Host-specific and community-driven dynamics of plasmid transfer and stability in activated sludge

Plasmid-mediated transfer of antibiotic resistance genes in activated sludge presents a significant public health concern, yet the dynamics of plasmid transfer within these complex microbial communities remain poorly understood. In this study, we investigated the conjugation behavior of RP4 plasmids in activated sludge, focusing on host range, stability and microbial community effects. By developing a novel identification method, we identified a range of different RP4 transconjugates, including Burkholderia, Escherichia, Klebsiella and Brevardimona from activated sludge. Serial passage experiments revealed that even under environmental stress, RP4 plasmids exhibit significant instability, with rapid loss within approximately 50-150 generations. Environmental stress increases reactive oxygen species, which increases cell membrane permeability and facilitates plasmid transfer, but may also lead to plasmid damage. Microcosm experiments demonstrated that activated sludge microbiomes enhance RP4 plasmid persistence possible by a "community buffering effect". These findings underscore the role of microbial communities in promoting plasmid retention in activated sludge systems. By elucidating species-specific and community-level mechanisms of plasmid maintenance, this study provides new insights into plasmid transfer in wastewater treatment systems with implications for mitigating associated public health risks.