Rapid genotyping of CTX-M extended-spectrum β-lactamases by denaturing high-performance liquid chromatograpy

Denaturing high-performance liquid chromatography (dHPLC) is a powerful technique which has been used extensively to detect genetic variation. This is the first report of the application of dHPLC for rapid genotyping of bacterial beta-lactamase genes. The technique was specifically developed to genotype members of all bla(CTX-M) DNA homology groups. Thirteen well-defined bla(CTx-M) extended-spectrum beta-lactamase (ESBL)-producing strains were used to develop and optimize the dHPLC genotyping assay. Further evaluation was carried out with a blinded panel of 62 clinical isolates. The results of bla(CTX-M) genotyping achieved by dHPLC were comparable to the typing results obtained by DNA sequencing. Applying the newly developed dHPLC-based genotyping method, we successfully genotyped all 73 bla(CTX-M) ESBL-producing strains from the 4-month survey study. Furthermore, we found the first reported cases in the United Kingdom of clinically significant disease caused by CTX-M-14- and CTX-M-1-producing Escherichia coli strains. We conclude that the novel dHPLC assay is highly accurate, rapid, and cost-effective for the genotyping of bla(CTX-M)-producing ESBLs and has great potential for determining the clinical relevance of different and new bla(CTX-M) genotypes, as well as for epidemiological studies and surveillance programs.