【摘要】 目的 利用浓度梯度倍增的氯法齐明(clofazimine,Cfz)体外诱导获得结核分枝杆菌(Mycobacterium tuberculosis,Mtb)耐药株,并进行耐药机制的初步研究。方法 选取Mtb标准株H37Rv,从亚最低抑菌浓度(subminimal inhibitory concentration,sub-MIC)E1/8 MIC(按照Cfz的MIC值为0.12μg/ml配置)、1/4 MIC、1/2 MIC…]开始,在Cfz浓度倍增的7H11固体培养基上进行传代培养,逐步诱导菌株对Cfz的耐药性,并通过药物敏感性试验和全基因组测序来进行验证;同时利用Alamar blue微板分析法(microplate Alamar blue assay,MABA)测定诱导耐药菌株对10种抗结核药物(INH、RFP、EMB、Mfx、Lzd、Cs、Cm、TMC207、TBI-166、Cfz)的MIC情况。结果 Mtb标准株经诱导后对Cfz的MIC值是诱导前的32~64倍(诱导前Cfz的MIC值是0.12μg/ml)。对10种抗结核药物敏感性试验显示,Cfz诱导耐药菌株对TMC207(Bendaquiline)及TBI-166产生交叉耐药。耐药株在7H9液体培养基中进行无药连续培养10代后,测定其MIC值无明显变化,MIC数值都稳定在32 MIC~64 MIC之间,对此诱导耐药株进行全基因组测序共检测到195个单核苷酸多态性(single nucleotide polymorphisma,SNP)突变和19个插入缺失(InDel)突变。结论 从亚MIC药物浓度开始,逐渐递增的长期药物刺激能够诱导结核分枝杆菌对氯法齐明的获得性耐药,获得的耐药株耐药性稳定。更多还原

【Abstract】 Objective To induct Mycobacterium tuberculosis(Mtb) resistant strains in vitro by using gradient multiplied concentration of clofazimine(Cfz) and study the preliminary mechanisms of resistance.Methods We subcultured Mtb standard strains H37 Rv to 7H11 solid medium with doubled Cfz concentrations from the beginning of sub-MIC concentrations(1/8 MIC,1/4 MIC,1/2 MIC...) and induced Cfz-resistance gradually.Than we tested the resistance through drug susceptibility testing and whole genome sequencing.Meanwhile,We measured the MICs of the induced resistant strains to ten anti-TB drugs(INH,RFP,EMB,Mfx,Lzd,Cs,Cm,TMC207,TBI-166,Cfz).Results MIC values of the Mtb standard strain after induction were 32 to 64 times higher than before(0.12 μg/ml),The drug susceptibility testing also showed that Cfz had a cross-resistance to TMC207(Bendaquiline) and TBI-166;MIC values of the Mtb resistent strain was stable among 32 MIC to 64 MIC after continuously cultivating 10 generations in drug-free 7H9 liquid medium.We also detected 195 single nucleotide polymorphism(SNP) mutations and 19 insertion deletion(InDel) mutations in this resistant strain by whole genome sequencing.Conclusion Cfz resistent strain can be obtained by the long-term drug-induced stimulation from the beginning of sub-MIC concentrations to gradually increasing the concentrations.The resistent strain had a stable characteristic.更多还原