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.

Staphylococcus aureus is one of the leading causes of food-borne illness worldwide. Raw milk and dairy products are often contaminated with enterotoxigenic strains of this bacterium. Some of these strains carry antimicrobial resistance, leading to a potential risk for consumers. The aim of this study was to characterize S. aureus strains circulating in raw milk and traditional dairy products for carriage of staphylococcal enterotoxin (se) genes and antimicrobial resistance. Overall, 62 out of 270 samples (23%) were contaminated with S. aureus, and 69 S. aureus strains were identified. We studied the enterotoxin genes using 2 multiplex PCR targeting 11 se genes. Seventeen (24.6%) isolates carried one or more genes encoding for staphylococcal enterotoxins. The most commonly detected se genes were seb and sep, followed by seh, sea, and see. Using the disk diffusion method, we found that resistance to penicillin G and tetracycline was the most common. Eleven isolates of methicillin-resistant S. aureus (MRSA) carried the mecA gene. All MRSA isolates belonged to the same spa type (t024) and sequence type (ST8), and carried the seb and sep enterotoxin genes. However, none of them carried the Panton Valentine leukocidin gene (lukF/S-PV). The presence of enterotoxigenic S. aureus strains, including MRSA, in raw milk and dairy products, raises a serious public health concern, because these strains may cause food poisoning outbreaks, be disseminated to the population, or both.

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