Linking Clinical and Environmental Multidrug Resistance Plasmids Captured from the Tama River Flowing Through the Tokyo Megalopolis

Background: Plasmid-mediated horizontal transfer of antimicrobial resistance genes (ARGs) is a major driver of resistance dissemination across clinical and environmental settings. Urban rivers flowing through densely populated megacities represent critical interfaces where human-associated and environmental microbiomes intersect; however, the genetic structures and functional characteristics of resistance plasmids circulating in such environments remain insufficiently resolved. Methods: In this study, we conducted detailed genomic and phenotypic analyses of 11 ARG-bearing plasmids previously captured from the Tama River, an urban river flowing through the Tokyo megalopolis. These plasmids belonged to IncN, IncU, IncQ2γ, IncC, and IncPγ groups. Whole-plasmid sequencing, comparative genomic analyses, conjugation assays, and antimicrobial susceptibility testing were employed to characterize plasmid backbones, accessory resistance regions, mobile genetic elements, and conjugative transferability. Results: A total of 11 plasmids belonging to five major incompatibility groups (IncN, IncC, IncU+IncQ2γ, and IncP) were analyzed. These plasmids collectively encoded ARGs conferring resistance to five major antimicrobial classes, including aminoglycosides, β-lactams, tetracyclines, chloramphenicol, and mercury, and frequently harbored class 1 integrons, ISCR1 elements, and Tn3-derived inverted-repeat miniature elements (TIME). Notably, two plasmids (IncN and IncC) exhibited high structural similarity to clinically reported plasmids from geographically distant regions, whereas multiple IncP plasmids and one multi-replicon IncU+IncQ2γ plasmid displayed accessory-region architectures characteristic of environmental plasmids and broad host-range transferability. Antibiotic susceptibility testing demonstrated that these plasmids substantially increased resistance levels in hosts. Conclusions: This study reveals that urban river environments can harbor both clinically related and environmentally unique multidrug resistance plasmids, shaped by diverse mobile genetic elements. By providing nucleotide-level structural and functional evidence, this study highlights urban rivers as potential ecological hubs linking clinical and environmental resistance plasmid pools and supports the importance of continued monitoring of resistance plasmids in megacity-associated river systems.