摘要目的 研究铜绿假单胞菌外膜通道蛋白OprD2的表达减弱或缺失,以及OprD2蛋白自身突变是否会影响铜绿假单胞菌对碳青霉烯类药物的耐药性.方法 收集分离自临床对亚胺培南(IPM)的最低抑菌浓度(MIC)值≥8μg/m1的铜绿假单胞菌共101株,采用肉汤稀释法检测菌株对比阿培南(BPM)、美罗培南(MEM)、帕尼培南(PEM)的MIC值;荧光定量RT-PCR检测铜绿假单胞菌膜通道蛋白oprD2基因的表达量情况;针对oprD2相对表达量正常并对亚胺培南耐药的铜绿假单胞菌,采用普通PCR的方法扩增oprD2全长基因并测序.结果 根据铜绿假单胞菌的OprD2蛋白相对表达量结果,将101株铜绿假单胞菌分成两组,组1为OprD2相对表达量降低组;组2为OprD2相对表达量正常组;组1与组2相比,对IPM、BPM、MEM、PEM的MIC值≥16μg/ml的菌株比例差异均有统计学意义(P＜0.01).组1中,28株同时对4种药物的MIC均≥16 μg/ml,其中有25株的OprD2的相对表达量明显减低(＜0.4);外膜孔蛋白OprD2转录水平与4种碳青霉烯类药物MICs之间呈负相关趋势.组2中,有16株9prD2基因发生突变,按照突变的类型主要分成4组;与PAO1相比,这些菌株对IPM、BPM、MEM、PEM的MIC值有不同程度的增加.结论 OprD2外膜蛋白的表达量减少或缺失是铜绿假单胞菌对亚胺培南耐药的主要机制,可能也与其他3种碳青霉烯类药物耐药有密切关系;铜绿假单胞菌的oprD2基因发生突变,可能会降低铜绿假单胞菌对这儿种碳青霉烯类药物的敏感性.

abstractsObjective To investigate the impact on the resistance of carbapenem with the expression of OprD2 or OprD2 mutation in Pseudomonas aeruginosa. Methods One hundred and one clinical strains of Pseudomonas aeruginosa with MIC for imipenem ≥8 μg/ml were studied. MIC were determined by the broth microdilution method, and the antibiotics tested were imipenem(IPM ), biapenem( BPM), meropenem(MEM) and panipenem(PEM). The expression of the oprD2 gene in Pseudomonas aeruginosa were analyzed by real-time reverse transcriptase PCR(RT-PCR). For the Pseudomonas aeruginosa with normal expression of OprD2 and resistance to imipenem, full-length oprD2 gene was amplified by PCR and the products were sequenced. Results According to the result of the expression of oprD2 gene, 101 strains of Pseudomonas aeruginosa were divided into two groups: group1 with diminished expression of OprD2, and group2 with normal expression of OprD2. Comparing isolates with MIC of 4 kinds of carbepenem agents ≥ 16 μg/ml in two groups. Data showed the amount of OprD2 expression were different between two groups(P ＜0.01 or P ＜ 0.05). In group1, there are 28 isolates with MIC ≥ 16 μg/ml of all the 4 kinds of carbapenems, among which 25 isolates have obviously diminished expression of OprD2 ( ＜ 0.4). Negative correlations tendency appeared between the level of OprD2 transcription and MICs of 4 kinds of carbepenem agents in Pseudomonas aeruginosa. In group2, 16 strains with OprD2 mutation divided into 4 types according to the pattern of alteration. Compared with PAO1, these strains have increased MIC with different degree to IPM,BPM, MEM and PEM. Conclusion The deletion or diminished expression of OprD2 resulted in resistance to imipenem in Pseudomonas aeruginosa. The level of OprD2 transcription and antimicrobial activities for carbapenem agents proved to be highly correlated in Pseudomonas aeruginosa. The mutation of OprD2 in Pseudomonas aeruginosa probably decreased the sensitivity of carbapenem agents against Pseudomonas aeruginosa.