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

• Published in: Journal of antimicrobial chemotherapy Vol. 56; no. 4; pp. 619 - 623
• Main Authors: Randall, L. P., Coldham, N. G., Woodward, M. J.
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01-10-2005; Oxford Publishing Limited (England)
• Subjects: Amino Acid Substitution; Anti-Bacterial Agents - pharmacology; Antibacterial agents; Antibiotics. Antiinfectious agents. Antiparasitic agents; Bacterial diseases; Bacterial diseases of the digestive system and abdomen; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Chromatography, High Pressure Liquid - instrumentation; ciprofloxacin; DNA Gyrase - genetics; DNA Mutational Analysis - methods; DNA Topoisomerase IV - genetics; fluoroquinolones; Fundamental and applied biological sciences. Psychology; Human bacterial diseases; Infectious diseases; Medical sciences; Microbiology; Mutation - genetics; Pharmacology. Drug treatments; Reproducibility of Results; resistance; S. enterica; Salmonella enterica; Salmonella enterica - drug effects; Salmonella enterica - genetics; Salmonella typhimurium; Temperature; Salmonella; HPLC chromatography; Instrumentation; Denaturing HPLC chromatogrphy; Standards; Infection; Chemokine; Resistance; Fluoroquinolone derivatives; Analysis method; Gene; S. enterica; Bacteriosis; Bacteria; Genetics; Salmonellosis; Mutation; fluoroquinolones; Antibacterial agent; Ciprofloxacin; Detection; Quinolone derivatives; Enterobacteriaceae
• ISSN: 0305-7453; 1460-2091
• DOI: 10.1093/jac/dki293

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.