title: Detection of mutations in Salmonella enterica gyrA, gyrB, parC and parE genes by denaturing high performance liquid chromatography (DHPLC) using standard HPLC instrumentation.
abstract: OBJECTIVE
  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.
authors.affiliation: Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB, UK. l.randall@vla.defra.gsi.gov.uk
authors.forename: L P
authors.initials: LP
authors.lastname: Randall
authors.forename: N G
authors.initials: NG
authors.lastname: Coldham
authors.forename: M J
authors.initials: MJ
authors.lastname: Woodward
mesh_terms: D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation; D027081:DNA Gyrase / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D027101:DNA Topoisomerase IV / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D015203:Reproducibility of Results; D019779:Salmonella enterica / Q000187:drug effects / Q000235:genetics*; D013696:Temperature
chemical_list: D000900:Anti-Bacterial Agents; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase