發(fā)布時(shí)間：2018-02-21 11:43

  本文關(guān)鍵詞： 豬 大腸桿菌 耐藥性 水平轉(zhuǎn)移　出處：《吉林農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：伴隨著現(xiàn)代化養(yǎng)殖模式的興起,在畜牧生產(chǎn)中抗生素的合理使用越來(lái)越受到重視�？股乜捎糜谥委熀皖A(yù)防疾病、提高飼料轉(zhuǎn)化率和促進(jìn)動(dòng)物生長(zhǎng)。盡管抗生素在實(shí)踐應(yīng)用中取得了良好的效果,但是多重耐藥菌也隨之出現(xiàn)并給感染治療帶來(lái)更多困難。大腸桿菌是重要的腸道共生菌,對(duì)維持腸道的平衡起到了重要作用。在養(yǎng)豬業(yè)中抗生素濫用情況嚴(yán)重,這使得豬源大腸桿菌耐藥形勢(shì)日趨嚴(yán)峻。豬源大腸桿菌已成為重要的耐藥細(xì)菌貯存庫(kù),它極有可能通過(guò)環(huán)境污染、直接接觸或食物鏈將耐藥菌傳染給人類,因此檢測(cè)豬源大腸桿菌的耐藥性并探討其公共衛(wèi)生意義十分必要。本研究于2013年在吉林省采集318份豬源樣品,分別來(lái)源于臨床健康豬、腹瀉仔豬以及市售豬肉,共分離鑒定大腸桿菌275株。以氨芐西林、頭孢噻肟等15種藥物進(jìn)行了藥物敏感性實(shí)驗(yàn),并以多重PCR方法進(jìn)行系統(tǒng)進(jìn)化分群。結(jié)果表明,健康豬樣品大腸桿菌分離株主要分屬A群和B1群,為非致病群;腹瀉仔豬大腸桿菌分離株B2和D群的比例顯著高于健康豬大腸桿菌。綜合分析,大腸桿菌分離株對(duì)四環(huán)素、氨芐西林和磺胺甲基異惡唑耐藥最嚴(yán)重,其中176株菌表現(xiàn)為對(duì)3類以上抗生素的多重耐藥,全部菌株對(duì)美洛培南、多粘菌素敏感。從仔豬腹瀉樣品分離的大腸桿菌對(duì)β-內(nèi)酰胺類抗生素、喹諾酮類抗生素、四環(huán)素、氯霉素及磺胺甲基異惡唑的耐藥率顯著高于健康豬和豬肉樣品分離株的耐藥率。針對(duì)具有較高耐藥率的豬源大腸桿菌檢測(cè)其耐藥基因,探討其分子遺傳基礎(chǔ)以及分子流行病學(xué)規(guī)律。采用PCR方法檢測(cè)CTX-M、TEM、SHV等3種ESBLs的基因和tet(A)、tet(B)、tet(C)、tet(D)、te t(M)、t et(W)等6種四環(huán)素耐藥基因。結(jié)果顯示275株大腸桿菌中,116株檢出TEM-1基因,該基因型為主要流行基因型;tet(A)、tet(B)、tet(C)、tet(M)耐藥基因的檢出率分別為64%、22%、1%、5%,t et(A)/tet(B)為主要流行基因型。采用質(zhì)粒接合轉(zhuǎn)導(dǎo)實(shí)驗(yàn)探究了質(zhì)粒在多重耐藥大腸桿菌耐藥性水平傳播中的作用。隨機(jī)選取50株豬源多重耐藥大腸桿菌為供體菌,用氨芐西林,四環(huán)素等12種抗生素對(duì)接合子進(jìn)行藥敏試驗(yàn),多重PCR方法進(jìn)行質(zhì)粒分型。結(jié)果顯示,50株供體菌中,18株接合轉(zhuǎn)導(dǎo)成功,接合轉(zhuǎn)導(dǎo)率達(dá)36%。與接合子耐藥譜分析結(jié)果表明,存在多種耐藥性共轉(zhuǎn)移的現(xiàn)象。18株接合子獲得質(zhì)粒復(fù)制子分型以FIB為主。研究表明,接合轉(zhuǎn)導(dǎo)型耐藥質(zhì)粒在豬源細(xì)菌耐藥性傳播中起到重要作用。本研究獲得了豬源大腸桿菌耐藥性的基本流行病學(xué)數(shù)據(jù),探討了多重耐藥性水平傳播的分子遺傳學(xué)機(jī)制,為指導(dǎo)養(yǎng)殖業(yè)的臨床用藥及耐藥性監(jiān)測(cè)提供科學(xué)依據(jù)。
[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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R446.5

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