Rapid detection of the fluoroquinolone resistance-associated ParC mutation in Neisseria gonorrhoeae using TaqMan probes.
BACKGROUND
In fluoroquinolone-resistant Neisseria gonorrhoeae, the amino acid mutations in the fluoroquinolone-resistant determining region (QRDR) of the parC gene are an important factor. The aim of the present study was to develop a rapid detection method of a serine 88 to proline substitution in parC which we previously showed as having significantly higher fluoroquinolone minimal inhibitory concentrations (MIC) using the TaqMan discrimination system.


METHODS
We extracted DNA from 90 urine or urethral swab samples obtained from male patients with urethritis caused by N. gonorrhoeae. After DNA extraction, they were subjected to real-time polymerase chain reaction (PCR) using a TaqMan discrimination system and compared with the results of conventional DNA sequencing.


RESULTS
Of the 90 samples, the TaqMan technique result showed 13 samples that were classified as having a serine 88 to proline mutation in parC, and 77 samples that did not have a serine 88 to proline mutation in parC. The classifications of all samples completely corresponded to those determined by conventional DNA sequencing. We also found that N. gonorrhoeae with a serine 88 to proline mutation in parC have a significantly higher MIC to ciprofloxacin than that without a serine 88 to proline mutant in parC.


CONCLUSIONS
The present genotyping method of real-time PCR using a TaqMan discrimination system could be applied to the rapid detection of a serine 88 to proline amino acid mutation in parC of N. gonorrhoeae. This point mutation is significant for the determination of fluoruquinolone resistance. This rapid detection system may lead to the prevention of use of noneffective antimicrobial agents and a decrease of resistant strains.
Shigemura
Katsumi
K
Division of Urology, Department of Organs Therapeutics, Faculty of Medicine, Kobe University School of Medicine, Kobe, Japan.
Shirakawa
Toshiro
T
Tanaka
Kazushi
K
Arakawa
Soichi
S
Gotoh
Akinobu
A
Fujisawa
Masato
M
D027101:DNA Topoisomerase IV / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D024841:Fluoroquinolones / Q000494:pharmacology*; D005838:Genotype; D006069:Gonorrhea / Q000188:drug therapy / Q000382:microbiology; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D009344:Neisseria gonorrhoeae / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D019914:Taq Polymerase; D014526:Urethritis / Q000188:drug therapy / Q000382:microbiology
D004269:DNA, Bacterial; D024841:Fluoroquinolones; D019914:Taq Polymerase; D027101:DNA Topoisomerase IV