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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Applicability of a multiplex PCR to detect O26, O45, O103, O111, O121, O145, and O157 serogroups of Escherichia coli in cattle feces.
Shiga toxin-producing Escherichia coli (STEC), particularly O157, are major food borne pathogens. Non-O157 STEC, particularly O26, O45, O103, O111, O121, and O145, have also been recognized as a major public health concern. Unlike O157, detection procedures for non-O157 have not been fully developed. Our objective was to develop a multiplex PCR to distinguish O157 and the 'top six' non-O157 serogroups (O26, O45, O103, O111, O121, and O145) and evaluate the applicability of the multiplex PCR to detect the seven serogroups of E. coli in cattle feces. Published sequences of O-specific antigen coding genes, rfbE (O157) and wzx and wbqE-F (non-O157), were analyzed to design serogroup-specific primers. The specificity of amplifications was confirmed with 138 known STEC strains and the reaction yielded the expected amplicons for each serogroup. In feces spiked with pooled 7 STEC strains, the sensitivity of the detection was 4.1 × 10(5)CFU/g before enrichment and 2.3 × 10(2) after 6h enrichment in E. coli broth. Additionally, 216 fecal samples from cattle were collected and tested by multiplex PCR and cultural methods. The multiplex PCR revealed a high prevalence of all seven serogroups (178 [O26], 108 [O45], 149 [O103], 30 [O111], 103 [O121], 5 [O145], and 160 [O157]) of 216 samples in fecal samples. Cultural procedures identified 33.1% (53/160) and 35.5% (11/31) of PCR-positive samples for E. coli O157 and non-O157 serogroups, respectively. Samples that were culture-positive were all positive by the multiplex PCR. The multiplex PCR can be used to identify serogroups of putative STEC isolates.
Paddock Z
，Shi X
，Bai J
，Nagaraja TG
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