淋病奈瑟菌分离株喹诺酮耐药机制的体外分析。
Analysis of quinolone resistance mechanisms in Neisseria gonorrhoeae isolates in vitro.
作者信息
Tanaka M, Sakuma S, Takahashi K, Nagahuzi T, Saika T, Kobayashi I, Kumazawa J
机构信息
Department of Urology, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
出版信息
Sex Transm Infect. 1998 Feb;74(1):59-62. doi: 10.1136/sti.74.1.59.
BACKGROUND AND OBJECTIVES
Gonococcal fluoroquinolone resistance is now a significant problem in Japan. We generated gonococcal mutants resistant to norfloxacin in vitro from norfloxacin sensitive isolates and analysed the contribution of three known mechanisms of quinolone resistance in Neisseria gonorrhoeae.
MATERIALS AND METHODS
Three clinical isolates of N gonorrhoeae susceptible to norfloxacin were exposed to increasing concentrations of norfloxacin. To identify mutations in the gyrA and parC genes of the gonococcal mutants, the quinolone resistance determining regions of the gyrA and parC genes were polymerase chain reaction (PCR) amplified and the PCR products were directly sequenced. Norfloxacin accumulation in the gonococcal cells was also measured.
RESULTS
The MICs of norfloxacin for three variants containing a single GyrA mutation were 16-fold higher than that for their parent isolates. A variant showing reduced norfloxacin accumulation in the cells, without mutations in the GyrA or ParC proteins, was also less sensitive to norfloxacin, with a 16-fold increase in the MIC, compared with the parent strain. The MIC of norfloxacin for a variant which contained a single GyrA mutation with reduced norfloxacin accumulation in the cells was 128-fold higher than for the parent strain. A variant containing mutations in both GyrA and ParC proteins with reduced accumulation of norfloxacin in the cells showed a 256-fold increase in the norfloxacin MIC compared with the parent strain. There was no variant containing a ParC mutation without the simultaneous presence of a GyrA mutation.
CONCLUSIONS
The results from this study suggest that not only a mutation in the gyrA gene but also reduced drug accumulation in cells contributes to the development of fluoroquinolone a mutation in the gyrA gene contributes to a high level of fluoroquinolone resistance in gonococci with decreases in accumulation in cells having an additional but lesser effect.
背景与目的
淋球菌对氟喹诺酮耐药目前在日本是一个重大问题。我们从对诺氟沙星敏感的淋球菌分离株体外诱导出对诺氟沙星耐药的突变株，并分析了淋病奈瑟菌中三种已知喹诺酮耐药机制的作用。
材料与方法
将三株对诺氟沙星敏感的淋病奈瑟菌临床分离株暴露于浓度递增的诺氟沙星中。为鉴定淋球菌突变株gyrA和parC基因中的突变，对gyrA和parC基因的喹诺酮耐药决定区进行聚合酶链反应（PCR）扩增，并对PCR产物直接测序。还测定了诺氟沙星在淋球菌细胞中的蓄积情况。
结果
三个含有单个GyrA突变的变体对诺氟沙星的最低抑菌浓度（MIC）比其亲本分离株高16倍。一个在细胞中诺氟沙星蓄积减少、GyrA或ParC蛋白无突变的变体对诺氟沙星的敏感性也较低，与亲本菌株相比，MIC增加了16倍。一个含有单个GyrA突变且细胞中诺氟沙星蓄积减少的变体对诺氟沙星的MIC比亲本菌株高128倍。一个GyrA和ParC蛋白均有突变且细胞中诺氟沙星蓄积减少的变体与亲本菌株相比，诺氟沙星MIC增加了256倍。不存在仅含ParC突变而无同时存在GyrA突变的变体。
结论
本研究结果表明，不仅gyrA基因突变，而且细胞内药物蓄积减少也有助于氟喹诺酮耐药的产生；gyrA基因突变导致淋球菌对氟喹诺酮高度耐药，而细胞内蓄积减少有额外但较小的影响。
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