耐药结核分枝杆菌感染动物模型所用菌株的筛选

Screening of drug-resistant Mycobacterium tuberculosis isolates for candidate standard strains for in vivo assays

本研究通过小鼠体内实验检测我国部分地区结核分枝杆菌耐药菌株的毒力,以筛选耐药结核分枝杆菌感染动物模型所用菌株。收集从我国部分省份98例结核病患者痰培养液中分离出的结核分枝杆菌,用比例法药敏试验进行结核分枝杆菌一线和二线药物的药敏试验,筛选出对二线药物敏感而对一线药物利福平或异烟肼耐药或敏感的菌株,然后进行小鼠体内毒力实验,对异烟肼耐药相关基因katG和利福平耐药相关基因rpoB测序并进行基因突变分析。从98株菌中筛选出药物敏感谱清晰的40株,进行小鼠体内毒力实验。结果显示,共35株半数死亡时间≤H37Rv的半数死亡时间,其中18株耐利福平合并耐异烟肼、5株单耐利福平,7株单耐异烟肼、5株对利福平和异烟肼均敏感。通过小鼠毒力研究,分别筛选出基因背景清晰,半数死亡时间≤7d的耐利福平合并耐异烟肼的菌株1株、半数死亡时间≤7d单耐利福平和异烟肼的菌株各1株,作为耐药结核分枝杆菌感染小鼠模型所用菌株及进一步进行豚鼠等其他动物模型感染用候选菌株。

To establish a panel of standard clinic strains as representative drug-resistant Mycobacterium tuberculosis （M . tuberculosis） ,the strains collected from tuberculosis patients in several provinces of China were subjected to this study .The drug susceptibility test with first-line and second-line anti-tuberculosis drugs and drug-resistant gene mutant analysis were performed in 98 clinic isolations .The strains with explicit drug resistant phenotype and mutant were selected for in vivo virulence experiment in mice .Among the 40 strains with explicit drug resistant patterns ,the half lethal time （LT50 ） of 35 strains were shorter than that of H37Rv in the virulence experiment in mice .18 in 35 strains were rifampin-resistant and isoniazid-resistant . 5 strains were rifampin-resistant ,7 strains were isoniazid-resistant , and 5 strains were sensitive to both isoniazid and rifampin .Standard strain with moderate virulence and explicit genotype was selected for each drug resistant phenotype .For rifampin-resistance combined with isoniazid-resistance ,one strain with LT50 ≤ 7 d was selected for its strong virulence .For rifampin-resistance or isoniazid-resistance ,a similar selection standard was applied .The selected panel of standard strains can be used as candidate standard strains used in M . tuberculosis infection model of guinea pig or other animals .