title: Application of a DNA hybridization-hydrophobic-grid membrane filter method for detection and isolation of verotoxigenic escherichia coli.
abstract: Verotoxigenic Escherichia coli (VTEC) strains were isolated from food and animal fecal samples by using PCR to screen for the presence of VTEC after broth enrichment and then filtering VTEC-positive cultures through hydrophobic-grid membrane filters (HGMFs) which were incubated on MacConkey agar. The filters were probed with a digoxigenin-labeled PCR product generated by amplification of a conserved verotoxin gene sequence. Replication of the growth on filters allowed probe-positive colonies to be picked. When ground beef samples were inoculated with VTEC strains, 100% of the strains were recovered, and the detection limit was 0.1 CFU per g. Similar results were obtained with seven types of artificially contaminated vegetables. A survey of 32 packages of vegetables and 23 samples of apple cider obtained at the retail level did not reveal the presence of VTEC. However, the intestinal fecal contents of a moose, 1 of 35 wild mammals and birds examined, contained E. coli O157:H7. The DNA hybridization-HGMF method was also used in a prevalence survey of 327 raw and 744 ready-to-eat products; VTEC strains were recovered from 4.9% of the raw products and 0.7% of the ready-to-eat products. No serotype O157:H7 strains were detected. This method is particularly suited for surveys in which low numbers of VTEC-positive samples are expected and isolates are required.
authors.affiliation: Bureau of Microbial Hazards, Food Directorate, Health Protection Branch, Health Canada, Ottawa, Ontario K1A 0L2. ewen_todd@hc-sc.gc.ca
authors.forename: E C
authors.initials: EC
authors.lastname: Todd
authors.forename: R A
authors.initials: RA
authors.lastname: Szabo
authors.forename: J M
authors.initials: JM
authors.lastname: MacKenzie
authors.forename: A
authors.initials: A
authors.lastname: Martin
authors.forename: K
authors.initials: K
authors.lastname: Rahn
authors.forename: C
authors.initials: C
authors.lastname: Gyles
authors.forename: A
authors.initials: A
authors.lastname: Gao
authors.forename: D
authors.initials: D
authors.lastname: Alves
authors.forename: A J
authors.initials: AJ
authors.lastname: Yee
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics*; D001628:Beverages / Q000382:microbiology; D001717:Birds; D003670:Deer; D019453:Escherichia coli O157 / Q000235:genetics* / Q000302:isolation & purification*; D005243:Feces / Q000382:microbiology*; D005516:Food Microbiology; D005638:Fruit / Q000382:microbiology; D008322:Mammals; D008460:Meat / Q000382:microbiology; D016133:Polymerase Chain Reaction; D022622:Shiga Toxin 1; D014675:Vegetables / Q000382:microbiology
chemical_list: D001427:Bacterial Toxins; D022622:Shiga Toxin 1