Detection of Shiga toxin-producing Escherichia coli serotypes O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7 in raw-milk cheeses by using multiplex real-time PCR.
Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are a diverse group of food-borne pathogens with various levels of virulence for humans. In this study, we describe the use of a combination of multiple real-time PCR assays for the screening of 400 raw-milk cheeses for the five main pathogenic STEC serotypes (O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7). The prevalences of samples positive for stx, intimin-encoding gene (eae), and at least one of the five O group genetic markers were 29.8%, 37.3%, and 55.3%, respectively. The H2, H7, H8, H11, and H28 fliC alleles were highly prevalent and could not be used as reliable targets for screening. Combinations of stx, eae variants, and O genetic markers, which are typical of the five targeted STEC serotypes, were detected by real-time PCR in 6.5% of the cheeses (26 samples) and included stx-wzx(O26)-eae-β1 (4.8%; 19 samples), stx-wzx(O103)-eae-ε (1.3%; five samples), stx-ihp1(O145)-eae-γ1 (0.8%; three samples), and stx-rfbE(O157)-eae-γ1 (0.3%; one sample). Twenty-eight immunomagnetic separation (IMS) assays performed on samples positive for these combinations allowed the recovery of seven eaeβ1-positive STEC O26:H11 isolates, whereas no STEC O103:H2, O145:H28, or O157:H7 strains could be isolated. Three stx-negative and eaeβ1-positive E. coli O26:[H11] strains were also isolated from cheeses by IMS. Colony hybridization allowed us to recover STEC from stx-positive samples for 15 out of 45 assays performed, highlighting the difficulties encountered in STEC isolation from dairy products. The STEC O26:H11 isolates shared the same virulence genetic profile as enterohemorrhagic E. coli (EHEC) O26:H11, i.e., they carried the virulence-associated genes EHEC-hlyA, katP, and espP, as well as genomic O islands 71 and 122. Except for one strain, they all contained the stx1 variant only, which was reported to be less frequently associated with human cases than stx2. Pulsed-field gel electrophoresis (PFGE) analysis showed that they displayed high genetic diversity; none of them had patterns identical to those of human O26:H11 strains investigated here.
Madic J
，Vingadassalon N
，de Garam CP
，Marault M
，Scheutz F
，Brugère H
，Jamet E
，Auvray F
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Application of a real-time PCR-based system for monitoring of O26, O103, O111, O145 and O157 Shiga toxin-producing Escherichia coli in cattle at slaughter.
Faecal samples were collected from 573 slaughtered cattle aged between three and 24 months in seven abattoirs. After enrichment (mTSB with novobiocin), samples were screened by real-time PCR first for stx and if positive, tested for the top-five Shiga toxin-producing Escherichia coli (STEC) serogroups using PCR assays targeting genes specific for serogroups O26, O103, O111, O145 and O157. Of 563 samples with available results, 74.1% tested positive for stx genes. Amongst them, the serogroups O145, O103, O26, O157 and O111 were detected in 41.9%, 25.9%, 23.9%, 7.8% and 0.8%, respectively. From 95 O26, 166 O145 and 30 O157 PCR-positive samples, 17 O26, 28 O145 and 12 O157 strains were isolated by colony hybridization after immunomagnetic separation. The 17 O26 strains were eae-positive, but only nine strains harboured stx (eight possessing stx1 and one stx2). Of the 28 O145 strains, ten were eae-positive including four harbouring stx1 or stx2, whereas 18 were negative for stx and eae. Five of the 12 O157 strains harboured stx2 and eae, did not ferment sorbitol, and were identified as STEC O157:H7/H⁻. The other seven O157 strains were negative for stx and eae or positive only for eae. Shiga toxin genes and the top-five STEC serogroups were frequently found in young Swiss cattle at slaughter, but success rates for strain isolation were low and only few strains showed a virulence pattern of human pathogenic STEC.
Hofer E
，Stephan R
，Reist M
，Zweifel C
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