Molecular characterization of O157:H7, O26:H11 and O103:H2 Shiga toxin-producing Escherichia coli isolated from dairy products.

Pathogenic Shiga toxin-producing E. coli (STEC) are recognized worldwide as environment and foodborne pathogens which can be transmitted by ingestion of ready-to-eat food such as raw milk-derived products. STEC show a prevalence rate in dairy products of 0.9%, yet comparably few outbreaks have been related to dairy products consumption. In this study, we used rt-qPCR to identify the virulence potential of O157, O26 and O103 STEC strains isolated from raw-milk dairy products by analyzing virulence-related gene frequencies and associations with O-island (OI) 44, OI-48, OI-50, OI-57, OI-71 and OI-122. Results showed that 100% of STEC strains investigated harbored genes associated with EHEC-related virulence profile patterns (eae and stx, with either espK, espV, ureD and/or Z2098). We also found similarities in virulence-related gene content between O157:H7 and O103:H2 dairy and non-dairy STEC strains, especially isolates from human cases. The O26:H11-serotype STEC strains investigated harbor the arcA-allele 2 gene associated with specific genetic markers. These profiles are associated with high-virulence seropathotype-A STEC. However, the low frequency of stx2 gene associated with absence of other virulence genes in dairy isolates of O26:H11 remains a promising avenue of investigation to estimate their real pathogenicity. All O26:H11 attaching-effacing E. coli (AEEC) strains carried CRISPRO26:H11SP_O26_E but not genetic markers espK, espV, ureD and/or Z2098 associated with the emerging potentially high-virulence "new French clone". These strains are potentially as "EHEC-like" strains because they may acquire (or have lost) stx gene. In this study, O157:H7, O103:H2 and O26:H11 STEC strains isolated from dairy products were assigned as potential pathogens. However, research now needs to investigate the impact of dairy product environment and dairy processing on the expression of their pathogenicity.

Shiga toxin-producing Escherichia coli strains isolated from dairy products - Genetic diversity and virulence gene profiles.

Shiga toxin-producing Escherichia coli (STEC) are widely recognized as pathogens causing food borne disease. Here we evaluate the genetic diversity of 197 strains, mainly STEC, from serotypes O157:H7, O26:H11, O103:H2, O111:H8 and O145:28 and compared strains recovered in dairy products against strains from human, meat and environment cases. For this purpose, we characterized a set of reference-collection STEC isolates from dairy products by PFGE DNA fingerprinting and a subset of these by virulence-gene profiling. PFGE profiles of restricted STEC total DNA showed high genomic variability (0.9976 on Simpson's discriminatory index), enabling all dairy isolates to be differentiated. High-throughput real-time PCR screening of STEC virulence genes were applied on the O157:H7 and O26:H11 STEC isolates from dairy products and human cases. The virulence gene profiles of dairy and human STEC strains were similar. Nevertheless, frequency-wise, stx1 was more prevalent among dairy O26:H11 isolates than in human cases ones (87% vs. 44%) while stx2 was more prevalent among O26:H11 human isolates (23% vs. 81%). For O157:H7 isolates, stx1 (0% vs. 39%), nleF (40% vs 94%) and Z6065 (40% vs 100%) were more prevalent among human than dairy strains. Our data point to differences between human and dairy strains but these differences were not sufficient to associate PFGE and virulence gene profiles to a putative lower pathogenicity of dairy strains based on their lower incidence in disease. Further comparison of whole-genome expression and virulence gene profiles should be investigated in cheese and intestinal tract samples.

Foodborne transmission of sorbitol-fermenting Escherichia coli O157:[H7] via ground beef: an outbreak in northern France, 2011.

Sorbitol-fermenting Escherichia coli O157:[H7] is a particularly virulent clone of E. coli O157:H7 associated with a higher incidence of haemolytic uraemic syndrome and a higher case fatality rate. Many fundamental aspects of its epidemiology remain to be elucidated, including its reservoir and transmission routes and vehicles. We describe an outbreak of sorbitol-fermenting E. coli O157:[H7] that occurred in France in 2011. Eighteen cases of paediatric haemolytic uraemic syndrome with symptom onset between 6 June and 15 July 2011 were identified among children aged 6 months to 10 years residing in northern France. A strain of sorbitol-fermenting E. coli O157:[H7] stx2a eae was isolated from ten cases. Epidemiological, microbiological and trace-back investigations identified multiply-contaminated frozen ground beef products bought in a supermarket chain as the outbreak vehicle. Strains with three distinct pulsotypes that were isolated from patients, ground beef preparations recovered from patients' freezers and from stored production samples taken at the production plant were indistinguishable upon molecular comparison. This investigation documents microbiologically confirmed foodborne transmission of sorbitol-fermenting of E. coli O157 via beef and could additionally provide evidence of a reservoir in cattle for this pathogen.

Shopper cards data and storage practices for the investigation of an outbreak of Shiga-toxin producing Escherichia coli O157 infections.

INTRODUCTION: An outbreak of shiga-toxin producing Escherichia coli infections occurred in southwest France in June 2012. The outbreak was investigated to identify the source of infection, and guide control measures. METHODS: Confirmed outbreak cases were patients who developed bloody diarrhoea or haemolytic uremic syndrome (HUS) between 28 May and 6 July 2012, with E. coli O157 isolates showing indistinguishable patterns on pulse field gel electrophoresis (PFGE). A standardized questionnaire was administered to patients to document food consumption and other risk exposures. Their purchase was checked through their supermarket shopper card data. RESULTS: Six patients (four with HUS and two with bloody diarrhea) were confirmed outbreak cases. Fresh ground beef burgers from one supermarket were the only common food exposure, identified by interviews and shopper card data. The PFGE profile of shiga toxin-producing E. coli O157 isolated from the suspected beef burgers was identical to those from the human cases. The suspected beef burgers were no longer on sale at the time of investigation but three patients confirmed as outbreak cases had deep-frozen some at home. CONCLUSION: Shopper card data was particularly useful to obtain precise and reliable information on the traceability of consumed food. Despite the expired use-by date, a recall was issued for the beef burgers. This contributed to preventing other cases among consumers who had deep-frozen the beef burgers.

Duplex real-time PCR detection of type III effector tccP and tccP2 genes in pathogenic Escherichia coli and prevalence in raw milk cheeses.

AIMS: To develop a duplex real-time PCR assay targeting enterohaemorrhagic Escherichia coli (EHEC) type III effector TccP/TccP2-encoding genes which are pivotal to EHEC-mediated actin cytoskeleton reorganization in human intestinal epithelial cells. METHODS AND RESULTS: The specificity of the assay was demonstrated with DNA from EHEC reference strains and non-E. coli bacterial species. The detection limit was determined as five tccP or tccP2 copies per reaction. The assay was then evaluated on a large collection of 526 E. coli strains of human, animal, food and environmental origins. The results showed that tccP was restricted to a limited number of serotypes (i.e. O5:H(-) , O55:H7, O125:H6 and O157:H7). The tccP2 gene was present in a higher number of serotypes including the five most frequent EHEC serotypes (i.e. O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7), and a few other serotypes that caused human infections (i.e. O4:H(-) , O45:H2 and O55:H7). A minority of O26:H11 and O103:H2 strains however tested negative for tccP2, though it is not known whether the lack of tccP2 affected their pathogenic potential. Real-time PCR analysis of 400 raw milk cheeses revealed the presence of tccP and/or tccP2 genes in 19·75% of the cheese enrichment suspensions. CONCLUSIONS: A highly specific and sensitive duplex real-time PCR method was developed for rapid and simultaneous detection of tccP and tccP2. Unpasteurized dairy products may be contaminated with E. coli strains carrying tccP and/or tccP2. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed real-time PCR assay represents a valuable alternative to conventional PCR tests and should be useful for characterization of the virulome of pathogenic E. coli strains.