外排蛋白介导的化脓隐秘杆菌多重耐药机制研究

Information indicated that drug resistance of A. pyogenes presented a critical problem, in particular the multidrug resistance has got increasing concern recently. However, there is no study carried out to investigate the mechanism of multidrug resistance of A. pyogene. In this study, we aimed to investigate the multidrug resistant mechanism of A.pyogenes mediated by efflux proteins. Initially, the genome DNA sequence of A.pyogenes will be measured to explore gene annotation and its function. In order to investigate the correlation between existence of efflux protein gene and multidrug resistance phenotype, the efflux protein genes which code NorA, MsrA, MefA, Bmr, P55, TetK and TetL from multidrug resistant A.pyogenes were amplified by PCR respectively. Besides, the correlations of mRNA expression levels of efflux proteins and multidrug resistance, the relative expression levels of efflux proteins and multidrug resistance were studied by prokaryotic expression and fluorescent quantitative PCR after the gene sequences of efflux proteins were measured to elucidate the functions of these efflux proteins and their expression-regulation mechanisms by bioinformatics method. Finally, wild type A pyogenes, the efflux gene been knocked out, will be obtained by DNA recombination technology, which can be confirmed by dot blot hybridization; and the multidrug resistant variations of A. pyogenes and A.pyogenes with efflux proteins deletion will be studied to find out the functions of efflux proteins in multidrug resistance A.pyogenes. This study will provide theoretical foundation in discovery and development of novel antibiotics, and it is also meaningful for preventing the emergence and dissemination of bacterial drug resistance and curing the A pyogenes infections.

国内外资料显示化脓隐秘杆菌耐药现象越来越严重，尤其是多重耐药现象已受到众多学者的关注，但尚未见有关其多重耐药机制的报道。本研究以多重耐药化脓隐秘杆菌为研究对象，采用PCR技术扩增编码外排蛋白NorA、MsrA、MefA、Bmr、P55、TetK和TetL的基因，结合基因组测序结果，利用生物信息学方法预测外排蛋白功能，并分析其表达调控机制。构建外排基因原核表达质粒，经Western-blotting和SDS-PAGE分析其相对表达量与耐药表型关系；采用荧光定量RT-PCR检测外排基因mRNA表达量，揭示其mRNA表达水平与多重耐药表型相关性。采用DNA重组技术构建化脓隐秘杆菌外排基因敲除菌株，通过斑点杂交证实，并测定基因敲出前后菌株耐药表型的变化，探明外排基因在化脓隐秘杆菌多重耐药中的作用。本研究较系统的分析和揭示外排蛋白介导化脓隐秘杆菌多重耐药机制，将为研发抗耐药菌新药奠定坚实的理论基础。

化脓隐秘杆菌可引起家畜、经济动物和人类的多种化脓性感染，如子宫内膜炎、乳房炎、肺炎、脓肿等，给人类及动物的健康带来极大的危害。化脓隐秘杆菌感染的防治主要依靠应用抗菌药物，临床常用的抗菌药物包括β-内酰胺类、大环内酯类、四环素类、氟喹诺酮类等。但随着抗菌药物的广泛使用，化脓隐秘杆菌的耐药性问题尤其是多重耐药现象越来越严重。深入研究化脓隐秘杆菌的耐药性产生机制，尤其是外排蛋白介导的多重耐药机制，可望对抗耐药菌新药的研发及科学防治化脓隐秘杆菌引起的感染性疾病具有重要意义。. 本项目按计划完成化脓隐秘杆菌基因组测序、基因注释和功能预测，完成外排蛋白介导化脓隐秘杆菌对大环内酯类药物多重耐药机制研究，初步完成外排蛋白介导化脓隐秘杆菌对四环素类药物多重耐药机制的研究。发表学术论文8篇，其中SCI收录2篇；指导青年教师1人，培养硕士研究生4名。. 经全体人员的团结协作，本项目基本完成了预期的研究计划，取得如下成果：测得了化脓隐秘杆菌的基因组DNA序列，完成了主要基因的功能注释。化脓隐秘杆菌的基因组为一条长2,152,080 bp的环形染色体，GC含量为59.49%，有1467个（73%）基因具有明确的生物学功能。基因注释功能发现了一些耐药相关的基因，包括多重药物转运蛋白编码基因、四环素类耐药相关基因、β-内酰胺类耐药相关基因、氨基糖苷类耐药相关基因、大环内酯类耐药相关基因、万古霉素耐药因子等。编码化脓隐秘杆菌外排蛋白的基因携带情况与其多重耐药性具有相关性。外排蛋白MsrA、MefA 蛋白可介导化脓隐秘杆菌对大环内酯类多重耐药，外排蛋白TetK、TetL可介导化脓隐秘杆菌对四环素类药物的多重耐药；MATE蛋白可介导化脓隐秘杆菌对β-内酰胺类、氨基糖苷类药物的多重耐药。本研究初步揭示了外排蛋白介导的化脓隐秘杆菌多重耐药机制，为新型抗耐药菌药物的研发提供了新的思路。