Abstract:

Objective To explore the mechanism of TBI-166 against drug-resistant Mycobacterium tuberculosis (MTB) by determining the reactive oxygen content and catalase activity of MTB.Methods H37Rv standard strains were treated with clofazimine (CFZ) or TBI-166 at 1, 2, 4, 8 times the minimal inhibitory concentration (MIC) for 24 hours. The levels of reactive oxygen species (ROS) in bacteria were determined with fluorescent probe 2',7'-dichloro fluorescent yellow diacetate ROS detection kit, and the changes of ROS levels in bacteria were compared before and after the medication of CFZ or TBI-166 with different concentrations. The catalase activities of 5 strains resistant to both CFZ and TBI-166, 3 strains resistant to CFZ but sensitive to TBI-166, 1 strain (13385) sensitive to both CFZ and TBI-166, and H37Rv standard strain were determined by the catalase activity detection kit. The difference in catalase activity between drug-resistant strains and sensitive strains to both CFZ and TBI-166 was compared. SPSS 19.0 software was used for statistical analysis. Spearman correlation analysis and linear regression analysis were performed upon the concentrations of TBI-166 and the fluorescence values. The significance level was 0.05.Results The ROS results showed that after 24 h of treatments with different concentrations CFZ and TBI-166, the rank order of the fluorescence value of samples were 1 MIC (6668.5) >8 MIC (2751.5) >2 MIC (2572.0) >4 MIC (2015.0), and 8 MIC (2637.5) >4 MIC (2297.0) >2 MIC (2085.5) >1 MIC (1896.0), respectively. The amount of ROS accumulated in the bacteria was at least 2 times higher than that in the negative control samples without drug (H37Rv standard strain+DCFH-DA, 878.5). Moreover, in the strains treated with different concentrations of TBI-166, the fluorescence values were positively correlated with the drug concentration (Spearman correlation analysis, r=0.976, P<0.001). The regression equation test results showed that the regression model was significant (F=92.576, P=0.011). The catalase activity results showed that the average catalase activity of the CFZ-resistant strains (0.87 U/10 ⁴ cell) was nearly twice that of the CFZ-sensitive strains (0.48 U/10 ⁴ cell). The average catalase activity of the TBI-166-resistant strains (0.88 U/10 ⁴ cell) was slightly higher than that of the TBI-166-sensitive strains (0.71 U/10 ⁴ cell), and was similar to that of CFZ-resistant strains. Conclusion Both CFZ and TBI-166 can induce the accumulation of intracellular ROS in MTB, and the two drugs may have similar mechanisms of action. Increased catalase activity may be associated with the drug resistance to CFZ and TBI-166 in MTB.

Key words: Mycobacterium tuberculosis, Drug resistance, Molecular mechanisms of pharmacological action, Drug evaluation, preclinical, Clofazimine, TBI-166