鼠伤寒沙门菌baeSR基因缺失株的构建及其对抗生素敏感性分析

Construction of baeSR gene knock-out in Salmonella typhimurium and analysis of its sensitivity to antibiotics

摘要:

【背景】沙门菌的多重耐药现象日渐严重，对其耐药机理的研究尤为迫切，双组分系统与细菌耐药性密切相关。【目的】构建鼠伤寒沙门菌baeSR基因缺失株及回补株，探究双组分系统BaeSR对鼠伤寒沙门菌耐药性的影响。【方法】以鼠伤寒沙门菌体外诱导耐药株CR为研究对象，通过自杀质粒pLP12介导的同源重组方法，以氯霉素抗性标记和阿拉伯糖诱导的致死基因vmt进行正、反向双重筛选，获得基因缺失株CRΔbaeSR，并将重组表达质粒pBAD-baeSR转化于CRΔbaeSR构建回补株CR CΔbaeSR。采用微量肉汤稀释法测定11种常见代表药物对野生株、缺失株及回补株的最小抑菌浓度(Minimum inhibitory concentration，MIC)，并测定3株菌的生长曲线、运动性及生物膜形成能力。【结果】与野生株相比，环丙沙星(CIP)、恩诺沙星(ENR)、沙拉沙星(SAR)、头孢噻呋(CEF)、庆大霉素(GEN)、阿米卡星(AMK)、安普霉素(APR)对缺失株的MIC值有所下降；缺失株的生长速率稍显缓慢，且最终浓度也相对较低，但并无统计学上的显著差异(P>0.05)；缺失株的运动性(P<0.05)及生物膜形成能力(P<0.01)均显著下降。【结论】鼠伤寒沙门菌baeSR基因缺失后，可通过影响其运动性及生物膜形成能力而对抗生素的敏感性产生影响。

Abstract:

[Background] The multi-drug resistance of Salmonella is becoming more and more serious. It is particularly urgent to study the mechanism of antibiotic resistance in Salmonella. The two-component system (TCS) is closely related to bacterial antibiotic resistance. [Objective] The baeSR gene deletion and complementary strains of Salmonella typhimurium were carried out to study the effect of BaeSR on antibiotic resistance in S. typhimurium. [Methods] S. typhimurium selected in vitro to obtain cipro?oxacin-resistant strain CR, and take it as the study object, deleted mutants CRΔbaeSR were obtained by homologous recombination mediated by suicide plasmid pLP12. And we used chloramphenicol and arabinose-induced vmt toxicity gene for selecting. In addition, the complemental strains of CR C△baeSR were generated through transformation of a recombinant expression plasmid of pBAD-baeSR into CR△baeSR. And minimum inhibitory concentration (MIC) of 11 antibiotics against CR, CR△baeSR and CR C△baeSR strains was determined by broth microdilution method. Growth curve, migration ability and biofilm formation ability of three strains were also measured. [Results] The results showed that the MICs of CIP, ENR, SAR, CEF, GEN, AMK, APR were lower than those of wild strains; The growth rate of CR△baeSR was slightly slower than CR, and the final concentration was relatively low, but there was no significant difference (P>0.05). The migration ability (P<0.05) and biofilm formation ability (P<0.01) of CR△baeSR decreased significantly. [Conclusion] The deletion of baeSR gene in S. typhimurium can affect the ability of migration and biofilm-forming to affect the sensitivity to antibiotics.