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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.
Appl Environ Microbiol. 2011 Mar; 77(6):2035-41.AE

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-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.

Authors+Show Affiliations

Anses-Laboratoire de Sécurité des Aliments, 23 Avenue du Général de Gaulle, 94706 Maisons-Alfort Cedex, France.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

21239543

Citation

Madic, Jordan, et al. "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." Applied and Environmental Microbiology, vol. 77, no. 6, 2011, pp. 2035-41.
Madic J, Vingadassalon N, de Garam CP, et al. 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. Appl Environ Microbiol. 2011;77(6):2035-41.
Madic, J., Vingadassalon, N., de Garam, C. P., Marault, M., Scheutz, F., Brugère, H., Jamet, E., & Auvray, F. (2011). 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. Applied and Environmental Microbiology, 77(6), 2035-41. https://doi.org/10.1128/AEM.02089-10
Madic J, et al. 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. Appl Environ Microbiol. 2011;77(6):2035-41. PubMed PMID: 21239543.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Madic,Jordan, AU - Vingadassalon,Noémie, AU - de Garam,Carine Peytavin, AU - Marault,Muriel, AU - Scheutz,Flemming, AU - Brugère,Hubert, AU - Jamet,Emmanuel, AU - Auvray,Frédéric, Y1 - 2011/01/14/ PY - 2011/1/18/entrez PY - 2011/1/18/pubmed PY - 2011/6/23/medline SP - 2035 EP - 41 JF - Applied and environmental microbiology JO - Appl Environ Microbiol VL - 77 IS - 6 N2 - 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. SN - 1098-5336 UR - https://www.unboundmedicine.com/medline/citation/21239543/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_ L2 - http://aem.asm.org/cgi/pmidlookup?view=long&pmid=21239543 DB - PRIME DP - Unbound Medicine ER -