Iron-Reversible Bactericidal Activity of Marine-Derived Aspergillus ostianus Hydroxamate Pyrazinones Against Replicating and Hypoxia-Induced Non-Replicating Mycobacterium smegmatis.

Tuberculosis therapy is prolonged partly because dormant subpopulations of Mycobacterium tuberculosis show reduced susceptibility to first-line drugs. Therefore, agents active against both replicating and non-replicating mycobacteria remain important to explore. Here, we investigated secondary metabolites from the Indonesian marine-derived fungus Aspergillus ostianus for activity against Mycobacterium smegmatis, a BSL-1 mycobacterial model, under aerobic and hypoxia-induced non-replicating conditions, and examined the underlying mechanism. Bioassay-guided fractionation and spectroscopic analysis identified three hydroxamate pyrazinones: neohydroxyaspergillic acid (NHAA), hydroxyaspergillic acid (HAA), and neoaspergillic acid (NAA). The MIC values were 1.56 µg/mL for NHAA and 3.13 µg/mL for HAA and NAA under both aerobic and hypoxic atmospheres. Time-kill kinetics showed ≥3-log10 CFU reductions within 24-72 h at 4-8× MIC under aerobic conditions and within 48-96 h at 4-8× MIC under hypoxia, with no regrowth at the final sampling point. Scanning electron microscopy and release of UV-absorbing intracellular material at OD260/OD280 were consistent with envelope disruption in both atmospheres. Antimycobacterial activity was attenuated in a concentration-dependent manner by exogenous Fe3+ and was reversed at 100 µM FeCl3, whereas isoniazid activity was unaffected, supporting iron-reversible and pyrazinone-specific killing. Together with the established Fe3+-binding hydroxamate pharmacophore shared by this compound class, these findings support iron sequestration as a plausible mechanism and identify fungal hydroxamate pyrazinones as scaffolds that retain bactericidal activity against hypoxia-adapted non-replicating mycobacteria, warranting further evaluation in M. tuberculosis models.