可移动元件整合子与细菌多重耐药性的关系研究

发布时间：2018-11-18 09:34

【摘要】：目的：调查多重耐药铜绿假单胞菌株中可移动性遗传元件的分布情况,并以整合子为对象深入研究,探究整合子中不同可变区启动子Pant对耐药基因盒和整合酶的表达以及整合频率的影响,了解启动子Pant在整合子介导的耐药机制中的作用,从而为控制细菌广泛耐药和新型抗菌药物的研发提供一定的理论基础。方法：从临床标本中分离并筛选出49株多重耐药的铜绿假单胞菌,采用聚合酶链反应(PCR)分析21种可移动遗传元件的分布情况,并对可移动元件的组合形式进行分析；构建高表达整合酶基因的重组质粒pET-INT和同时含有无耐药基因盒整合子和aadA2基因盒的重组质粒pACYC-IN-AAD,通过表型筛选法测定其整合频率；通过点突变法构建含有不同可变区启动子的重组质粒,通过实时荧光定量PCR比较其aadA2基因和intI1基因的表达量。结果：49株多重耐药铜绿假单胞菌菌株中,共检出11种可移动遗传元件相关遗传标记基因,4个种类的可移动性遗传元件均有检出。阳性率最高的为IS26(77.6%),其次为intI1(67%)。于国内首次在多重耐药铜绿假单胞菌中检出traF基因；本研究成功构建了整合子剪切和捕获耐药基因盒的体外模型,研究发现,在BL21(DE3)菌株中,含有高表达整合酶的整合子对耐药基因盒的整合能力是无高表达整合酶整合子的9.41倍；不同可变区启动子下游基因盒中基因的转录水平有显著差异,4种质粒的aadA2基因转录水平从高到低依次为：MU2pACYC-IN-AADMU3MU1,最高者是最低者的114.7倍。结论：可移动遗传元件在铜绿假单胞菌株中广泛存在,插入序列和整合子在其耐药基因水平传播中起主要的作用。可变区启动子的启动强度与下游的耐药基因盒表达水平关系密切,启动强度越高,基因盒表达越强,反之亦然。图14幅,表14个,参考文献42篇。
[Abstract]:Objective: to investigate the distribution of transportable genetic elements in multidrug resistant Pseudomonas aeruginosa strains and to study the integron. To explore the effect of different variable region promoter Pant on the expression and integration frequency of drug resistance gene box and integrase in integron, and to understand the role of promoter Pant in integron mediated drug resistance mechanism. It provides a theoretical basis for the control of bacterial extensive drug resistance and the development of new antimicrobial agents. Methods: 49 strains of multidrug resistant Pseudomonas aeruginosa were isolated and screened from clinical specimens. The distribution of 21 kinds of movable genetic elements were analyzed by polymerase chain reaction (PCR). The frequency of integration was determined by phenotypic screening method in the construction of recombinant plasmid pET-INT with high expression of integrase gene and the recombinant plasmid pACYC-IN-AAD, containing both cassette integron and aadA2 gene box. The recombinant plasmids containing different variable region promoters were constructed by point mutation method. The expression levels of aadA2 gene and intI1 gene were compared by real-time fluorescence quantitative PCR. Results: among 49 strains of multidrug resistant Pseudomonas aeruginosa, 11 kinds of mobile genetic elements were detected, and 4 kinds of mobile elements were detected. The highest positive rate was IS26 (77.6%), followed by intI1 (67%). TraF gene was first detected in multidrug resistant Pseudomonas aeruginosa in China. In this study, we successfully constructed an in vitro model of integron shearing and capturing drug resistance gene cassette. It was found that in BL21 (DE3) strain, The integration ability of integron containing high expression integrase to drug resistant gene box was 9.41 times as high as that of non-high expression integrase integron. The transcriptional level of aadA2 gene in the cassette of different variable region promoters was significantly different. The transcription level of aadA2 gene in the four plasmids was 114.7 times higher than that in the lowest one. Conclusion: mobile genetic elements are widely present in Pseudomonas aeruginosa. Insertion sequences and integrons play a major role in the horizontal transmission of drug resistance genes. The priming intensity of variable region promoter was closely related to the expression level of drug resistance gene box downstream. The higher the starting intensity, the stronger the gene box expression, and vice versa. There are 14 figures, 14 tables and 42 references.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R378

【共引文献】