摘要
目的研究美罗培南耐药铜绿假单胞菌的多药外排泵机制。方法采用琼脂稀释法测定美罗培南对141株铜绿假单胞菌的最低抑菌浓度(MIC),同时观察加入泵抑制剂MC207110后的美罗培南对141株铜绿假单胞菌MIC变化,检测多药外排泵机制。实时定量PCR检测外排泵mRNA水平的表达。PCR扩增外排泵编码基因及调控基因并测序分析。结果外排泵表型试验显示美罗培南对69.1%(65/94)菌株的MIC降低至原来的1/4～1/512。20株美罗培南耐药铜绿假单胞菌中有4种外排泵基因mRNA表达水平有增高。其中以MexEF-OprN为最多,共有10株。其次为MexCD-oprJ,有5株。MexAB-OprM有4株,MexXY-OprM有3株。其中有2株细菌的MexA和MexC基因mRNA表达水平同时增高。20株美罗培南耐药铜绿假单胞菌的4种外排泵调控基因测序结果显示MexAB-OprM过度表达的菌株中有3株系mexR突变,1株系nalC突变所致,未发现nalD突变。5株MexCD-OprJ过度表达的菌株均发生了nfxB突变,10株MexEF-OprN高表达株均有mexT突变,3株MexXY-OprM高表达株均有mexZ基因突变。20株菌株外排泵编码基因(MexB、MexD、MexF、MexY)均未发生突变。结论外排泵机制可能是造成铜绿假单胞菌对美罗培南耐药的主要原因之一。外排泵系统的过度表达主要是由于其相应的调节基因突变所致。
Objective To study the multi-drug efflux pump of meropenem-resistant Pseudornonas aeruginosa. Methods The minimum inhibitory concentrations （MICs） of meropenem against 141 strains of P. aeruginosa were determined by agar dilution method with or without the presence of efflux pump inhibitor MC207110 （20 mg/L）. The expression level of efflux pumps mRNA were analyzed by real-time PCR. The coding genes and regulatory genes of efflux pumps were amplified and analyzed by PCR and DNA sequencing. Results The results of efflux pump phenotype testing showed that MIC values of meropenem de- creased to 1/4 to 1/512 for 69. 1% of the strains tested. The mRNA expression level of 4 efflux pump genes increased in 20 strains of meropenem-resistant P. aeruginosa, primarily MexEF-OprN （10 strains）, followed by MexCD-oprJ （5）, MexAB- OprM （4）, and MexXY-OprM （3）. The mRNA expression of both MexA and MexC genes increased in 2 of these strains. Sequencing results of the 4 efflux pump genes showed that MexAB-OprM overexpression was due to MexR mutation in 3 strains, and nalC mutation in 1 strain, nMD mutation was not identified. All the 5 strains with MexCD-OprJ overexpression showed nfxB mutation. All the 10 strains with MexEF-OprN overexpression had mexT mutation. All the 3 strains with MexXY-OprM overexpression showed mexZ gene mutation. The coding genes mexB, mexD, mexF, mexY of efflux pump did not mutate in the 20 strains. Conclusions Efflux pump may be one of the main mechanisms of P. aeruginosa resistance to meropenem. The overexpression of the efflux pump system is primarily due to the mutation of corresponding regulatory genes
出处
《中国感染与化疗杂志》
CAS
2011年第6期446-451,共6页
Chinese Journal of Infection and Chemotherapy
基金
国家973资助项目(2005CB523101)
国家自然科学基金项目(30772619)
