【摘要】：目的 了解產(chǎn)CTX-M型酶的大腸埃希菌和肺炎克雷伯菌對14種抗菌素的耐藥性，以指導(dǎo)臨床用藥。 了解合肥市部分醫(yī)院CTX-M酶的基因型，研究新的CTX-M酶的生化及耐藥特性。材料與方法菌株來源： 1999年至2000年收集的合肥市四家大型醫(yī)院的臨床產(chǎn)ESBLs大腸埃希菌和肺炎克雷伯菌，共98株。 方法 以98株ESBLs~+菌株總DNA為模板，使用bla_(CTX-M)通用引物進(jìn)行PCR擴增以篩選出CTX-M~+菌株；再將CTX-M~+菌株與E．coliC600共孵，行轉(zhuǎn)移接合試驗；采用瓊脂平皿稀釋法對野生株與接合子進(jìn)行MIC測定。 根據(jù)通用引物擴增結(jié)果，設(shè)計兩對全編碼區(qū)擴增引物，為方便后續(xù)試驗，5｀端分別加EcoRI和BamHI酶切位點。 將全編碼區(qū)PCR產(chǎn)物，交送生物公司進(jìn)行直接序列分析，結(jié)果至Genbank進(jìn)行比對，確定CTX-M酶的基因型。 在EcoRI和BamHI酶切之后，將新基因型全編碼基因克隆入表達(dá)載體pHSG_(398)，轉(zhuǎn)化于感受態(tài)細(xì)胞E．coliDH_(5α)，用瓊脂平皿稀釋法測定14種抗生素的MIC值。 采用超聲破碎法進(jìn)行產(chǎn)新酶細(xì)菌的接合子的粗酶提取，等電聚焦電泳測定其pI。 采用堿裂解法抽取產(chǎn)新酶的細(xì)菌的質(zhì)粒，PstI消化，進(jìn)行質(zhì)粒分析。 采用脈沖場凝膠電泳(pulsed field gel electrophoresis，PFGE)分析產(chǎn)新酶細(xì)菌的同源性。 結(jié)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 偽. 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
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R446.5;R378

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