Multi-drug-resistant (MDR) strains demonstrate extensive antibiotic resistance. Antibiotics were added at day 1 to the 3-D model and Mtb growth measured with luminescence. First-line drugs: rifampicin at 1 μg/ml (RIF 1, dark red), isoniazid at 0.25 μg/ml (INH 0.25, navy blue), pyrazinamide at 500 μg/ml (PZA 500, dark green), and ethambutol at 4 μg/ml (EMB 4, orange). Second-line drugs: moxifloxacin at 5 μg/ml (MOXI 5, light brown), D-cycloserine at 20 μg/ml (DCS 20, lilac) and 200 μg/ml (DCS 200, dark purple), and linezolid at 24 μg/ml (LNZ 24, midpurple). Emerging drugs: bedaquiline at 5 μg/ml (BDQ 5, beige) and 10 μg/ml (BDQ 10, dark brown) and pretomanid at 3 μg/ml (PA-824 3, light blue). (a) 1471A lux strain confirmed resistance to rifampicin, isoniazid, and ethambutol and showed partial sensitivity to pyrazinamide. (b, c) Growth of this multi-drug-resistant clinical isolate was unaffected by the lower concentration of D-cycloserine and bedaquiline (both concentrations tested). 0940Y lux strain was resistant to all (d) first-line antibiotics and (e) D-cycloserine at the lower concentration. (f) 0940Y had partial sensitivity to bedaquiline. Bacteria were fully inhibited by all other antibiotics examined. Mtb growth was unaltered by adding DMSO, used as solvent for rifampicin, linezolid, bedaquiline, and PA-824. Crosses (x) designate background level of luminescence. Black arrow shows antibiotic addition. The experiment was performed in triplicate on two separate donors, and a representative experiment is shown. Statistical analyses were carried out using 2-way ANOVA with Tukey’s multiple comparison test; .