摘要
目的探讨亚胺培南(IPM)对bla_(NDM-1)阳性Escherichia coli耐药性及其内膜secY、secE和secG转录水平的影响。方法以重组质粒pET28a(+)-bla_(NDM-1)转化菌株E.coli DH5α-bla_(NDM-1)和E.coli BL21(DE3)-bla_(NDM-1)为研究对象,在梯度浓度增加或撤消IPM暴露下传代培养菌株,检测17种抗生素对IPM暴露菌株的MIC值;SDS-PAGE凝胶电泳检测NDM-1表达;蛋白活性实验检测NDM-1活性;qRT-PCR检测bla_(NDM-1)及内膜secY、secE和secG转录水平。结果12μg/mL和0_(20)μg/mL IPM暴露的E.coli DH5α-bla_(NDM-1)菌株CFZ、CXM、FOX、CRO、CAZ、FEP等头孢类药物的MIC值(≥8μg/mL/≥8μg/mL、≥32μg/mL/≥32μg/mL、≥32μg/mL/≥32μg/mL、≥64μg/mL/=32μg/mL、≥32μg/mL/≥32μg/mL、≥32μg/mL/=8μg/mL)与0μg/mL IPM暴露MIC值(≤2μg/mL、=16μg/mL、≤8μg/mL、≤1μg/mL、≤4μg/mL、≤2μg/mL)相比均显著增高,而IPM和MEM的MIC值与0μg/mL IPM暴露(≤1μg/mL和≤1μg/mL)相比也显著增高(=8μg/mL/=8μg/mL和=4μg/mL/=4μg/mL)。12μg/mL和0_(20)μg/mL IPM暴露的E.coli BL21(DE3)-bla_(NDM-1)菌株FOX、CRO、FEP等头孢类药物的MIC值(≥32μg/mL/≥32μg/mL、≥64μg/mL/≥64μg/mL、=16μg/mL/=16μg/mL)与0μg/mL IPM暴露(≤8μg/mL、≤1μg/mL、≤2μg/mL)相比也均显著增高,而IPM和MEM的MIC值与0μg/mL IPM暴露(≤1μg/mL和≤1μg/mL)相比也均显著增高(≥16μg/mL/≥16μg/mL和≥16μg/mL/≥16μg/mL)。SDS-PAGE显示,随12μg/mL IPM暴露时间的延长,菌株NDM-1水解IPM活性增加。qRT-PCR显示,12μg/mL IPM暴露的E.coli DH5α-bla_(NDM-1)和E.coli BL21(DE3)-bla_(NDM-1)的bla_(NDM-1)、secY、secE和secG转录水平分别上调2.31/2.5、3.05/1.96、2.83/1.24和2.71/1.45倍。结论IPM可使bla_(NDM-1)阳性E.coli由碳青霉烯类敏感变为耐药且保持稳定,细菌内膜SecYEG跨膜通道蛋白参与菌株耐药性调控,这为认识抗生素压力下肠杆菌科细菌耐药性变化及指导临床合理用药提供理论支撑。
Objective To investigate the effects of imipenem(IPM)on the resistance of bla_(NDM-1) positive Escherichia coli and the transcription levels of secY,secE and secG in intima.Method E.coli DH5α-bla_(NDM-1) and E.coli BL21(DE3)-bla_(NDM-1) transformed by recombinant plasmid pET28a(+)-bla_(NDM-1) were used as the research objects,and subcultured under the condition that the gradient concentration was increased or IPM exposure was canceled.The MIC values of 17 kinds of antibiotics against the IPM exposed strains were detected.The expression of NDM-1 was detected by SDS-PAGE gel electrophoresis.NDM-1 activity was detected by the protein activity test.The transcription levels of bla_(NDM-1),secY,secE and secG in intima were detected by qRT-PCR.Result The MIC values of CFZ,CXM,FOX,CRO,CAZ,FEP,other cephalosporins on E.coli DH5α-bla_(NDM-1) strain exposed by 12μg/mL and 0_(20)μg/mL IPM(≥8μg/mL/≥8μg/mL,≥32μg/mL/≥32μg/mL,≥32μg/mL/≥32μg/mL,≥64μg/mL/=32μg/mL,≥32μg/mL/≥32μg/mL,≥32μg/mL/=8μg/mL)were significantly increased,compared with 0μg/mL IPMexposed strain(≤2μg/mL,=16μg/mL,≤8μg/mL,≤1μg/mL,≤4μg/mL,≤2μg/mL),and the MIC values of IPM and MEM were also significantly increased(=8μg/mL/=8μg/mL and=4μg/mL/=4μg/mL),compared with 0μg/mL IPM-exposed strain(≤1μg/mL and≤1μg/mL).The E.coli BL21(DE3)-bla_(NDM-1) strain induced by 12μg/mL and 0_(20)μg/mL IPM(≥32μg/mL/≥32μg/mL,≥64μg/mL/≥64μg/mL,=16μg/mL/=16μg/mL)were also significantly increased,compared with 0μg/mL IPM-exposed strain(≤8μg/mL,≤1μg/mL,≤2μg/mL).The MIC values of IPM and MEM were also significantly increased(≥16μg/mL/≥16μg/mL and≥16μg/mL/≥16μg/mL),compared with 0μg/mL IPM-exposed strain(≤1μg/mL and≤1μg/mL).Compared with 0μg/mL IPM-exposed strain,qRTPCR showed the expression of bla_(NDM-1),secY,secE,secG in E.coli DH5α-bla_(NDM-1) and E.coli BL21(DE3)-bla_(NDM-1) induced by 12μg/mL IPM were increased by 2.31/2.5,3.05/1.96,2.83/1.24 and 2.71/1.45 times respectively.Conclusion IPM can make bla_(NDM-1) positive E.coli changed from carbapenem sensitive to drug resistant and remained stable,SecYEG transmembrane channel protein in bacterial intima participated in the regulation of bacterial resistance,which provided theoretical support for understanding the change of Enterobacteriaceae bacterial resistance under antibiotic pressure and guiding clinical rational drug use.
作者
吴兆猛
赵琼
吴玲玲
王祖华
余春芳
金志雄
Wu Zhaomeng;Zhao Qiong;Wu Lingling;Wang Zuhua;Yu Chunfang;Jin Zhixiong(Department of Basic Medical Science,Hubei University of Medicine,Shiyan 442000;Department of Clinical Laboratory,Dongfeng Hospital,Hubei University of Medicine,Shiyan,442008;Department of Blood Transfusion,Taihe Hospital,Hubei University of Medicine Shiyan,442000)
出处
《中国抗生素杂志》
CAS
CSCD
北大核心
2023年第9期1048-1056,共9页
Chinese Journal of Antibiotics
基金
湖北省卫生健康委项目(No.WJ2021F049)
“十四五”湖北省高等学校优势特色学科群(生物与医药)项目资助(No.2022BMXKQY1)。
