牛源大肠杆菌16S rRNA甲基化酶基因的检测及扩散机制的研究

发布时间：2018-12-31 15:45

【摘要】：氨基糖苷类抗生素是对细菌抗菌活性较好的一类广谱、高效的抗生素,然而该类抗生素在临床应用的普遍性,其耐药新机制——16S rRNA甲基化酶的出现,导致在临床现象中耐药菌株出现增多,耐药率也越来越高,并且还出现了多重耐药菌的β-内酰胺类和喹诺酮类抗菌药物耐药性同时存在现象,使得在临床中对氨基糖苷类类抗生素的应用造成了极其严重的影响,因而研究宁夏地区牛源大肠杆菌16S rRNA甲基化酶的扩散机制,不仅对临床合理用药具有指导意义,而且有利于降低和控制耐药大肠杆菌的扩散。本研究主要材料是采集宁夏地区部分奶牛场的乳房炎乳样及犊牛腹泻粪便样,采用革兰氏染色镜检、大肠杆菌显色培养基以及PCR扩增16S rDNA基因等方法进行大肠杆菌的分离鉴定,再根据CLSI标准委员会推荐的肉汤微量稀释法测定16种药物对245株大肠杆菌分离株的MIC值,并采用药敏纸片法进行ESBLs的表型确证试验。结果显示,共分离鉴定出245株牛源大肠杆菌,分离率为19.48%;药敏试验结果显示,245株牛源大肠杆菌对氨苄西林的耐药率最高,达到51.84%,其次是链霉素(37.96%)、多西环素(36.33%)、四环素(35.51%)以及头孢唑林(30.20%)。对多粘菌素、氟苯尼考、氨基糖苷类、头孢类及喹诺酮类抗菌药物则相对比较敏感,敏感率都在60%以上。较多的菌株都表现为对16种抗菌药物的耐药。经过ESBLs表型确证试验确定,245株牛源大肠杆菌中检出60株ESBLs菌株,检出率为24.49%(60/245)。运用PCR方法对分离出的245株牛源大肠杆菌进行16S rRNA甲基化酶基因及ESBLs基因的检测,结果显示,16S rRNA甲基化酶基因只检测出rmtB基因,检出率为5.3% (13/245); ESBLs基因blaTEM、bla CTX-M、blaOXA的检出率分别为23.27%(57/245)、11.02%(27/245)、2.86%(7/245)；检出的13株rmtB阳性大肠杆菌中还检出了blaTEM-1、blaCTX-M基因,并检出了2株blaOXA-1基因,检出率分别为100.0%(13/13),100.0%(13/13)和15.38%(2/13)。最后采用PFGE分子分型试验、接合转移试验和Southern杂交等方法研究16S rRNA甲基化酶基因的扩散机制。试验数据显示,宁夏地区牛源大肠杆菌中rmtB基因的扩散方式主要通过垂直和水平传播两种方式扩散。综上所述,本研究通过对宁夏地区牛源大肠杆菌耐药性分析及16S rRNA甲基化酶基因扩散机制的研究,初步揭示了大肠杆菌在宁夏地区奶牛场的耐药情况和分子流行病学特征,以及牛源大肠杆菌16S rRNA甲基化酶基因的传播扩散机制,以期为降低和控制耐药大肠杆菌的扩散及指导临床合理用药提供理论依据。
[Abstract]:Aminoglycoside antibiotics are a kind of broad-spectrum and high-efficient antibiotics with good antibacterial activity against bacteria. However, the prevalence of these antibiotics in clinical application and the emergence of 16s rRNA methylase, a new mechanism of drug resistance, are common in clinical application of aminoglycoside antibiotics. As a result, the resistant strains appeared more and more in clinical phenomenon, and the resistance of 尾-lactams and quinolones were also found in the multidrug resistant bacteria, and the drug resistance of 尾-lactam and quinolone antibiotics existed simultaneously, and the drug resistance of 尾-lactam and quinolone antibiotics were also found at the same time. Therefore, the study on the diffusion mechanism of 16s rRNA methylase of bovine Escherichia coli in Ningxia area is not only of guiding significance to the rational use of drugs in clinical practice, but also to the clinical application of aminoglycoside antibiotics. And it is beneficial to reduce and control the spread of drug resistant Escherichia coli. The main materials of this study were collected milk samples from dairy farms in Ningxia area and fecal samples from calves with diarrhea. E. coli was isolated and identified by Gram staining, coloration medium of Escherichia coli and PCR amplification of 16s rDNA gene. The MIC values of 16 drugs against 245 strains of Escherichia coli were determined by broth dilution method recommended by CLSI Standard Committee, and the phenotypic confirmatory test of ESBLs was carried out by drug sensitive disk method. The results showed that 245 strains of bovine Escherichia coli were isolated and the isolation rate was 19.48%. The drug sensitivity test showed that 245 strains of bovine Escherichia coli had the highest resistance to ampicillin (51.84%), followed by streptomycin (37.96%) and doxycycline (36.33%). Tetracycline (35.51%) and cefazolin (30.20%). Polymyxin, florfenicol, aminoglycosides, cephalosporins and quinolones were more sensitive than 60%. More strains showed resistance to 16 antimicrobial agents. By ESBLs phenotypic confirmation test, 60 strains of ESBLs were detected in 245 strains of bovine Escherichia coli, the detection rate was 24.49% (60 / 245). 16s rRNA methylase gene and ESBLs gene were detected by PCR method. The results showed that only rmtB gene was detected by 16s rRNA methylase gene, and the detection rate was 5.3% (13 / 245). The detection rate of ESBLs blaTEM,bla CTX-M,blaOXA was 23.27% (57 / 245), 11.02% (27 / 245) and 2.86% (7 / 245) respectively. BlaTEM-1,blaCTX-M gene was also detected in 13 rmtB positive Escherichia coli strains and two blaOXA-1 genes were detected. The detection rates were 100.0% (13 / 13), 100.0% (13 / 13) and 15.38% (2 / 13), respectively. Finally, the diffusion mechanism of 16s rRNA methylase gene was studied by PFGE molecular typing test, conjugation transfer test and Southern hybridization. The experimental data showed that the diffusion of rmtB gene in bovine Escherichia coli in Ningxia area was mainly carried out by vertical and horizontal transmission. In conclusion, the drug resistance and molecular epidemiological characteristics of Escherichia coli in dairy farms in Ningxia were preliminarily revealed by analyzing the drug resistance of bovine Escherichia coli and the gene diffusion mechanism of 16s rRNA methylase. And the transmission and diffusion mechanism of 16s rRNA methylase gene of bovine Escherichia coli, in order to provide theoretical basis for reducing and controlling the spread of drug-resistant Escherichia coli and guiding rational drug use in clinic.
【学位授予单位】：宁夏大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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