头孢菌素敏感或中介的铜绿假单胞菌对亚胺培南耐药机制的研究  

Study on mechanism of imipenem resistance of cephalosporin-susceptible or intermediate Pseudomonas aeruginosa

作　　者：曾章锐[1,2] 邵海枫[2] 王卫萍[2] 黄梅[2] 史利宁[2] 王颖[2] 范明[2] 

机构地区：[1]江苏大学基础医学与医学技术学院,江苏镇江212001 [2]南京军区南京总医院临床中心实验科,南京210002

出　　处：《临床检验杂志》2013年第1期24-27,共4页Chinese Journal of Clinical Laboratory Science

摘　　要：目的探讨临床新检出的对碳青霉烯类药物耐药、头孢他啶(CAZ)和/或头孢吡肟(FEP)不耐药的铜绿假单胞菌耐药机制。方法琼脂稀释法测定5种抗菌药物对铜绿假单胞菌的MIC。改良三维试验检测细菌耐药酶的产生。SDS-PAGE观察对亚胺培南(IPM)耐药的铜绿假单胞菌分子量(Mr)46 000外膜蛋白OprD的改变。PCR扩增oprD基因并进行序列分析。结果 29株分离菌对IPM均为中、低度耐药,对CAZ为敏感或者中介(除46号菌外)。改良三维试验显示29株细菌均未产碳青霉烯酶。SDS-PAGE显示外膜蛋白缺失可分为3种类型:7株为外膜蛋白整体缺失或者减少,4株外膜蛋白无明显变化,其他均为Mr 46 000外膜蛋白缺失或者减少。按3种缺失类型选择了12株菌测序,发现:其中1株不同位置有多个点突变,导致出现终止密码子;7株出现碱基的少量缺失;3株有新碱基插入;1株为大片段的碱基置换。结论由编码外膜蛋白的oprD基因缺失、突变以及插入导致的外膜蛋白缺失或改变是引起该表型铜绿假单胞菌对碳青酶烯类药物耐药的主要原因。Objective To investigate the imipenem-resistance mechanism of the new clinical detectable Pseudomonas aeruginosa,which was resistant to carbapenems but susceptible or intermediate to ceftazidime and/or cefepime.Methods Minimal inhibitory concentrations（MIC） of 29 strains of P.aeruginosa to imipenem and meropenem were determined by agar dilution method.Carbapenemases were detected by the modified three-dimensional test.Outer membrane proteins were extracted from the isolates and analyzed by SDS polyacrylamide gel electrophoresis（SDS-PAGE）.The gene encoding outer membrane OprD was analyzed by PCR and DNA sequencing.Results All the 29 isolates but number 46 exhibited moderate or low resistance to imipenem and susceptibility or intermediate to ceftazidime,and none of them produced carbapenemases.The SDS-PAGE profiles showed that 7 isolates lost all the bands of outer membrane proteins,18 isolates lost or reduced the outer membrane protein with the band of 46 000,and the outer membrane protein of the other 4 did not change.Twelve isolates were selected from the above three deletion mutation types of outer membrane protein for DNA sequencing.The sequencing result showed that,of these isolates,1 isolate presented multiple point mutations,leading to the stop cordon at position 615.Another isolate presented large fragment base substitution.The results also revealed that 7 isolates lost a small number of bases,and 3 isolates turned up bases insertion.Conclusion The deletion,mutation and insertion of oprD gene caused the loss or reduction of outer membrane protein.This founding may provide insight into the resistance of the phenotype of P.aeruginosa to imipenem.

关 键 词：铜绿假单胞菌 碳青霉烯类 耐药机制 外膜蛋白 

分 类 号：R446.5[医药卫生—诊断学] R37[医药卫生—临床医学]