铜绿假单胞菌对碳青霉烯类抗生素的耐药机制

Mechanism of resistance of Pseudomonas aeruginosa to carbapenems

目的探讨临床分离的铜绿假单胞菌对碳青霉烯类抗生素耐药机制。方法收集该院58株耐亚胺培南和美罗培南铜绿假单胞菌,①在M-H琼脂平板中分别加入β内酰胺酶抑制剂和外排泵抑制剂,形成改良K-B法,进行药敏试验;②用改良三相水解试验检测β内酰胺酶;③用聚合酶链反应(PCR)检测金属酶基因(IMP、VIM)以及外膜膜孔蛋白基因(OprD2)。结果58株耐碳青霉烯类抗生素铜绿假单胞菌中,53株主动外排系统过度表达合并持续高产AmpC酶,其中15株伴有外膜膜孔蛋白OprD2缺失,1株产超广谱β内酰胺酶;在5株既不过度表达主动外排系统又不产β内酰胺酶细菌中,有1株OprD2基因缺失;未检出产金属酶菌株。结论该院铜绿假单胞菌对碳青霉烯类抗生素耐药的主要机制是主动外排系统的过度表达并伴有持续高产AmpC酶,其中16株菌株伴有外膜膜孔蛋白OprD2基因缺失。

Objective carbapenems. Methods To investigate the mechanism of resistance of clinical isolates of Pseudomonas aeruginosa to Fifty-eight strains of imipenem- and meropenem-resistant Pseudomonas aeruginosa were isolated. Modified K-B technique was adopted in the susceptibility test by adding β-lactamases inhibitors and efflux pumps inhibitors to M-H agar plates. The refined three-dimensional extract test was used to detect β-lactamases. The genes of metallo-enzyme IMP, VIM, as well as outer membrane porin D2 （OprD2） were analyzed with polymerase chain reaction. Results Among the 58 strains of carbapenems-resistant Pseudomonas aeruginosa, 53 produced over-expressed active efflux and continuously produced large amont of AmpC enzyme, 15 of which were accompanied by the loss of OprD2, and 1 of which were accompanied by extended spectrum β-lactamases （ESBLs）. Among the 5 strains which neither produced over-expressed active efflux nor β-lactamases, only 1 was found with OprD2 gene deletion. Metallo-enzyme was not detected in any of the 58 strains. Conclusion The mechanism of resistance of Pseudomonas aeruginosa to carbapenems was mainly the production of the over-expressed active efflux combined with the continuous production of large amount of AmpC enzyme. Sixteen of the strains were accompanied by the loss of OprD2 gene.