Construction of quinolone resistant model of Klebsiella pneumoniae in vitro and the association of the mutations of gyrA and parC gone with resistance

Abstract

Objective To construct quinolone resistant model of Klebsiella pneumoniae in vitro and investigate the association of mutations of gyrA and parC gone with the quinolone resistance. Methods Klebsialla pneumoniae were isolated and cultured with levofloxacin at subinhibitory concentration in media first, then the induced drug was increased at double concentration until klebsialla pneumoniae grew well on the plate containing high concentration of levofloxacin. Resistant strains were harvested and detected for mutations of quinolone resistance determining regions (QRDR)in gyrA gone and parC gone by PCR amplification and DNA sequence. Results Quinolone resistant model of Klebsiella pneumoniae in vitro was constructed successfully at high concentration. All the strains had mutations at the QRDR, most of which had double mutations of both gyrA and parC. Mutations of gyrA related to quinolone resistance primarily included Ser83 and Asp87 (Ser83→Ile, Asp87→Arg), some of the strains mutated at 86 position amino acid(Tyr86→Ser). Most of the strains parC mutated from Ser to Ile at 80 position amino acid. Conclusions Long-term use of quinolone at low concentration can contribute to resistance of Klebsiella pneumoniae. The mutation of QRDR is closely associated to the quinoione resistance. The double mutations of gyrA and parC can result in high resistance, but other unknown mechanism of drug resistance can not be excluded. Key words: KlebsieUa pneumoniae; Quinolones; Drug tolerance; DNA gyrase; DNA topoisomerase Ⅳ ; Quinolone resistance-determining region