Illuminating Antarctic Microbial Dark Matter: In situ Cultivation Reveals Rare Taxa with Broad Resistome and Virulence Phenotypes.

Glacial forelands in Maritime Antarctica are rapidly expanding due to deglaciation, thereby increasing ecological connectivity among previously isolated microbial reservoirs. From a One Health perspective, this process is relevant because environmental microorganisms represent long-standing reservoirs of antibiotic resistance and virulence-associated traits. However, current knowledge is mainly limited to dominant, fast-growing taxa recovered by conventional cultivation, leaving the functional potential of slow-growing and rare Antarctic bacteria largely unexplored. Here, we compared traditional plating methods (PM) and in situ cultivation (ISC) for recovering bacteria from permafrost, moraine, and rhizosphere soils, quantified total culturable diversity using 16S rRNA metabarcoding, and assessed antibiotic susceptibility and phenotypic virulence across 158 nonredundant isolates. Cultured communities were dominated by Pseudomonadota, Actinomycetota, Bacteroidota, and Bacillota, with Pseudomonas among the most prevalent genera, although its relative abundance varied across soil compartments and cultivation strategies. ISC expanded the taxonomic breadth of cultured bacteria and enabled the recovery of rare or underrepresented genera absent from PM collections, including Bosea, Rathayibacter, Rugamonas, Massilia, Phyllobacterium, Polaromonas, and Curtobacterium. Functional profiling revealed a widespread environmental resistome, with high frequencies of resistance to β-lactams, cephalosporins, and oxazolidinones. Notably, isolates affiliated with Flavobacterium, Brucella, Pseudomonas, and Curtobacterium exhibited extreme multidrug resistance, with individual strains resistant to more than 20 antibiotics. Virulence-associated phenotypes were broadly distributed across cultured taxa, including lecithinase and hemolytic activity (>35% of isolates), pyocyanin production (∼20%), and DNase activity (∼7%). Together, these results demonstrate that in situ cultivation provides access to underrepresented components of Antarctic microbial diversity and reveals resistance- and virulence-associated traits beyond those of dominant taxa. Antarctic glacier foreland soils, therefore, represent underexplored environmental reservoirs of health-relevant microbial functions, underscoring the value of culturomics for environmental health research amid climate-driven change.