Fig 1. Schematic representation of the long-term intracellular M. tuberculosis culture protocol.

For each infection cycle, a frozen stock of THP-1 cells was thawed and cells were cultured for 4 passages. Cells were differentiated by PMA stimulation and infected with M. tuberculosis. After 18 h of infection, medium was replaced, and infected cells were incubated for 6 days. At day 7, THP-1 cells were lysed to release the intracellular bacteria, which were diluted in cell culture medium and subjected to clump disaggregation as described in Methods. Approximately 1/10 to 1/20 of this bacterial suspension was used to infect a new THP-1 cell culture. A total of 10 serial infection cycles were performed, and bacterial CFU and THP-1 cells were counted at 18 h and 7 days of infection for each cycle. An aliquot of the initial bacterial inoculum and of the intracellular bacteria collected at the end of the tenth cycle were diluted and spread onto agar plates for single-colony isolation and whole genome sequencing.