临床分离食品动物源多重耐药大肠杆菌耐药分子特征研究

Characterization of multiple resistant Escherichia coli isolates from food-producing animals

本研究从主动外排机制、膜孔蛋白缺失及氟喹诺酮类药物作用靶位改变等几个方面探讨临床分离的20株动物源性多重耐药大肠杆菌的耐药分子特征。实验结果表明,20株临床分离大肠杆菌gyrA83、gyrA87、parC80的突变率分别为95%、85%、55%。gyrA和parC共同突变的有11株,突变率为55%;20株多重耐药菌大肠杆菌普遍存在主动外排机制,主要介导对部分氨基糖苷类、四环素、氟苯尼考和氟喹诺酮类药物耐药,当添加外排泵抑制剂PAβN后多数菌株庆大霉素、新霉素、四环素、氟苯尼考及氟喹诺酮类药物的MIC都降低了2倍～256倍。利用建立的ELISA方法检测外排泵AcrA蛋白的表达水平,结果证实所有临床分离菌外排泵表达都增高;20株分离大肠杆菌中,部分菌株缺失OmpC或OmpF蛋白,同时缺失这两个蛋白的只有3株。部分菌株OmpF蛋白条带附近存在有多重耐药相关蛋白(Mar)。本研究结果揭示多重耐药大肠杆菌对常用抗菌药物高水平的耐药表型是主动外排机制、药物作用靶位的改变、外膜通透性的改变及其它机制共同作用的结果。

Twenty multi-drug resistant E.coli isolates were analyzed for mechanisms of drug resistance. It was shown that the occurrence of fluoroquinolones resistant related mutation in gyrA83, gyrA87 and parC80 were 95 %, 85 % and 55 %, respectively. Co-mutations in GyrA and ParC subunits were found in 11 of 20 isolates. MIC of most isolates for gentamicin, neomycin, tetracycline, florfenicol and fluoroquinolones decreased 2-256 folds in presence of effiux pump inhibitor PA β N, which confirmed the existence of efflux pump among the most isolates. An increased expression of efflux protein in outer membrane of 20 E.coli isolates could be detected with AcrA antibody by ELISA. There were some isolates that had lost OmpC or OmpF, and 3 isolates were found lost both OmpC and OmpF, Mrp activation could be detected in resistance/multi-resistance development. We concluded that the high resistance phenotype to common used antibiotics among E.coli were the results of concomitant resistance mechanisms.