致犊牛腹泻耐万古霉素粪肠球菌的分离鉴定及部分生物学特性研究

发布时间：2018-09-09 09:32

【摘要】：犊牛腹泻致病因素多,发病快,会造成犊牛生长发育不良、甚至死亡,犊牛腹泻是危害犊牛生长发育过程最严重的疾病之一。其中,由于动物免疫力低下而致粪肠球菌的机会感染屡见不鲜,随着耐万古霉素粪肠球菌的出现,其机会感染和特殊的耐药机制引起了全世界的广泛关注和重视。粪肠球菌为革兰阳性球菌,是人和动物肠道内的正常菌群之一,普遍散布于自然界中,通常不引起发病,为重要的条件病原菌。近几年,有关粪肠球菌引发多钟动物发病和死亡的报道逐渐增多。尤其伴随着耐万古霉素粪肠球菌的产生,使得粪肠球菌的治疗变得更为棘手。为此,本研究以正常犊牛和腹泻犊牛肠道中的粪肠球菌为研究对象,采取常规微生物学检测方法结合16S rRNA分子生物学手段将分离株鉴定到种,并对其生理生化特性、培养特性、分子特性、耐药性以及毒力基因进行研究,研究结果如下:(1)采用常规微生物学检测方法结合16S rRNA分子生物学手段等试验,从48份正常及腹泻犊牛肛拭子中共分离到23株粪肠球菌,其中从新疆石河子市某规模化牛场犊牛腹泻肛拭子中分离1株耐万古霉素粪肠球菌,分离菌株的培养、生理及生化特性粪肠球菌的特征相同;通过与NCBI中其他已有肠球菌序列的同源性比对,结果表明与粪肠球菌NBRC100480及ATCC 19433的同源性高达100%。(2)根据CLSI推荐的K-B法检测该菌对9种抗生素的耐药表型,结果发现其对所检测的8种抗生素(阿莫西林、环丙沙星、链霉素、红霉素、氨苄西林、万古霉素、四环素、青霉素)表现为耐药,对呋喃妥因敏感,尤其是对于兽医临床上极少使用的万古霉素也表现为耐药。(3)3种耐万古霉基因(vanA、vanB、vanC)的PCR检测结果中只检测到vanC基因,将PCR产物通过回收、纯化、测序,序列结果和其在NCBI检索基因序列比对同源性为100%。(4)5种毒力因子(聚集物质Asa1、Asa373;胶原结合蛋白黏附素Ace;溶血素CylA;心内膜炎抗原EfaA)的PCR检测结果表明,该分离株只携带Ace毒力基因,该毒力基因主要在粪肠球菌早期感染中体现作用,说明分离株粪肠球菌可能为该犊牛早期感染的病原之一。研究结果揭示,本地区已经存在耐万古霉素粪肠球菌的感染,尽管分离率较低4.3%(1/23),但为了防止耐万古霉素粪肠球菌通过动物源性食品向人类的传播,应加强该耐药指示菌的监测工作。
[Abstract]:Calf diarrhea is one of the most serious diseases affecting the growth and development of calves. Among them, the opportunistic infection of Enterococcus faecalis caused by low animal immunity is common. With the emergence of vancomycin resistant Enterococcus faecalis, its opportunistic infection and special drug resistance mechanism have attracted worldwide attention. Enterococcus faecalis is a gram-positive cocci. It is one of the normal flora in the intestinal tract of human and animal. It is widely distributed in nature and usually does not cause disease and is an important conditional pathogen. In recent years, there have been more and more reports of Enterococcus faecalis causing many animal diseases and deaths. With the production of vancomycin resistant Enterococcus faecalis, the treatment of Enterococcus faecalis becomes more difficult. Therefore, in this study, Enterococcus faecalis in the intestines of normal calves and diarrhea calves were studied. The isolated strains were identified by conventional microbiological detection method combined with 16s rRNA molecular biology, and their physiological and biochemical characteristics and culture characteristics were analyzed. Molecular characteristics, drug resistance and virulence genes were studied. The results are as follows: (1) conventional microbiological methods combined with 16s rRNA molecular biological methods were used. A total of 23 strains of Enterococcus faecalis were isolated from anal swab of 48 normal and diarrhea calves. One strain of Enterococcus faecalis resistant to vancomycin was isolated from a large scale calf in Shihezi city of Xinjiang. The physiological and biochemical characteristics of Enterococcus faecalis were the same. The results showed that the homology with Enterococcus faecalis NBRC100480 and ATCC 19433 was 100. (2) according to the K-B method recommended by CLSI, the resistance phenotypes of the bacteria to 9 kinds of antibiotics were detected, and the results showed that it was resistant to 8 kinds of antibiotics (amoxicillin, ciprofloxacin, streptomycin). Erythromycin, ampicillin, vancomycin, tetracycline, penicillin) are resistant and sensitive to furantoin. In particular, vancomycin, which is rarely used in veterinary clinic, also shows drug resistance. (3) only vanC gene was detected in the PCR detection results of three vancomyces resistant genes (vanA,vanB,vanC), and the PCR products were recovered, purified and sequenced. (4) PCR detection of five virulence factors (Asa1,Asa373; collagen-binding protein adhesin Ace; hemolysin CylA; endocarditis antigen EfaA) showed that the isolated strain only carried Ace virulence gene. The virulence gene mainly plays an important role in the early infection of Enterococcus faecalis, indicating that the isolated strain of Enterococcus faecalis may be one of the pathogens of early infection of the calf. The results revealed that vancomycin resistant Enterococcus faecalis had been infected in the region, although the isolation rate was 4.3% (1 / 23), but in order to prevent the transmission of vancomycin resistant Enterococcus faecalis to humans through animal-derived food, The monitoring of the drug resistance indicator bacteria should be strengthened.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.23

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