鼠伤寒沙门菌cpxR基因缺失株的构建及其对抗菌药物敏感性分析

Construction of cpxR gene deleted Salmonella typhimurium and analysis of its susceptibility to antibiotics

为研究鼠伤寒沙门菌双组分信号系统的应答调节蛋白基因cpxR对菌株药物敏感性的影响,本研究以p KD4质粒为模板,利用PCR扩增两侧与cpxR基因上下游同源且含有卡那霉素抗性(kanr)基因的线性打靶DNA片段,在p KD46质粒的辅助下进行Red同源重组,利用线性打靶DNA片段替换鼠伤寒沙门菌CVCC541(JS)株的cpxR基因,再利用p CP20质粒消除FRT位点间的kanr基因,构建基因缺失株JS△cpxR。并将重组表达质粒p BAD-cpxR转化于JS△cpxR中,制备回补菌株JS△cpxR/pcpxR。利用微量稀释法测定12种代表性抗菌药物对亲本株JS、JS△cpxR和回补菌株JS△cpxR/pcpxR的最小抑菌浓度(MIC)。结果显示,庆大霉素(GEN)、阿米卡星(AMK)、头孢噻呋(CEF)和头孢曲松(CRO)对亲本株JS的MIC值分别为JS△cpxR的4、4、4和2倍;而随诱导剂L-阿拉伯糖浓度的增高,这4种药物对回补菌株JS△cpxR/pcpxR的MIC值均逐渐恢复到亲本株JS的水平,呈剂量依赖性关系,而且高浓度诱导时4种药物对回补菌株的MIC分别为JS△cpxR的16、8、8和16倍,其他药物的MIC值未出现明显的变化。本研究结果表明:cpxR能够调控鼠伤寒沙门菌对GEN、AMK、CEF和CRO的敏感性。

To explore the influence of the regulation protein encoding by cpxR gene on the antibacterial susceptibility of Salmonella typhimurium, the cpxR gene deleted S.typhimudum CVCC541 （JS） strain （JS△cpxR） was constructed via h-Red homologous recombinant system with the DNA fragment amplified from the plasmid pKD4 by PCR, which contained kanamycin resistance gene and homologues arms of the cpxR gene. In addition, the complemental strain of JS△cpxR/pcpxR was generated through transformation of a recombinant expression plasmid of pBAD-cpxR into JS△ cpxR. The minimal inhibitory concentrations （MICs） of 12 kinds of antibiotics for JS, JS△cpxR and JS△cpxR/pcpxR were determined with broth microdilution method. The results indicated that the JS△cpxR showed 4-, 4-, 4-, and 2-fold decreased in the MICs to Gentamicin （GEN）, Amikacin （AMK）, Cefliofur （CEF） and Cettriaxone （CRO）, respectively, compared with the parental strain of JS. The MICs of the four drugs to JS △cpxR/pcpxR returned gradually to the levels of parental strain JS with the increasing concentration of the inducer （L-arabinose） in a dose-dependent manner. Moreover, JS△cpxR/pcpxR revealed 16-, 8-, 8-, and 16- fold increased in the MICs of the four drugs compared with those of the mutant JS△cpxR under the higher concentration of inducer. However, no significant variation was found in the MICs of the other selected antibacterial agents among these strains. In conclusion, cpxR was able to regulate the antimicrobial susceptibility of S. typhimurium to GEN, AMK, CEF and CRO.