Organization for Standardization confirmation protocols used in Europe compared with strategies provided by the USDA Microbiology Laboratory Guidebook (MLG). Generally, results from the USDA confirmation plates (modified Rainbow agar) were better than the European Union confirmation plates (MacConkey agar with or without rhamnose). In summary, detection of non-O157 STEC in 375 g of beef trim can be performed by any of the three methods on the market evaluated in the study.

Prevalence of carriage of Shiga toxin-producing Escherichia coli serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 among slaughtered adult cattle in France.

The main pathogenic enterohemorrhagic Escherichia coli (EHEC) strains are defined as Shiga toxin (Stx)-producing E. coli (STEC) belonging to one of the following serotypes: O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28. Each of these five serotypes is known to be associated with a specific subtype of the intimin-encoding gene (eae). The objective of this study was to evaluate the prevalence of bovine carriers of these "top five" STEC in the four adult cattle categories slaughtered in France. Fecal samples were collected from 1,318 cattle, including 291 young dairy bulls, 296 young beef bulls, 337 dairy cows, and 394 beef cows. A total of 96 E. coli isolates, including 33 top five STEC and 63 atypical enteropathogenic E. coli (aEPEC) isolates, with the same genetic characteristics as the top five STEC strains except that they lacked an stx gene, were recovered from these samples.O157:H7 was the most frequently isolated STEC serotype. The prevalence of top five STEC (all serotypes included) was 4.5% in young dairy bulls, 2.4% in young beef bulls, 1.8% in dairy cows, and 1.0% in beef cows. It was significantly higher in young dairy bulls (P<0.05) than in the other 3 categories. The basis for these differences between categories remains to be elucidated. Moreover,simultaneous carriage of STEC O26:H11 and STEC O103:H2 was detected in one young dairy bull. Lastly, the prevalence of bovine carriers of the top five STEC, evaluated through a weighted arithmetic mean of the prevalence by categories, was estimated to 1.8% in slaughtered adult cattle in France.

Colonization of the meat extracellular matrix proteins by O157 and non-O157 enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) are anthropozoonotic agents that range third among food-borne pathogens respective to their incidence and dangerousness in the European Union. EHEC are Shiga-toxin producing E. coli (STEC) responsible for foodborne poisoning mainly incriminated to the consumption of contaminated beef meat. Among the hundreds of STEC serotypes identified, EHEC mainly belong to O157:H7 but non-O157 can represent 20 to 70% of EHEC infections per year. Seven of those serogroups are especially of high-risk for human health, i.e. O26, O45, O103, O111, O121, O145 and O104. While meat can be contaminated all along the food processing chain, EHEC contamination essentially occurs at the dehiding stage of slaughtering. Investigating bacterial colonization to the skeletal-muscle extracellular matrix (ECM) proteins, it appeared that environmental factors influenced specific and non-specific bacterial adhesion of O157 and non-O157 EHEC as well as biofilm formation. Importantly, mechanical treatment (i.e. shaking, centrifugation, pipetting and vortexing) inhibited and biased the results of bacterial adhesion assay. Besides stressing the importance of the protocol to investigate bacterial adhesion to ECM proteins, this study demonstrated that the colonization abilities to ECM proteins vary among EHEC serogroups and should ultimately be taken into consideration to evaluate the risk of contamination for different types of food matrices.

Slaughterhouse effluent discharges into rivers not responsible for environmental occurrence of enteroaggregative Escherichia coli.

Enteroaggregative Shiga-toxin-producing Escherichia coli strains were responsible for a massive outbreak in Europe in 2011, and had been previously isolated from French patients. The objective of this study was to investigate the presence of enteroaggregative E. coli (EAEC) in slaughterhouse effluents (wastewater, slurry, sludge and effluents), and in river waters near these slaughterhouses. A total of 10,618 E. coli isolates were screened by PCR for the presence of EAEC-associated genetic markers (aggR, aap and aatA). None of these markers was detected in E. coli isolated from slaughterhouse samples. A unique enteroaggregative E. coli (EAEC) O126:H8 was detected in river water sampled upstream from slaughterhouse effluent discharge. These results confirmed that animals might not be reservoirs of EAEC, and that further studies are required to evaluate the role of the environment in the transmission of EAEC to humans.

Intimin gene (eae) subtype-based real-time PCR strategy for specific detection of Shiga toxin-producing Escherichia coli serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 in cattle feces.

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 are known to be associated with particular subtypes of the intimin gene (eae), namely, γ1, ß1, ε, θ, and γ1, respectively. This study aimed at evaluating the usefulness of their detection for the specific detection of these five main pathogenic STEC serotypes in cattle feces. Using real-time PCR assays, 58.7% of 150 fecal samples were found positive for at least one of the four targeted eae subtypes. The simultaneous presence of stx, eae, and one of the five O group markers was found in 58.0% of the samples, and the five targeted stx plus eae plus O genetic combinations were detected 143 times. However, taking into consideration the association between eae subtypes and O group markers, the resulting stx plus eae subtype plus O combinations were detected only 46 times. The 46 isolation assays performed allowed recovery of 22 E. coli strains belonging to one of the five targeted STEC serogroups. In contrast, only 2 of 39 isolation assays performed on samples that were positive for stx, eae and an O group marker, but that were negative for the corresponding eae subtype, were successful. Characterization of the 24 E. coli isolates showed that 6 were STEC, including 1 O157:H7, 3 O26:H11, and 2 O145:H28. The remaining 18 strains corresponded to atypical enteropathogenic E. coli (aEPEC). Finally, the more discriminating eae subtype-based PCR strategy described here may be helpful for the specific screening of the five major STEC in cattle feces.