A Preliminary Study On The Mechanism Of Phenazine Compounds

Objective To induce mycobacterium tuberculosis(Mtb) resistant strains to clofazimine in vitro, and to preliminary study its mechanisms of resistance. Methods H37 Rv strain was successive subcultured on 7H11 solid medium from containing the minimal concentration(1/8MIC) to the maximal concentration(64MIC) of clofazimine(1/8 MIC, 1/4 MIC, 1/2 MIC, ……, 64 MIC) and then valided with DST and whole genome sequencing. Meanwhile MIC of the induced strain to 10 antituberuclousis drugs(INH、RFP、EMB、Mfx、LZD、Cs、Cm、Bdq、TBI-166、Cfz) was detected with Microplate Alamar Blue Assay(MABA). Results MIC values of the Mtb standard strain after induction were 32 to 64 times higher than before(0.12μg/ml). The cross-resistance to Bendaquiline(Bdq, TMC207) and TBI-166 were confirmed with the result of DST for the mentioned 10 anti-TB drugs. The MIC value of the induced resistant strain was no significant change after sucssessive culture in drug-free 7H9 liquid medium for 10 generations. 195 single nucleotide polymorphism(SNP) and 19 insertion deletion(In Del) mutations were detected from the resistant strain by whole genome sequencing. Conclusions The resistant strain of Cfz is acquired using successive subcultured on medium from containing the minimal concentration(1/8 MIC) to the maximal concentration(64 MIC) of clofazimine step by step, and the strain resistance is stable. The mechanism of resistance to Cfz may be related with efflux pump regulating, respiratory and metabolic process regulating.Objective To explore mechanism of anti-TB on TBI-166 according to the change of gene expression detected before and after treatment with TBI-166 in H37 Rv strain.Methods H37 Rv standard strain was selected and cultured for log phase in 7H9 liquid medium, and was inoculated it in duplicated 7H9 medium containing 4 MIC and 10 MIC TBI-166 for 4 hours and 24 hours, respectively. The total RNA was extracted after culture. The different expression of m RNA from different H37 Rv strain treated with 4MIC TBI-166 for 4h and 24 h, and wtih 10 MIC TBI-166 for 4h were screened using c DNA microarray technology, in order to explore the possible target of TBI-166 against mycobacterium tuberculosis. Results The gene expression of heat sensitive,protein folding, protein disulfide oxidoreductase activity were increased in H37 Rv strain treated with TBI-166 for 4h and the value of up-regulated expression was more than 6 times among Rv3066、Rv2622、Rv2621c、Rv3418c. And there were no obvious differences between 10MIC-4h and 4MIC-4h groups except the lower fold changes.While the gene expression of flavin adenine dinucleotide-binding, aromatic compound catabolic processwere increased in H37 Rv strain treated with TBI-166 for24 h and the value of up-regulated expression was more than 26 times in Rv3066.Conclusion It indicates that up-regulated expression of Rv3066, Rv2622, Rv2621 c,Rv3418c may be related to TBI-166 against mycobacterium tuberculosis. TBI-166 may activate these genes expression as inducer. Some of these genes still need to be verified further in the future.