Revisiting the STEC Testing Approach: Using espK and espV to Make Enterohemorrhagic Escherichia coli (EHEC) Detection More Reliable in Beef

Abstract

Current methods for screening Enterohemorrhagic Escherichia coli (EHEC) O157 and non-O157 in beef enrichments typically rely on the molecular detection of stx, eae, and serogroup-specific wzx or wzy gene fragments. As these genetic markers can also be found in some non-EHEC strains, a number of "false positive" results are obtained. Here, we explore the suitability of five novel molecular markers, espK, espV, ureD, Z2098, and CRISPRO26:H11 as candidates for a more accurate screening of EHEC strains of greater clinical significance in industrialized countries. Of the 1739 beef enrichments tested, 180 were positive for both stx and eae genes. Ninety (50%) of these tested negative for espK, espV, ureD, and Z2098, but 12 out of these negative samples were positive for the CRISPRO26:H11 gene marker specific for a newly emerging virulent EHEC O26:H11 French clone. We show that screening for stx, eae, espK, and espV, in association with the CRISPRO26:H11 marker is a better approach to narrow down the EHEC screening step in beef enrichments. The number of potentially positive samples was reduced by 48.88% by means of this alternative strategy compared to the European and American reference methods, thus substantially improving the discriminatory power of EHEC screening systems. This approach is in line with the EFSA (European Food Safety Authority) opinion on pathogenic STEC published in 2013.

Keywords: CRISPR; O157; STEC; Z2098; espK; espV; non-O157; ureD.

Figure 1

Distribution of the genetic markers stx, eae, espK, espV, and Z2098 among 1739 beef samples. (A) (stx1/stx2, eae, and espK/Z2098) is Method A, (B) (stx1/stx2, eae, and espK/espV) is Method B, (C) (stx1/stx2, eae, and espK/ureD) is Method C. stx1/stx2⁺ for samples giving a positive result for stx1 and/or stx2, eae⁺ for samples giving a positive result for eae, espK/Z2098⁺ for samples giving a positive result for espK and/or Z2098, espK/espV⁺ for samples giving a positive result for espK and/or espV, espK/ureD⁺ for samples giving a positive result for espK and/or ureD.

Figure 2

Comparison of Methods A–C on 180 beef samples that tested positive for both stx and eae genes. stx⁺ for samples giving a positive result for stx1 and/or stx2; eae⁺ for samples giving a positive result for eae; espK/Z2098⁺ for samples giving a positive result for espK and/or Z2098; espK/espV⁺ for samples giving a positive result for espK and/or espV; espK/ureD⁺ for samples giving a positive result for espK and/or ureD. Sector ❶ (stx⁺, eae⁺, Z2098⁺), sector ❷ (stx⁺, eae⁺, Z2098⁺, espV⁺), sector ❸ (stx⁺, eae⁺, espK/Z2098⁺, espK/espV⁺, espK/ureD⁺), sector ❹ (stx⁺, eae⁺, Z2098⁺, ureD⁺), sector ❺ (stx⁺, eae⁺, espV⁺), sector ❻ (stx⁺, eae⁺, espV⁺, ureD⁺), sector ❼ (stx⁺, eae⁺, ureD⁺) and sector ❽ (stx⁺, eae⁺). [n = x] is the number of samples that tested positive by the CRISPR_O26:H11 PCR assay detecting the new O26 clone, N = x is the total number of samples per sector.