Journal Article

Detection of mutations in <i>Salmonella enterica</i> <i>gyrA</i>, <i>gyrB</i>, <i>parC</i> and <i>parE</i> genes by denaturing high performance liquid chromatography (DHPLC) using standard HPLC instrumentation

L. P. Randall, N. G. Coldham and M. J. Woodward

in Journal of Antimicrobial Chemotherapy

Published on behalf of British Society for Antimicrobial Chemotherapy

Volume 56, issue 4, pages 619-623
Published in print October 2005 | ISSN: 0305-7453
Published online September 2005 | e-ISSN: 1460-2091 | DOI: http://dx.doi.org/10.1093/jac/dki293
Detection of mutations in Salmonella enterica gyrA, gyrB, parC and parE genes by denaturing high performance liquid chromatography (DHPLC) using standard HPLC instrumentation

Show Summary Details

Preview

Aims: Quinolone antibiotics are the agents of choice for treating systemic Salmonella infections. Resistance to quinolones is usually mediated by mutations in the DNA gyrase gene gyrA. Here we report the evaluation of standard HPLC equipment for the detection of mutations (single nucleotide polymorphisms; SNPs) in gyrA, gyrB, parC and parE by denaturing high performance liquid chromatography (DHPLC).

Methods: A panel of Salmonella strains was assembled which comprised those with known different mutations in gyrA (n = 8) and fluoroquinolone-susceptible and -resistant strains (n = 50) that had not been tested for mutations in gyrA. Additionally, antibiotic-susceptible strains of serotypes other than Salmonella enterica serovar Typhimurium strains were examined for serotype-specific mutations in gyrB (n = 4), parC (n = 6) and parE (n = 1). Wild-type (WT) control DNA was prepared from Salmonella Typhimurium NCTC 74. The DNA of respective strains was amplified by PCR using Optimase® proofreading DNA polymerase. Duplex DNA samples were analysed using an Agilent A1100 HPLC system with a Varian Helix™ DNA column. Sequencing was used to validate mutations detected by DHPLC in the strains with unknown mutations.

Results: Using this HPLC system, mutations in gyrA, gyrB, parC and parE were readily detected by comparison with control chromatograms. Sequencing confirmed the gyrA predicted mutations as detected by DHPLC in the unknown strains and also confirmed serotype-associated sequence changes in non-Typhimurium serotypes.

Conclusions: The results demonstrated that a non-specialist standard HPLC machine fitted with a generally available column can be used to detect SNPs in gyrA, gyrB, parC and parE genes by DHPLC. Wider applications should be possible.

Keywords: ciprofloxacin; S. enterica; fluoroquinolones; resistance

Journal Article.  3290 words.  Illustrated.

Subjects: Medical Oncology ; Critical Care

Full text: subscription required

How to subscribe Recommend to my Librarian

Users without a subscription are not able to see the full content. Please, subscribe or login to access all content.