Screening the Antibiotic Activity of Cave Actinobacteria Against Multidrug‐Resistant Strains of Pseudomonas aeruginosa and Methicillin‐Resistant Staphylococcus aureus

Many antibiotics originate from soil‐inhabiting Actinobacteria, especially from the diverse genus Streptomyces . However, the emergence of antibiotic resistance poses a significant global challenge to treating infectious diseases. Therefore, the search for Actinobacteria, particularly from less‐explored environments, as potential sources of novel antimicrobial compounds, is of great importance. This study sampled various biofilms growing on cave structures within Deer Cave and Lagang Cave, located in the UNESCO World Heritage Site of Gunung Mulu National Park (GMNP; Sarawak, Malaysia). From this relatively untapped niche in caves, we identified and screened actinobacterial isolates for their potential antimicrobial properties against drug‐resistant Pseudomonas aeruginosa and Staphylococcus aureus strains. Of 48 isolates, 24 showed inhibition of one or both drug‐resistant strains in the antimicrobial assays conducted using cross‐streak and agar well diffusion methods. The ethyl acetate extracts containing potential secondary metabolites demonstrated effective inhibition, particularly against the drug‐resistant Gram‐negative P. aeruginosa . In contrast, the supernatants obtained from aerobic cultivation exhibited comparatively better activity against Gram‐positive S. aureus strains. 16S rRNA gene sequencing analysis of the isolates revealed that all except one isolate belonged to the genus Streptomyces . Maximum likelihood bootstrap tree analysis strongly supported the correct clustering of the Streptomyces isolates with well‐known bioactive compound producers, such as S. gardneri , S. laurentii , and S. zaomyceticus . Notably, Deer Cave Isolate D3‐12 exhibited inhibitory activity against both drug‐resistant strains and, therefore, represents a promising candidate for future studies involving the characterization of its bioactive compounds. The remaining actinobacterial isolate exhibited 100% sequence homology to soil‐inhabiting Rhodococcus pedecola , known for its antibacterial properties. These findings suggest that the caves of GMNP harbor untapped ecological niches of diverse cave‐dwelling Actinobacteria, which may serve as sources of antimicrobial compounds effective against emerging antibiotic‐resistant pathogens.