ATP burst is the dominant driver of antibiotic lethality in Mycobacterium smegmatis

Antibiotic-tolerant bacteria, due to their unique physiology, are refractory to antimicrobial killing and pose challenges for infection control. Incomplete knowledge of how bactericidal antibiotics work limits our understanding of partial resistance due to phenotypic tolerance in mycobacteria, a driver for developing genetic resistance. Using proteomics, ¹³C isotopomer analysis, genetic and biochemical assays, we investigated the physiological response of M. smegmatis challenged with aminoglycoside and fluoroquinolone antibiotics. Two distinct classes of antibiotics elicited remarkably similar responses and increased flux through the TCA cycle, causing enhanced respiration, ROS generation, and ATP burst. We observed that excessive ATP levels and not ROS dominantly contribute to cidality, which may in part be conferred by sequestration of divalent metal ions by ATP. Consequently, ¹³C isotope tracing indicated TCA cycle flux deviation from its oxidative arm as a bacterial adaptive mechanism, which also included activated intrinsic resistance and a higher propensity to develop antibiotic resistance. Our study provides a new understanding of the intricate mechanisms of antibiotic-induced cell death and expands the current paradigm for antibiotic action.