PDF《Verotoxigenic Escherichia coli》

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 0099-2240/99/$04.

00?0 Nov. 1999, p. 4775C4780 Vol. 65, No.

11 Copyright ? 1999, American Society for Microbiology. All Rights Reserved. Application of a DNA HybridizationCHydrophobic-Grid Membrane Filter Method for Detection and Isolation of Verotoxigenic Escherichia coli E. C. D. TODD,1 * R. A. SZABO,1 J. M. MACKENZIE,1 ? A. MARTIN,2 K. RAHN,3 C. GYLES,4 A. GAO,4 D. ALVES,5 AND A. J. YEE6 Bureau of Microbial Hazards, Food Directorate, Health Protection Branch, Health Canada, Ottawa, Ontario K1A 0L2,1 Education, Research and Laboratories Division, Ontario Ministry of Agriculture, Food and Rural Affairs, Guelph, Ontario N1G 4Y2,2 Guelph Laboratory, Health Protection Branch, Health Canada, Guelph, Ontario, N1G 3W4,3 Department of Pathobiology, University of Guelph, Guelph, Ontario N1G 2W1,4 Livestock Technology Health Management, Ontario Ministry of Agriculture, Food and Rural Affairs, Fergus, Ontario N1M 2W3,5 and Laboratory Services Division, University of Guelph, Guelph, Ontario N1H 8J7,6 Canada Received

12 April 1999/Accepted

4 August

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 ?ltering VTEC-positive cultures through hydrophobic-grid membrane ?lters (HGMFs) which were incubated on MacConkey agar. The ?lters were probed with a digoxigenin-labeled PCR product generated by ampli?cation of a conserved verotoxin gene sequence. Replication of the growth on ?lters 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 arti?cially 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. More than

200 serotypes of verotoxigenic Escherichia coli (VTEC), also known as Shiga toxin-producing E. coli, have been isolated from cattle (12), and approximately

50 of these serotypes have been identi?ed as causative agents of hemor- rhagic colitis (HC) and hemolytic uremic syndrome (HUS) (1, 3, 11, 13, 25, 26, 36). The E. coli strains which are implicated in HC and HUS in humans are called enterohemorrhagic E. coli (EHEC) strains, but for most serotypes of VTEC it is not known whether the organisms can cause HC and HUS in humans. The serotype most frequently associated with disease outbreaks is E. coli O157:H7, but members of other serotypes, including O26, O104, O111, and O145, have also been respon- sible for serious illnesses and deaths resulting from consump- tion of contaminated food or water. Non-O157 VTEC strains are found in cattle in Ontario at a higher frequency than O157 strains are found (13). Strains of Citrobacter freundii containing verotoxin (VT) genes have also been implicated in food-borne disease (4, 33, 40). VT or Shiga toxins are the main virulence factors in VTEC strains that cause HC and HUS (14), and there is presently no marker other than serotype that can be used to predict the virulence of an isolate (2C4, 12, 13, 18, 20). Therefore, there is a need to identify all VT-positive organ- isms, not just E. coli O157:H7 strains, as all VT-positive or- ganisms need to be treated as potential pathogens. Potential virulence factors, such as the 60-MDa plasmid, Eae, and EHEC hemolysin, have been investigated as virulence mark- ers. Although these factors are correlated with virulence, they cannot be used to assess whether an isolate has the potential to cause disease, as some pathogenic VTEC may lack these genes and proteins (12, 17, 20, 32). PCR methods for detection of VT genes (21C23, 42) can detect VTEC in mixed cultures, but con?rmation of positive results by isolating VTEC is challenging. Broth cultures must be streaked onto selective agar, and individual colonies or colony sweeps must be picked and tested for VT production (7). This is a time-consuming process without any guarantee of success, especially if the food and environmental samples being tested have low concentrations of VTEC. In this paper we describe a DNA probe hybridization procedure in which we used hydrophobic-grid membrane ?lters (HGMFs), herein- after called the DNA-HGMF method. This procedure has the advantage that the HGMFs can be mechanically replicated so that individual bacterial colonies in hydrophobic-grid cells on a replicate correspond to colonies on the original ?lter (34). This procedure was successfully used to isolate E. coli O157 and Salmonella strains from food and environmental samples with speci?c monoclonal antibodies (6, 34, 37C39). A similar meth- od in which a DNA probe hybridization procedure is used with HGMFs to isolate Listeria monocytogenes has also been de- scribed (19). With these methods, not only can speci?c patho- gens be isolated, but there is the additional advantage that multiple strains can be picked, because each HGMF consists of 1,600 separate cells in a hydrophobic grid. * Corresponding author. Mailing address: Bureau of Microbial Haz- ards, Food Directorate, Health Protection Branch, Sir Frederick G. Banting Research Centre, Building Locator 2204A2, Ottawa, Ontario K1A 0L2, Canada. Phone: (613) 957-0887. Fax: (613) 941-0280: E- mail: [email protected]. ? Deceased.