Journal
LETTERS IN APPLIED MICROBIOLOGY
Volume 53, Issue 3, Pages 264-270Publisher
WILEY-BLACKWELL
DOI: 10.1111/j.1472-765X.2011.03100.x
Keywords
Detection; E. coli; environmental; food safety; identification
Categories
Funding
- Ministry of Education, Culture, Sports, Science and Technology of Japan [18602001]
- Ministry of Health, Labour and Welfare of Japan
- Grants-in-Aid for Scientific Research [18602001, 21241005] Funding Source: KAKEN
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Aims: The present study aimed to develop a colony hybridization method for the exhaustive detection and isolation of diarrhoeagenic Escherichia coli (DEC) from samples containing numerous coliform bacteria. Methods and Results: Digoxigenin-labelled DNA probes were designed to detect seven pathotypes of DEC based on type-specific genes. A total of 615 meat, food and faeces samples identified as DEC-positive by multiple real-time PCR for the virulence genes (eae, stx, elt, est, virB, aggR, afaB and astA) were analysed by a colony hybridization method, which involved filtering enrichment cultures through hydrophobic grid-membrane filters. DEC were isolated from 72.5% (446/615) of samples by the colony hybridization method but were only detected in 26.3% (162/615) of samples by a conventional culture method. The hybridization method was particularly effective for isolating low-level contaminants, such as enterotoxigenic and Shiga toxin-producing E. coli, which were isolated from 51.8% (58/112) of samples identified as positive by PCR for the enterotoxin genes, in contrast to only 4 5% (5/112) of samples analysed by the conventional method. Conclusions: The developed colony hybridization system allows for the efficient and simultaneous isolation of all DEC pathotypes. Significance and Impact of the Study: The colony hybridization system described here permits the sensitive isolation of DEC and represents a suitable tool for ecological investigations of DEC.
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