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

Abstract

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-␤1 (4.8%; 19 samples), stx-wzx O103 -eae-(1.3%; five samples), stx-ihp1 O145 -eae-␥1 (0.8%; three samples), and stx-rfbE O157 -eae-␥1 (0.3%; one sample). Twenty-eight immunomagnetic separation (IMS) assays performed on samples positive for these combinations allowed the recovery of seven eae␤1-positive STEC O26:H11 isolates, whereas no STEC O103:H2, O145:H28, or O157:H7 strains could be isolated. Three stx-negative and eae␤1-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.

Prevalence of Shiga toxin-producing Escherichia coli in food products of animal origin as determined by molecular methods

“…This difference for the dairy samples is less evident, 42 % positive by qPCR and 36 % positive by identification of isolates. These results confirm previous findings concerning the higher percent of prevalence of STEC when determined by PCR methods applied directly in food samples compared to culture based methods (Auvray et al, 2009;Madic et al, 2011;Pradel et al, 2000). Such deviation may partly be explained by the fact that isolates were randomly selected and that for most samples, a relatively low STEC load was detected.…”

“…It has been suggested that such a discrepancy could be attributed to the stress to which bacteria are subjected during processing (Pradel et al, 2000). It should also be noted that a difficulty in STEC isolation has been previously reported (Auvray et al, 2007;Madic et al, 2011).…”

“…It has become evident that the information regarding presence and viability of pathogens obtained by culture-independent approaches is, in many cases, different from the information gathered by culture-dependent approaches (Auvray et al, 2009;Madic et al, 2011;Pradel et al, 2000). Furthermore, in a 'farm-to-fork' study, taking into consideration the prevalence of STEC throughout the lamb food chain, it was concluded that qPCR detected virulent populations in sampling points (environmental and meat samples) where conventinal microbiological methods failed (Oses et al, 2010).…”

Microbial quality of industrial and retail market grated parmesan cheese in the State of São Paulo, Brazil

“…Thus, the presence of these microorganisms poses a risk to the consumer's health and contributes to the deterioration of the product (MONTANHINI et al, 2013). High amounts of these microorganisms also indicated a greater possibility of pathogens being present in the food as most pathogens are mesophilic bacteria (MADIC et al, 2011).…”

Prevalence of pathogenic and antibiotics resistant Escherichia coli from effluents of a slaughterhouse and a municipal wastewater treatment plant in Dakar

“…E. coli isolates positive for stx1 and/or stx2 genes were classified as STEC, isolates positive for eae gene, and negative for stx1, stx2, bfpA and EPEC adherence factor (EAF) plasmid were classified as atypical EPEC (Trabulsi et al, 2002). The five major serotypes associated with EHEC (O157:H7, O26:H11, O145:H28, O111:H8 and O103:H2) were investigated in STEC and potential EPEC isolates, by real-time PCR with conditions and control strains described elsewhere (Madic et al, 2011;Perelle et al, 2004Perelle et al, , 2005.…”