Abstract: Objective: To evaluate the interaction between pyrifazimine (TBI-166) and bedaquiline (Bdq), moxifloxacin (Mfx), delamanid (Dlm), SQ109 and PBTZ169 on clinical isolates of Mycobacterium tuberculosis (MTB), and primarily explore the synergistic mechanism of TBI-166 and Bdq, so as to provide basis for TBI-166 combination therapy.Methods: MTB standard strain H37Rv (ATCC 27294), two drug-sensitive MTB clinical isolates (strain numbers: 30031 and 30091), and one clinical isolate of pre-extensively drug-resistant Mycobacterium tuberculosis (strain number: 13385) were selected. Checkerboard method was used to determine the interaction between TBI-166 and Bdq, Mfx, Dlm, SQ109, and PBTZ169 on MTB clinical isolates clinical isolates. Time sterilization curve method was used to evaluate the antibacterial activity of the synergistic of the two drug combinations. Bacterial reactive oxygen species (ROS) detection kit and adenosine triphosphate (ATP) detection kit were used to determine the ROS and ATP levels in H37Rv standard strain 24 hours after being treated with different concentrations of TBI-166 and Bdq.Results: After combining with Bdq, the minimum inhibitory concentration (MIC) of TBI-166 on MTB clinical isolates No. 30031, 30091 and 13385 were 0.08, 0.05 and 0.08 μg/ml, respectively, all of which decreased to 1/2 of the MIC values of TBI-166 alone. The fractional inhibitory concentration index (FICI) values of the two-drug combination of TBI-166 and Bdq on strains No. 30031, 30091 and 13385 were 1.00, 0.75 and 1.00, respectively. The time sterilization experiment showed that compared with TBI-166 alone, after being treated with TBI-166 and Bdq for 14 days, the viable bacteria of strains No. 30031, 30091 and 13385 decreased by 1.59log₁₀ CFU/ml, 1.27log₁₀ CFU/ml and 1.70log₁₀ CFU/ml, respectively. Compared with the 0.3 μg/ml Bdq group, the ATP content of H37Rv standard strain 24 h after being treated with 0.4 μg/ml TBI-166 in combination with 0.3 μg/ml Bdq had a significant decrease (relative light unit value, 115160.67±19129.79 vs. 208599.20±24078.74; F=75.109, P=0.013), while the ROS content increased significantly (the fluorescence value, 21014.33±1189.19 vs. 13715.00±907.93; F=115.403, P<0.001).Conclusion: The combination of TBI-166 and Bdq had synergistic bactericidal activity, and the mechanism of synergistic bacteria might be related to the augmenting accumulation of ROS and the block of ATP synthesis.

Key words: Antitubercular agents, Drug interactions, Mycobacterium tuberculosis, Pyrifazimine, Bedaquiline