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Escherichia coli O157:H7 can cause hemorrhagic colitis and other diseases through consumption of food and water and by human-to-human transmission (5). Infection by E. coli O157:H7 has become a very important food-borne disease in developed countries (4). One of the common modes of transmission appears to be the consumption of contaminated beef and related products (5). Isolation rates of E. coli O157:H7 from ground-beef samples in North America ranged from none to 3.7% (5). There have been reports on the isolation of this organism in other parts of the world (4). In Asia, isolation of E. coli of serogroup O157 has so far been reported in Japan, India, and China (5, 6, 15). The strains isolated in Asian countries other than Japan have not been well characterized. Other Asian countries do not seem to be exempt from the E. coli O157:H7 infection, although a study in Thailand failed to isolate E. coli of serogroup O157 from beef samples (14). We therefore investigated in this study whether E. coli O157:H7 is distributed in retailed beef in Malaysia.

Isolation of E. coli O157:H7. Beef originally imported from India through an importer and sold as tenderloin was purchased from four retail stores in Malaysia at certain intervals. Twenty-five beef samples thus obtained were examined in this study. A 25-g portion of each beef sample was homogenized in a stomacher with 225 ml of medium (Difco Laboratories, Detroit, Mich.) for 2 min and then incubated statically at 37°C for 4 h. The culture was diluted in tryptone water (1% tryptone, 0.5% NaCl), inoculated onto MacConkey agar (Oxoid, Ltd., Basingstoke, England), and incubated overnight at 37°C. Twenty to 50 colonies per sample were selected and screened for lactose fermentation (blue-black colony with a greenish metallic sheen) on eosin methylene blue agar (Oxoid) and for sorbitol nonfermentation (colorless colony) on sorbitol MacConkey agar (Oxoid) and the same medium containing cefixime (50 µg/liter) and potassium tellurite (2.5 mg/liter) (2, 16). The isolates thus selected were subjected to the standard biochemical tests for identification of E. coli. The tests included conventional indole-methyl red-Voges-Proskauer-citrate and lysine decarboxylase tests, TSI reactions, and examination with an API20E test strip (Biomerieux Vitek, Inc., Hazelwood, Mo.). The identified strains were then screened for the absence of -glucuronidase with sorbitol MacConkey agar with added 5-bromo-4-chloro-3-indoxyl--D-glucuronic acid cyclohexylammonium salt (0.1 mg/ml) and Fluorocult O157:H7 medium (Merck, Darmstadt, Germany). The -glucuronidase-negative strains were further screened for the presence of the O157 antigen by the latex agglutination test with the E. coli O157 test kit (Oxoid) and the Serobact test kit E. coli O157 (Medvet Science Pty Ltd., Adelaide, Australia) according to the manufacturers' specifications.

Virulence-associated traits. E. coli O157:H7 has important virulence attributes: production of Shiga toxin (Stx, synonymous with Vero toxin and Shiga-like toxin [3]) and attaching and effacing adherence (5). These characteristics of the test strains were examined as follows. To examine the ability to produce Shiga toxin, the test strain was grown in CAYE medium (2% Casamino Acids, 0.6% yeast extract, 0.25% NaCl, 0.87% K₂HPO₄, 0.005% MgSO₄, 0.0005% FeCl₃) with shaking (130 rpm) at 37°C for 15 h. The culture supernatant was then tested for the presence or absence of Stx1 and Stx2 by a reversed passive latex agglutination test with a commercially available kit (Verotox-F SEIKEN; Denka Seiken Co.). The presence or absence of the stx₁ and stx₂ genes in the test strain was tested by a PCR method. The test strain was grown on tryptic soy agar (Difco), and the growth was suspended in distilled water to achieve a slight turbidity (ca. 10⁸ cells/ml). PCR was performed with a PCR amplification kit and EVT-1, EVT-2, EVS-1, and EVS-2 primers purchased from TaKaRa Biomedicals (Tokyo, Japan), and the amplicons of the specified sizes were detected by 1.5% agarose gel electrophoresis according to the manufacturer's specifications. The presence or absence of the eae gene, which is necessary for attaching and effacing adherence (5), was examined by the DNA colony hybridization method with a polynucleotide probe. The 1-kb HindIII fragment isolated from pCDV443 was used as the probe. pCVD443 was constructed by the isolation of the 1-kb SalI-KpnI fragment of pCVD434 (8), the addition of the HindIII linkers to the isolated fragment by PCR, and the cloning of the fragment into the HindIII site of pUC19 in James B. Kaper's laboratory (9). The probe DNA was labeled with [-³²P]dCTP, and hybridization was performed under high-stringency conditions as described previously (10). In addition, E. coli O157:H7 carries a plasmid of 60 MDa, a putative factor involved in adherence (5). We examined the presence or absence of the 60-MDa plasmid in the test strains. Plasmid DNA was extracted by the alkaline lysis method with a commercially available kit (FlexiPrep kit; Pharmacia Biotech Inc., Uppsala, Sweden) according to the manufacturer's specifications, except that the DNA purification step with Sephaglas FP slurry was omitted. The DNA was resolved on a 0.7% agarose gel and visualized by staining with ethidium bromide and photographed with short-wave UV light. For detection of the eae gene and the 60-MDa plasmid, strain EDL933, an isolate from an outbreak in Michigan in 1982 (11, 13), was employed as the control strain. The results of the tests for the stx genes are presented in Table 1. The results of the detection of the Stxs and the respective genes for the 12 O157:H7 strains agreed (data not shown in Table 1). Seven of the 12 O157:H7 strains had the ability to produce both Stx1 and Stx2. The remaining five strains were capable of producing Stx2 only. The 12 strains had the eae gene (Table 1) and a 60-MDa plasmid (Fig. 1, lanes 3 to 14). Two non-O157 strains, MA38 and MA39, were unable to produce Stx and had neither the eae gene nor the 60-MDa plasmid.

View larger version (73K): [in this window] [in a new window]   FIG. 1.   Results of the plasmid assay. Lanes: 1, phage  DNA digested with HindIII (molecular size markers); 2, EDL933 (control strain for 60-MDa plasmid); 3, MA1; 4, MA6; 5, MA7; 6, MA17; 7, MA40; 8, MA43; 9, MA51; 10, MA53; 11, MA59; 12, MA21; 13, MA29; 14, MA32; 15, MA38; and 16, MA39. The 60-MDa plasmid is indicated by the open triangle. The two smears at lower positions (above the largest [23 kb]  HindIII marker) are residual chromosomal DNA. An apparently smaller plasmid in strains MA1, MA6, and MA7 is indicated by the solid triangle. These plasmids overlap the smear of chromosomal DNA (lanes 3 to 5).

Diversity of the strains. The 12 O157:H7 strains differed regarding some other characteristics. Of the five strains producing only Stx2, three strains had an additional plasmid apparently smaller than 60 MDa (Fig. 1, lanes 3 to 5). The antibiograms of the 12 strains were examined. Resistance or sensitivity of the test strain was examined on Mueller-Hinton agar (Oxoid) by the standard single-disk method (1) with each of the following antibiotic disks: ceftriazone (30 µg/ml, BBL Sensi-Disk; Becton Dickinson, Franklin Lakes, N.J.) ceftrizomine (30 µg/ml, BBL Sensi-Disk), carbenicillin (100 µg/ml, BBL Sensi-Disk), cefuroxime (30 µg/ml, BBL Sensi-Disk), tetracycline (30 µg/ml; Oxoid), cephalotin (30 µg/ml; Oxoid), nalidixic acid (30 µg/ml; Oxoid), amoxicillin (30 µg/ml, BBL Sensi-Disk), gentamicin (10 µg/ml, BBL Sensi-Disk), kanamycin (30 µg/ml; Oxoid), chloramphenicol (30 µg/ml; Oxoid), streptomycin (10 µg/ml; Oxoid), ampicillin (10 µg/ml; Oxoid), rifampin (15 µg/ml, BBL Sensi-Disk), norfloxacin (10 µg/ml, BBL Sensi-Disk), and vancomycin (30 µg/ml, BBL Sensi-Disk). The tests were done three times, and identical results were obtained for each strain and for all antibiotics. The results are summarized in Table 1. None of the strains showed identical antibiograms. The genetic difference among the strains was examined by an arbitrarily primed PCR (AP-PCR) method. Primer 1 (5'-d[GGTGCGGGAA]-3'), primer 2 (5'-d[GTTTCGCTCC]-3'), primer 3 (5'-d[GTAGACCCGT]-3'), primer 4 (5'-d[AAGAGCCCGT]-3'), primer 5 (5'-d[AACGCGCAAC]-3'), and primer 6 (5'-d[CCCGTCAGCA]-3'), included in RAPD analysis primer set (Pharmacia Biotech, Inc.), and the total DNAs extracted from the test strains were used for the AP-PCR assay as described previously (12). All 12 O157:H7 strains and the control strain, EDL933, had many amplicon bands in common, but strain-to-strain variation could be detected by the presence or absence of some other bands. Representative AP-PCR profiles are shown in Fig. 2. A detailed comparison of the AP-PCR profiles obtained with the six different primers indicated that the 12 O157:H7 strains could be classified into 11 different genetic types, as shown in Table 2 (genetic type numbers 2 to 12).

View larger version (87K): [in this window] [in a new window]   FIG. 2.   Representative results of the AP-PCR assay. The results obtained with primer 1 and primer 5 are shown in panels A and B, respectively. Lanes: 1, mixture of phage  DNA digested with HindIII and phage X174 DNA digested with HaeIII (molecular size markers); 2, EDL933 (a control strain); 3, MA1; 4, MA6; 5, MA7; 6, MA17; 7, MA40; 8, MA43; 9, MA51; 10, MA53; 11, MA59; 12, MA21; 13, MA29; 14, MA32; 15, MA38; and 16, MA39.