Detection of mutations in gyrB using denaturing high performance liquid chromatography (DHPLC) among Salmonella enterica serovar Typhi and Paratyphi A

Abstract

Background: Fluoroquinolone resistance is mediated by mutations in the quinolone-resistance determining region (QRDR) of the topoisomerase genes. Denaturing high performance liquid chromatography (DHPLC) was evaluated for detection of clinically important mutations in gyrB among Salmonella.

Methods: Salmonella Typhi and S. Paratyphi A characterised for mutation in QRDR of gyrA, parC and parE were studied for mutation in gyrB by DHPLC and validated by sequencing.

Results: The DHPLC analysis was able to resolve the test mutant from isolates with wild type gyrB and distinguished mutants from other mutant by peak profile and shift in retention time. Three sequence variants were detected at codon 464, and a novel mutation Ser→Thr was also detected. gyrB mutation was associated with non classical quinolone resistance (NALS-CIPDS) in 34 isolates of S. Typhi only and was distinct from classical quinolone resistance associated with gyrA mutations (NALR-CIPDS).

Conclusions: DHPLC is effective for the detection of mutation and can reduce the need for sequencing to detect clinically significant gyrB mutations.

Genbank accession nos: KF993966, KF993965 and KF993964.

Keywords: DHPLC; Decreased ciprofloxacin susceptibility; Salmonella Paratyphi A; Salmonella Typhi; gyrB mutation.