临床铜绿假单胞菌多重耐药株外排泵机制研究

The mechanism of efflux pumps in clinical isolated multidrug-resistant Pseudomonas aeruginosa strains

目的研究临床分离的多重耐药铜绿假单胞菌MexA-MexB-OprM外排泵的外膜蛋白OprM的表达及泵调节基因mexR突变情况。方法筛选铜绿假单胞菌多重耐药株13株和敏感菌株5株进行外膜蛋白SDS-PAGE分析,比较OprM的表达差异。聚合酶链反应(PCR)法扩增mexR基因,鉴定产物,测序。结果多重耐药菌株组OprM蛋白在相对分子量为49 kD处的各电泳条带较敏感菌株和质控菌株铜绿假单胞菌ATCC 27853的灰度明显增强,其光密度值多重耐药菌株组为157.79,敏感菌株组为132.03,两组比较,差异有显著性(t=19.345,P<0.01);多重耐药菌株mexR基因产物测序,除个别碱基插入外,以A-96→G,G-411→A,C-308→G,T-377→A 4个核苷酸点突变为主,后二者还导致了其编码的氨基酸发生改变,分别为Ala-103→Gly,Val-126→Glu。结论OprM表达增强提示MexA-MexB-OprM外排泵耐药机制参与多重耐药形成。多重耐药菌株的mexR基因均存在点突变,提示mexR基因突变引起MexA-MexB-OprM外排泵表达增强。

Objective To explore the expression of outer membrane protein OprM of MexA-MexB-OprM efflux pump and the mutation of regulation gene mexR of MexA-MexB-OprM in multidrug-resistant Pseudomonas aeruginosa isolated from clinic. Methods Thirteen multidrug-resistant and 5 antimicroblal susceptible Pseudomonas aeruginosa isolates from clinical specimens were screened. Outer membrane proteins were analysed with SDSPAGE. MexR was amplified with PCR and PCR products were analyzed with DNA sequencing. Results The grey density of multidrug-resistant bacterial OprM bands near 49 kD increased obviously compared with that of susceptibile and control strain ATCC 27853, the optical density of multidrug-resistant strains and susceptible strains was 157. 79 and 132. 03 respectively, there was significant difference between two groups （ t = 19. 345, P d0. 01）; The main sites of point mutation were A-96→G, G-411→A, C-308→G , T-377→A, the latter two mutations resulted in the change of amino acid sequence, which was Ala-103→Gly, Val-126→Glu respectively. Conclusion The increased expression of OprM suggests that MexA-MexB-OprM efflux pump involves in multidrug resistance. The mexR genes of multidrug-resistant bacterial strains have point mutation, which sugget that mexR gene mutation can result in high level expression of MexA-MexB-OprM.