Background/Objectives: Recently, antimicrobial-resistant pathogens and cancers have emerged as serious global health problems, highlighting the immediate need for novel therapeutics. Consequently, we aimed to isolate and characterize endophytic Streptomyces strains from the rhizospheres of the Himalayan region of Nepal and identify specialized metabolites with antibacterial, antifungal, and cytotoxic potential. Methods: To isolate Streptomyces sp., we collected two soil samples and cultured them on an ISP4 medium after pretreatment. We isolated and identified the strains PY108 and PY109 using a combination of morphological observations and 16S rRNA gene sequencing. Results: The BLAST results showed that PY108 and PY109 resembled Streptomyces hundungensis PSB170 and Streptomyces sp. Ed-065 with 99.28% and 99.36% nucleotide similarity, respectively. Antibacterial assays of ethyl acetate (EA) extracts from both isolates PY108 and PY109 in a tryptic soy broth (TSB) medium were conducted against four pathogenic bacteria. They showed significant antibacterial potential against Staphylococcus aureus and Klebsiella pneumoniae. Similarly, these extracts exhibited moderate antifungal activities against Saccharomyces cerevisiae and Aspergillus niger. Cytotoxicity assays on cervical cancer cells (HeLa) and breast cancer cells (MCF-7) revealed significant potential for both extracts. LC-MS/MS profiling of the EA extracts identified 27 specialized metabolites, including diketopiperazine derivatives, aureolic acid derivatives such as chromomycin A, and lipopeptide derivatives. In comparison, GC-MS analysis detected 34 metabolites, including actinomycin D and γ-sitosterol. Furthermore, a global natural product social molecular networking (GNPS)-based molecular networking analysis dereplicated 24 metabolites in both extracts. Conclusions: These findings underscore the potential of endophytic Streptomyces sp. PY108 and PY109 to develop new therapeutics in the future.

Metabolic profiling of actinomycetes isolated from Nepalese soil has uncovered promising candidates for the development of new antimicrobial and anticancer agents. This research examines the potential of actinobacteria isolated from soil, namely strains PY40, PY118, and PY122, as promising sources for biological applications. These strains were identified using 16S rRNA gene sequencing as Streptomyces sp. PY40, Streptomyces sp. PY118, and Amycolatopsis sp. PY122. Comprehensive bioassays were carried out on ethyl acetate (EA) extract originating from the fermentation broths of these species to assess their antimicrobial and anticancer activities. The results showed noteworthy antifungal and anticancer properties of Streptomyces sp. PY40 extract, thereby indicating its possible use in the synthesis of pharmaceuticals. Furthermore, potent antifungal activity was observed with an extract from Amycolatopsis sp. PY122. These isolates showed strong inhibition against several multi-drug-resistant strains of bacteria, including Shigella sonnei ATCC 25931, Klebsiella pneumoniae ATCC 700603, Escherichia coli ATCC 25922, and, Staphylococcus aureus ATCC 43300, suggesting significant antibacterial activity of the extracts. Gas Choursomatography-Mass Spectrometry (GC-MS) analysis revealed particularly volatile chemical constituents in the extracts. Antibiotic agents such as diketopiperazines and hexahydropyrrolo[1,2-a]pyrazine-1,4-dione were prevalent in the extracts of these isolates. This study enhances our comprehension of the bioactive potential of these microorganisms and underscores their value as a source for creating novel therapeutics to address multi-resistant infections and cancer demands. These findings show that actinomycetes from Nepalese soil have high prospects for biological applications.