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

摘要

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 Compact 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 inhibito-ry 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 )se-quencing analysis showed that the mutations were mainly frame-shifting mutation characteristic of base (C)insertion at position 941 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-OprJ 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-OprJ efflux systems may contribute to the resistance to both ciprofloxacin and levofloxacin in clinical isolates of P .aeruginosa.