化脓隐秘杆菌β-内酰胺类抗生素耐药导致的PBPs氨基酸变化对毒力影响的研究

化脓隐秘杆菌是位于动物黏膜表面的一种革兰氏阳性菌，常引起多种动物如牛、羊、猪等多器官的化脓感染。近年来，化脓隐秘杆菌对抗菌药物特别是对β-内酰胺类药物耐药性日益严重，而该类药物耐药跟PBPs变化密切相关。毒力因子是细菌致病的关键因素，研究化脓隐秘杆菌PBPs变化对毒力的影响，对阐明化脓隐秘杆菌耐药机制具有重要意义。本研究利用化脓隐秘杆菌分离株，采用PCR方法扩增对β-内酰胺类抗生素耐药菌株的pbp基因，预测PBPs氨基酸变化情况；采用同源重组技术，构建化脓隐秘杆菌ATCC 19411-pUC-pbp菌株。分析重组菌株的PLO、NanH、NanP和CbpA等毒力因子的表达量变化；观察重组菌株细胞形态变化；测定菌株对HeLa细胞的黏附力和对小鼠的致病力，分析菌株的致病力变化情况，阐明化脓隐秘杆菌PBPs氨基酸变化对毒力的影响，为研究化脓隐秘杆菌β-内酰胺类抗生素耐药对毒力的影响奠定基础。

Trueperella pyogenes (T. pyogenes) is a Gram-positive bacteria locating on the surface of mucous membrane in the animal, and can cause a suppurative infection to many organs in many domestic animals, such as cow, sheep, swine and so on. Recently, this bacteria has become resistant to many kinds of antimicrobial agents especially β-lactams, and the studies indicated that the substitutions on PBPs were relevant with the emergence of β-lactams resistance in bacteria. Virulence plays an important role in infection caused by bacteria, therefore it is crucial for illustrating the mechanism of antibiotic resistance to study the influence of the substitutions of PBPs on virulence in T. pyogenes. In this study, PCR will be hired to amplify the pbp genes of T. pyogenes, and the substitutions of PBPs will be analyzed after sequence analysis of pbp genes. And the ATCC 19411-pUC-pbp mutants will be constructed according homologous recombination to identify the influence of the changes in sequence of amino acids in PBPs on virulence in T. pyogenes. The expression of virulences including PLO, NanH, NanP, and CbpA will be measured by western blot in mutants, and the cellular morphology will also be observed. Besides, in order to determine the pathogenicity of mutants, the adhesion to HeLa cells and pathogenicity to mice will be carried out. Finally, the research will illustrate the the influence of PBPs substitutions on virulence in T. pyogenes, which will be beneficial to study the effect of β-lactams resistance on virulence in T. pyogenes.