5,000-Year-Old Cave Bacterium Resists Antibiotics and Kills Pathogens

By Pooja Toshniwal PahariaReviewed by Lauren HardakerFeb 26 2026

A millennia-old microbe preserved in cave ice reveals a surprising double life, harboring a broad array of resistance genes while producing compounds that suppress modern-day pathogens.

Image credit: dirtyclown/Shutterstock.com

In a recent study published in Frontiers in Microbiology, researchers uncovered a remarkable microbial survivor preserved in 5,000-year-old ice from Romania’s Scărișoara Ice Cave. The team isolated and sequenced a novel strain of Psychrobacter, SC65A.3, revealing an ancient, ice-adapted bacterium, with an unexpected dual identity: it carries multidrug resistance (MDR) genes while also demonstrating antimicrobial activity.

Whole-genome sequencing (WGS) analysis showed a layered resistance architecture, highlighting ice caves as overlooked reservoirs of ancient resistomes and biologically active traits with potential clinical relevance.

Cave Ice Preserves Ancient Multidrug Resistance Genes

Frozen habitats cover vast areas of the planet, making cold-adapted microorganisms vital for understanding life in extreme environments, particularly amid accelerating climate change. Ancient ice deposits can preserve diverse psychrophilic microbes with unique metabolic traits and significant biotechnological promise. However, compared with permafrost and polar regions, cave-associated ice systems remain relatively understudied.

These environments may also shelter ancient antimicrobial resistance (AMR) determinants, offering insight into the evolutionary origins of resistance amid today’s global AMR crisis. Notably, species within the genus Psychrobacter combine cold-active enzymatic potential with emerging evidence of pathogenicity, although their antibiotic resistance profiles remain poorly explored.

Genomic and Phenotypic Profiling of SC65A.3

In the present study, researchers performed a comprehensive phenotypic and genomic characterization of Psychrobacter sp. SC65A.3 to investigate the biological and resistance features of the ancient isolate.

The team isolated the strain at 4 °C and assessed its growth range between 4 °C and 15 °C and salinity conditions, tolerating up to approximately 1.9 M NaCl and 0.9 M MgCl₂, to evaluate extremotolerance. They extracted genomic deoxyribonucleic acid (DNA) for sequencing the 16S ribosomal ribonucleic acid (rRNA) gene to confirm taxonomic identity.

The researchers determined antimicrobial susceptibility using the Kirby–Bauer disk diffusion method against 28 antibiotics from 17 classes. They interpreted zone diameters using breakpoints established by the European Committee on Antimicrobial Susceptibility Testing (EUCAST 2020) and the Clinical and Laboratory Standards Institute (CLSI 2021).

They applied criteria from closely related taxa, including Acinetobacter, Moraxella catarrhalis, and Pseudomonas species, when species-specific standards were unavailable, noting that no Psychrobacter-specific clinical breakpoints exist and that resistance classifications should therefore be interpreted cautiously.

The team further evaluated antimicrobial activity by testing SC65A.3 extracts against reference and clinical bacterial strains, assessing inhibition zone formation after 18 hours at 37 °C. They profiled enzymatic activity to quantify substrate hydrolysis. Lastly, genome assembly and annotation enabled the identification of coding sequences and resistance determinants. Researchers used the evolutionary gene genealogy Non-supervised Orthologous Groups (EggNOG) database for functional classification, average nucleotide identity (ANI) values for species comparison, and the Genome Taxonomy Database (GTDB) for phylogenetic characterization.

Ancient Strain Resists Drugs Yet Inhibits Pathogens

Psychrobacter SC65A.3 grew at low temperatures (4 °C and 15 °C) and tolerated elevated salinity, including 0.9 M MgCl₂ and 1.9 M NaCl. The findings are consistent with a psychrophilic and moderately halophilic lifestyle. 16S rRNA gene sequencing revealed 97% similarity with P. glaciei and P. cryohalolenti, supporting an evolutionary link to polar and marine environments. Phylogenomic analysis and ANI values (~96.6%) supported assignment within P. cryohalolentis rather than designation as a new species.

Functionally, SC65A.3 expressed several hydrolytic enzymes, including phosphatases, lipases, and esterases. The strain also demonstrated metabolic versatility through urea hydrolysis, citrate assimilation, nitrate reduction, and β-glucosidase activity. The diverse enzymatic profile highlights its potential for applications in biotechnological, pharmaceutical, and environmental applications.

Functional assays demonstrated MDR, with resistance to 10 of 28 antibiotics tested across 8 antimicrobial classes, confirming a multidrug-resistant phenotype. WGS identified 107 AMR-associated genes, including those linked to β-lactams, tetracyclines, fluoroquinolones, aminoglycosides, and colistin. Although the genome encoded the colistin resistance gene mcr1, colistin susceptibility was not phenotypically tested.

Genomic findings further revealed determinants associated with resistance to heavy metals and active drug efflux systems. Compared with other Psychrobacter bacteria listed in the National Center for Biotechnology Information (NCBI) Taxonomy Database, SC65A.3 exhibits one of the broader resistance gene repertoires reported among currently sequenced Psychrobacter strains, encompassing genes associated with antibiotic and toxic compound resistance.

The SC65A.3 strain inhibited multiple ESKAPE pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa, but showed no inhibitory effect against the tested Acinetobacter baumannii strains. Overall, extracts inhibited 14 of the 22 strains tested, including reference and clinical isolates. The findings align with genomic signatures indicative of antimicrobial agents such as bacitracin and glycopeptides.

The genome also encoded 45 genes involved in stress adaptation, including htpG, htpX, and pka. Most genes are associated with heat-shock rather than cold-shock processes, suggesting complex thermal adaptation strategies. Although many annotated genes were related to amino acid transport, metabolism, and energy production, several genes were classified as having unknown functions. The findings underscore the untapped biological potential of this psychrophilic Psychrobacter from an underexplored icy habitat.

Frozen Ecosystems Hold Untapped Biomedical Potential

The study provides the first combined genomic and functional analysis of a millennia-old cave-ice resistome derived from Psychrobacter sp. SC65A.3, phylogenetically placed within P. cryohalolentis, recovered from Scărișoara Ice Cave. This psychrophilic bacterium exhibits a unique combination of MDR, antimicrobial activity, cold-active enzymes, and distinctive thermal adaptation genes.

Its broad AMR repertoire highlights the role of environmental bacteria as potential reservoirs of clinically relevant resistance determinants, particularly under selective pressure.

As the first whole-genome sequence of a cave-ice member of the genus Psychrobacter, SC65A.3 advances understanding of the evolution of ancient resistomes while highlighting the medical and biotechnological promise of understudied icy ecosystems.

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Journal Reference

Paun, V. I., Itcus, C., Lavin, P., Chifiriuc, M. C., & Purcarea, C. (2026). First genome sequence and functional profiling of Psychrobacter SC65A.3 preserved in 5,000-year-old cave ice: Insights into ancient resistome, antimicrobial potential, and enzymatic activities. Frontiers in Microbiology, 16, 1713017. DOI: 10.3389/fmicb.2025.1713017. https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2025.1713017/full