Tuberculosis (TB) still claims more human lives each year than any other bacterial infection1. The latest report from the World Health Organization revealed signs of progress against drug-susceptible TB; however, the incidence rates of multidrug-resistant TB (MDR-TB) have sharply increased, thereby threatening global TB control programs1 2. Recent clinical data from two controlled phase 2 trials have revealed the efficacy of a novel anti-TB drug, bedaquiline (BDQ, marketed as Sirturo), in treatment of MDR-TB3 4. On the basis of the surrogate end point of time-to-sputum culture conversion, BDQ was granted accelerated approval by the US Food & Drug Administration for the treatment of pulmonary MDR-TB as part of combination therapy in adults5 6. This marks the first regulatory approval of an anti-TB drug since the introduction of rifampin in 1971. BDQ is a first-in-class ATP synthase inhibitor, displaying high selectivity for mycobacterial ATP synthase7 8 9, thus highlighting the key role of energy metabolism as a novel drug target pathway in mycobacteria10 11 12. BDQ exhibited potent bactericidal activity both in mouse models of TB infection7, and also when given for either 2 or 6 months in combination with a background regimen in MDR-TB patients3 4. However, its bactericidal activity in extended early bactericidal activity (eBA) studies showed a delayed onset, with the decline in bacterial sputum counts observed only from day 4–6 onwards13 14 15. This delay in onset of bactericidal activity is not simply due to the inability of the drug to reach steady state levels in patients, as a similar delay was seen in in vitro studies7 16. Similar to BDQ, the front-line anti-TB drug pyrazinamide also displays almost no bactericidal activity during the first 2–4 days of treatment, but nonetheless it kills consistently thereafter12. However, pyrazinamide was developed in 1950s in a different regulatory environment. Today, eBA is used for establishing a quick clinical proof of concept, and unfavourable eBA is regarded as an impediment for drug development. eBA studies are traditionally carried out over a period of 2–7 days, consequently eBA may underestimate the potency of drugs with a delayed onset of bactericidal activity. As such, insight into microbiological and molecular factors underlying the delayed onset of bactericidal activity by BDQ may provide valuable information for the design and interpretation of eBA studies on new drug candidates. In the current report, we aim at understanding the reason for the delayed onset of killing by BDQ. We investigate individual cells of M. tuberculosis in microfluidic devices and show the absence of bacteriolysis in mycobacteria during the initial days of BDQ treatment. We employ multiple biochemical and molecular approaches and elucidate temporal changes in bacterial metabolic pathways upon BDQ exposure. We show bacteria tend to minimize the consumption of cellular ATP and at same time enhance the capacity of ATP-generating pathways, which contributes to maintaining bacterial viability in spite of antibiotic stress. We also investigate the impact of the employed energy source on mycobacterial susceptibility to BDQ and demonstrate that mycobacteria grown on lipids display enhanced BDQ mediated killing. These data reveal how metabolic remodeling upon drug exposure can enable transient bacterial survival. Our results also indicate that inhibitors of mycobacterial ATP synthesis may prove powerful drugs for eradicating mycobacteria growing in lipid-rich environments in the human host. This shows that non-fermentable energy sources can be explored for in vitro characterization of new molecules targeting mycobacterial energy pathways. Finally, these data highlight that drugs specifically targeting the mycobacterial respiratory chain components like ATP synthase or cytochrome bc1, may exhibit a delayed onset of bactericidal activity both in vitro as well as in clinical eBA type studies within TB patients. Results Delayed killing of M. tuberculosis by BDQ We correlated the killing activity of BDQ with its impact on cellular ATP levels, in particular during the initial days of drug exposure. We confirmed that BDQ was highly bactericidal on M. tuberculosis over a period of 18 days in vitro, leading to a drop of ~4 log10 units in colony forming units (CFUs) by day 14 (Fig. 1a). However, during the first 2 days BDQ showed very little bactericidal activity, with 1,500 proteins and determined their differential expression 6 and 24 h after BDQ exposure (10 × MIC) (detailed in Supplementary Data 2–4). For a global overview of key changes in metabolism upon BDQ exposure proteins displaying high differential regulation (top 100 proteins at each time point) were grouped into functional categories based on the TubercuList server classification28. At both 6 and 24 h time points, BDQ treatment most prominently affected intermediate metabolism and respiration (Fig. 4a), reflecting the drug’s unique mechanism of action. Prominent differential regulation was also observed for proteins involved in cell wall and cell processes, information pathways and lipid metabolism as well as for conserved hypothetical proteins (Fig. 4a). On the basis of these top-line findings, we subsequently focused on metabolic pathways connected to synthesis or consumption of cellular ATP. Proteins belonging to these metabolic pathways were selected and their differential regulation was presented in a heat map (Fig. 4b). We regarded proteins showing an increase or decrease with a fold change (FC)>1.5 as strongly regulated, those with fold change 1.3