Integrated in vitro and in silico Evaluation of the Antimicrobial and Bioactive Properties of Natural Honey and Its Enhanced Derivatives Against Multidrug-Resistant Pathogens.

BACKGROUND: Multidrug-resistant (MDR) bacteria remain difficult to treat, creating a need for alternative antimicrobial strategies. This study evaluated standardized natural honey, enzymatically activated honey, and Moringa oleifera-fortified honey against MDR pathogens using integrated in vitro and in silico approaches. METHODS: A single-batch polyfloral Apis mellifera honey was characterized and modified either by controlled 1:1 aqueous activation to maximize glucose oxidase-mediated hydrogen peroxide production or by fortification with hydroethanolic M. oleifera leaf extract. Samples were tested at 25%, 50%, and 75% (w/v) against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae using agar diffusion, MIC, MBC, antibiofilm, DPPH, ABTS, FRAP, GC-MS, and molecular docking assays, with ascorbic acid included as the antioxidant positive control. RESULTS: At 75% (w/v), natural honey produced inhibition zones of 18 +/- 1.1, 14 +/- 1.0, 12 +/- 0.9, and 11 +/- 0.8 mm against S. aureus, E. coli, P. aeruginosa, and K. pneumoniae, respectively. Enzymatic activation increased these zones to 24 +/- 1.3, 19 +/- 1.1, 17 +/- 1.0, and 16 +/- 0.9 mm, while M. oleifera fortification produced the strongest activity: 26 +/- 1.4, 21 +/- 1.2, 19 +/- 1.1, and 18 +/- 1.0 mm, with MIC/MBC values as low as 10/20 mg/mL. Fortified honey also showed the highest antibiofilm inhibition and the strongest antioxidant performance across DPPH, ABTS, and FRAP assays. GC-MS retention-index verification and docking were consistent with possible contributions from phenolic acids, flavonoids, and terpenoid-like compounds, but did not establish target-specific biological activity. CONCLUSION: Enzymatic activation and Moringa fortification improved honey bioactivity under the tested laboratory conditions. These findings justify further mechanistic, cytocompatibility, in vivo, and formulation studies but do not establish therapeutic efficacy.