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 Jordan Madic, Noémie Vingadassalon, Carine Peytavin de Garam, Muriel Marault, Flemming Scheutz, Hubert Brugère, Emmanuel Jamet, and Frédéric Auvray
Appl. Environ. Microbiol. March 2011 77: 2035-2041
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-wzxO26-eae-β1 (4.8%; 19 samples), stx-wzxO103-eae- (1.3%; five samples), stx-ihp1O145-eae- 1 (0.8%; three samples), and stx-rfbEO157-eae- 1 (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.
Discussion snippets: The prevalence of the stx-positive cheeses analyzed here was similar to those reported previously (2, 22) but higher than those found in other studies (5.7% to 13%) (42, 50, 53). However, direct comparison of the results is difficult, as the last studies were based on the use of distinct stx detection methods (i.e., conventional PCR) and included analysis of hard cheeses. In contrast, samples selected for this study were uncooked and soft cheeses, all made from raw milk and thus associated with higher risk of STEC infection than other types of cheeses derived from manufacturing processes more effective in eliminating STEC. E. coli O26:H11 has been frequently detected in calves and cattle (26), and contamination of raw milk and raw-milk cheeses with STEC O26:H11 has also been described (1, 18). In terms of risk to human health, the significance of the prevalence found for typical EHEC combinations of markers in raw-milk cheeses (i.e., 6.5%) deserves further investigation. In particular, the pathogenic potential of cheese STEC isolates belonging to EHEC serotypes, such as O26:H11 and O91:H10, needs to be further examined. The hypothetical loss of stx genes during STEC isolation from foods should also be investigated, as loss of stx could result in foodstuffs being falsely considered uncontaminated by pathogenic STEC and therefore safe.
(1). Allerberger, F., et al. 2003. Hemolytic-uremic syndrome associated with enterohemorrhagic Escherichia coli O26:H_ infection and consumption of unpasteurized cow’s milk. Int. J. Infect. Dis. 7:42–45.
(2). Auvray, F., C. Lecureuil, F. Dilasser, J. Tache, and S. Derzelle. 2009. Development of a real-time PCR assay with an internal amplification control for the screening of Shiga toxin-producing Escherichia coli in foods. Lett. Appl. Microbiol. 48:554–559.
(18). Espie, E., et al. 2006. Shiga-toxin producing Escherichia coli O26 infection and unpasteurised cows cheese, France, 2005, poster. In J. Sofronidis (ed.), Progr. Abstr. 6th Int. Symp. Shiga Toxin (Verocytoxin)-Producing Escherichia coli Infect., Melbourne, Australia, 2006. Cambridge Publishing, West Leederville, W.A., Australia. http://www.invs.sante.fr/surveillance/shu/poster_melbourne_o26.pdf.
(22). Fach, P., S. Perelle, F. Dilasser, and J. Grout. 2001. Comparison between a PCR-ELISA test and the Vero cell assay for detecting Shiga toxin-producing Escherichia coli in dairy products and characterization of virulence traits of the isolated strains. J. Appl. Microbiol. 90:809–818.