gyrA、gyrB和外排系统共同介导铜绿假单胞菌对喹诺酮类耐药机制研究

Mechanisms of gyrA,gyrB and efflux system jointly mediating quinolone resistance in clinical isolates of Pseudomonas aeruginosa

目的探讨临床分离的对环丙沙星和左氧氟沙星均耐药的铜绿假单胞菌耐药机制。方法收集临床分离经VITEK-2(C0mpact细菌鉴定仪检测环丙沙星和左氧氟沙星均耐药的铜绿假单胞菌,琼脂稀释法测定环丙沙星和左氧氟沙星的MIC值,PCR扩增DNA促旋酶基因gyrA和gyrB以及DNA拓扑异构酶Ⅳ的parC和parE基因,实时-RT-PCR分析细菌外排系统表达情况。结果琼脂稀释法检测结果与VITEK-2 Compact细菌鉴定仪检测结果相符。PCR扩增测序发现以DNA促旋酶基因gyrA(在位点941处插入碱基C)和gyrB(3株在位点1588处缺失碱基A,其他菌株在位点1543处插入碱基T)基因缺失或者插入导致移码突变为主,parE基因有3株在位点1895处插入碱基C。实时定量PCR检测发现以mexA和mexC表达增加为主。结论检出的耐环丙沙星和左氧氟沙星铜绿假单胞菌是由于DNA促旋酶基因gyrA和gyrB基因的突变和mexAB-OprM和mexCD-OprJ表达增加共同作用的结果。

Objective To investigate the mechanisms of both levofloxacin and ciprofloxacin resistance in clinical strains of Pseudomonas aeruginosa isolated from our hosptial.Methods Twenty P.aeruginosa isolates resistant to both levofloxacin and ciprofloxacin as tested by VITEK-2 Compact were collected.Agar dilution method was used to confirm their minimum inhibitory concentrations（MICs） of levofloxacin and ciprofloxacin.DNA gyrase（gyrA and gyrB） and topoisomerase Ⅳ（parC and parE） were analyzed by PCR amplification.The expression of efflux systems were analyzed by real-time RT-PCR.Results The MIC results were consistent between Agar dilution method and Vitek-2 Compact system.DNA gyrase（gyrA and gyrB） sequencing analysis showed that the mutations were mainly frame-shifting mutation characteristic of base（C） insertion at position941 in gyrA gene,deletion of base（A） at position 1588 in gyrB gene,and insertion of base（T） at position 1543 in gyrB gene.Insertion of base（C） at position 1895 in parE gene was identified in 3 strains.Overexpression of MexAB-OprM and MexCD-Opr]was detected by real-time RT-PCR.Conclusions The insertion and/or deletion of bases in DNA gyrase（gyrA and gyrB） genes and overproduction of MexAB-OprM and MexCD-Opr]efflux systems may contribute to the resistance to both ciprofloxacin and levofloxacin in clinical isolates of P.aeruginosa.