Microplate Alamar Blue assay (MABA) for antibiotic susceptibility testing

M. tuberculosis pknA, pknB, pknA+pknB depletion strains were grown with ptc induction at 37°C in 7H9-ADC-Tw to OD₆₀₀ ~0.8. The cells were pelleted, washed twice with 7H9-ADC-Tw and diluted to a calculated OD₆₀₀ 0.001 in7H9-ADC-Tw. Each strain was placed in a 96-well clear bottom plate with 2-fold dilutions of ptc across the columns and 2-fold dilutions of antibiotic across the rows. Dilutions were made in 7H9-ADC-Tw and all wells contained 10 μg/ml clavulanic acid. Plates were incubated for 7–8 days and inhibitory concentrations of antibiotics were determined as previously described, based on color change following overnight incubation after addition of Alamar Blue, with pink indicating growth and blue indicating absence of growth. The lowest antibiotic concentration that results in absence of growth (blue well) correlates with minimal inhibitory concentrations obtained using traditional methods [58]. For the kinase depletion strains, we defined the lowest inhibitory concentration as the lowest antibiotic concentration that inhibited growth at the lowest ptc concentration that resulted in full growth in the absence of antibiotic. Biological duplicates were performed for each experiment.