Mycobacterium tuberculosis Growth Assay

Mtb H37Rv was grown in Middlebrook 7H9 (Becton, Dickinson and Company) liquid medium supplemented with 10% (v/v) Albumin–Dextrose Complex (ADC), 0.2% (v/v) glycerol and 0.1% (w/v) Tween 80 at 37°C with shaking or in SMM containing hygromycin with or without pristinamycin. SMM comprised of 0.3 M sucrose, 20 mM MgSO₄, 0.1% Tween 80 (w/v), 10% (v/v) ADC in standard 7H9 broth. Bacterial growth was measured by absorbance at 580nm, or by colony-forming unit (CFU) counting on 7H10 agar (Becton, Dickinson and Company), or by most probable number (MPN) counting using established protocols (Loraine et al., 2016 (link)) and the MPN calculator program (Jarvis et al., 2010 (link)). Escherichia coli OverExpress™ C41(DE3) and DH5α were grown in Lysogeny broth. For protein expression, E. coli was grown to mid-log phase (OD₆₀₀ 0.6-0.8) at 37°C with shaking at 200 rpm before adding 0.5 mM isopropyl β-D-1-thiogalactopyranoside followed by incubation at 18°C overnight. Antibiotics were used at the following concentrations (μg/ml): pristinamycin, 0.5; kanamycin, 50; hygromycin, 50; ampicillin, 50. Wayne model of non-replicating persistence was set up as previously described (Wayne and Sramek, 1994 (link)). CFU and MPN counts were determined at 0-, 7- and 12-week time points.