一株铜绿假单胞菌临床菌株耐药的分子机制研究

Mechanisms of imipenem resistance of a Pseudomonas aeruginosa clinical isolate

目的研究1株铜绿假单胞菌临床分离菌株对亚胺培南耐药的分子机制。方法采用药敏分析系统(VITEK32)和纸片扩散法分析菌株的耐药性,实时定量PCR分析相关耐药基因的表达,PCR和测序法分析相关耐药基因的序列。结果药敏分析结果显示,该菌株对亚胺培南耐受,对美罗培南中介,对其余抗生素均表现为敏感。与对照菌株相比,外膜通道基因oprD的相对表达量仅为0.000 17,有明显下降;而外排泵编码基因mexE的相对表达量为0.60,无显著下降;oprD全基因测序发现第372位甲硫氨酸变成异亮氨酸,氨基酸极性没有发生变化。结论该临床菌株对亚胺培南的耐药性可能是由外膜通道基因oprD表达量下降造成,但不能完全排除氨基酸突变的影响。

Objective To study the molecular mechanisms underlying the imipenem resistance in a Pseudomonas aeruginosa clinical isolate.Methods Antibiotic resistance of the clinical isolate was detected by VITEK32 and Kirby-Bauer method.The expression level of antibiotic resistance related genes were analyzed by quantitative real time PCR method.PCR was performed to amplify the antibiotic resistance related genes and the amplified products were subject to sequencing for further analysis.Results The results of antimicrobial susceptibility analysis showed that the clinical isolate exhibited resistance to imipenem but susceptible to other tested antibiotics.Compared to the control strain,the expression level of the outer membrane channel encoding gene oprD was significantly decreased（0.000 17）;while no obvious change was observed in the expression level of one of the RND efflux pump encoding gene mexE（0.60）.There was a single amino acid substitution（Met372Ile）in protein OprD and no change in amino acid polarity.Conclusions The expression decrease of gene oprD may contribute to the imipenem resistance of the clinical isolate.However,the effect of the amino acid substitution could not be excluded completely.