合肥市部分医院产CTX-M型超广谱β-内酰胺酶细菌的耐药性分析及其分子生物学特征

发布时间：2018-11-26 10:20

【摘要】：目的了解产CTX-M型酶的大肠埃希菌和肺炎克雷伯菌对14种抗菌素的耐药性，以指导临床用药。了解合肥市部分医院CTX-M酶的基因型，研究新的CTX-M酶的生化及耐药特性。材料与方法菌株来源： 1999年至2000年收集的合肥市四家大型医院的临床产ESBLs大肠埃希菌和肺炎克雷伯菌，共98株。方法以98株ESBLs~+菌株总DNA为模板，使用bla_(CTX-M)通用引物进行PCR扩增以筛选出CTX-M~+菌株；再将CTX-M~+菌株与E．coliC600共孵，行转移接合试验；采用琼脂平皿稀释法对野生株与接合子进行MIC测定。根据通用引物扩增结果，设计两对全编码区扩增引物，为方便后续试验，5｀端分别加EcoRI和BamHI酶切位点。将全编码区PCR产物，交送生物公司进行直接序列分析，结果至Genbank进行比对，确定CTX-M酶的基因型。在EcoRI和BamHI酶切之后，将新基因型全编码基因克隆入表达载体pHSG_(398)，转化于感受态细胞E．coliDH_(5α)，用琼脂平皿稀释法测定14种抗生素的MIC值。采用超声破碎法进行产新酶细菌的接合子的粗酶提取，等电聚焦电泳测定其pI。采用碱裂解法抽取产新酶的细菌的质粒，PstI消化，进行质粒分析。采用脉冲场凝胶电泳(pulsed field gel electrophoresis，PFGE)分析产新酶细菌的同源性。结果
[Abstract]:Objective to investigate the antibiotic resistance of Escherichia coli and Klebsiella pneumoniae producing CTX-M-type enzyme to 14 antimicrobial agents. To investigate the genotypes of CTX-M enzyme in some hospitals of Hefei, and to study the biochemical and drug resistance characteristics of the new CTX-M enzyme. Materials and methods: 98 strains of Escherichia coli and Klebsiella pneumoniae were collected from four large hospitals in Hefei from 1999 to 2000. Methods the total DNA of 98 ESBLs~ strains was used as template, and PCR amplification was carried out with bla_ (CTX-M) universal primers to screen out CTX-M~ strains, and then CTX-M~ was co-incubated with E.coliC600, and then transferred and conjugated. Agar plate dilution method was used to determine the MIC of wild strain and conjugate. According to the results of universal primer amplification, two pairs of full coding region amplification primers were designed. In order to facilitate the subsequent test, EcoRI and BamHI endonuclease sites were added to the 5'end respectively. The PCR products of the whole coding region were handed over to the biological company for direct sequence analysis. The results were compared with Genbank to determine the genotype of CTX-M enzyme. After digested with EcoRI and BamHI, the new gene was cloned into the expression vector pHSG_ (398) and transformed into E. coli DH5 伪. The MIC values of 14 antibiotics were determined by Agar plate dilution method. The pI. of the conjugate of new enzyme producing bacteria was determined by isoelectric focusing electrophoresis. The plasmids of new enzyme producing bacteria were extracted by alkaline lysis and digested by PstI, and the plasmids were analyzed. The homology of new enzyme producing bacteria was analyzed by pulsed field gel electrophoresis (pulsed field gel electrophoresis,PFGE). Result
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R446.5;R378

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