Isolation of Nepalese Soil Actinomycetes and RSM-Driven Optimization of Fermentation Conditions for Maximum Antimicrobial Production

Actinomycetes are filamentous gram-positive bacteria renowned for producing diverse antimicrobial compounds. This study isolated and evaluated actinomycetes from soils across twelve ecologically and altitudinally diverse regions of Nepal (129–4,023 m) for their potential against pathogenic bacteria. Using selective starch casein agar supplemented with nalidixic acid and cycloheximide, 27 isolates were obtained. Morphological and physiological characterization, including spore formation, pigment production, geosmin odor, and growth under varying pH, temperature, and salinity, confirmed their identity and adaptability. Preliminary screening via perpendicular streak method identified five isolates (D4, X1, J3, G2, D1) with notable activity against Staphylococcus aureus and other pathogens. Optimization of fermentation conditions using Response Surface Methodology enhanced antimicrobial production, with the most potent strain (D4) showing a 23 mm zone of inhibition. Crude ethyl acetate extracts exhibited minimum inhibitory concentrations of 6.25–25 mg/mL. These findings highlight the rich diversity of Nepalese actinomycetes and their potential as sources of novel antimicrobial agents. These findings demonstrate the feasibility of employing Nepalese actinomycetes in industrial fermentation processes for scalable production of antimicrobial agents and also provides a foundation for further compound characterization and development of actinomycete-derived therapeutics to combat pathogenic bacteria.