Dubos medium

Frozen aliquots of mycobacteria were thawed at room temperature and passed through a syringe for single cell separation. 200 µL bacterial culture was added to 5 mL Middlebrook 7H9 broth (Difco, Detroit, MI, USA) containing 0.2% glycerol, 0.05% Tween 80, and 10% oleic acid-albumin-dextrose-catalase (OADC) growth enrichment (Becton Dickinson, Cockeysville, MD, USA). Cultures were incubated at 37 °C until they reached an OD of ~ 0.2. Two mL pre-culture (at OD = 0.2) were added to 15–18 mL Dubos medium (Becton, Dickinson) supplemented with 0.05% Tween 80 and 10% OADC and incubated in roller bottles (Corning) at 37 °C for 7–10 days. At an OD of 0.4–0.5 (log phase) the cultures were diluted to OD = 0.004 in Dubos complete medium and used for dormancy experiments. Colony forming unit (CFU) analyses were calculated by serial dilutions plated on Middlebrook 7H11 agar containing 0.5% glycerol and 10% OADC growth enrichment. All CFU values in this study represent CFU/mL culture. A list of all used clinical isolates can be found in Supplementary Table S3.

M. tuberculosis H37Rv, M. celatum ATCC 51130 (strain 1908), M. fortuitum ATCC 6841, and a clinical isolate of M. abscessus (previously identified by rRNA 16S sequencing) were cultured in Dubos medium with 10% of albumin-dextrose-catalase supplement (bovine albumin fraction V 50 g/L, dextrose 20 g/L, and catalase 0.04 g/L) (Becton Dickinson, Franklin Lakes, NJ, USA) and incubated at 37°C. The bacteria were then pelleted by centrifugation and resuspended in RPMI 1640 medium (Gibco, Life Technologies, Grand Island, NY, USA) [17 ]. Viability was evaluated by counting the colony-forming units (CFUs), and the bacterial suspension was aliquoted and frozen at −70°C.