Abstract

Introduction: Ancient cryospheric environments may preserve overlooked reservoirs of antimicrobial resistance (AMR) and bioactive potential. This study reports the first whole-genome sequencing and functional characterization of Psychrobacter sp. SC65A.3 isolated from 5,000-year-old ice from Sc & abreve;risoara Ice Cave, revealing a multidrug-resistance phenotype alongside antimicrobial activity. Methods: Whole-genome sequencing combined with phenotypic characterization for extremotolerance, antibiotic susceptibility and biochemical profile were used to identify and functionally characterize the ancient Psychrobacter sp. SC65A.3. Results: SC65A.3 is a polyextremophile, growing up to 15 degrees C and tolerating 1.9 M NaCl and 0.9 M MgCl2. Phylogenetic analysis classified it within P. cryohalolentis. Functional assays showed broad hydrolytic activity and resistance to 10 antibiotics across 8 classes, including third-generation cephalosporins, fluoroquinolones, aminoglycosides, and rifampicin. Whole-genome analysis identified >100 AMR-associated genes, including clinically relevant determinants (e.g., ampC, gyrA, gyrB, parC, parE, dfrA, rpoB, tetA, tetC, and mcr-1), as well as multiple heavy-metal resistance and multidrug efflux genes. SC65A.3 inhibited 14 ESKAPE-group pathogens (including MRSA, Enterococcus faecium, Enterobacter sp., Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii), consistent with genes linked to antimicrobial compounds such as glycopeptides and bacitracin. In addition, 45 stress-response genes related to cold/heat adaptation were detected, including distinctive htpX, htpG, and pka genes among cold-adapted Psychrobacter. Discussion: SC65A.3 represents an ancient, ice-adapted Psychrobacter with a dual profile of multidrug resistance and antimicrobial activity, highlighting ice caves as underexplored reservoirs of ancient resistomes and bioactive traits. To our knowledge, this is the first genome analysis of a Psychrobacter isolate from an ice cave and the first characterization of an ancient resistome from this environment, supporting future ecological, biotechnological, and medical exploration.