PDF《Verotoxigenic Escherichia coli》

4775 on February 22,

2019 by guest http://aem.asm.org/ Downloaded from Whereas it has been proven that monoclonal antibodies can be used to detect speci?c pathogens, such antibodies are not always available or may be licensed to a supplier. In contrast, the DNA sequences of speci?c primers are usually published, and PCR products that are used for probes can be made in a standard manner. In this paper we describe a method in which a DNA hybridization procedure is used in combination with HGMFs to isolate VTEC from food and from animal intestinal and fecal samples. This technique can be used in surveys in combination with PCR, a Vero cell assay, or other screening tests and is particularly valuable when low frequencies of pos- itive samples are expected. In this assay samples are screened for VTEC by PCR, and the positive isolates are identi?ed on HGMFs. MATERIALS AND METHODS Media. The following media were purchased from Difco Laboratories, Detroit, Mich.: tryptic soy broth, tryptic soy agar (TSA), brain heart infusion (BHI) broth, BHI agar, MacConkey agar, and MacConkey broth. Other media were prepared from ingredients obtained from Difco Laboratories;

these media included HC broth (20 g of tryptone, 1.12 g of bile salts no. 3,

5 g of NaCl,

10 g of sorbitol, 0.015 g of bromcresol purple,

1 liter of distilled water;

(pH 7.0), Institut Pasteur maintenance medium (5 g of beef extract,

10 g of peptone,

3 g of NaCl,

2 g of Na2HPO4,

10 g of agar,

1 liter of distilled water;

(pH 7.0), and TSA-Nal (TSA supplemented with

30 ?g of nalidixic acid/ml). Bacterial strains and culture conditions. The strains used for inoculation of foods were obtained from the research laboratory culture collection of the Health Protection Branch, Health Canada, and were maintained on TSA slants or Institut Pasteur maintenance medium at room temperature (38). For routine use, strains were inoculated into BHI broth, incubated overnight at 35°C, and diluted to a concentration of approximately

101 CFU/ml in 0.1% peptone water before they were added to foods. Master libraries on HGMFs were prepared as described by Todd et al. (38) and were probed directly as described below. These libraries contained

50 E. coli O157:H7 strains,

3 E. coli O157:NM (nonmotile) strains,

19 other VTEC strains,

2 Shigella dysenteriae strains, and

268 non-VTEC gram-negative bacterial strains (members of Citrobacter, Enterobacter, Klebsiella, Proteus, Salmonella, Shigella, and Yersinia spp.). The E. coli strains used to generate the VTEC probe (H30, E32511, 412, H.I.8, and 933W), as described by Yee et al. (42), were obtained from the Laboratory Services Division, University of Guelph (formerly the Ontario Ministry of Agriculture, Food and Rural Af- fairs). E. coli H30 and ATCC

25922 were used as positive and negative controls on the HGMFs, respectively. Nalidixic acid-resistant O157:H7 VTEC strain E318 from the Health of Animals Laboratory (now Guelph Laboratory, Health Canada), Guelph, Ontario, Canada, was used in the limit-of-detection experi- ment. Arti?cial inoculation of food samples. Arti?cially inoculated foods were pre- pared and tested with the DNA-HGMF method. In order to establish the lowest level of detection (limit of detection) for the assay, locally purchased freshly ground beef, a sample of which had previously tested negative for VTEC as described below, was spiked with VTEC. A total of

111 25-g samples were inoculated individually with between

9 and

18 CFU of

99 different VTEC strains,

10 non-VT-producing E. coli strains, and two S. dysenteriae strains (Table 1) that had been grown overnight at 35°C in BHI broth. The 25-g samples were placed in sterile plastic bags, and the bags were kneaded by hand to distribute the bacteria evenly throughout the meat;