宁夏地区牛源大肠杆菌优势血清型和毒力基因检测及耐药性分析

发布时间：2018-03-05 14:46

本文选题：大肠杆菌　切入点：耐药性　出处：《宁夏大学》2015年硕士论文　论文类型：学位论文

【摘要】：大肠杆菌(Escherichia coli, E. coli),兽医临床上最常见的病原菌之一,可引起人和家畜禽的多种疾病,如引起幼畜严重腹泻和败血症、猪水肿病、人的出血性结肠炎-溶血性尿毒综合征、新生儿脑膜炎及肾炎等多种疾病。随着养殖业的逐渐规模化,抗菌药物的过度使用,导致大肠杆菌的耐药性及其多重耐药性已严重影响了兽医临床疾病的治疗,因此对本地区奶牛乳房炎大肠杆菌进行毒力基因检测和耐药性分析及耐药性分子流行病学的调查,不但对大肠杆菌引起的疾病防治具有指导意义,而且有利于控制耐药菌株的产生和传播。本研究采用微生物学和分子生物学方法对宁夏地区部分奶牛场临床型及隐性乳房炎奶样,进行大肠杆菌分离鉴定,应用CLSI(2012)推荐的纸片扩散法(K-B)对其进行抗菌药物敏感性试验,采用O抗原平板凝集试验鉴定大肠杆菌优势血清型,运用PCR扩增方法对大肠杆菌毒力基因及耐药基因进行检测。1.2011-2013年采集宁夏地区部分奶牛场临床型及隐性乳房炎奶样,进行大肠杆菌的分离鉴定。通过观察大肠杆菌显色培养基上细菌菌落形态、革兰氏染色镜检和PCR扩增16S rDNA等方法进行大肠杆菌的鉴定,最终分离鉴定出197株大肠杆菌。2.根据有关文献,选取38种大肠杆菌O抗原血清,对197株奶牛乳房炎大肠杆菌进行O抗原血清鉴定。鉴定结果显示,共有138株大肠杆菌被鉴定出血清型,优势血清型有0127(35/138)0158(16/138)和044(12/138),有6株为自凝,53株未定型。3.对197株奶牛乳房炎大肠杆菌进行16种常用抗菌药物的敏感性试验,结果显示,所分离的大肠杆菌对氨苄西林耐药率最高,达到了54.69%,其次对多西环素(39.59%)、四环素(39.09%)和链霉素(38.58%)的耐药率也较高。而对头孢类、多粘菌素、氟苯尼考和喹诺酮类药物则比较敏感,敏感率都在60%以上。分离株最多耐受15种抗菌药物。4.采用PCR方法对197株奶牛乳房炎大肠杆菌进行了毒力基因和耐药基因的检测,结果显示,毒力基因astA、eaeA、hlyA、escV和sepA以及耐药基因tetB、tetC、GyrA、GyrB和ParC都被检出。其中astA检出率是21.13%,hlyA检出率最低,只有1.02%。四环素类的2种耐药基因tetC的阳性率为14.72%,tetB的阳性率是7.61%；氟喹诺酮类的GyrA、GyrB和ParC三种耐药基因检出率非常高,分别为97.46%、98.98%和98.48%。综上所述,本研究通过对宁夏地区奶牛乳房炎大肠杆菌血清型、毒力基因和耐药基因的检测及耐药性分析,初步揭示了本地区牛源大肠杆菌的耐药情况及毒力基因的流行情况,为指导抗菌药物在临床上的合理应用以及预防和控制耐药菌传播提供了科学依据。
[Abstract]:E. coli Escherichia coli, E. coli, one of the most common pathogens in veterinary practice, can cause many diseases in humans and domestic animals, such as severe diarrhea and septicemia in young animals, edema in pigs, hemorrhagic colitis and hemolytic uremic syndrome in human beings. Neonatal meningitis and nephritis and other diseases. With the gradual scale of aquaculture, the excessive use of antimicrobial agents, leading to the drug resistance of Escherichia coli and multidrug resistance has seriously affected the treatment of veterinary clinical diseases. Therefore, the detection of virulence gene, the analysis of drug resistance and the investigation of molecular epidemiology of drug resistance in dairy cow mastitis Escherichia coli in this area are not only of guiding significance for the prevention and treatment of diseases caused by E. coli. In this study, microbiological and molecular biological methods were used to isolate and identify clinical and recessive mastitis samples from some dairy farms in Ningxia. The antimicrobial susceptibility test was carried out by using the disk diffusion method (K-B-) recommended by CLSI (2012), and the predominant serotype of Escherichia coli was identified by O antigen plate agglutination test. The virulence genes and drug resistance genes of Escherichia coli were detected by PCR amplification. 1. Clinical and recessive mastitis samples were collected from dairy farms in Ningxia from 2011 to 2013. The colony morphology of Escherichia coli on color-forming medium was observed, microscopical examination of Gram stain and 16s rDNA amplification by PCR were used to identify Escherichia coli. Finally, 197 strains of Escherichia coli were isolated and identified. According to the related literature, 38 kinds of Escherichia coli O antigen serum were selected and 197 strains of cow mastitis Escherichia coli were identified by O antigen serum. A total of 138 strains of Escherichia coli were identified as serotypes. The predominant serotypes were 01277 / 35 / 138 / 015816 / 138) and 044 / 12 / 138C, and 6 strains were self-coagulant 53 strains of untyped .3.The sensitivity tests of 16 common antimicrobial agents against 197 strains of dairy cow mastitis Escherichia coli were carried out, and the results showed that, The highest resistance rate of Escherichia coli to ampicillin was 54.69, followed by doxycycline 39.59, tetracycline 39.09) and streptomycin 38.58), but sensitive to cephalosporins, polymyxin, florfenicol and quinolone. The sensitivity rate was above 60%. The isolates were resistant to 15 antimicrobial agents. The virulence genes and drug resistance genes were detected by PCR method. The virulence gene, astAeae, hly AescV and sepA, as well as the resistant gene tetBntetCnGyrB and ParC were detected, and the detection rate of astA was the lowest, and the detection rate of astA was the lowest. The positive rate of tetC was 14.72% for tetB and 7.61 for fluoroquinolones GyrAgyrB and ParC, respectively, which were 97.46% and 98.48%, respectively. In this study, the serotypes, virulence genes and drug resistance genes of E. coli from dairy cattle in Ningxia area were detected and drug resistance was analyzed, and the situation of drug resistance and the prevalence of virulence genes were preliminarily revealed. It provides a scientific basis for the rational application of antimicrobial agents in clinic and the prevention and control of the spread of drug resistant bacteria.
【学位授予单位】：宁夏大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.612

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