Ajoene: a natural compound with enhanced antimycobacterial and antibiofilm properties mediated by efflux pump modulation and ROS generation against M. Smegmatis.

Tuberculosis (TB) continues to be a primary worldwide health concern due to relatively ineffective treatments. The prolonged duration of conventional antibiotic therapy warrants innovative approaches to shorten treatment courses. In response to challenges, the study explores potential of Ajoene, a naturally occurring garlic extract-derived compound, for potential TB treatment. Mycobacterium smegmatis as a model organism for M. tuberculosis (M. tb) to investigate Ajoene's efficiency. In vitro techniques like antimicrobial susceptibility, antibiofilm, EtBr accumulation assay, and ROS assay evaluate the potency of Ajoene and conventional TB drugs against Mycobacterium smegmatis. An in-silico study also investigated the interaction between Ajoene and quorum-sensing proteins, specifically regX3, MSMEG_5244, and MSMEG_3944, which are involved in biofilm formation and sliding activity. In vitro findings revealed that Ajoene exhibited significant antibacterial activity by inhibiting growth and showing bactericidal effects. It also demonstrated additive interactions with common antibiotics such as Isoniazid and Rifampicin. Furthermore, Ajoene demonstrated a comparative interaction with commonly used antibiotics, such as Isoniazid and Rifampicin, and reduced M. smegmatis motility, both alone and in combination with these antibiotics. In silico analysis shows that Ajoene exhibited a higher binding affinity with regX3, a protein orthologous to the regX3 gene in M.tb. Ajoene also demonstrated consistent antibiofilm effects, particularly when combined synergistically with Isoniazid and Rifampicin. Mechanistic investigations demonstrated Ajoene's potential to inhibit efflux pumps and promote ROS generation in bacteria, suggesting a potential direct killing mechanism. Collectively, the findings emphasize Ajoene's effectiveness as a novel antimycobacterial and antibiofilm molecule for TB treatment.