鼠伤寒沙门菌cpxR和acrB双基因缺失菌株的构建及其对抗菌药物敏感性分析  

Construction of cpxR and acrB Double Gene Deletion Strain of Salmonellaenterica Serovar Typhimurium and Analysis of Its Susceptibility to Antibacterial Agents

作　　者：黄慧[1] 刘保光[1] 孙亚伟[2] 马彩珲 陈丽鹏 苑丽[1] 潘玉善[1] 胡功政[1] 

机构地区：[1]河南农业大学牧医工程学院,郑州450002 [2]河南科技学院动物科技学院,新乡453001

出　　处：《畜牧兽医学报》2016年第3期595-602,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31372481)

摘　　要：为了探索cpxR基因对鼠伤寒沙门菌多重耐药性的调控机制,同时避免外排泵acrB的存在掩盖cpxR对其他外排泵或外膜蛋白的调控作用,利用基于Red同源重组系统的一步法失活染色体基因,用含有同源臂的线性打靶DNA片段替换目的基因,构建鼠伤寒沙门菌JS株的acrB基因缺失株JSΔacrB。然后利用噬菌体转导技术,以前期构建的cpxR缺失株JSΔcpxR∷kan为供体菌,本研究新构建的JSΔacrB为受体菌,在P22噬菌体的作用下构建双基因缺失菌株JSΔacrBΔcpxR∷kan,同时制备cpxR基因回补菌株JSΔacrBΔcpxR∷kan/pcpxR。最后用微量稀释法测定10种临床常用代表性抗菌药物对JS和各缺失株的最小抑菌浓度(MIC)。结果表明,成功构建鼠伤寒沙门菌的双基因缺失菌株JSΔacrBΔcpxR∷kan以及其cpxR基因互补菌株JSΔacrBΔcpxR/pcpxR。MIC结果显示,acrB基因缺失后,多西环素、庆大霉素、阿米卡星、环丙沙星、恩诺沙星、氟苯尼考、头孢曲松和头孢噻呋的MIC值分别降低32、4、4、16、32、4、2和128倍;双基因缺失后,多西环素、庆大霉素、阿米卡星和头孢噻呋的MIC值进一步降低2、8、2和2倍;而将cpxR基因回补之后,这4种抗菌药物的MIC值会升高4、16、2和4倍。结果表明,cpxR能调控鼠伤寒沙门菌对多西环素、庆大霉素、阿米卡星和头孢噻呋的敏感性。The aim of this study was to explore the role of cpxRgene in the multidrug resistance of Salmonella enterica Serovar Typhimurium and exclude a possibility that acrB masks the effect of cpxR on the other efflux pumps or outer membrane proteins.A acrB gene deletion strain JSΔacrB was constructed by the method of one-step inactivation of chromosomal genes on basis of Red homologous recombination system.A double gene deletion strain JSΔacrBΔcpxR∷kan was constructed by phage P22-mediated transduction with strain JSΔcpxR∷kan constructed in the previous study as the donor and strain JSΔacrBas the recipient.Meanwhile,the cpxRcomplemental strain JSΔacrBΔcpxR∷kan/pcpxR was prepared through introducting the expression plasmid pBAD-CpxR into the double gene mutant.At last the minimal inhibitory concentrations（MICs）of 10 kinds of commonly used representative antibacterial agents for JS and the abovestrains constructed in this study were determined with broth microdilution method.The double gene deletion strain and related cpxRcomplemental strain were constructed successfully.Strain JSΔacrBshowed 32-,4-,4-,16-,32-,4-,2-and 128-fold decrease in the MICs of doxycycline,gentamycin,amikacin,ciprofloxacin,enrofloxacin,florfenicol,ceftriaxone and ceftiofur respectively,compared to the parent strain JS.Strain JSΔacrBΔcpxR∷kan showed 2-,8-,2-and 2-fold decrease in the MICs of doxycycline,gentamicin,amikacin,and ceftiofur compared to strain JSΔacrB.While the complemental strain JSΔacrBΔcpxR∷kan/pcpxRshowed 4-,16-,2-and 4-fold increase in the MICs of the above four kinds of antibacterial agents compared to strain JSΔacrBΔcpxR∷kan.In conclusion,cpxRis able to regulate the antimicrobial susceptibility of S.enterica Serovar Typhimurium to doxycycline,gentamicin,amikacin,and ceftiofur.

关 键 词：鼠伤寒沙门菌 cpxR ACRB 双基因缺失 MIC 

分 类 号：S852.612[农业科学—基础兽医学]