【作者】 杜艳；

【导师】 贾文祥； 陈端；

【作者基本信息】 四川大学 ， 病原微生物， 2005， 博士

【摘要】 目的:多重耐药是一个全球性的问题,其严重后果是影响临床疗效,增加治疗成本,缩短新药的应用周期,增大新药的研究与开发成本,直接威胁着人体健康。近年来,抗生素耐药机制的分子特征新进展阐明了整合子(integron)基因结构的存在,细菌通过整合子-基因盒(Gene cassette)系统,在整合酶的作用下,捕获外来耐药基因,并在位于整合子上游的启动子作用下表达,使细菌具有耐药及多重耐药性。本研究旨在了解昆明地区革兰阴性杆菌临床多重耐药菌株中整合子的携带情况,研究整合子与多重耐药的关系,为进一步明确细菌多重耐药的新机制奠定基础。 方法:1对昆明地区临床常见革兰阴性杆菌进行药物更多还原

【Abstract】 Objectives: Multidrug resistance is a world problem. Its significant result is to affect clinical treatment, increase the treatment cost, shorten the applying period of a new drug, add the researching and developing cost of a new drug and threaten directly the health of human. Recently, the molecular characteristic advance of antibiotic resistance mechanism has stated the existence of integron gene structure. Bacteria were affected by integrase captureexternal drug-resistant gene through integron-gene cassette system and were expressed by the promoter of integron upstream, which enable bacteria to have drug resistance and multidrug resistance. This study is aimed to understand the carrying of integron in gram negative clinical multidrug resistant strains of Kunming area, investigate the relation of integron and multidrug resistance, which may establish a foundation of clarifying fiirtherly the new mechanism of bacteria multidrug resistance.Methods: 1 Antibiotic susceptive analyse of gram negative clinical bacilli in Kunming area, 2 Using PCR and Southern blotting to detect the integron relative gene in clinical strains, 3 plasmid transformation and the analysis of drug resistance shifting.Results : 1 Much clinical strains show multidrug resistance to three or above three antibiotics. 2 The PCR amplication positive ratio of the integron conserved segament is 30%~50%, intll integrase gene is high(80 % ~ 97 % ),intI2 integrase gene is found only inPseudomonas aeruginosa and Enterobacter cloacae,the positive ratio is 6.25% and 13.5% differently.The positive ratio of sul 1 gene in class 1 integron 3 ’ conserved segament is above 90%, qacEAl gene is above 80%, aadB is 10%~50%o Southern blotting verify the intll, sul 1 and qacEAl gene of class 1 integron. 3 The biochemical reaction of a transconjugant is similar to the receptor bacterium, which is Escherichia coli identified by VITEK2. The drug-resistant scope of a transconjugant is identical to the donator bacterium.Conclusions: The multidrug resistance of bacteria has significant relation with integron. The relative genes of the integron show a high homogenicity in spite of genus or species. The sul 1 and qacEAl gene may seem as the marker genes of class 1 integron .The integron may be carried by the plasmid. The drug-resistant genes carried by the integron may transfer through the transformation test.Objectives: AmpC enzyme was produced by gram negative bacilli and was chromosome-mediated cephalosporin enzyme. It was encoded by ampC, which can inactive many β -lactam antibiotics including the third cephalosporin. The study was aimed to eatablish an efficient method about routine detection of AmpC enzyme in clinical laboratory. AmpC enzyme structural gene ampCand regular gene ampD were analysed for determine their location in integron.We are groping for the relation between AmpC enzyme amd integron, analyzing the bacterial resistance mechanim and guiding the clinics to rational drugs. Doing so can provide the theorial base for the clinics in treating infection induced by enzyme-produced strains.Methods: Five detection methods were applied(Cefoxitin screening test, phenotype screening test, flucloxacillin double inhibitors diffuse synergy test, three dimensional test and gene detection of AmpC enzyme). PCR mapping was used to analyse AmpC enzyme structural gene ampC and regular gene ampD for determine their location in integron.Results: The detection of AmpC enzyme showed: The positive ratio of Cefoxitin screening test in Enterobacter cloacae and Pseudomonas aeruginosa are both 100%. Phenotype screening test suggested 35.1% (26/74) and 37.5%(30/80) respectively. Flucloxacillin doubleinhibitors diffuse synergy test results were 37.8% (28/74) and 40%(32/80) respectively. The positive ratio of three dimensional test were 28.4% (21/74) and 30% (24/80) respectively. The positive ratio of PCR amplication ampC gene were 89.2 % (66/74) and 91 % (73/80) respectively. The positive ratio of PCR amplication ampD gene were 86.5 % (64/74) and 87.5%(70/80) respectively. The positive ratio of PCR mapping was 33.3%, and the positive results were 400bp to 800bp polypeptides , smaller than lOOObp. Therefore the insertive resistant gene ampC and ampD may have located in the up stream of integron.Conclusions: The appearance of AmpC enzyme may have much to do with the integron gene structure.更多还原