Insights into the assessment of highly pathogenic Shiga toxin-producing Escherichia coli in raw milk and raw milk cheeses by High Throughput Real-time PCR

Current methods for screening Enterohemorrhagic Escherichia coli (EHEC) O157 and non-O157 serogroups in raw milk products typically rely on the molecular detection of stx, eae, and serogroup-specific wzx or wzy genes. As these genetic markers can also be carried by non-EHEC strains, a number of ‘false positive’ results are obtained during the screening step. The suitability of new EHEC markers (espK, espV, ureD, Z2098, and CRISPRO26:H11) were tested as candidates for a more accurate screening of EHEC in dairy products. High-throughput PCR analysis of 1451 DNA extracts from milk and raw milk cheeses positive for both stx and eae demonstrated that addition of these new markers in the detection scheme resulted in a higher selectivity with a systematic reduction of the number of presumptive positive samples that require further O-group testing and confirmation by strain isolation. This reduction is more important (26% to 52%, depending on the animal production species) in the absence of prior IMS treatment of the enriched culture for the Top7 EHEC serotypes. However, even with prior treatment of the enriched cultures by IMS, the reduction rate varied between 5% and >25%. Analysis of eae-subtype, stx-subtypes indicated strong differences in the STEC (Shiga toxin producing E. coli) flora between animal species (goat, sheep, and cow). This study also pointed toward the possible presence of EHEC O80 (a new emerging EHEC serogroup in Europe) in cow's raw milk cheeses, which warrants further investigations. •Detection of highly pathogenic Shiga toxin-producing Escherichia coli (STEC) in raw milk and raw milk cheeses•Optimization of a novel PCR method adapted from the ISO/TS 13136:2012•A study designed to assess the contribution of new genetic markers to refine the first screening of highly pathogenic STEC•A new method providing a significant reduction of ‘presumptive positive’ in dairy samples.•A study pointing toward the possible presence of STEC O80 (an emerging pathogen in Europe) in cow raw milk cheeses