The escalating threat of antimicrobial resistance (AMR) has created an urgent need for new antimicrobial agents. Antimicrobial peptides (AMPs) are promising alternatives to conventional antibiotics due to their broad-spectrum activity and reduced risk of resistance development. While most AMP discovery efforts have focused on terrestrial microbes, extreme environments remain largely untapped. Deep-sea hydrothermal vent biofilms, such as those from the Arctic Mid-Ocean Ridges (AMOR), are unique ecosystems characterized by high pressure, temperature gradients, and chemical extremes. These conditions select for microorganisms with specialized adaptations, including the production of bioactive compounds that confer survival advantages. Such peptides may exhibit enhanced stability and novel mechanisms of action, making hydrothermal biofilms an exceptional resource for next-generation antimicrobials. Using metagenomic and metatranscriptomic datasets from nine recently published AMOR biofilms, we predicted 961 AMP sequences with Macrel, of which 873 were unique and showed no identity to entries in the Antimicrobial Peptide Database (APD). AMPs were distributed across 51 microbial phyla, including underrepresented archaeal groups such as Asgardarchaeota, Nanoarchaeota, and Micrarchaeota. Transcriptomic profiling detected AMP expression in 25 phyla, including low-abundance candidate taxa, highlighting active AMP production. In silico minimum inhibitory concentration (MIC) prediction using APEX 1.1 suggested that 16.7% of AMPs may inhibit at least one clinically relevant pathogen, with Acinetobacter baumannii emerging as the most susceptible. Four peptides were synthesized for experimental validation; AMP OLKFNNDA_52_10 exhibited moderate in vitro activity against Staphylococcus aureus and weak activity against Escherichia coli, while showing low cytotoxicity toward human HEK293 cells. Other tested peptides displayed weak or no activity, underscoring discrepancies between computational predictions and biological outcomes. Our study reveals extensive taxonomic and structural diversity of AMPs in Arctic hydrothermal vent biofilms and identifies novel candidates withbioactive potential. These findings emphasize the importance of integrating metagenomics, transcriptomics, machine learning, and experimental validation to uncover bioactive compounds from underexplored microbial ecosystems. Overall, AMOR biofilms represent a rich and untapped source of AMPs, offering new opportunities for antimicrobial drug discovery in the fight against AMR.
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