Investigating the distribution of antibiotic resistance genes in relation to bacterial, fungal, and functional diversity in a hay field

amplicon sequencing to measure bacterial and fungal diversity, respectively, in combination with whole-genome sequencing to characterize functional and ARG diversity. We find increasing bacterial and functional diversity along the transect, as well as distinct community structures for both bacteria and fungi. While we find that the diversity of environmental ARGs is significantly correlated with both bacterial and fungal diversity, clinical ARG diversity significantly decreased as fungal diversity increased. Our results suggest that while bacterial diversity increases with distance from the road and stream, this diversity is correlated with the diversity of the environmental ARGs, this trend is not observed for ARGs of clinical relevance, which appear to be largely driven by the variety of fungal groups in the environment.IMPORTANCEAntibiotic resistance is often studied in hospitals and clinical settings, but much less is known about how resistance genes are distributed in everyday environments, such as agricultural soils. Hay fields are widespread, actively managed ecosystems that sit at the interface of natural microbial communities, farming practices, and food systems. In this study, we examine how antibiotic resistance genes are associated with broader patterns of bacterial diversity, fungal communities, and soil functional potential in a working hay field. By integrating resistance gene profiles with ecological measures of microbial diversity, we move beyond simply cataloging resistance and instead place it within a community and ecosystem context. This approach helps clarify whether resistance genes are linked to specific microbial groups, overall biodiversity, or functional traits related to soil processes. Our findings provide insight into how antibiotic resistance persists and is structured in low-input agricultural systems, contributing to a more complete understanding of environmental reservoirs of resistance and informing discussions about sustainable land management and public health risk.