Rapid detection of gyrA and parC mutations in fluoroquinolone-resistant Neisseria gonorrhoeae by denaturing high-performance liquid chromatography.
The detection of DNA sequence variation is fundamental to the identification of the genomic basis of phenotypic variability. Denaturing high-performance liquid chromatography (DHPLC) is a novel technique that is used to detect mutations in human DNA. This is the first report that this technique is used as a tool to detect mutations in genes encoding fluoroquinolone resistance in Neisseria gonorrhoeae. Eighty-one strains of N. gonorrhoeae were used in this study. Genomic DNA from each strain was subjected to PCR amplification of 225 bp in gyrA and 166 bp in parC spanning the fluoroquinolone-resistance determining regions (QRDRs). After we performed DNA sequencing of these amplicons and identification of mutations in the QRDRs, DHPLC was undertaken to investigate whether its results correlate the distinctive chromatogram with their DNA mutations pattern. The profilings detected by DHPLC completely corresponded to the results of the DNA sequencing in mutation patters in gyrA and parC genes. They resulted in the following amino acid substitutions: Ser-91Phe, Asp-95Gly, and Asp-95Asn in gyrA; and Gly-85Asp, Asp-86Asn, Ser-87Arg, and Ser-88Pro in parC, respectively. These mutations existed alone or as combinations, and we identified five mutations patterns in gyrA and six in parC including wild-type. These mutations and their patterns could be rapidly and reproducibly identified from the PCR products using DHPLC, producing specific peak patterns that correlate with genotypes. This novel detection system facilitates the detection of resistance alleles, providing a rapid (5 min per sample), economic (96 sample per run), and reliable technique for characterizing fluoroquinolone resistance in N. gonorrhoeae.
Division of Urology, Department of Organs Therapeutics, Faculty of Medicine, Kobe University Graduate School of Medicine, Japan.
Katsumi
K
Shigemura
Toshiro
T
Shirakawa
Hiroshi
H
Okada
Kazushi
K
Tanaka
Tohru
T
Udaka
Sadao
S
Kamidono
Soichi
S
Arakawa
Akinobu
A
Gotoh
D000900:Anti-Bacterial Agents / Q000494:pharmacology* / Q000627:therapeutic use; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D027081:DNA Gyrase / Q000235:genetics*; D027101:DNA Topoisomerase IV / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics; D024841:Fluoroquinolones / Q000494:pharmacology* / Q000627:therapeutic use; D006801:Humans; D008297:Male; D008826:Microbial Sensitivity Tests; D009344:Neisseria gonorrhoeae / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D017354:Point Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014526:Urethritis / Q000382:microbiology
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D024841:Fluoroquinolones; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase