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

Abstract

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. 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. The developed colony hybridization system allows for the efficient and simultaneous isolation of all DEC pathotypes. The colony hybridization system described here permits the sensitive isolation of DEC and represents a suitable tool for ecological investigations of DEC.