Bacterial Strains, Growth Conditions and Determination of Minimal Inhibitory Concentrations (MICs)

The reference laboratory strain M. tuberculosis H37Rv (ATCC 25618) and a panel of eight genetically distinct clinical strains of M. tuberculosis were used for drug susceptibility testing. This included strain GC1237, a highly transmissible strain of the Beijing lineage. A derivative of the H37Rv strain containing plasmid vector pSUM36 (Ainsa et al., 1996) was used for testing the in vivo effect of alkaloids on DNA supercoiling. To determine the mechanism of action of topoisomerase inhibitors, Mycobacterium smegmatis mc²155 (Snapper et al., 1990) was used along with its derivative MsPptrtopoI conditional knock-down mutant (Ahmed et al., 2015), in which levels of topoisomerase I (MsTopoI) can be reduced by addition of anhydrotetracycline (ATc). All strains were grown in Middlebrook 7H9 broth (Becton Dickinson) supplemented with 10% ADC (Becton Dickinson) and 0.05% Tween 80 (Sigma). Kanamycin (50 mg/L) was added to ensure the maintenance of plasmid pSUM36. Minimal inhibitory concentrations (MICs) were determined by microdilution as previously reported for M. tuberculosis (Palomino et al., 2002); MICs of drugs for M. smegmatis were determined by the same method except that plates were incubated for 3 days. The MIC was defined as the lowest concentration of drug that prevented change of resazurin from its oxidized form (blue) into the reduced one (pink), which is indicative of bacterial growth. Imipramine, a well-known topoisomerase-poison described previously (Godbole et al., 2015) was included as a control. For the time-kill kinetics experiments, a bacterial inoculum of 10⁷ CFU/ml was incubated in the presence of inhibitory concentrations of N-SCN (8 × MIC) for 24 h at 37°C; then, the culture was serially diluted in order to determine the number of viable bacteria, by plating on Middlebrook 7H10 plates; relative CFUs in comparison with untreated control cultures were determined.