Rapid determination of Escherichia coli O157:H7 lineage types and molecular subtypes by using comparative genomic fingerprinting

Abstract

In this study, variably absent or present (VAP) regions discovered through comparative genomics experiments were targeted for the development of a rapid, PCR-based method to subtype and fingerprint Escherichia coli O157:H7. Forty-four VAP loci were analyzed for discriminatory power among 79 E. coli O157:H7 strains of 13 phage types (PT). Twenty-three loci were found to maximize resolution among strains, generating 54 separate fingerprints, each of which contained strains of unique PT. Strains from the three previously identified major E. coli O157:H7 lineages, LSPA6-LI, LSPA6-LI/II, and LSPA6-LII, formed distinct branches on a dendrogram obtained by hierarchical clustering of comparative genomic fingerprinting (CGF) data. By contrast, pulsed-field gel electrophoresis (PFGE) typing generated 52 XbaI digestion profiles that were not unique to PT and did not cluster according to O157:H7 lineage. Our analysis identified a subpopulation comprised of 25 strains from a closed herd of cattle, all of which were of PT87 and formed a cluster distinct from all other E. coli O157:H7 strains examined. CGF found five related but unique fingerprints among the highly clonal herd strains, with two dominant subtypes characterized by a shift from the presence of locus fprn33 to its absence. CGF had equal resolution to PFGE typing but with greater specificity, generating fingerprints that were unique among phenotypically related E. coli O157:H7 lineages and PT. As a comparative genomics typing method that is amenable for use in high-throughput platforms, CGF may be a valuable tool in outbreak investigations and strain characterization.

FIG. 1.

The dendrogram created when the 23-locus binary fingerprint of each of the 79 E. coli O157:H7 strains and non-O157:H7 strain K-12 MG1655 were clustered using Bionumerics version 5.1. A black square represents the presence of a locus and a white square represents the absence of a locus, as determined by PCR. Sequence data for EDL933 indicate that locus fprn16 of OI#93/S-loop#153 is homologous to that of Sakai; however, repeated efforts to amplify fprn16 in EDL933 were unsuccessful. Similarly, previous work with the Sakai strain used in this study has shown a deletion of fprn29 in OI#57/S-loop#108.

FIG. 2.

The dendrogram created when the XbaI PFGE banding pattern of each of the 79 E. coli strains studied were clustered by UPGMA using the Dice coefficient in Bionumerics version 5.1, with an optimization of 1.5% and a tolerance of 1.5%.