耐喹诺酮类铜绿假单胞菌的耐药机制研究

目的探讨广州地区铜绿假单胞菌对喹诺酮类药物的耐药机制。方法对喹诺酮耐药株的gyrA和parC基因进行限制性片段长度多态性分析（PCR-RFLP）,并对其中的高水平耐药株gyrB和parE基因进行测序;用琼脂稀释法测定加入碳酰氰基-对-氯苯腙（CCCP）前后环丙沙星的最小抑菌浓度（MIC）;同时用SDS-PAGE对高水平耐药株的外膜蛋白进行电泳分析。结果有72.7%（72/99）的菌株发生gyrA突变,主要为Thr-83-Ile;25.3%（25/99）的菌株发生parC突变,主要为Ser-87-Leu,且均是gyrA和parC双基因突变;gyrB和parE突变较少见。53.3%（53/99）的菌株的MIC可被CCCP逆转,其MIC能明显降低;7%（7/10）的高水平耐药株的外膜蛋白在43～67kDa间条带增多,其蛋白含量有差异。结论抗菌药物作用靶位的改变和外排泵机制是本地区铜绿假单胞菌对喹诺酮类耐药的重要机制。

Study on the Mechanisms of Quinolone Resistance in Pseudomonas aeruginosa

Objective To study the molecular mechanism of quinolone resistance in P.aeruginosa in Guangzhou area.Methods Polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） and DNA sequencing method was used for detecting the mutations in the gyrA,parC,gyrB,and parE genes.The dilution method was used to determine the minimum inhibitory concentration （MIC） with or without the efflux pump inhibitor carbonylcyanide-pchlorophenyl hydrazone （CCCP）.The outer membrane proteins were analyzed by SDS-PAGE.Results gyrA mutations （mostly Thr-83-Ile） could be observed in 72.7% （72/99） strains,and parC mutations （mostly Ser-87-Leu） were identified in 25.3% （25/99） strains,while mutations in gyrB or parE were relatively infrequent.The MlC of 53.3% （53/99） strains were reduced significantly by adding CCCP.7%（7/10） of all the high level quinolone resistance strains showed increased expression of outer membrane proteins between 43-67 kDa.Conclusion The mutation of two types of topoisomerases and efflux pump may be the mainly causes of quinolone resistance in P.aeruginosa.