BACKGROUND: Antibiotic resistance genes (ARGs) are proliferating in wastewater microbiomes, yet the biotic forces shaping their diversity remain poorly understood. Here, we integrate 14 months of metagenomic and metatranscriptomic data from a wastewater treatment plant to reveal that viruses and microeukaryotes, long-overlooked trophic actors, may play an important role in shaping bacterial and ARG diversity.

RESULTS: We show that viral and microeukaryotic communities exhibit strong seasonal dynamics that cascade through the microbial food web, significantly structuring prokaryotic communities and subsequently ARG profiles. Crucially, we find that viral and microeukaryotic diversity are positively associated with bacterial diversity, which in turn shapes ARG diversity, underscoring the regulatory potential of ecological interactions.

CONCLUSIONS: Our findings challenge the abiotic-centric paradigm and establish the central role of multi-trophic interactions in shaping ARG dynamics in wastewater ecosystems. Video Abstract.