阴沟肠杆菌质粒介导耐喹诺酮类药物的机制研究_药学论文

阴沟肠杆菌质粒介导耐喹诺酮类药物的机制研究_药学论文 阴沟肠杆菌质粒介导耐喹诺酮类药物的机制研究_药学论文 陆坚 邓启文 张扣兴 【摘要】 目的 了解深圳地区阴作者:吴创鸿 沟肠杆菌中qnrA基因的流行情况、基因定位及其介导喹诺酮耐药性 产生的机制。方法 收集临床分离的阴沟肠杆菌45株，采用PCR法结 合测序的技术筛查qnrA基因，大质粒提取技术、Southern杂交和接 合传递试验进行质粒定位，琼脂对倍稀释法进行药物敏感性检测。结 果 45株阴沟肠杆菌中，7株PCR法及测序证实为qnrA基因。7株菌 中6株菌所携质粒被成功提取并进行Southern杂交，qnrA基因定位 于80,200kb大小的低拷贝数天然质粒上。4株菌成功进行接合传递 试验，使接合子对环丙沙星的MIC值提高了32,64倍。结论 质粒介 导的喹诺酮耐药基因qnrA在深圳地区阴沟肠杆菌中具有较高的流行 率，可能是导致阴沟肠杆菌对喹诺酮类抗菌药获得性耐药的重要原 因。 【关键词】 qnrA基因 阴沟肠杆菌 喹诺酮 耐药 质粒 Plasmidmediated quinolone resistance in clinical isolates ABSTRACT Objective To investigate the prevalence and location of qnrA gene in clinical isolates of Enterobacter cloacae and its function in the development of quinolone resistance. Method Fortyfive Enterobacter cloacae were screened for the qnrA gene by PCR and then conformed with direct sequencing. Large plasmid extraction and Southern hybridization were done to determine the location of qnrA. Conjugation experiments were done to determine the transferability and its effects in the development of quinolone resistance. Results qnrA were detected in 7 (15.6%) of 45 strains. The qnrA sequence matched that of NCBI GenBank with 100%. Plasmid extraction and Southern hybridization were succeed in 6 stains. The qnrA were located in plasmids in size of 80,200kb. Conjugation experiments were done in 4 strains. Transconjugants had 32,64fold increases in the MIC of ciprofloxacin relative that of the recipient but were still susceptible to ciprofloxacin according to CLSI stardard. Conclusion Transferable plasmidmediated quinolone resistance associated with qnrA were high prevalent in clinical isolates of Enterobacter cloacae from Shenzhen city and may contribute to the quinolone resistance of thiese bacteria. KEY WORDS qnrA; Enterobacter cloacae; Quinolone; Drug resistance; Plasmid 1998年，Martinez等首次证实肺炎克雷伯菌对喹诺酮类抗菌药存在 质粒介导的耐药机制，并将该耐药基因命名为qnr(现命名为qnrA) ,1,。之后我国学者王明贵等研究发现qnrA基因在耐喹诺酮类抗菌 药大肠埃希菌中的检出率高达7.7%，提示qnrA基因在介导肠杆菌科 细菌耐药机制方面可能发挥着重要作用，引起广泛关注,2,。 目前阴沟肠杆菌已成为医院内感染的重要病原菌，且对喹诺酮类抗 菌药的耐药性迅速上升，给临床抗感染治疗带来严峻挑战,3，4,。 为了解qnrA基因在介导阴沟肠杆菌耐药性播散方面的作用，本研究 采用PCR技术对2004,2005年我院临床分离的阴沟肠杆菌进行了 qnrA基因携带率的筛查，DNA测序证实并与已报道的qnrA序列进行 比较，并通过Southern杂交及接合传递试验对qnrA基因进行了质粒 定位，现报道如下。 1 材料和方法 1.1 菌株来源及鉴定 2003,2004年我院及深圳市人民医院临床分离的非重复阴沟肠杆菌 45株。采用法国BioMerieux公司API 20E系统进行种属鉴定。 研究用菌株为E.coli J53/pMG252作为qnrA基因PCR检测和Southern 杂交检测的阳性质控株，E.coli J53 Azr(耐叠氮钠)作为接合传递实 验受体菌及MICs参照株，均由美国Jacoby教授提供。 1.2 抗菌药物敏感性检测 采用琼脂稀释法检测临床分离菌及接合子对环丙沙星的MICs值，其 它抗菌药物的敏感性采用KB法检测。MH培养基和药敏纸片均为Oxoid 公司产品。结果按照美国临床和实验室委员会(CLSI)2005年版 推荐的标准进行解释。质控菌株为大肠埃希菌ATCC25922和阴沟肠杆 菌ATCC13047。 1.3 qnrA扩增和测序 采用PCR法并结合测序进行PCR引物参照文献,4,委托上海英骏生 TCAGCAAGAGGATTTCTCA3′，下物有限公司合成。上游引物5′ 游引物5′GGCAGCACTATTACTCCCA3′，增片段为627bp。PCR循 环参数为:94?预变性5min，95? 30s，45? 60s，72? 60s，循 环35次，最后72?延伸5min。PCR产物送上海英骏生物有限公司纯 化后直接进行双向测序。阳性对照菌株为E.coli J53 pMG252。 1.4 qnrA序列比较 所测序列用DNAstar软件进行序列拼接、校正后，采用美国国家信 息中心(NCBI)在线分析工具Blastn(【参考文献】 Plasmidmediated quinolone resistance in clinical isolatesof Enterobacter cloacaeWu Chuanghong1, Lu Jian2, Deng Qiwen1, Zhang Kouxing1,Zhang Guoliang2 and Pan WeiGuang1(1 Department of Infectious Diseases, The Sixth People′s Hospital, Shenzhen 518052;ABSTRACT Objective To investigate the prevalence and location of qnrA gene in clinical isolates of Enterobacter cloacae and its function in the development of quinolone resistance. Method Fortyfive Enterobacter cloacae were screened for the qnrA gene by PCR and then conformed with direct sequencing. Large plasmid extraction and Southern hybridization were done to determine the location of qnrA. Conjugation experiments were done to determine the transferability and its effects in the development of quinolone resistance. Results qnrA were detected in 7 (15.6%) of 45 strains. The qnrA sequence matched that of NCBI GenBank with 100%. Plasmid extraction and Southern hybridization were succeed in 6 stains. The qnrA were located in plasmids in size of 80,200kb. Conjugation experiments were done in 4 strains. Transconjugants had 32,64fold increases in the MIC of ciprofloxacin relative that of the recipient but were still susceptible to ciprofloxacin according to CLSI stardard. Conclusion Transferable plasmidmediated quinolone resistance associated with qnrA were high prevalent in clinical isolates of Enterobacter cloacae from Shenzhen city and may contribute to the quinolone resistance of thiese bacteria.KEY WORDS qnrA; Enterobacter cloacae; Quinolone; Drug resistance; Plasmid1998年，Martinez等首次证实肺炎克雷伯菌对 喹诺酮类抗菌药存在质粒介导的耐药机制，并将该耐药基因命名为 qnr(现命名为qnrA),1,。之后我国学者王明贵等研究发现qnrA基 因在耐喹诺酮类抗菌药大肠埃希菌中的检出率高达7.7%，提示qnrA 基因在介导肠杆菌科细菌耐药机制方面可能发挥着重要作用，引起广 泛关注,2,。目前阴沟肠杆菌已成为医院内感染的重要病原菌，且对 喹诺酮类抗菌药的耐药性迅速上升，给临床抗感染治疗带来严峻挑战 ,3，4,。为了解qnrA基因在介导阴沟肠杆菌耐药性播散方面的作用， 本研究采用PCR技术对2004,2005年我院临床分离的阴沟肠杆菌进 行了qnrA基因携带率的筛查，DNA测序证实并与已报道的qnrA序列 进行比较，并通过Southern杂交及接合传递试验对qnrA基因进行了 质粒定位，现报道如下。1 材料和方法1.1 菌株来源及鉴定2003, 2004年我院及深圳市人民医院临床分离的非重复阴沟肠杆菌45株。 采用法国BioMerieux公司API 20E系统进行种属鉴定。研究用菌 株为E.coli J53/pMG252作为qnrA基因PCR检测和Southern杂交检 测的阳性质控株，E.coli J53 Azr(耐叠氮钠)作为接合传递实验受体 菌及MICs参照株，均由美国Jacoby教授提供。1.2 抗菌药物敏感性 检测采用琼脂稀释法检测临床分离菌及接合子对环丙沙星的MICs 值，其它抗菌药物的敏感性采用KB法检测。MH培养基和药敏纸片均 为Oxoid公司产品。结果按照美国临床和实验室标准委员会 (CLSI)2005年版推荐的标准进行解释。质控菌株为大肠埃希菌 ATCC25922和阴沟肠杆菌ATCC13047。1.3 qnrA扩增和测序采用PCR 法并结合测序进行PCR引物参照文献,4,委托上海英骏生物有限公 司合成。上游引物5′TCAGCAAGAGGATTTCTCA3′，下游引物5′ GGCAGCACTATTACTCCCA3′，增片段为627bp。PCR循环参数为:94?预变性5min，95? 30s，45? 60s，72? 60s，循环35次，最后72?延伸5min。PCR产物送上海英骏生物有限公司纯化后直接进行双向测序。阳性对照菌株为E.coli J53 pMG252。1.4 qnrA序列比较所测序列用DNAstar软件进行序列拼接、校正后，采用美国国家信息中心(NCBI)在线分析工具Blastn(