铜绿假单胞菌亚胺培南异质性耐药的机制

Mechanisms of heterogeneous drug resistance against imipenem in Pseudomonas aeruginosa

目的:探讨铜绿假单胞菌(Pseudomonas aeruginosa,P.aeruginosa)亚胺培南异质性耐药的机制。方法:分别采用常规的KB法和MIC法检测310株铜绿假单胞菌对亚胺培南的敏感性,双纸片协同试验检测金属β-内酰胺酶,实时定量PCR实验检测外排泵基因Mex AB和Mex CD的表达水平,半定量结晶紫染色法检测生物膜的形成量,PCR扩增法检测细菌膜孔蛋白OPRD2基因是否缺失。结果:在临床分离的310株铜绿假单胞菌中,检出244株亚胺培南敏感菌株和66株非敏感菌株,244株中有36株铜绿假单胞菌对亚胺培南具有异质性耐药,总体检出率为11.6%。36株异质性耐药铜绿假单胞菌均不产生金属β-内酰胺酶,但高表达Mex AB外排泵,且生物膜的形成量也较高,其中6株检出膜孔蛋白OPRD2基因缺失。结论:临床存在部分异质性耐药的铜绿假单胞菌,它们通过形成生物膜、高表达多重耐药外排泵基因和基因缺失等机制介导耐药。

Objective： To explore the mechanisms of the heterogeneous drug resistance against imipenem in Pseudomonas aeruginosa（ P. aeruginosa） isolated from clinical specimens. Methods： The sensitivity of 310 strains of P. aeruginosa to imipenem（ IMP） was evaluated with conventional KB method and the instrument method. The metal β-lactamase phenotype was analyzed with double disc inhibitory synergy test. The expression level of efflux pump gene in bacteria was detected with reverse transcription real-time quantitative PCR. The biofilm formation was evaluated with semi-quantitative analysis of crystal violet. Furthermore,drug-resistant genes of bacteria OPRD2 was detected with PCR. Results： There were 244 IMP sensitive strains and 66 insensitive strains in the 310 clinically isolated P. aeruginosa,and36 strains had heteroresistance to IMP among 244 IMP sensitive strains. The total ratio of strains of P.aeruginosa with heteroresistance to IMP was 11. 6%. Among the 36 heteroresistance P. aeruginosa,none produced metal β-lactamase,and they all expressed high level in Mex AB efflux pump and had high level biofilm formation. There were 6 strains showed OPRD2 gene deletion. Conclusion： There are many strains of P. aeruginosa with heterogeneous drug resistance against IMP in clinical samples. The mechanism of the heterogeneous drug resistance against IMP may be related to high level Mex AB efflux pump,high level biofilm formation and resistant genes.