铜绿假单胞菌对碳青霉烯类抗生素的耐药性及其耐药表型与外膜蛋白OprD2的关系

The resistance to carbapenems and the correlation between resistance phenotype and the amounts of OprD2 of Pseudomonas aeruginosa

目的 了解铜绿假单胞菌（Pseudomonas aeruginosa，Pa）临床分离株对几种常用抗铜绿假单胞菌药物的敏感性；探索铜绿假单胞菌临床分离株对亚胺培南和美罗培南耐药性差异与其膜孔蛋白0prD2变化的关系。方法琼脂对倍稀释法测定142株Pa对亚胺培南、美罗培南等8种常用抗假单胞菌药物的最低抑菌浓度，从中筛出32株对亚胺培南和美罗培南不同耐药表型的Pa，提取外膜蛋白并采用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）法分离，检测OprD2相对含量。结果耐药率最高的是亚胺培南（54．23％），其次是环丙沙星（45．07％）、美罗培南（28，87％）、氨曲南（19．72％）、阿米卡星（17.61％）、哌拉西林／三唑巴坦（14．08％）和头孢他啶（11．97％），头孢哌酮／舒巴坦耐药率最低（9．86％）。呈现多重耐药的菌株占38．73％（55／142），交叉耐药率高达40．84％（58／142）。在对亚胺培南耐药的菌株中，合并有其他肛内酰胺类抗生素耐药的菌株达到62．34％（48／77）。32株Pa外膜蛋白电泳结果显示OprD2相对含量在耐药组为0～3．5％（其中有一株为8.70％），敏感组为6．20％～10.20％。在碳青霉烯类耐药组与敏感组之间统计分析P〈0．05，耐碳青霉烯类组低于敏感组，但是在亚胺培南耐药、美罗培南敏感组与亚胺培南、美罗培南均耐药组之间差异无统计学意义。结论铜绿假单胞菌耐药菌株常见，且常呈多重耐药和交叉耐药。亚胺培南耐药率高于美罗培南。膜孔蛋白OprD2减少或缺失是我院Pa对亚胺培南耐药的主要原因之一。但在美罗培南耐药中所起的作用，有待进一步研究。

Objective To study the activities of some commonly used antibiotics against Pseudomonas aeruginosa, investigate the correlation between the resistance phenotypes and the amounts of outer membrane protein （OpD2） of P. aeruginosa, and discuss the mechanism of different resistant phenotypes in P. aeruginosa. Methods The minimal inhibitory concentrations （MICs） of 8 antimicrobial agents against 142 clinical isolates were determined by agar dilution method. Membrane proteins of 32 strains with different resistant phenotypes to imipenem and meropenem were obtained by SDS-PAGE electrophoresis method and the relative amounts of OprD2 were determined. Results The resistant rate of imipenem was the highest （54.23%）, followed by ciprofloxacin （45.07%）, meropenem （28.87%）, aztreonam （19.72%）, amikacin （17.61%）, piperacillin/tazobactam （14.08%）, ceftazidime （11.97%） and cefoperazone/sulbactam （9.86%）. The cross resistance rate to β-lactams was 40.84% （58/142）, muhi-drug resistance rate was 38.73% （55/142）. It accounted for 62.34% （48/77）, resistant to the other β-lactams in all isolates resistant to imipenem. Membrane proteins of 32 strains were isolated and determined by SDS-PAGE electrophoresis method. Result showed that the relative amounts of OprD2 ranged from 0 to 3. 5% in strains resistant to carbapenems （excepta strain which OprD2 was 8.70%）; others were from 6.20% to 10.20% in susceptible strains. The statistic results showed the relative amounts of OprD2 of 27 strains resistant to carbapenems were much than susceptible strains. Conclusions P. aeruginosa was frequently resistant to clinical antimicrobial agents, the most were cross resistant and multi-drug resistant strains. Resistance rate of imipenem was higher than that of meropenem. The loss or diminishment of OprD2 was the main reason for the imipenem resistance of P. aeruginosa in our hospital, but its effect on meropenem resistance deserved further study.