【摘要】：犢牛腹瀉致病因素多,發(fā)病快,會造成犢牛生長發(fā)育不良、甚至死亡,犢牛腹瀉是危害犢牛生長發(fā)育過程最嚴重的疾病之一。其中,由于動物免疫力低下而致糞腸球菌的機會感染屢見不鮮,隨著耐萬古霉素糞腸球菌的出現(xiàn),其機會感染和特殊的耐藥機制引起了全世界的廣泛關(guān)注和重視。糞腸球菌為革蘭陽性球菌,是人和動物腸道內(nèi)的正常菌群之一,普遍散布于自然界中,通常不引起發(fā)病,為重要的條件病原菌。近幾年,有關(guān)糞腸球菌引發(fā)多鐘動物發(fā)病和死亡的報道逐漸增多。尤其伴隨著耐萬古霉素糞腸球菌的產(chǎn)生,使得糞腸球菌的治療變得更為棘手。為此,本研究以正常犢牛和腹瀉犢牛腸道中的糞腸球菌為研究對象,采取常規(guī)微生物學檢測方法結(jié)合16S rRNA分子生物學手段將分離株鑒定到種,并對其生理生化特性、培養(yǎng)特性、分子特性、耐藥性以及毒力基因進行研究,研究結(jié)果如下:(1)采用常規(guī)微生物學檢測方法結(jié)合16S rRNA分子生物學手段等試驗,從48份正常及腹瀉犢牛肛拭子中共分離到23株糞腸球菌,其中從新疆石河子市某規(guī)模化牛場犢牛腹瀉肛拭子中分離1株耐萬古霉素糞腸球菌,分離菌株的培養(yǎng)、生理及生化特性糞腸球菌的特征相同;通過與NCBI中其他已有腸球菌序列的同源性比對,結(jié)果表明與糞腸球菌NBRC100480及ATCC 19433的同源性高達100%。(2)根據(jù)CLSI推薦的K-B法檢測該菌對9種抗生素的耐藥表型,結(jié)果發(fā)現(xiàn)其對所檢測的8種抗生素(阿莫西林、環(huán)丙沙星、鏈霉素、紅霉素、氨芐西林、萬古霉素、四環(huán)素、青霉素)表現(xiàn)為耐藥,對呋喃妥因敏感,尤其是對于獸醫(yī)臨床上極少使用的萬古霉素也表現(xiàn)為耐藥。(3)3種耐萬古霉基因(vanA、vanB、vanC)的PCR檢測結(jié)果中只檢測到vanC基因,將PCR產(chǎn)物通過回收、純化、測序,序列結(jié)果和其在NCBI檢索基因序列比對同源性為100%。(4)5種毒力因子(聚集物質(zhì)Asa1、Asa373;膠原結(jié)合蛋白黏附素Ace;溶血素CylA;心內(nèi)膜炎抗原EfaA)的PCR檢測結(jié)果表明,該分離株只攜帶Ace毒力基因,該毒力基因主要在糞腸球菌早期感染中體現(xiàn)作用,說明分離株糞腸球菌可能為該犢牛早期感染的病原之一。研究結(jié)果揭示,本地區(qū)已經(jīng)存在耐萬古霉素糞腸球菌的感染,盡管分離率較低4.3%(1/23),但為了防止耐萬古霉素糞腸球菌通過動物源性食品向人類的傳播,應(yīng)加強該耐藥指示菌的監(jiān)測工作。
[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.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S858.23

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