Longitudinal cross-species transmission of microbiomes and resistomes across farmers, animals and environment.

Understanding the acquisition and dissemination of microbiomes and antimicrobial resistance genes (ARGs) that circulate across human-animal-environment interfaces remains a central One Health challenge, largely because of complex ecological interactions and multiple confounding factors. Although occupational exposure is known to influence the microbiomes and resistomes of farmers, how environmental compartments involve in this system is unclear. Here, we conducted a one-year longitudinal study combining strain-resolved metagenomics (500 metagenomes) with isolate-based whole-genome sequencing (28 isolates) in an ecologically managed, antibiotic-free farming ecosystem spanning animals, farmers, environmental compartments and non-exposed individuals. Assembling 6,075 species-level genomes, we show that animal-associated occupancy reshapes the microbiome and resistome of occupationally exposed farmers and their surrounding environments. Animals and their associated habitats formed the dominant interface for both strain sharing and ARG dissemination across connected ecological compartments, whereas village residents and surrounding river samples - used as ecological controls - showed limited integration into this sharing network. Tracking a frequently shared lineage further revealed within-lineage genetic turnover together with selection-consistent changes following cross-species spread, suggestive of ecological selection across hosts and habitats. Finally, we identify Klebsiella pneumoniae as the most widespread ESKAPE pathogen in this ecosystem, with repeated occurrence across animal, human and environmental compartments, consistent with a neglected but clinically critical broad profile of ecological generalist. Together, these findings identify animals as central interfaces for microbiome and resistome sharing and show how agricultural ecosystems can sustain circulation of opportunistic pathogens and resistance determinants across human-animal-environment interfaces even in the absence of routine antibiotic use.