ANALYSIS OF QUINOLONE RESISTANCE MECHANISMS IN NEISSERIA-GONORRHOEAE ISOLATES IN-VITRO

Background and objectives: Gonococcal fluoroquinolone resistance is no w a significant problem in Japan. Fire generated gonococcal mutants re sistant to norfloxacin in vitro from norfloxacin sensitive isolates an d analysed the contribution of three known mechanisms of quinolone res istance in Neisseria gonorrhoeae. Materials and methods: Three clinica l isolates of N gonorrhoeae susceptible to norfloxacin were exposed to increasing concentrations of norfloxacin. To identify mutations in th e gyrA and parC genes of the gonococcal mutants, the quinolone resista nce determining regions of the gyrA and parC genes were polymerase cha in reaction (PCR) amplified and the PCR products were directly sequenc ed. Norfloxacin accumulation in the gonococcal cells was also measured . Results: The MICs of norfloxacin for three variants containing a sin gle GyrA mutation were 16-fold higher than that for their parent isola tes. A variant showing reduced norfloxacin accumulation in the cells, without mutations in the GyrA or ParC proteins, was also less sensitiv e to norfloxacin, with a 16-fold increase in the MIG, compared with th e parent strain. The MIC of norfloxacin for a variant which contained a single GyrA mutation with reduced norfloxacin accumulation in the ce lls was 128-fold higher than for the parent strain. A variant containi ng mutations in both GyrA and ParC proteins with reduced accumulation of norfloxacin in the cells showed a 256-fold increase in the norfloxa cin MIC compared with the parent strain. There was no variant containi ng a ParC mutation without the simultaneous presence of a GyrA mutatio n. Conclusions: The results from this study suggest that not only a mu tation in the gyrA gene but also reduced drug accumulation in cells co ntributes to the development of fluoroquinolone resistance in gonococc i and that a mutation in the parC gene with the simultaneous presence of a mutation in the gyrA gene contributes to a high level of fluoroqu inolone resistance in gonococci with decreases in accumulation in cell s having an additional but lesser effect.