The mycobacterial ATP burst is a lysis artifact and serves as an assay for drug-induced cell wall damage

Antibiotics targeting the mycobacterial cell wall are a cornerstone of tuberculosis treatment, yet how these drugs facilitate bacterial killing remains incompletely understood. Studies using the BacTiter-Glo luminescence assay have reported increased mycobacterial ATP levels following treatment with cell wall inhibitors such as isoniazid, a phenomenon referred to as an “ATP burst.” This is proposed to contribute to drug-induced killing. Here, we show the ATP burst is not a biological response but rather an experimental artifact resulting from enhanced cell lysis induced by cell-wall-targeting drugs. Mechanical lysis by bead beating abolishes the ATP burst, enabling more reliable assessment of ATP levels. We demonstrate the utility of this approach as a functional readout for identifying compounds that disrupt the mycobacterial cell wall and for screening synergistic or antagonistic interactions with cell wall inhibitors. These findings clarify the mechanistic basis of the ATP burst and provide a practical tool for antimycobacterial drug discovery.