High-throughput Screening of Compounds against Autoluminescent NonreplicatingMycobacterium tuberculosisunder Diverse Conditions

Abstract

ABSTRACTThe screening of new anti-mycobacterial chemicals is primarily focused on inhibiting the active growing bacteria. However, a major challenge in tuberculosis control is the ability ofMycobacterium tuberculosisto enter a nonreplicating state for extended periods, rendering it resistant to many clinical drugs and complicating eradication efforts. Existing low-oxygen-recovery assays designed for screening compounds targeting nonreplicatingM. tuberculosishave limitations, including the colony-forming unit counting for non-luminousM. tuberculosisand the instability of the free plasmid carryingluxABgenes in luminescentM. tuberculosis, along with exogenous substrate requirements for light producing. Moreover, these assays fail to accurately replicate the growth conditions of nonreplicatingM. tuberculosis in vitro, thus resulting in less convincing results. To address these challenges, we have developed an autoluminescence-based, cholesterol-enriched culture evaluation model to assess 17 anti-tuberculosis drugs of different classes against nonreplicatingM. tuberculosis. Our findings indicate that the relative light unit, measured in real-time, serves as a reliable surrogate marker for colony-forming unit, which typically becomes available one month later. This suggests the utility of our model for the rapid determination of drug susceptibility dynamically. The autoluminescentM. tuberculosis, harbouringluxCDABEgene cluster within its genome, can emit blue-green light stably and autonomously without requiring an external substrate supplement. The minimal inhibitory concentrations of all the drugs tested under anaerobic conditions are significantly different from that detected in aerobic environment. Our model allows for rapid, precise, and efficient assessment of drug activity under anaerobic conditions, thereby enabling a more comprehensive evaluation of anti-mycobacterial efficacy. Overall, our model represents a significant advancement in anti-tuberculosis drug discovery and pharmaceutical development.