Mapping plasmid-host networks and tracking persistent plasmids in wastewater microbiomes

Background Plasmids are major drivers of horizontal gene transfer (HGT), playing a central role in disseminating antimicrobial resistance in water resource recovery facilities (WRRFs). Most studies have focused on model or clinical plasmids in simplified settings, leaving their in situ dynamics in complex environmental communities poorly understood. To address this gap, we applied Hi-C metagenomic sequencing to systematically resolve plasmid-host associations and evaluate how environmental and operational factors influence plasmid persistence and host range across treatment stages and geographic regions in municipal WRRFs. Results We identified 944 plasmid clusters across influent, activated sludge, and effluent samples from three WRRFs located in three U.S. states, revealing distinct plasmid distribution patterns and plasmid-host associations across facilities and treatment stages. While overall bacterial community composition remained relatively stable across treatment stages, plasmid-host interactions varied, indicating that environmental conditions and treatment processes influenced plasmid retention and transfer. Plasmids in influent and activated sludge exhibited broader host ranges relative to effluent, where, plasmids were associated with a narrower set of bacterial hosts, likely reflecting the impacts of disinfection. Notably, certain plasmids persisted across treatment stages but exhibited substantial shifts in their associated bacterial hosts. Key taxa such as Burkholderiaceae and Rhodocyclaceae remained abundant throughout, indicating that shifts in plasmid-host associations were not solely driven by community turnover, but also suggesting HGT. Supporting this, functional profiling of effluent plasmids revealed enrichment in conjugation-related genes and virulence factors, including oxidative stress resistance, which may facilitate plasmid persistence and dissemination. Conclusions Our findings reveal that while wastewater microbial communities are diverse, plasmid-associated hosts are restricted to a few dominant bacterial families. Plasmid host range consistently narrows across treatment stages, reflecting the effectiveness of WRRFs in limiting plasmid persistence. However, shifts in host associations among highly similar plasmid clusters suggest potential HGT events. These results highlight the role of specific bacterial groups in plasmid dissemination and underscore the need for future studies to identify keystone taxa and mechanisms driving plasmid stability in complex microbial communities.