Exhaustive isolation of diarrhoeagenic Escherichia coli by a colony hybridization method using hydrophobic grid-membrane filters in combination with multiplex real-time PCR.

摘要

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