The Escherichia coli O149 rfb gene cluster and its use for the detection of porcine E. coli O149 by real-time PCR

Abstract

PCR or hybridization assays are widely used for the identification and detection of various Escherichia coli serogroups and serotypes. This study explored this approach for the detection of E. coli O149 in pigs, a dominant serogroup among those associated with porcine post-weaning diarrhea (PWD) worldwide. For this purpose, we sequenced the O antigen (rfb) gene cluster of a representative enterotoxigenic E. coli (ETEC) O149:H10 strain and identified a 2kbp region specific for the O149 rfb gene cluster. This region lacked similarity with sequences in GenBank, except for the rfb gene cluster of the human pathogen Shigella boydii O1 with which it shares 99% sequence identity. Southern blot hybridization with 91 E. coli strains belonging to 62 serogroups confirmed the specificity of the identified region. Restriction analysis of the rfb gene cluster of 38 geographically and chronologically diverse E. coli O149 strains with the endonucleases HindIII, PstI and XhoI demonstrated that this cluster is highly conserved across the serogroup O149. Analysis of the O antigen of a representative O149:H10 ETEC strain by nuclear magnetic resonance confirmed the identity of the O antigen structure in recent and older O149 ETEC strains. Finally, we developed a real-time PCR assay for the detection of E. coli O149 in pig fecal samples by targeting the part of the rfb sequence specific for this serogroup. This method was suitable for a sensitive qualitative detection of O149 E. coli in pig fecal samples after enrichment, but further investigations are needed to find a suitable method for a direct, reproducible and sensitive quantification of O149 E. coli in pig feces.