亚抑菌浓度万古霉素对质粒接合转移的促进作用  

Promoting Effect of Sub-inhibitory Concentration of Vancomycin on Plasmid Conjugation Transfer

作　　者：游祖姣 曾福鑫 王志强[1,2] 肖霞 YOU Zujiao;ZENG Fuxin;WANG Zhiqiang;XIAO Xia(College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Jiangsu University Collaborative Innovation Center for the Prevention and Control of Important Animal Diseases and Zoonosis,Yangzhou 225009,China)

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]江苏高校动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医杂志》2024年第5期78-85,共8页Chinese Journal of Veterinary Medicine

基　　金：国家自然科学基金项目(32273055)。

摘　　要：为了分析亚抑菌浓度万古霉素对质粒接合转移的影响,本试验建立了以大肠杆菌(E.coli)J53作为受体菌,携带RP4-7质粒的E.coli DH5α和携带mcr-1阳性IncI2质粒的临床菌株E.coli LD67-1为供体菌的接合转移模型,测定亚抑菌浓度的万古霉素对接合转移的影响,并通过细胞膜通透性检测、扫描电子显微镜观察、活性氧(ROS)检测和实时荧光定量PCR(RT-qPCR)方法探究其内在机制。结果显示,与空白对照组相比,亚抑菌浓度万古霉素可显著提高RP4-7质粒和mcr-1阳性IncI2质粒的接合转移频率(P<0.05);N-苯基-1-萘胺(NPN)和碘化丙啶(PI)探针检测显示,亚抑菌浓度万古霉素可使受体菌细胞内、外膜通透性增加,供体菌细胞外膜通透性增加;扫描电子显微镜观察显示,经1/4最小抑菌浓度(MIC)万古霉素处理后接合细菌细胞膜发生损伤;ROS检测结果显示,与空白对照组相比,虽然经1/4 MIC万古霉素处理的受体菌的ROS表达水平显著上调(P<0.05),但1/8 MIC万古霉素处理后,供体菌和受体菌的ROS水平均无显著变化(P>0.05);RT-qPCR结果显示,与空白对照组相比,亚抑菌浓度万古霉素显著上调了供体菌和受体菌的孔蛋白基因ompF和ompC的mRNA表达水平(P<0.05),极显著上调了trbBp基因(调控细菌间形成“连接桥”)的mRNA表达水平(P<0.01),显著上调了细菌SOS响应(SOS response)相关基因recA、recN、ruvA和lexA的mRNA表达水平(P<0.05)。结果表明,亚抑菌浓度的万古霉素可能通过提高细菌细胞膜通透性,触发SOS响应,促进接合转移“连接桥”形成等方式促进RP4-7质粒和mcr-1阳性IncI2质粒的接合转移过程。To analyze the effect of sub-inhibitory concentration of vancomycin on plasmid conjugation transfer,this study established a conjugation transfer model using Escherichia coli(E.coli)J53 as the recipient strain,E.coli DH5αcarrying RP4-7 plasmid and clinical strain E.coli LD67-1 carrying mcr-1 positive IncI2 plasmid as donor strains.The influence of sub-inhibitory concentration of vancomycin on conjugation transfer was determined,and the underlying mechanisms were explored through tests of cell membrane permeability,scanning electron microscopy observation,detection of reactive oxygen species(ROS),and real-time quantitative PCR(RT-qPCR).The results showed that compared with the blank control group,sub-inhibitory concentration of vancomycin significantly increased the conjugation transfer frequency of RP4-7 plasmid and mcr-1 positive IncI2 plasmid(P<0.05);N-phenyl-1-naphthylamine(NPN)and propidium iodide(PI)probe tests revealed that sub-inhibitory concentration of vancomycin increased the permeability of both the inner and outer membranes of recipient cells,as well as the outer membrane of donor cells;scanning electron microscopy observation demonstrated membrane damage in conjugative bacterial cells after treatment'with 1/4 minimal inhibitory concentration(MIC)of vancomycin;ROS detection results showed that although the expression levels of ROS in recipient bacteria treated with 1/4 MIC of vancomycin were significantly upregulated compared with the blank control group(P<0.05),there were no significant changes in ROS levels in donor and recipient bacteria when treated with 1/8 MIC of vancomycin(P>0.05);RT-qPCR results showed that sub-inhibitory concentration of vancomycin significantly upregulated the mRNA expression levels of pore protein genes ompF and ompC in both donor and recipient bacteria(P<0.05),markedly upregulated the mRNA expression level of trbBp gene(which regulates the formation of"connecting bridge"between bacteria)(P<0.01),and significantly upregulated the mRNA expression levels of bacteria SOS resp

关 键 词：亚抑菌浓度 万古霉素 质粒接合转移 mcr-1 

分 类 号：S859.7[农业科学—临床兽医学]