Insights into the riverine metaplasmidome: The role of anthropogenic factors in shaping microbial communities

The environmental plasmidome, encompassing the collective plasmids within an ecosystem, plays a key role in microbial adaptation and evolution. Plasmids, as mobile DNA molecules, enable the transfer of beneficial traits among bacteria, especially in response to environmental stresses. In this study, we developed a novel protocol combining PlasmidSafe DNase and REPLI-g amplification to selectively enrich and characterize plasmidic DNA from polluted environments. We applied this approach to the Virilla River (Costa Rica), a system heavily impacted by urban and industrial activities, analyzing water and sediment samples across seasons and pollution gradients. Our method enabled the recovery of longer, circular contigs confidently identified as plasmids through multiple independent tools, with 28,043 circular sequences primarily linked to Gammaproteobacteria. Contaminated sites, particularly during the rainy season, showed higher plasmid diversity and a marked increase in antibiotic resistance genes (ARGs). This heightened diversity in contaminated environments has implications for the dissemination of antibiotic and metal-resistance genes, as well as other adaptive traits within microbial communities. This research underscores the need to study plasmids in natural environments to better comprehend their ecological roles and potential risks in polluted ecosystems. • A novel plasmid extraction and analysis protocol was developed for polluted environments. • Plasmid diversity and antibiotic resistance genes increased with pollution levels. • The study reveals how human impact shapes plasmid-driven microbial adaptation.