铜绿假单胞菌MexABOprM主动外排表型筛选与外排基因检测.docVIP

铜绿假单胞菌MexABOprM主动外排表型筛选和外排基因检测 作者：曹敬荣 沈定霞 钱妙玲 罗燕萍 徐雅萍 田芳 【摘要】 目的 研究 解放军总 医院 临床分离的多重耐药铜绿假单胞菌的主动外排系统，为指导临床合理选择抗生素提供依据。 方法 采用外排泵抑制剂(苯丙氨酸精氨酸β萘酰胺)与喹诺酮类抗生素，以微量肉汤稀释法测定加与不加外排泵抑制剂时环丙沙星与左氧氟沙星的最低抑菌浓度(MIC)值，并 计算 MIC值的变化，根据加与不加外排泵抑制剂MIC值降低3个稀释度及以上筛选主动外排表型阳性菌株；PCR扩增外排表型阳性菌的耐药基因oprM、mexB和mexR，并对3株菌进行mexR基因测序 分析 ；RTPCR检测oprM基因的mRNA水平，分析耐药基因的表达情况。结果 苯丙氨酸精氨酸β萘酰胺能极大的降低左氧氟沙星和环丙沙星的MIC值，70株多重耐药铜绿假单胞菌中46株表型阳性(65.7%)，其中PCR检测oprM、mexB和mexR基因同时阳性菌株42株(91.3%)，仅oprM基因阳性2株(4.3%)，oprM、mexB和mexR基因均阴性2株(4.3%)。RTPCR产物测定A值(A260/A280)与PAO1对照阳性菌株13株，以PAO1的oprM/rpoD的A值为对照阳性菌株4株，同时满足A值(A260/A280)和oprM/rpoD比值均高于PAO1的菌株3株。测序结果与GenBank中已知的mexR基因比对，其中2株同源性为100%，1株第434位核苷酸插入2bp(AT)。结论 中国 人民解放军总医院多重耐药铜绿假单胞菌大多存在主动外排的耐药机制，其耐药与外排泵过度表达相关。 【关键词】 铜绿假单胞菌 主动外排 外排泵抑制剂 苯丙氨酸精氨酸β萘酰胺 MexABOprM RTPCR Detection of the phenotype and genes of active efflux pump ABSTRACT Objective To study the active efflux pumps of clinical isolates of multidrug resistant Pseudomonas aeruginosa in PLA hospital and offer the theoretical evidences to clinicians. Methods The microdilution method of 96 wells was used to determine the minimum inhibitory concentrations (MICs) of levofloxacin, ciprofloxacin alone and the effect of the presence of efflux pump inhibitor (PheArgβnaphthylamide). Based on the variety of MIC to decrease 8fold in the presence of PAβN to choose the positive phenotype strains. PCR was used to detect the resistant genes of efflux pumps: oprM, mexB and mexR based on the phenotypic selection. DNA sequencing was adopted to analyze the resistancerelated mexR genes for 3 strains. RTPCR was used to test the levels of mRNA and analyze the expression of the resistance genes. Results Forty six strains were positive in phenotype tests from 70 multidrug resistant P.aeruginosa (65.7%), and they were used as templates to amplify oprM, mexB and mexR genes. Among them, 42 strains were detected in specific bands of oprM, mexB and mexR simultaneously (91.3%). Two strains were only detected in