This study investigates the plasmidome of Costa Rica's heavily polluted Virilla River, which is significantly affected by urbanization and industrial activities. Water and sediment samples were collected from multiple sites, resulting in a total of 12 samples analyzed in this study. The plasmidome sequencing was performed using the Illumina NovaSeq 6000 platform.

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. This study examines the plasmidome of Costa Rica's heavily polluted Virilla River, impacted by urbanization and industrial activities. We analyzed water and sediment samples across various sites to understand how pollution affects plasmid diversity and characteristics. By employing protocols for plasmidic DNA extraction, high-throughput sequencing, and ad hoc bioinformatics pipelines, we discovered that contaminated sites exhibit increased plasmid diversity, with the highest species richness observed at the most polluted site during the rainy season. Different bioinformatics methods were applied at each step, enhancing the accuracy, reliability, and robustness of the results, which in turn provided more reliable biological insights. The most effective circular detection method identified 28,043 circular contigs, predominating plasmid hosts associated with Gammaproteobacteria. Our findings suggest that polluted urban environments may promote the formation of longer plasmidic contigs. The study also revealed a significant increase in antibiotic resistance genes (ARGs) at highly polluted sites, particularly in freshwater samples and during the rainy season. 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.