耐喹诺酮铜绿假单胞菌药物作用靶位改变的研究

Study on the Mechanism of Quinolone Resistance in Pseudomonas Aeruginosa

目的：探讨铜绿假单胞菌对喹诺酮类药物的耐药机制。方法30株对环丙沙星耐药的铜绿假单胞菌，采用 PCR方法扩增DNA解旋酶和拓扑异构酶Ⅳ的基因 gyrA，gyrB，parC和 parE，再测序查找是否存在位点突变，同时用脉冲场电泳（pulsed-field gel electrophoresis，PFGE）对菌株进行同源性分析。结果30株菌中，28株菌出现 gyrA基因扩增片段的137位点均有C→T突变，导致T83I改变；17株菌gyrB基因扩增片段的351位出现G→C突变，导致G466A改变；21株菌的 parC基因扩增片段的277位点有 C→U 突变导致 S87L 改变；2株菌 parE 基因在不同位点出现 C→U 突变，导致A425V和 A473V改变。30株菌可分为6个克隆，其中 A克隆4株，仅有 gyrA突变；B克隆7株，有 gyrA和 parC两种基因发生突变；C克隆3株，有gyrA和gyrB两种基因发生突变；D克隆14株，同时有 gyrA，gyrB和 parC三种基因发生突变；其他克隆2株，仅在 parE位点发生突变。结论该组菌株的靶位突变型与流行克隆型密切相关，同一流行型的菌株药物作用靶位的改变相同，并与环丙沙星的 MICs值的高低呈正比，突变基因数越多，MICs 值越高。4种基因中 gyrA基因突变频率最高，且该突变比其他靶位的突变对药物与靶位结合的影响更大，是需要关注的重点。

Objective 30 Pseudomonasaeruginosa mechanism of resistance to quinolones.Methods For the determination of ciprofloxacin MIC by agar dilution method.Used PCR on DNA gyrase and topoisomerase Ⅳ,resistance genes gyrA,gyrB, parC and parE were amplified,and BLAST,to determine whether there was resistance to bits mutation point;using pulsed-field gel electrophoresis （PFGE）of these 30 strains homology analysis.Results The 28 bacterial strains gyrA gene ampli-fied fragment of 137 points were C→T mutation causes T83I;17 strains gyrB gene amplified fragment of 351 G→C lead to G466A;parC gene amplification 21 bacteria fragment 277 point increase with C→U mutation causes S87L change two differ-ent strains parE gene locus C→U mutation A425V and A473V cause change.PFGE results：30 Pseudomonas aeruginosa could be divided into six clones,Aclone 4,B clone 7,C clone 3,D clone 14,and two other single clones.Conclusion The tar-get mutant strains closely related to the epidemic clone type,the same changes in the same pop-type strains of drug targets, and proportional to the level of ciprofloxacin MICs value,the more the number of mutated genes,MICs value higher.GyrA gene most prone to mutation,the mutation was also the first to be discovered,more than any other target of the mutation mutations on binding of drugs and targets that would be the focus of concern.