A Mycobacterium marinum zone of inhibition assay as a method for screening potential antimycobacterial compounds from marine extracts

A novel screening method for anti mycobacterial agents using Mycobacterium marinum was developed. M. marinum was selected as a model organism because it has a close phylogenetic relationship to M. tuberculosis, a relatively rapid doubling time, similar drug susceptibilities to M. tuberculosis, and less stringent safety requirements. More than 1000 crude marine and plant extracts were screened against M. marinum in a Zone of Inhibition (ZOI) assay, and twenty-one target extracts were identified. The crude organic extract of the marine sponge, Haliclona sp.10, was chosen for further investigation as it yielded a ZOI of 20 mm at a concentration of 80 mu g/disk. Following bioassay-guided fractionation, (-)-papuamine was isolated, and yielded a 15 mm ZOI at a concentration of 25 mu g/disk. In standard assays using M. marinum, (-)-papuamine exhibited both an MIC and an MBC95 of 6.25 mu g/mL. This is the first report of anti mycobacterial activity for (-)-papuamine. In addition, when (-)-papuamine and other natural product extracts were tested for activity against both M. marinum and M. tuberculosis, activity was comparable between the two species. These data indicate that (-)-papuamine is a promising lead for the development of new antimycobacterial agents and that M. marinum is a useful surrogate for the screening of antimycobacterial compounds.