Application of a DNA hybridization-hydrophobic-grid membrane filter method for detection and isolation of verotoxigenic Escherichia coli

• Published in: Applied and environmental microbiology 1999-11, Vol.65 (11), p.4775-4780
• Main Authors: Todd, E.C.D, Szabo, R.A, Mackenzie, J.M, Martin, A, Rahn, K, Gyles, C, Gao, A, Alves, D, Yee, A.J
• Format: Article
• Language: English
• Subjects: Animals; Bacterial Toxins - biosynthesis; Bacterial Toxins - genetics; Bacteriological methods and techniques used in bacteriology; Bacteriology; Beverages - microbiology; Biological and medical sciences; Birds; Deer; DNA hybridization; Escherichia coli; Escherichia coli O157 - genetics; Escherichia coli O157 - isolation & purification; Feces - microbiology; food contamination; Food Microbiology; Fruit - microbiology; Fundamental and applied biological sciences. Psychology; Mammals; Meat - microbiology; microbial contamination; Microbiology; Polymerase Chain Reaction; Shiga Toxin 1; Vegetables - microbiology; verocytotoxins; verotoxins
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.65.11.4775-4780.1999

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.