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.
Anses-Laboratoire de Sécurité des Aliments, 23 Avenue du Général de Gaulle, 94706 Maisons-Alfort Cedex, France.
Jordan
J
Madic
Noémie
N
Vingadassalon
Carine Peytavin
CP
de Garam
Muriel
M
Marault
Flemming
F
Scheutz
Hubert
H
Brugère
Emmanuel
E
Jamet
Frédéric
F
Auvray
D018829:Adhesins, Bacterial / Q000235:genetics; D000818:Animals; D002611:Cheese / Q000382:microbiology*; D016521:Electrophoresis, Gel, Pulsed-Field; D019453:Escherichia coli O157 / Q000145:classification / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics; D005516:Food Microbiology; D016133:Polymerase Chain Reaction; D022621:Shiga Toxin / Q000235:genetics; D054323:Shiga-Toxigenic Escherichia coli / Q000145:classification / Q000235:genetics* / Q000378:metabolism*
D018829:Adhesins, Bacterial; D029968:Escherichia coli Proteins; C073819:eaeA protein, E coli; D022621:Shiga Toxin
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