Streptomyces sp. TBSA08 From Western Ghats Forest Soil: Antibacterial Activity Against Multidrug‐Resistant Pathogens and Synergy With Ciprofloxacin

Antimicrobial resistance (AMR) represents an urgent global health challenge, necessitating the discovery of new antibacterial agents. This study investigated the antibiotic‐producing potential of Actinomycetes isolated from forest soil of the Thattekad Bird Sanctuary, Kerala, India, using complementary metagenomic and culture‐dependent approaches. Metagenomic sequencing revealed a diverse microbial community, with Actinomycetota (Actinobacteria) as the second most abundant phylum and genes linked to antibiotic biosynthesis, including vancomycin pathways. A total of 45 Actinomycetes isolates were obtained, of which 15 showed antibacterial activity against the tested standard bacterial strains. The most potent strain, Streptomyces sp. TBSA08, showed strong inhibition against multiple standards and selected multidrug‐resistant (MDR) clinical isolates, including Acinetobacter baumannii MDR (26.0 ± 0.50 mm), Enterobacter cloacae MDR (19.9 ± 1.00 mm), methicillin‐resistant Staphylococcus aureus (20.0 ± 0.60 mm) and vancomycin‐resistant Enterococcus faecalis (23.2 ± 0.00 mm). The TBSA08 extract exhibited MIC values of 3.906 μg/mL against most tested standard strains and 7.812–15.625 μg/mL against the tested MDR clinical isolates. Its secondary metabolites demonstrated notable synergism with ciprofloxacin against the tested MDR clinical isolates, with FIC index values ≤ 0.5. Chemical profiling using GC/MS and UPLC/Q‐TOF‐MS tentatively annotated several metabolites based on mass‐spectral/database matching. Docking studies indicated favourable interactions of selected TBSA08 metabolites, including 1,3‐Diolein and Oleoyl chloride, with bacterial topoisomerase IV (6WAA) and DNA gyrase (4Z2D), providing a computational basis for further investigation of their possible antibacterial mechanisms. These findings identify Thattekad forest soil as a source of antibiotic‐producing Actinomycetes and highlight Streptomyces sp. TBSA08 as a promising candidate for further antibacterial investigation. The observed activity against selected MDR pathogens and synergy with ciprofloxacin support further bioassay‐guided isolation and experimental validation of the active metabolites.