碳青霉烯类抗生素耐药铜绿假单胞菌外排泵和外膜孔蛋白表达水平的研究

Level of MexAB-OprM and OprD_2 expression in carbapenem-resistant Pseudomonas aeruginosa

目的探讨我国临床分离的碳青霉烯类抗生素耐药铜绿假单胞菌外排泵MexAB-OprM和外膜孔蛋白OprD2表达水平及其与碳青霉烯类抗生素耐药性之间的关系。方法收集我国15个省市28所医院临床分离的铜绿假单胞菌655株,琼脂稀释法测定该菌加入外排泵抑制剂羟基氰氯苯腙(CCCP)前后对亚胺培南和美罗培南的MIC;RT-PCR检测外排泵MexAB-OprM和孔蛋白OprD2的表达水平;十二烷基硫酸钠-聚丙酰胺凝胶电泳(SDS-PAGE)和蛋白印迹(Western-blot)检测外膜孔蛋白OprD2。结果收集到的655株铜绿假单胞菌菌株对亚胺培南和美罗培南敏感率分别为52.5%和59.2%,加入CCCP前后其对亚胺培南和美罗培南的敏感率无明显变化;52.0%(51/98)美罗培南耐药株MexAB-OprM显著高表达(P<0.05),43.7%(59/135)亚胺培南耐药株OprD2显著低表达(P<0.05),14.8%(20/135)亚胺培南耐药株OprD2表达缺失;SDS-PAGE和Western-blot显示与标准菌株PAO1相比,OprD2表达水平减低菌株ZZ20显影减低,表达缺失菌株BJ1不显影。结论MexAB-OprM表达升高和OprD2表达降低甚至缺失是我国铜绿假单胞菌对美罗培南和亚胺培南耐药的重要机制。

Objective To investigate the expression level of efflux pump MexAB-OprM and porin protein OprD2 in P. aeruginosa and the relationship between their expression level and resistance to carbapenems. Methods A total of 655 clinical P. aeruginosa isolates were collected from 28 hospitals in 15 Chinese cities. Agar dilution technique was used to determine the MICs of imipenem and meropenem against P. aeruginosa before and after addition of efflux pump inhibitor CCCP. Real-time-RT-qPCR was used to test the expression level of MexAB-OprM and OprD2. SDS-PAGE and Western-blot were used to identify OprD2. Results The percentage of the collected P. aeruginosa isolates susceptible to imipenem and meropenem was 52.5% and 59.2% respectively. These figures did not change obviously after adding CCCP. MexAB-OprM was over-expressed in 52. 0% （51/98） of the meropenem resistant P. aeruginosa isolates （P〈0.05）. The expression of OprD2 was lower in 43.7% （59/135） of the imipenem-resistant P. aeruginosa isolates （P〈0.05）. And 14.8%（20/135） of the imipenem-resistant P. aeruginosa isolates had no OprD2 expression. SDS-PAGE and Western-blot confirmed this finding. Conclusions Both over-expression of MexAB-OprM and decreased or no expression of OprD2 are important mechanisms underlying the resistance of P. aeruginosa to meropenem and imipenem.