猪源大肠杆菌分离鉴定及耐药性研究

发布时间：2018-02-21 11:43

本文关键词： 猪大肠杆菌耐药性水平转移　出处：《吉林农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：伴随着现代化养殖模式的兴起,在畜牧生产中抗生素的合理使用越来越受到重视。抗生素可用于治疗和预防疾病、提高饲料转化率和促进动物生长。尽管抗生素在实践应用中取得了良好的效果,但是多重耐药菌也随之出现并给感染治疗带来更多困难。大肠杆菌是重要的肠道共生菌,对维持肠道的平衡起到了重要作用。在养猪业中抗生素滥用情况严重,这使得猪源大肠杆菌耐药形势日趋严峻。猪源大肠杆菌已成为重要的耐药细菌贮存库,它极有可能通过环境污染、直接接触或食物链将耐药菌传染给人类,因此检测猪源大肠杆菌的耐药性并探讨其公共卫生意义十分必要。本研究于2013年在吉林省采集318份猪源样品,分别来源于临床健康猪、腹泻仔猪以及市售猪肉,共分离鉴定大肠杆菌275株。以氨苄西林、头孢噻肟等15种药物进行了药物敏感性实验,并以多重PCR方法进行系统进化分群。结果表明,健康猪样品大肠杆菌分离株主要分属A群和B1群,为非致病群;腹泻仔猪大肠杆菌分离株B2和D群的比例显著高于健康猪大肠杆菌。综合分析,大肠杆菌分离株对四环素、氨苄西林和磺胺甲基异恶唑耐药最严重,其中176株菌表现为对3类以上抗生素的多重耐药,全部菌株对美洛培南、多粘菌素敏感。从仔猪腹泻样品分离的大肠杆菌对β-内酰胺类抗生素、喹诺酮类抗生素、四环素、氯霉素及磺胺甲基异恶唑的耐药率显著高于健康猪和猪肉样品分离株的耐药率。针对具有较高耐药率的猪源大肠杆菌检测其耐药基因,探讨其分子遗传基础以及分子流行病学规律。采用PCR方法检测CTX-M、TEM、SHV等3种ESBLs的基因和tet(A)、tet(B)、tet(C)、tet(D)、te t(M)、t et(W)等6种四环素耐药基因。结果显示275株大肠杆菌中,116株检出TEM-1基因,该基因型为主要流行基因型;tet(A)、tet(B)、tet(C)、tet(M)耐药基因的检出率分别为64%、22%、1%、5%,t et(A)/tet(B)为主要流行基因型。采用质粒接合转导实验探究了质粒在多重耐药大肠杆菌耐药性水平传播中的作用。随机选取50株猪源多重耐药大肠杆菌为供体菌,用氨苄西林,四环素等12种抗生素对接合子进行药敏试验,多重PCR方法进行质粒分型。结果显示,50株供体菌中,18株接合转导成功,接合转导率达36%。与接合子耐药谱分析结果表明,存在多种耐药性共转移的现象。18株接合子获得质粒复制子分型以FIB为主。研究表明,接合转导型耐药质粒在猪源细菌耐药性传播中起到重要作用。本研究获得了猪源大肠杆菌耐药性的基本流行病学数据,探讨了多重耐药性水平传播的分子遗传学机制,为指导养殖业的临床用药及耐药性监测提供科学依据。
[Abstract]:With the rise of modern breeding mode, more and more attention has been paid to the rational use of antibiotics in livestock production. Antibiotics can be used to treat and prevent diseases. Raise feed conversion rate and promote animal growth. Although antibiotics have achieved good results in practical use, multidrug resistant bacteria have also appeared and made it more difficult to treat infection. E. coli is an important enteric symbiotic bacterium. It plays an important role in maintaining the balance of intestinal tract. The situation of antibiotic abuse is serious in pig industry, which makes the situation of drug resistance of porcine Escherichia coli more and more serious. The porcine Escherichia coli has become an important reservoir of drug-resistant bacteria. It is highly likely to transmit drug-resistant bacteria to humans through environmental pollution, direct contact or the food chain, Therefore, it is necessary to detect the drug resistance of porcine Escherichia coli and to explore its public health significance. In 2013, 318 porcine samples were collected from clinical healthy pigs, diarrhea piglets and marketable pork. A total of 275 strains of Escherichia coli were isolated and identified. Fifteen drugs, such as ampicillin and cefotaxime, were used for drug sensitivity test, and the multiplex PCR method was used for phylogenetic grouping. The Escherichia coli isolates of healthy pigs mainly belong to group A and group B1, which are non-pathogenic group, and the proportions of group B2 and group D of diarrhea piglets Escherichia coli are significantly higher than those of healthy swine Escherichia coli. Comprehensive analysis shows that the percentage of tetracycline in Escherichia coli isolates is higher than that in healthy pigs. Ampicillin and sulfamethoxazole were the most resistant strains, 176 strains showed multidrug resistance to more than 3 kinds of antibiotics, and all strains were resistant to melopenem. E. coli isolated from diarrhea samples of piglets is sensitive to 尾 -lactam antibiotics, quinolones antibiotics, tetracycline. The resistance rates of chloramphenicol and sulfamethoxazole were significantly higher than those of healthy pig and pork isolates. To study its molecular genetic basis and molecular epidemiology, six tetracycline resistance genes were detected by PCR method, including three ESBLs genes and six tetracycline resistance genes. The results showed that TEM-1 gene was detected in 116 strains of E. coli. This genotype is the main prevalent genotype, the detection rate of the drug resistance gene of the main prevalent genotype, AtetShe Atetetor, is 64 / 22, respectively. The role of plasmids in the horizontal transmission of drug resistance in multidrug resistant Escherichia coli was investigated by using plasmid conjugation transduction experiment, which was used to investigate the role of plasmids in the horizontal transmission of drug resistance of multi-heavy drug resistant Escherichia coli (MDR). 50 strains of porcine multidrug resistant Escherichia coli were randomly selected as donor bacteria. The drug sensitivity test was carried out with 12 antibiotics, such as ampicillin and tetracycline, and plasmid typing was carried out by multiple PCR method. The results showed that 18 of the 50 strains of donor bacteria were successfully transduced by conjugation. The rate of conjugation transduction was 36%. The results of drug resistance spectrum analysis with the conjugate showed that there was a phenomenon of multiple drug resistance cotransfer. The plasmid replicas were obtained from 18 strains of conjugates. The results showed that FIB was the main type of plasmid replicas. Conjugate transductive drug resistance plasmids play an important role in the transmission of drug resistance of porcine bacteria. In this study, the basic epidemiological data of drug resistance of porcine Escherichia coli were obtained, and the molecular genetic mechanism of multidrug resistance transmission was discussed. To provide scientific basis for guiding clinical drug use and drug resistance monitoring in aquaculture.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5

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