FIG 5.

Colony formation in cultures of wild-type (WT) and katG loss-of-function mutant M. bovis BCG strains treated with 2,2′-bipyridyl (BP) (80 μM), SA23 (0.16 μM and 0.32 μM, equivalent to 0.5× and 1× the MIC₉₀ value, respectively) and combinations of BP and SA23 (SA23+BP) at the stated concentrations. Drug treatments were carried out in catalase free 7H9 broth at 37°C and 110 rpm for 5 days. The aliquots were then withdrawn, serially diluted, and plated out on 7H10 agar for colony-forming unit (CFU) counting. DF D0 and DF D5 were untreated, drug free controls that were monitored at day 0 and day 5 which correspond to the start and end of drug exposure. No significant differences were observed between the gray and red bars at each time point, indicating that the WT and katG mutant strains displayed similar growth patterns over the 5-day period. “BP” refers to WT/katG mutant cultures treated with BP at 80 μM. The CFU of the “DF D5” and “BP” treatment arms (WT, katG mutant) were comparable, indicating that BP was not bactericidal at 80 μM on either strain. In the case of SA23, the cotreatment arm (SA23+BP) was compared to the control arm (SA23) as described in the text. MBC₉₀, MBC₉₉, and MBC_99.9 are the concentrations required to reduce the CFU by 10×, 100× and 1,000× compared to DF D0. The solid line represents the approximate limit for CFU detection. The experiment was repeated thrice independently, with one representative set of results shown. **, P < 0.01; Student’s t test; GraphPad Prism Ver. 8.4.3. The CFU counts are inversely related to antibacterial activity. SA23 exhibited greater activity on the katG mutant compared to the WT strain. In the presence of BP, the increase in activity was particularly pronounced in the katG mutant strain treated with SA23 at 1× MIC₉₀, at which the CFU counts were reduced to a level below the detection limit (indicated by a red star).