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

Verotoxigenic Escherichia coli (VTEC) strains were isolated from food and a nimal fecal samples by using PCR to screen for the presence of VTEC after b roth enrichment and then filtering VTEC-positive cultures through hydrophob ic-grid membrane filters (HGMFs) which were incubated on MacConkey agar. Th e filters were probed with a digoxigenin-labeled PCR product generated by a mplification of a conserved verotoxin gene sequence. Replication of the gro wth on filters allowed probe-positive colonies to be picked. When ground be ef samples were inoculated with VTEC strains, 100% of the-strains were reco vered, and the detection limit was 0.1 CFU per g. Similar results were obta ined with seven types of artificially contaminated vegetables. A survey of 32 packages of vegetables and 23 samples of apple:cider obtained at the ret ail level did not reveal the presence of VTEC. However, the intestinal feca l 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 preval ence survey of 327 raw and 744 ready-to-eat products; VTEC strains were rec overed 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 sui ted for surveys in which low numbers of VTEC-positive samples are expected and isolates are required.