嗜麦芽寡养单胞菌临床株外排泵SmeDEF诱导表达的研究论文.docVIP

　　嗜麦芽寡养单胞菌临床株外排泵SmeDEF诱导表达的研究论文 孙二琳 宋诗铎 祁伟 王玉宝 【摘要】 目的 了解嗜麦芽寡养单胞菌的临床株H407和A26在抗菌药物诱导下耐药性和SmeDEF外排泵表达的变化。方法 比较有或无环丙沙星诱导时嗜麦芽寡养单胞菌H407和A26的MIC和EOP值，对在亚抑菌浓度环丙沙星中培育后的菌株smeD基因进行RT-PCR扩增，监测表达水平。结果 对抗生素敏感的H407和A26泵抑制呈阳性或阴性反应。2株菌经环丙沙星过夜孵育后测得的对氯霉素和环丙沙星MIC值比正常非诱导时的MIC值高1～3个稀释度；加泵抑制剂CCCP后，2个诱导株的MIC值均有明显下降。H407和A26经环丙沙星诱导后氯霉素的EOP分别有4.8和近4倍的增长。2株菌在抗菌药物过夜和指数期添加生长时smeD的mRNA明显高于无抗菌药物培养株。结论 2株嗜麦芽寡养单胞菌临床株在环丙沙星诱导下对部分抗菌药物的耐药性提高，与SmeDEF泵的诱导表达增加有关。 【关键词】 嗜麦芽寡养单胞菌； 外排泵； 耐药性； 诱导 ABSTRACT Objective To investigate changes of antibiotic resistance of the clinical isolates of S.maltophilia, and the repression level of efflux pump SmeDEF by the induction of antibiotics. Methods To examine the resistance profile of the clinical strains by agar dilution and the efficiency of plate (EOP) to antimicrobes, and their reactions to pump inhibitor CCCP, and to investigate the mRNA level of smeD by RT-PCR, in the presence or not of ciprofloxacin. Results The antibiotic-sensitive strains H407 and A26 p inhibitor respectively. MICs of ciprofloxacin and chloramphenicol induced by ciprofloxacin phenicol in H407 and A26 increased by 4.8 and nearly 4 times respectively, and the level of mRNA of smeD in the 2 antimicrobe-induced strains rose as icrobes in the taltophilia could be induced by ciprofloxacin. The possible antibiotic resistance mechanisms seemed likely by the mvolvement of the increased transcription level of smeDEF. KEY H和LB琼脂，溴化乙啶(EB)为Sigma公司产品。SV Total RNA Isolation System、RT-PCR系统(Cat.#1260)和琼脂糖购自Promega公司产品。分子量标准DL-2000购自TaKaRa公司。 1.2 方法 (1)抗菌药物诱导和非诱导时的MIC值 取LB平皿上过夜生长的单菌落，分别接种于含环丙沙星(浓度为1/3MIC)的LB肉汤，孵育过夜；离心沉淀后重溶，多点接种于含和不含25μg/ml CCCP的环丙沙星和氯霉素的倍比稀释MHA平皿上，浓度105CFU/spot，35℃孵育24h。对照组过夜孵育的LB肉汤无抗菌药物，同样接种于含和不含CCCP的抗菌药物倍比稀释平皿上，质控和判断标准均依据美国临床实验室标准化研究所(CLSI/NCCLS)2004年颁布的准则。 (2)抗菌药物诱导的菌株耐药性—平皿效率实验(EOP) H407和A26同时进行以下两组实验。一组取LB平皿上过夜孵育的菌落，105CFU/ml 2.5μl涂布于含氯霉素(浓度为0.8MIC)的MHA平皿和空白对照平皿。另一组在含环丙沙星(浓度为1/3MIC)的LB平皿过夜孵育的菌落，稀释后同样涂布于含氯霉素的平皿和空白对照平皿，35℃孵育48h。计数最终的菌落数，EOP为含氯霉素的实验平皿上的CFU滴度除以空白对照平皿的比值，参照文献［6］。 (3