牛源大腸桿菌16S rRNA甲基化酶基因的檢測(cè)及擴(kuò)散機(jī)制的研究
發(fā)布時(shí)間：2018-12-31 15:45
【摘要】：氨基糖苷類抗生素是對(duì)細(xì)菌抗菌活性較好的一類廣譜、高效的抗生素,然而該類抗生素在臨床應(yīng)用的普遍性,其耐藥新機(jī)制——16S rRNA甲基化酶的出現(xiàn),導(dǎo)致在臨床現(xiàn)象中耐藥菌株出現(xiàn)增多,耐藥率也越來越高,并且還出現(xiàn)了多重耐藥菌的β-內(nèi)酰胺類和喹諾酮類抗菌藥物耐藥性同時(shí)存在現(xiàn)象,使得在臨床中對(duì)氨基糖苷類類抗生素的應(yīng)用造成了極其嚴(yán)重的影響,因而研究寧夏地區(qū)牛源大腸桿菌16S rRNA甲基化酶的擴(kuò)散機(jī)制,不僅對(duì)臨床合理用藥具有指導(dǎo)意義,而且有利于降低和控制耐藥大腸桿菌的擴(kuò)散。本研究主要材料是采集寧夏地區(qū)部分奶牛場(chǎng)的乳房炎乳樣及犢牛腹瀉糞便樣,采用革蘭氏染色鏡檢、大腸桿菌顯色培養(yǎng)基以及PCR擴(kuò)增16S rDNA基因等方法進(jìn)行大腸桿菌的分離鑒定,再根據(jù)CLSI標(biāo)準(zhǔn)委員會(huì)推薦的肉湯微量稀釋法測(cè)定16種藥物對(duì)245株大腸桿菌分離株的MIC值,并采用藥敏紙片法進(jìn)行ESBLs的表型確證試驗(yàn)。結(jié)果顯示,共分離鑒定出245株牛源大腸桿菌,分離率為19.48%;藥敏試驗(yàn)結(jié)果顯示,245株牛源大腸桿菌對(duì)氨芐西林的耐藥率最高,達(dá)到51.84%,其次是鏈霉素(37.96%)、多西環(huán)素(36.33%)、四環(huán)素(35.51%)以及頭孢唑林(30.20%)。對(duì)多粘菌素、氟苯尼考、氨基糖苷類、頭孢類及喹諾酮類抗菌藥物則相對(duì)比較敏感,敏感率都在60%以上。較多的菌株都表現(xiàn)為對(duì)16種抗菌藥物的耐藥。經(jīng)過ESBLs表型確證試驗(yàn)確定,245株牛源大腸桿菌中檢出60株ESBLs菌株,檢出率為24.49%(60/245)。運(yùn)用PCR方法對(duì)分離出的245株牛源大腸桿菌進(jìn)行16S rRNA甲基化酶基因及ESBLs基因的檢測(cè),結(jié)果顯示,16S rRNA甲基化酶基因只檢測(cè)出rmtB基因,檢出率為5.3% (13/245); ESBLs基因blaTEM、bla CTX-M、blaOXA的檢出率分別為23.27%(57/245)、11.02%(27/245)、2.86%(7/245)；檢出的13株rmtB陽性大腸桿菌中還檢出了blaTEM-1、blaCTX-M基因,并檢出了2株blaOXA-1基因,檢出率分別為100.0%(13/13),100.0%(13/13)和15.38%(2/13)。最后采用PFGE分子分型試驗(yàn)、接合轉(zhuǎn)移試驗(yàn)和Southern雜交等方法研究16S rRNA甲基化酶基因的擴(kuò)散機(jī)制。試驗(yàn)數(shù)據(jù)顯示,寧夏地區(qū)牛源大腸桿菌中rmtB基因的擴(kuò)散方式主要通過垂直和水平傳播兩種方式擴(kuò)散。綜上所述,本研究通過對(duì)寧夏地區(qū)牛源大腸桿菌耐藥性分析及16S rRNA甲基化酶基因擴(kuò)散機(jī)制的研究,初步揭示了大腸桿菌在寧夏地區(qū)奶牛場(chǎng)的耐藥情況和分子流行病學(xué)特征,以及牛源大腸桿菌16S rRNA甲基化酶基因的傳播擴(kuò)散機(jī)制,以期為降低和控制耐藥大腸桿菌的擴(kuò)散及指導(dǎo)臨床合理用藥提供理論依據(jù)。
[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.
【學(xué)位授予單位】：寧夏大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.61
本文編號(hào)：2396790
[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.
【學(xué)位授予單位】：寧夏大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.61
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