Isolation and whole-genome sequencing of antibiotic-resistant bacteria revealed the reservoir for indigenous antibiotic resistance genes in the deepest ocean sediment of the Challenger Deep.

Understanding the occurrence of antibiotic-resistant bacteria (ARB) and associated antibiotic resistance genes (ARGs) in remote marine environments is crucial for accessing treats of ARG pollution on a border ecological scale. While most studies focused on anthropogenically disturbed settings, the Challenger Deep, as the deepest ocean habitat, offers a unique opportunity to investigate minimally disturbed resistomes. We revived 123 bacterial isolates from the Challenger Deep sediment, assessed their antibiotic susceptibility, and identified their taxonomy via 16S rRNA gene sequencing. Among them, 96 strains (78.0%) were resistant to at least one antibiotic, with high prevalence observed in Halomonas, Idiomarina, Flagellimonas, and Microbacterium. Resistance was most common to ampicillin (73.2%), followed by sulfadiazine (30.1%) and nalidixic acid (4.9%). Untargeted metabolomics identified 359 metabolites in the sediment sample, including 6-aminopenicillanic acid, suggesting local microbial antibiotic production and selective pressure of resistance. Anthropogenic contaminants like nalidixic acid were also detected. Whole-genome sequencing of eight representative ARB strains revealed 77 copies of 26 ARG subtypes, predominantly associated with multidrug resistance and efflux pump mechanisms. Notably, no mobile genetic elements were linked to ARGs, indicating limited horizontal gene transfer. Phylogenetic analyses showed host species specificity of ARGs, independent of geography or environmental context, supporting vertical inheritance from ancestral lineages. This study offers the first culture-based evidence of ARB and ARGs in the Challenger Deep, suggesting that resistance may represent an adaptive trait to extreme conditions and underscoring its ancient, intrinsic origin. Our findings provide critical implications for understanding the revolution and dissemination of resistance in deep-sea environments.