【作者】 陈雅莉；

【导师】 林居纯；

【作者基本信息】 四川农业大学 ， 基础兽医学， 2010， 硕士

【摘要】 伴随着抗菌药物在兽医临床的广泛使用,耐药菌株迅速出现,多重耐药现象十分普遍,已经引起全世界的广泛关注。做好耐药性监测,从而指导兽医临床合理用药已刻不容缓。对于细菌耐药性检测,传统的方法主要是进行耐药表型监测,随着分子生物技术的发展,PCR技术在耐药基因型的检测及新耐药机制的发现方面发挥着重要的作用。本研究首先从四川省不同养殖场收集食品动物源大肠杆菌病样本,按常规的细菌分离鉴定方法进行大肠杆菌菌株的鉴定。按照CLSI推荐方法及结果判断标准,应用微量肉汤稀释法测定细菌对于不同抗菌药物的最小抑菌浓度(MIC),从而对四川省食品动物源大肠杆菌的耐药情况进行监测。对于多重耐药菌株,采用多重PCR技术对其携带的3类整合子进行检测,同时对整合子阳性菌株进行基因盒分析。本次研究分2部分完成。1.不同食品动物源性大肠杆菌耐药性的监测从四川不同地区规模较大的养殖场收集食品动物源(鸡源、鸭源、牛源、猪源)大肠杆菌病样本,分离鉴定出705株大肠杆菌。采用微量肉汤稀释法进行了25种抗菌药物的药敏实验。结果显示：705株大肠杆菌对25种抗菌药物表现出程度不同的耐药性,特别是对兽医临床长期使用的甲氧苄啶、复方新诺明、利福平的耐药率接近100%；其次是对氨苄西林、四环素、氯霉素、氟喹诺酮类药物的耐药率均超过了70%；对头孢唑啉、头孢噻呋、头孢曲松也表现出一定的耐药性,耐药率分别为34.34%、41.30%、26.25%,但中介率较高,可见大肠杆菌对β-内酰胺类药物存在耐药率上升的趋势；本次监测发现所有大肠杆菌只有对阿米卡星保持有一定的敏感性,敏感率超过67%。多重耐药性分析显示,705株菌均为多重耐药菌株,主要表现出15-24耐,在各种耐药谱型中,23耐菌株所占比率最大,其次为21耐。不同动物源菌株耐药性比较显示,禽源菌株的耐药情况较猪源菌株,牛源菌株严重。不同地区来源菌株耐药性比较可见,不同地区来源菌株对所检测的25种药物耐药性分布大体一致,对部分药物表现出一定地域差异。2.食品动物源性大肠杆菌的整合子—基因盒检测采用多重PCR方法同时扩增327株大肠杆菌整合酶Ⅰ型、Ⅱ型和Ⅲ型基因。多重PCR检测结果显示,在32Ⅳ株菌株中,294株细菌携带有Ⅰ型整合酶基因,其检出率为89.91%,本次检测未发现携带有Ⅱ型和Ⅲ型整合酶基因的菌株。随机抽取Ⅰ型整合酶阳性菌株81株,进行基因盒扩增,扩增结果显示14株Ⅰ型整合酶阳性菌株未扩增出基因盒片段,其余67株扩增出845bp-2731bp的基因盒片段。测序结果显示,67株菌中含有6种不同类型的基因盒,即dfrA17+aadA5、dfrA17、aadA22、dhfrAI+ aadA2、dfrA17+orfF+aadA2、aacA4+catB3+dfrA1,编码甲氧苄啶-磺胺类药物、氨基糖苷类药物和氯霉素耐药。本次研究发现在2株鸡源大肠杆菌株中,发现了通过整合子-基因盒携带的一种介导氨基糖苷类药物耐药的新基因aadA22,这是首次在四川分离菌株中发现该基因的存在,其介导的耐药性有待进一步的研究。更多还原

【Abstract】 With the worldwide use of antimicrobial agents in veterinary medicine, the incidence of resistance and multidrug-resistance among pathogenic bacteria has been increasing, which attracts global attentions. Surveillance of antimicrobial resistance is very urgent to clinical medicine. The traditional method used in surveillance of resistance is to test resistant phenotype. With the development of molecular biological technology, PCR technology have been extensivly used in detection of resistant genotype and novel resistant mechanism.In this study, The samples of colibacillosis were collected from food animals in different farms in Sichuan province, E.coli isolates were identified by conventional test. Antimicrobial susceptibility of these isolates was examined using the broth microdilution method and the results interpreted according to Clinical and laboratory standards institute (CLSI). Multiplex PCR was used to detect 3 different classes of integron in multi-resistant strains, and PCR-sequencing method was used to analysis gene cassettes. The study was classified into two parts as follows.1. Surveillance of different antimicrobial resistance among E.coli isolates from food animals.The samples of colibacillosis were collected from food animals, including chickens, ducks, cattles and pigs, in different farms in Sichuan province,705 isolates were identified in the study. Antimicrobial susceptibility of these isolates to 25 different agents indicated: The resistance of 705 isolates to 25 agents were different, more than 100% of the 705 E.coli isolates were resistant to TMP, Trimethoprim-sulfamethoxazole and Rifampin respectively; Then, above 70% of isolates were resistance to Ampicillin,Tetracyline, Chloramphenicol and Fluoroquinolone,respectively. Isolates exhibited relatively low resistant level toβ-lactams, resistane rate of Cephazolin,Ceftiofur and Ceftriaxone, was 34.34%,41.30% and 26.25%, respectively. However, the medium rates of 3 kinds ofβ-lactams were very high, which indicated that the resistant changing trends was ascendant; All of isolates showed different susceptible level to Amikacin, susceptible rates exceeded 67%.Multidrug-resistance was very serious among isolates, All of 705 isolates exhibited multiple resistance to 15-24 antimicrobial agents. The rate of resistant strains to 23 agents was at most, then to 21 agents. Resistant rate of Avian strains was the most serious among different animal isolates; Although the resistant levels of the partial agents were different in isolates from different locals, however, resistant patterns among E.coli isolates from different geographical origins was similar.2. Detection of integron-gene cassettes among Escherichia coli isolates from food-animalsMultiplex PCR was used to detect 3 different classes of integron in 327 isolates. The results of multiplex PCR indicated 294 of 327 isolates harbored intI1 integrase genes, the detection rate of intI1 was 89.91%, intI2 and intI3 genes were not discoveried in the study. Amplification of Gene cassettes in 81 intI1-positive strains choosed randomly was carried out. The results showed that amplicons of 845bp-2731bp were yielded in 67 intI1-positive strains. The sequencing of gene cassettes indicated that 6 kinds of gene cassettes were harbored in 67 strains. The gene cassettes arrays were dfrA17+aadA5,dfrA17, aadA22, dhfrAI+aadA2, dfrA17+orfF+aadA2 and aacA4+catB3+dfrA1, which were encoding resistant to TMP+Sulfonamides, Aminoglycosides and chloramphenicol, respectively. aadA22, which was novel gene encoding resistant to Aminoglycosides, was first acquired in avian E.coli strains from Sichuan province. The study of aadA22 gene will be carried out in future.更多还原