不同环境胁迫因子对猪源耐药大肠杆菌生长的影响  

Effects of different environmental stress factors on the growth of pig-origin drug-resistant Escherichia coli

作　　者：廖书丹 谭艾娟[2] 孙海峰 向润 杨剑 张元鑫 宋旭琴 吕世明[1] LIAO Shu-dan;TAN Ai-juan;SUN Hai-feng;XIANG Run;YANG Jian;ZHANG Yuan-xin;SONG Xu-qin;LYU Shi-ming(College of Animal Science,Guizhou University,Guiyang,Guizhou 550025,China;College of Life Sciences,Guizhou University,Guiyang,Guizhou 550025,China;Department of Agriculture and Rural Affairs of Guizhou Province,Guiyang,Guizhou 550005,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州大学生命科学学院,贵州贵阳550025 [3]贵州省农业农村厅,贵州贵阳550005

出　　处：《南方农业学报》2022年第4期946-957,共12页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31760749);贵州省科技计划项目(黔科合平台人才[2017]5788号,黔科合基础-ZK[2022]一般129号);贵州大学自然科学专项(特岗)科研基金项目(贵大特岗合字[2020]25号)。

摘　　要：【目的】探究环境胁迫因子对猪源多重耐药大肠杆菌生存适应性的影响,为创造多重耐药菌株带来高适应代价的环境及加速降低种群耐药水平提供理论依据。【方法】以分离自贵州省规模化猪场的多重耐药大肠杆菌QL15为研究对象,以大肠杆菌质控(敏感)菌株ATCC25922为对照,经体外竞争试验测定不同环境(培养基浓度、碳源、氮源、pH和温度)胁迫下耐药菌株QL15相对于敏感菌株ATCC25922的适应性,并通过重测序变异位点和实时荧光定量PCR测定分析耐药基因表型及外排泵和外膜孔道蛋白基因表达与细菌适应性代价的关系。【结果】耐药菌株QL15在体外独立及混合培养时,除在稀释LB培养基(1/4、1/8和1/16)中的长势较敏感菌株ATCC25922低外,在低碳源(0.1%和0.5%)、低氮源(0.01%和0.05%)、酸碱性(p H=5.0、pH=6.0和pH=8.0)及低温(20℃)和高温(42℃)胁迫下的生长均优于敏感菌株ATCC25922,且混合培养结果优于独立培养。将耐药菌株QL15基因组序列与敏感菌株ATCC25922基因组序列进行比对,共检测到509个InDel突变位点,其中Delete突变位点233个、Insert突变位点276个;共注释到71个功能基因,以细胞壁与细胞膜相关基因突变最多(10个),占14.1%。与敏感菌株ATCC25922相比,耐药菌株QL15在不同环境胁迫下其外排泵与外膜孔道蛋白相关基因emr B、acr B、tol C和omp C呈下调表达,而omp F基因呈上调表达。【结论】多重耐药大肠杆菌耐药性的获得促使其具有更强的适应性,但对可直接利用营养物质的获取弱于敏感菌株,即处于竞争劣势。耐药大肠杆菌的多重耐药性主要是由多种外排泵和外膜孔道蛋白介导完成,其中,omp F基因在pH胁迫、全营养胁迫和高、低温胁迫下均处于上调表达状态,故推测omp F基因的高表达有利于大肠杆菌在上述胁迫条件下生长。【Objective】To investigate the effects of environmental stress factors on the survival adaptability of pig-origin multi-drug resistant Escherichia coli,so as to provide a theoretical basis for creating an environment where multidrug resistant strains could present a high cost of adaptability and for accelerating the reduction of resistance level in the population.【Method】Multi-drug resistant E.coli QL15 isolated from a large-scale pig farm in Guizhou Province was taken as the study object,and E.coli quality control(sensitive)strain ATCC25922 was taken as the control.The adaptability of drug-resistant strain QL15 and that of sensitive strain ATCC25922 under different environmental stress(medium concentration,carbon source,nitrogen source,p H and temperature)was determined by in vitro competition assay.The relationship between drug-resistant gene phenotype and gene expression of efflux pump and outer membrane pore protein with the cost of bacterial adaptation was analyzed by resequencing variant loci and fluorescence quantitative PCR.【Result】Except that the growth of drug-resistant strain QL15 was lower in dilute LB medium(1/4,1/8 and 1/16)compared with the sensitive strain ATCC25922,the growth of drug-resistant strain QL15 was better than that of sensitive strain ATCC25922 in in vitro independent and mixed cultures under low carbon sources(0.1%and 0.5%),low nitrogen sources(0.01%and 0.05%),acid-base property(p H=5.0,pH=6.0 and pH=8.0),low and high temperature(20℃and 42℃).Besides,the results of mixed culture were better than that of the independent culture.The genome sequence of drug-resistant strain QL15 was compared with that of sensitive strain ATCC25922,and a total of 509 InDel mutation sites were detected,including 233 delete mutation sites and 276 insert mutation sites;a total of 71 functional genes were annotated,most of which were mutations related to cell wall and cell membrane(10),accounting for 14.1%.Compared with the sensitive strain ATCC25922,in the drug-resistant strain QL15,genes relat

关 键 词：多重耐药大肠杆菌 猪源 环境胁迫因子 相对适应性 耐药性 

分 类 号：S852.612[农业科学—基础兽医学]