抑制E.coli的SOS应答对恩诺沙星抗药性的影响  

Effect of SOS-response Inhibition on Enrofloxacin Resistance in Escherichia coli

作　　者：王进文[1,2] 胡明[1,2] 白华[1,2] 颜世敢[1,2] 李璐璐[1,2] 骆延波[1,2] 齐静[1,2] 韩哲[3] 刘玉庆[1,2] 

机构地区：[1]山东大学农学院,济南250101 [2]山东省农业科学院畜牧兽医研究所,济南250101 [3]山东省科学院新材料研究所,济南250014

出　　处：《中国科学：生命科学》2015年第5期498-506,共9页Scientia Sinica(Vitae)

基　　金：国家自然科学基金(批准号:81271886);山东省科技攻关计划(批准号:2012GGC02012);山东省自然科学基金青年基金(批准号:ZR2013CQ037);国家科技重大专项(批准号:2013ZX10004217-003);山东省农业重大应用技术创新课题资助

摘　　要：通过敲除SOS应答启动蛋白基因rec A,探讨SOS应答对E.coli恩诺沙星抗药性的影响,并体外评价Rec A抑制剂和恩诺沙星联用对细菌协同抑制作用的影响.利用Red重组系统,构建E.coli ATCC 25922的rec A缺失菌株E.coli ATCC 25922/?rec A;在恩诺沙星压力下,利用荧光定量PCR测定SOS应答系统相关基因rec A和umu C表达量的变化.用微量肉汤稀释法测定恩诺沙星等常用抗生素对两个菌株的MIC变化;利用梯度平板法测定恩诺沙星对两个菌株抗药性变异的影响;合成Rec A抑制剂,并评估其与恩诺沙星联合抑制E.coli生长及其抗药性的作用.结果表明,E.coli ATCC 25922/?rec A菌株对恩诺沙星的最低抑菌浓度值降低至原始菌株的1/8;经药物处理后,在梯度平板上,rec A缺失菌株较野生型不易产生抗药性;荧光定量PCR表明,rec A缺失菌株或在Rec A抑制剂作用下,SOS应答系统受到一定的抑制.敲除rec A,使菌株对恩诺沙星的抗药性和抗药率均明显降低;Rec A抑制剂在一定程度上能抑制SOS应答,起到协同抑菌作用.To explore the effect of SOS response on enrofloxacin resistance in E. coli, we deleted the rec A gene, which encodes the inducer of SOS response, and evaluated the combined inhibitory effect of Rec A inhibitor and enrofloxacin on E. coli in vitro. By Red recombination, a deficient strain E. coli ATCC 25922/△rec A was obtained. The expression of rec A and umu C under the pressure of enrofloxacin was measured by real-time PCR. The minimum inhibitory concentration（MIC） of E. coli ATCC 25922 and E. coli ATCC 25922/△rec A to many kinds of antibiotics, including enrofloxacin, were detected by broth dilution method. And the effect of rec A deficiency on resistance variant to enrofloxacin was investigated by gradient plate method. N6-（1-naphthyl）-ADP, the inhibitor of SOS response, was synthesized and its influence combined with enrofloxacin on bacterial growth and resistance was evaluated. As results, E. coli ATCC 25922/△rec A was established successfully and its MIC was decreased to an eighth of original strain. Moreover, the rec A deficient strain was less prone to develop resistance than E. coli ATCC 25922 when exposed to enrofloxacin. The results of real-time PCR showed that SOS response was inhibited in rec A-deleted strain or with the action of Rec A inhibitor. The rec A-deleted E. coli ATCC 25922 was more susceptive to enrofloxacin and the rate of resistant variation was lower than that in wild-type strain, and the inhibitor of Rec A can inhibit SOS response to a certain degree and assist enrofloxacin to inhibit bacteria growth.

关 键 词：E.COLI SOS应答 RECA 恩诺沙星 抗药性 RECA 抑制剂 

分 类 号：R96[医药卫生—药理学]