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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.
The effect of SOS response on the resistance to enrofloxacin in E. coli was explored by knocking out the recA of the promoter protein SOS in response to SOS and the effect of rec A inhibitor and enrofloxacin on the synergistic inhibition of the bacteria was evaluated in vitro Red recombination system to construct rec A deletion strain E.coli ATCC 25922 /? Rec A of E.coli ATCC 25922. The expression levels of rec A and umu C related genes of SOS response system were measured by fluorescence quantitative PCR under the pressure of enrofloxacin The change of MIC of two strains of enrofloxacin and other commonly used antibiotics was measured by the broth microdilution method.The effect of enrofloxacin on the resistance variation of two strains was assayed by gradient plate method.The Rec A inhibitor was synthesized, And evaluated its combined effect with enrofloxacin to inhibit the growth and drug resistance of E.coli.The results showed that the minimum inhibitory concentration of enrofloxacin of E.coli ATCC 25922 / 1/8; after treatment with rec A, the strains lacking rec A were less susceptible than the wild-type strains on the gradient plates. Fluorescence quantitative PCR showed that the rec A deletion strain or the Rec A inhibitor affected the SOS response system to a certain extent Inhibition. Rec A was knocked out to make the strain resistant to enrofloxacin And resistant was significantly decreased; Rec A inhibitors can be suppressed to some extent SOS response, a synergistic antimicrobial effect.