含qnrA基因质粒介导不同水平环丙沙星耐药性的机制研究  

Mechanism of the different levels of ciprofloxacin resistance in qnrA-containing transconjugants

作　　者：徐晓刚[1] 吴湜[1] 叶信予[1] 石万良[1] 张婴元[1] 王明贵[1] 

机构地区：[1]复旦大学附属华山医院抗生素研究所,上海200040

出　　处：《中华微生物学和免疫学杂志》2008年第3期203-207,共5页Chinese Journal of Microbiology and Immunology

基　　金：973计划项目（2005CB523101）;国家自然科学基金资助项目（30440061,30572229）

摘　　要：目的研究4个含qnrA基因质粒在大肠埃希菌接合子中介导环丙沙星耐药水平不同的发生机制。方法以大肠埃希菌J53作为受体菌，通过接合试验从4株qnrA阳性的临床菌株中获得4株接合子。采用Etest方法测定环丙沙星最低抑菌浓度（MIC）；以PCR法检测aac（6’）-Ib-cr基因，采用实时RT-PCR法测定qnrA的mRNA表达水平，通过启动子探针载体pKK232-8测定qnrA启动子强度，测定、比较启动子周边序列。结果环丙沙星对仅含qnrA质粒pHS4及pHS5的接合子的MIC为0．094μg/ml及0．125mCml，同时携带qnrA及aac（6’）-Ib-cr质粒pHS3及pUS6的接合子的环丙沙星MIC为0．25μg/ml及1．00μg/ml。含pHS6接合子qnrA相对表达水平较其他接合子高13～32．5倍，pHS6的qnrA上游序列启动子活性最强，比另3个质粒高12倍，序列分析发现与pHS3比较，pHS6中qnrA转录启始位点与启始密码之间有7bp（GTrAGCA）的缺失。结论1个质粒同时携带qnrA和aac（6’）-Ib-cr2个耐药基因及qnrA高表达是导致接合子对环丙沙星的耐药性不同的原因。Objective To investigate the mechan-ism of tile-different levels of ciprofloxacin resistance in qnrA-containing transconjugants. Methods E. coli J53Az^R as the recipient, 4 qnrA-containing transconjugants were constructed by conjugation from 4 qnrA-carrying clinical isolates. MICs of the transconjugants were measured by E test. aac（6＇）-Ib-cr was detected by PCR, and qnrA mRNA expression level was determined by real-time RT-PCR. The promoter sequences of qnrA were amplified by PCR from qnrA-bearing plasmids and cloned into plasmid pKK232-8, then transformed into HB101. All promoter fragments were sequenced. Results The MICs of ciprofloxacin against 4 transconjugants demonstrated a 10-fold difference from 0. 094μg/ml to 1. 000μg/ml. Of 4 qnrA-bearing plasmids in E. coli J53, ciprofloxacin MICs of pHS4 and pHS5 were 0. 094 μg/ml and 0. 125μg/ml, respectively; pHS3, which contained the aac（6＇）-Ib-cr gene as well, MIC was 0.25μg/ml; and pHS6, which had a high expression level ofqnrA and the aac（6＇）-Ib-cr gene, MIC was 1.00μg/ml. The relative expression levels of qnrA mRNA in J53 pHS6 was 32.5, much higher than the other 3 transconjugants （ from 1.0 to 2.5 ）. The promoter in plasmid pHS6 was 12-fold stronger than that in the other 3 plasmids. Compared with pHS3, there was 7 bp （GTTAGCA） deletion between the transcription initiation site and the start of qnrA in pHS6. Contusion Co-existence of qnrA and aac （6＇）-Ib-cr in a single plasmid and high level of qnrA expression can account for the different levels of ciprofloxacin resistance in transconjugants.

关 键 词：环丙沙星 质粒介导 耐药 qnrA 

分 类 号：R686[医药卫生—骨科学]