新疆牛羊源布鲁氏菌耐药基因流行特点及传播分子机制研究

Brucellosis is a zoonotic infectious disease caused by brucella. The sickened animal is a main source of infection .Xinjiang is the old epidemic of brucellosis。 RecentLY, the epdemic situation of brucellosis is rough increasing in Xinjiang. Antibiotic therapy is the first choice to treat the Brucella-infected patients. It is a big problem for antibiotic resistance during the treatment of infectious diseases. Therefore, how to choose antimicrobial agents for the effective treatment of brucellosis is a problem which is required to solve immediately. This project aims (1) to investigate the resistance information of brucella,which will be collected from cattle and sheep in Xinjiang, to common antibiotics, rifampcin,tetracyclines,macrocyclic lactones,aminoglycosides by the E-test method; (2) to detect the resistance genes of resistant strains to the four antibiotics;Analysis of the resistance diffence between the Br.abortus and Br.melitensis (3) to determine the genetic relationship of positive strains by PFGE; and (4) to study the molecular mechanisms by which resistance is transferred by southern hybridization, analysis of plasmid transferability and incompatibility, mapping and analysis of genetic elements which mediate the transfer of resistance genes. This project may reveal the epidemic dissemination mechanisms of resistance genes.It provide scientific theory basis for brucellosis treatment in xinjiang. The results suggested that it is important for prevention and control of brucellosis in the Xinjiang.

布鲁氏菌病是由布鲁氏菌引起的人畜共患性传染病。患病动物是主要传染源。新疆是布病老疫区，近几年，新疆布病发生率呈逐年急剧上升趋势。目前人布病治疗以抗生素为根本治疗，但一些用药策略的不合理现象导致布鲁氏菌对抗菌素出现了耐药性，给布病防控带来很大困难。本研究采用E-test法获取了新疆分离的牛羊源布鲁氏菌进行药物敏感性实验，获取菌株对常用抗生素的耐药信息，对利福平、大环内酯类、氨基糖苷类、四环素类耐药基因检测，分析新疆牛种布鲁氏菌及羊种布鲁氏菌种间的耐药性差异；应用PFGE 技术研究阳性菌株的亲缘关系，采用southern杂交定位、质粒可转移性分析、介导耐药基因转移的遗传元件结构图绘制及解析来探明其在布鲁氏菌中传播的分子机制。上述研究能揭示多类耐药基因的流行传播机制，以此为新疆布病治疗的给药方案的制定提供科学理论依据，对完善新疆地区布病预防控制体系及技术规范具有重要应用价值。

布鲁氏菌病是由布鲁氏菌引起的人畜共患性传染病。患病动物是主要传染源。新疆是布病老疫区，近几年，新疆布病发生率呈逐年急剧上升趋势。目前人布病治疗以抗生素为根本治疗 ，但一些用药策略的不合理现象导致布鲁氏菌对抗菌素出现了耐药性，给布病防控带来很大困难。应用AMOS-PCR法和MLVA-16方法对新疆牛羊源布鲁菌进行分子分型，基本摸清新疆地区牛羊布病流行病学特征，即新疆家畜布病以羊种3型为优势基因型，牛种3型基因型次之，而且布鲁菌显示出丰富的基因多态性现象。.应用K-B法对氨基糖苷类、大环内酯类、磺胺类、四环素类、β-内酰胺类、氟喹诺酮类、氯霉素类和利福霉素类8大类抗生素的19种药物对新疆地区分离的牛羊源布鲁菌进行药物敏感试验。结果表明新疆地区牛羊源布鲁菌对四环素类、氨基糖苷类、β-内酰胺类、氟喹诺酮类及氯霉素类药物较敏感,而对阿奇霉素、克拉霉素、复方新诺明耐药性较高.应用MIC法筛选获得耐利福平布鲁菌4株（≥4.0mg/L），耐复方新诺明耐药株17株（≥2/38mg/L）。新疆牛羊源布鲁氏菌耐复方新诺明耐药基因型主要是由sul1、sul2、dfrA18和intI基因所介导，而且整体上以sul2和dfrA18基因为主。耐利福平布鲁菌耐药机制主要由编码rpoB基因突变而导致耐药。本研究中耐利福平布鲁菌株的rpoB基因共7处密码子突变，其中526位密码子与536位密码子突变直接与利福平耐药性相关。Glu-846-Leu、Arg-876-缺失、His-879-Pro、Arg-885-Pro突变也导致利福平耐药。.以新疆家畜布病流行病学调查数据为基础，分别建立了牛布病A19疫苗与自然感染布鲁菌的多重PCR鉴别诊断技术；建立了鉴别区分牛布病A19-ΔVirB12疫苗与自然感染抗体的血清学诊断方法；建立了鉴定Rev.1疫苗与羊种布鲁菌的PCR-RFLP 方法，为动物布病的检疫和防控提供了技术支撑，为新疆制定布鲁氏菌病的综合防制策略奠定了基础。