鼠伤寒沙门氏菌baeR过表达株的构建及其耐药性  

Construction and antibiotic resistance of a Salmonella typhimurium strain overexpressing bae R

作　　者：高海侠 卢芳 姜西迪 魏祺灵 漆彩丽 张临 付恒峰 李琳[1] GAO Haixia;LU Fang;JIANG Xidi;WEI Qiling;QI Caili;ZHANG Lin;FU Hengfeng;LI Lin(College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,Anhui,China)

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036

出　　处：《微生物学通报》2022年第2期659-678,共20页Microbiology China

基　　金：安徽农业大学研究生创新基金(2020ysj-24);国家自然科学基金面上项目(31772802)。

摘　　要：[背景]鼠伤寒沙门氏菌(Salmonella typhimurium)是一种重要的人兽共患病原菌,其多重耐药性问题日益严重,双组分系统可调控鼠伤寒沙门氏菌的耐药性。[目的]通过构建鼠伤寒沙门氏菌baeR过表达株及回补株探究BaeSR双组分系统对鼠伤寒沙门氏菌耐药性的影响。[方法]在BaeSR双组分系统和AcrB外排泵双缺失株(CRΔbaeSRΔacrB)的基础上构建baeR过表达株(CRpbaeRΔbaeSRΔacrB)及baeR回补株(CRcbaeRΔbaeSRΔacrB),测定双缺失株、回补株和过表达株的最小抑菌浓度(minimum inhibitory concentration,MIC),并对其生长特性、生物膜形成能力及运动性进行分析。采用转录组学技术筛选与耐药相关的差异表达基因,RT-qPCR验证耐药相关基因。[结果]构建了鼠伤寒沙门氏菌baeR过表达株和baeR回补株。与双缺失株相比,过表达株对氧氟沙星、恩诺沙星、氟苯尼考、乙酰甲喹、头孢他啶、头孢噻呋、阿莫西林和氨苄西林的MIC分别升高2-256倍,对大观霉素、安普霉素的MIC下降了50%;与双缺失株相比,回补株对头孢他啶、头孢噻呋、氟苯尼考、恩诺沙星的MIC分别升高了2倍;与双缺失株相比,过表达株和回补株的生长速率无明显变化,生物膜形成能力及运动能力极显著升高(P<0.01)。转录组学分析表明CRpbaeRΔbaeSRΔacrB/CRΔbaeSRΔacrB比较组共筛选到743个差异表达基因(上调724个,下调19个),差异表达基因主要富集在β-内酰胺抗性、群体感应系统和双组分系统等通路中;CRcbaeiRΔbaeSΔtacrB/CRΔbaeSRΔacrB比较组共筛选到3073个差异表达基因(上调1467个,下调1606个),差异表达基因主要富集在碳代谢、氨基酸的生物合成和抗生素的生物合成等通路中。筛选出15个外排泵、鞭毛、生物膜、双组分系统及外膜蛋白等耐药相关差异表达基因,随机选取5个差异表达基因进行;T-qPCR验证,趋势与转录组一致。[结论]baeR过表达后通过调控生物膜形成及运�[Background]Salmonella typhimurium is a major zoonotic pathogen,and its multi-drug resistance is becoming increasingly serious.The two-component system can regulate the resistance of S.typhimurium.[Objective]We constructed the baeR-overexpressing strain and complementary strain of S.typhimurium to study the effect of BaeSR on the antibiotic resistance of S.typhimurium.[Methods]A complementary strain(CRcbaeR?baeSR?acrB)and an overexpression strain(CRpbaeR?baeSR?acrB)were constructed based on a baeSR and acr B double-deletion strain of S.typhimurium(CR?baeSR?acr B),and their antimicrobial susceptibility and biological characteristics were tested.RNA-Seq was used to screen the differentially expressed genes(DEGs)related to drug resistance,and RT-qPCR was conducted to verify some drug-related genes.[Results]The baeR-overexpressing strain and complementary strain were successfully constructed.Compared with that in CR?baeSR?acrB,the minimum inhibitory concentrations(MICs)of ofloxacin,enrofloxacin,florfenicol,mequindox,ceftazidime,ceftiofur,amoxicillin,and ampicillin increased by 2–256 folds while that of spectinomycin and apramycin decreased by 50%in CRpbaeR?baeSR?acrB.The MIC of ceftazidime,ceftiofur,florfenicol,and enrofloxacin was two-fold higher in CRcbaeR?baeSR?acrB than in CR?baeSR?acrB.The growth curves of CR?baeSR?acr B,CRpbaeR?baeSR?acr B,and CRcbaeR?baeSR?acr B showed no significant difference.However,the biofilm-forming ability and motility of CRpbaeR?baeSR?acr B and CRcbaeR?baeSR?acr B were significantly higher than those of CR?baeSR?acrB(P<0.01).A total of 743 DEGs were identified between CRpbaeR?baeSR?acr B and CR?baeSR?acr B,including 724 upregulated genes and 19 downregulated genes.Bioinformatics analysis showed that the DEGs were mainly enriched in pathways such asβ-lactam resistance,quorum sensing,and two-component system.A total of 3073 DEGs were identified between CRcbaeR?baeSR?acrB and CR?baeSR?acrB,including 1467 upregulated genes and 1606 downregulated genes.These DEGs were mainly enriched in

关 键 词：鼠伤寒沙门氏菌 双组分系统 耐药性 基因过表达 转录组学 

分 类 号：S852.61[农业科学—基础兽医学]