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- 2018
鼠伤寒沙门菌质粒毒力基因对耐药表型的影响
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Abstract:
目的:探讨沙门菌质粒毒力基因(spv)表达改变对鼠伤寒沙门菌耐药表型的影响。方法:在eGFP基因序列上下游增加启动子及终止子并在两端位点上、下游设计酶切位点,以pACYC184-eGFP为载体,构建spv基因回补质粒,电转化至前期构建的鼠伤寒沙门菌spvB及spvC基因共同缺陷株获得基因回补株,检测菌株间耐药性的差异及acrAB表达的差异。结果:成功构建鼠伤寒沙门菌spvB、spvC基因荧光回补株,连续培养12代质粒未发生丢失。与野生型菌株及回补株相比,spv基因缺陷变异株对哌拉西林他唑巴坦、头孢噻肟、头孢西丁、四环素、氯霉素及环丙沙星的MIC值下降。spv基因缺陷变异株AcrA周质蛋白水平低于野生菌株及回补株(P<0.05)。结论:在spvB及spvC基因共同缺陷鼠伤寒沙门菌基础上的基因回补株构建成功,spv下调能改变鼠伤寒沙门菌的耐药表型,这可能是通过影响外排泵AcrA来完成的。
Aim: To observe the influence of spv on the antibiotic resistance of Salmonella typhimurium isolated from the faeces of the diarrhea children.Methods: The promoter was added to the upstream and the terminator was added to the downstream of the sequence of eGFP published in GeneBank as well as restriction sites was designed,and finally pACYC184-eGFP was synthesized using multiple PCR.The spv-complementing plasmid was constructed using pACYC184-eGFP vector,then electroporated into the spvB and spvC gene-deleted mutant to acquire the corresponding strain, and then drug resistance and the expression of resistance related genes of different strains were examined.Results: PCR and gene sequencing confirmed successful construction of Salmonella typhimurium spvB, spvC gene complemented mutant strain with fluorescence. Low copy plasmid does not lose after 12 generation continuous culture. Compared with the wide type strain and the compensating strain,the MICs of spvB and spvC gene-deleted mutant strain descended original MIC values for piperacillin/tazobactam, cefotaxime, cefoxitin, tetracycline, chloramphenicol and ciprofloxacin.The expression of periplasmic protein AcrA in spvB and spvC gene-deleted mutant strain was lower than that of wide type strain and the compensating strain(P<0.05).Conclusion: The spv complementation strain has been constructed successfully on the basis of spvB and spvC-deleted mutant strain,the downregulation of spv can change the antibiotic resistance phenotype of Salmonella typhimurium,which may due to the downregulation of AcrA
