铜绿假单胞菌在碳青霉烯类治疗过程中由敏感株发展为耐药株的机制研究

摘要

Objective To explore the mechanism of sensitivity to resistance of Pseudomonas aeruginosa to carbpenem in the process of treatment using carbpenem. Methods Two strains Pseudomonas aeruginosa were collected in the process of treatment using carbpenem.Then case was consulted and homology was investigated by PFGE. PorinoprD2 was analyzed by SDS - PAGE. The minimum inhibitory concentrations (MIC) of lmipenem and Meropenem with or without MC207110 were determined by agar dilution method. Expression level of efflux pumps mRNA was tested by realtime PCR. Metallo - β - lactamase (MBLs) was screened by EDTA - disk synergy test. The encoding genes of MBLs were amplified by PCR and analyzed DNA sequencing. Results Two strains in the process of treatment belonged to the same PFGE type. SDS - PAGE electrophoresis profile of 2 strains Paernginosa showed membrane protein 46kD OprD2 of IMPRMEMR type strain was lost, and OprD2 of I IMPSMEMs strain was normal. With or without MC207110, MIC of carbpenem - resistand P. aeruginosa decreaced by 4 fold. However, MIC of carbpenem - sensitive P. aeruginosa didnt. Compared with carbpenem - sensitive strain, the expression level of MexX gene mRNA of carbpenem - resistand strain increased by 102.5 fold, but the expression level of the other type efflux pumps mRNA didn 1 increase markedly. Both of carbpenem - resistand and carbpenem - sensitive P. aeruginosa didn 1 produce Metallo - β - lactamase.Conclusion Mechanism of sensitivity to resistance of Pseudomonas aeruginosa to carbpenem in the process of treatment using carbpenem are due to the lost of Porin oprD2 and the increase of expression level of MexXY - OprM of carbpenem - resistand strain.%目的 研究1例铜绿假单胞菌感染患者在接受抗菌药物治疗过程中其感染病原菌铜绿假单胞菌对碳青霉烯类抗菌药物由敏感株发展为耐药株的耐药机制.方法 收集感染患者治疗过程中分离的铜绿假单胞菌敏感株和耐药株共2株,查阅病史并对该2株菌进行:①PFGE分析其同源性,②SDS-PAGE分析外膜孔蛋白oprD2的改变,③泵抑制剂MC207110联合亚胺培南和美罗培南协同试验观察两药的MIC变化,④RealtimePCR检测外排泵基因的表达,⑤EDTA协同试验及PCR试验检测产金属β-内酰胺酶.结果 PFGE显示该患者在治疗前和过程中分离的铜绿假单胞菌敏感株(亚胺培南 4 mg/L和美罗培南4 mg/L)和耐药株(亚胺培南16 mg/L和美罗培南64 mg/L)系同一克隆;SDS-PAGE结果显示该克隆治疗前分离获得的敏感株具有oprD2蛋白带,而在治疗过程中分离获得的耐药株显示外膜蛋白oprD2缺失;泵抑制剂MC207110能使耐药株对美罗培南的MIC降低了3/4,敏感株未见明显下降.Realtime PCR检测发现该耐药克隆外排泵MexX基因mRNA表达水平较敏感株增高102.5倍,而其余外排泵系统的相关基因(MexA、MexC、MexE)mRNA表达水平未见明显变化;对敏感株和耐药株金属β-内酰胺酶表型初筛试验及PCR试验检测金属β-内酰胺酶基因均呈阴性.结论 在抗菌药物治疗过程中铜绿假单胞菌由对碳青霉烯类的敏感株发展为对碳青霉烯类的耐药株,其机制可能是由于细菌的外膜蛋白OprD2的缺失并合并外排泵MexXY-OprM系统表达增高所致.