From Glacier to In Silico: Integrated LC-MS Metabolomics and Multi-Level Computational Prioritization of...

Antibiotic resistance is a global threat requiring new potential antimicrobial sources. Secondary metabolites from cold-adapted microorganisms may provide unique antimicrobial compounds. Ethyl-acetate extract of Alcaligenes pakistanensis LTP10 from Passu glacier was explored for antimicrobial potential against clinical isolates. Cytotoxicity was determined by Brine shrimp lethality assay. The extract is analyzed by LC-MS/MS and the data is processed by MZmine. Important compounds were evaluated by molecular docking and in silico study. The extract demonstrated activity against clinical isolates Staphylococcus aureus, Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, and Candida albicans with zone of inhibition ranging from 16 mm to 24 mm, with no killing of nauplii suggested non-cytotoxic nature. LC-MS/MS analysis established presence of important putative antimicrobial metabolites (E)-3-(acetyloxymethyl)-5-(2-formyl-4-hydroxy-5,5,8a-trimethyl-1,4,4a,6,7,8-hexahydronaphthalen-1-yl)pent-2-enoic acid, cyclizidine-F, neovasipyridone-G, paenibacillin-A, and tricholomenyn-A. Molecular docking study of these metabolites by AutoDock Vina against dihydrofolate reductase of S. aureus demonstrated binding affinities from -6.8 kcal/mol to -8.5 kcal/mol, while against enoyl-ACP reductase of E. coli showed binding affinity values from -6.3 kcal/mol to -7.9 kcal/mol. In silico analysis predicted considerable absorption, distribution, metabolism, excretion, safety, and druglikeness properties. These results suggest that metabolites from A. pakistanensis LTP10 possess antimicrobial potential and warrant advanced post-docking validation via molecular dynamics, free-energy, and mechanistic analyses for future antibiotic development.