Patrick Fach

Trends in Molecular Detection and Characterization of Enterohemorrhagic E. Coli

Patrick Fach¹, Sabine Delannoy¹, Lothar Beutin²

Molecular methods for screening Enterohemorrhagic E. coli (EHEC) O157 and non-O157 in beef products typically rely on the molecular detection of stx, eae, and the Top5 or Top7 EHEC serogroups in mixed bacterial enrichments as described in the ISO/TS 13136 (EU) and MLG 5B (US) reference methods. Refinement of EHEC test systems is occurring via the incorporation of additional gene targets but remains complicated by the presence of non-EHEC isolates such as EPEC that may harbor various combinations of these target genes. Whole genome sequencing and high throughput qPCR allowed selection of genetic markers (espK, espV) which dominate in stx- and eae-positive E. coli (typical EHEC) whereas they are less represented in other pathogroups (EPEC, STEC) and non-pathogenic E. coli. Hence, 98% of the Top7 EHEC serogroups which are regulated in the US were tested positive for espK and/or espV. The only few EHEC O26:H11 strains which tested negative for espK and espV were associated with a new highly virulent clone which is positive for stx2 only. Such strains can be detected via specific CRISPR sequences. Database of CRISPR sequences were obtained and CRISPR based PCR tests were designed for targeting the Top7 EHEC serogroups and the German clone O104:H4 (eae, espK and espV negative) responsible for a large outbreak in 2011. Identification of these additional gene markers and CRISPRs to better distinguish EHEC from other E. coli pathogroups would substantially enhance the power of EHEC test systems providing a significant reduction of 'presumptive positive' in beef samples. Validation of these tests in food and feces samples is under progress to propose a new approach which would be more performant and in line with the EFSA opinion published in 2013.

1) Anses (French Agency for Food, Environmental and Occupational Health and Safety), Food Safety Laboratory, IdentyPath platform, 14 rue Pierre et Marie Curie, Fr-94700 Maisons-Alfort, France.

2) National Reference Laboratory for Escherichia coli, Federal Institute for Risk Assessment (BfR), Diedersdorfer Weg 1, D-12277 Berlin, Germany.