Mutations in the gyrA and parC genes and in vitro activities of fluoroquinolones in 114 clinical isolates of Pseudomonas aeruginosa derived from urinary tract infections and their rapid detection by denaturing high-performance liquid chromatography

Abstract

Fluoroquinolone (FQ) resistance in Pseudomonas aeruginosa has spread. The purpose of this study was to investigate the correlation between representative FQ, i.e. levofloxacin (LVX), resistance and mutations in the gyrA and parC genes of P. aeruginosa clinical isolates from the urine of urinary tract infection patients and their rapid detection by denaturing high-performance liquid chromatography (DHPLC). The susceptibility to LVX of 114 clinical isolates was measured and the quinolone resistance-determining regions (QRDRs) in the gyrA and parC genes of these isolates were sequenced. DHPLC was undertaken to correlate the distinctive chromatograms with their DNA mutation patterns. Among 114 isolates tested, 22 isolates (19.3%) were resistant to LVX. Six amino acid mutations were detected (Thr83Ile, Asp87Tyr and Asp87Asn in gyrA and Ser87Leu, Ser87Trp and Glu91Arg in parC), existing alone or in combination. There were 10 kinds of mutation patterns. The presence of two or more kinds of mutation significantly correlated with LVX resistance compared with the wild-type or a single mutation (P<0.0001). DHPLC data identified the number of amino acid mutations with reproducibility distinguishable by peak number and profile of the DHPLC chromatogram. In conclusion, two or more mutations in gyrA and parC were significantly related to LVX resistance in P. aeruginosa. DHPLC facilitated the detection of resistant alleles, providing a rapid (5 min per sample), economical (96 samples per run) and reliable technique for characterising LVX resistance in P. aeruginosa. This rapid detection system could forecast LVX resistance by the DHPLC profile.