兔波氏杆菌的分离鉴定及耐药性研究

发布时间：2018-04-16 04:26

本文选题：波氏杆菌 + 分离鉴定　；参考：《山东农业大学》2015年硕士论文

【摘要】：兔波氏杆菌病是由支气管败血波氏杆菌引发的一种常见兔呼吸道疾病,主要引起成年兔的支气管炎、鼻炎和脓疱性肺炎,断乳仔兔和哺乳仔兔的急性死亡等。近年来,伴随着我国规模化兔业养殖的迅速发展,支气管败血波氏杆菌的感染率明显升高,并常与葡萄球菌、多杀性巴氏杆菌等混合感染,已成为导致家兔发病率和死亡率增加的重要原因之一,给养兔业造成重大经济损失。抗生素治疗在畜禽养殖中是一种重要的防制手段,但由于药物滥用,细菌耐药性日趋严重,常造成药物治疗失败。所以增加对当地细菌耐药性及药物敏感性状况的认知,对于正确分析耐药性的发展趋势及对临床上指导合理用药意义重大。针对当前严重的多重耐药现象,探究耐药机制,针对耐药原因找出对应方法来延长已有抗生素的敏感期和研究新药同为重要。本实验在分离鉴定细菌基础上,对药物敏感性进行了检测,并对耐药基因进行了鉴定,对耐药传播机制进行了初步研究。研究主要包括以下内容:1.本实验2012~2014年自浙江省部分兔场采集病兔的鼻腔分泌物,根据细菌形态、培养特性、生化试验,并结合PCR方法对分离菌株进行鉴定,分离鉴定出22株兔波氏杆菌;K-B氏纸片法检测细菌对18种常用抗生素的药物敏感性,药敏结果显示,分离菌多重耐药严重,对青霉素G、头孢拉定、链霉素、林可霉素、克林霉素、甲氧嘧啶耐药严重,而对多黏菌素B、左氟沙星、强力霉素、四环素等药物敏感。2.针对耐药表型对应的耐药基因,设计特异性引物,使用PCR法对22株兔波氏杆菌分离株进行耐药基因检测,并进行测序分析。其中所有分离株均检测到β-内酰胺类耐药基因blaTEM,检测率为100%;17株检测到磺胺类药物耐药基因sul1,检测率为77%;分离菌株检测到的这两种耐药基因与耐药表型相符。17株检测到Ⅰ类整合子的整合酶基因int1,检测率为77%,说明整合子在兔波氏杆菌耐药传播机制中发挥作用。20株检测到多重耐药基因marC,检测率为91%,因marC基因检出率较高,推测波氏杆菌耐药性与染色体基因突变介导的主动外排系统有关。9株检测到转座子遗传标志基因merA,检测率为41%,merA基因的存在及反向PCR结果显示TEM耐药基因的传播转座子存在相关联系。3.对本研究所分离的22株兔波氏杆菌进行了质粒提取及消除实验,结果表明,所有22株分离菌均携带质粒,质粒消除后细菌部分耐药性状发生变化,对氨苄青霉素、头孢唑林、四环素、多粘菌素B等药物的敏感性增强。证明了质粒介导了兔波氏杆菌分离菌株的部分耐药性。本研究结果表明,兔波氏杆菌是引起兔呼吸道疾病的主要病原菌;分离菌耐药率较高,并呈现多重耐药;检测到的耐药基因与耐药表型相符;质粒、整合子、转座子这三种转移因子的检出说明兔波氏杆菌存在多种耐药传播机制。
[Abstract]:Bordetella rabbit disease is a common respiratory tract disease caused by Bordetella bronchus. It mainly causes bronchitis, rhinitis and pustular pneumonia in adult rabbits, acute death of weaning rabbits and lactation rabbits, and so on.In recent years, with the rapid development of large-scale rabbit farming in China, the infection rate of Bordetella bronchus has increased significantly, and it is often mixed with Staphylococcus, Pasteurella multocida and so on.It has become one of the important reasons for the increase of morbidity and mortality of rabbits, and has caused great economic losses to rabbit farming.Antibiotic therapy is an important means of prevention and control in livestock and poultry breeding. However, drug abuse and drug resistance of bacteria are becoming more and more serious, which often lead to drug treatment failure.Therefore, it is of great significance for correctly analyzing the development trend of drug resistance and guiding rational drug use in clinic to increase the understanding of drug resistance and drug sensitivity of local bacteria.In view of the serious phenomenon of multidrug resistance at present, it is important to explore the mechanism of drug resistance, to find corresponding methods to prolong the sensitive period of existing antibiotics and to study new drugs.On the basis of isolation and identification of bacteria, the drug sensitivity was detected, the resistance gene was identified, and the mechanism of drug resistance transmission was studied.The research mainly includes the following contents: 1: 1.The nasal secretions of infected rabbits were collected from some rabbit farms in Zhejiang Province from 2012 to 2014. The isolated strains were identified by bacterial morphology, culture characteristics, biochemical tests and PCR method.A total of 22 strains of Bacillus vulgaris were isolated and identified by K-B disk method to detect the susceptibility of bacteria to 18 common antibiotics. The results of drug sensitivity showed that the isolates were multidrug resistant to penicillin G, cefradine, streptomycin, lincomycin, clindamycin, lincomycin, clindamycin, penicillin, cefradine, streptomycin and clindamycin.Methoxypyrimidine is highly resistant to polymyxin B, levofloxacin, doxycycline, tetracycline and so on.A specific primer was designed to detect the drug resistance genes of 22 isolates of Boxerobacterium rabbit by PCR method, and the results were analyzed by sequencing.Among them, 尾 -lactam resistance gene blaTEM was detected in all isolates, the detection rate was 100% and 17 strains detected sulfan resistance gene sul1, the detection rate was 770.The two resistant genes detected by the isolated strains were consistent with the phenotype of drug resistance.The integrase gene int1 of class I integron was detected, and the detection rate was 77. It indicated that integron played an important role in the transmission mechanism of drug resistance of Bacillus pipii. 20 strains of integron detected multidrug resistance gene marC.The detection rate was 91%, because the detection rate of marC gene was high.It is speculated that resistance of Bacillus bovis is related to the active efflux system mediated by chromosome mutation. A transposon genetic marker gene merA has been detected in 9 strains. The detection rate is 41% and the result of reverse PCR shows that the transmission of TEM resistance gene is transmissible.There is a correlation between the transposons.The plasmid extraction and elimination experiments were carried out on 22 strains of Bordetella rabbit isolated in this study. The results showed that all the 22 isolates carried plasmids. After the elimination of plasmids, some drug resistance characters of bacteria were changed, and ampicillin and cefazolin were detected.The sensitivity of tetracycline and polymyxin B was enhanced.It was proved that plasmid mediated partial drug resistance of the isolated strains.The results showed that Bacillus vulgaris was the main pathogen causing respiratory tract diseases in rabbits. The drug resistance rate of isolates was high and showed multidrug resistance. The detected resistant genes were consistent with the phenotype of drug resistance; plasmids, integrons, plasmids, integrons, plasmids, integrons, plasmids, integrons, plasmids, integrons,The detection of transposon these three transfer factors showed that there were many mechanisms of drug resistance transmission in B. rabbit.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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