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

Background. The global spread of antimicrobial-resistant bacteria is largely driven by plasmid-mediated transfer of antimicrobial resistance genes (ARGs). While plasmids carrying ARGs have been extensively characterized in clinical settings, their distribution, diversification processes, and dissemination dynamics in urban river environments, key interfaces between natural and human-associated microbiomes, remain poorly resolved. Methods. To elucidate the characteristics of environmental resistance plasmids, we performed detailed genomic and phenotypic analyses of 11 ARG-bearing plasmids previously captured from the Tama River, Japan. These plasmids belonged to IncN, IncU, IncQ2γ, IncC, and IncPγ groups. We examined plasmid backbones, accessory resistance regions, mobile genetic elements, and conjugative transfer ability, and compared these features with plasmids reported from clinical, agricultural, and environmental sources worldwide. Results. The captured plasmids harbored diverse ARGs, including those conferring resistance to aminoglycosides, β-lactams, tetracyclines, chloramphenicol, and mercury, and frequently carried transposons (ISCR1 elements) and class 1 integrons, and Tn3-derived inverted repeat miniature elements (TIMEs) that assembled multidrug resistance regions. Intriguingly, an IncN plasmid (pMNBM072) and an IncC plasmid (pMNBL073) exhibited highly similar backbone and accessory resistance structures to clinically reported plasmids from geographically distant regions. In contrast, the multi-replicon plasmid pMNBM065-2 (IncU+IncQ2γ) and several IncPγ plasmids displayed accessory region architectures characteristic of environmental plasmids and showed broad host-range transferability. Antibiotic susceptibility testing demonstrated that these plasmids substantially increased resistance levels in Escherichia coli hosts. Conclusions. This study reveals that urban river environments harbor both clinically related and environmentally unique multidrug resistance plasmids, shaped by diverse mobile genetic elements. More importantly, our findings provide new evidence linking clinical environments and urban river environments, highlighting the urgent need for global surveillance of environmental plasmids and ARGs in urban rivers.