Native Aquatic Plastispheres in a River-Wastewater Catchment: Carbapenem-Resistant Bacteria Isolation and Microscopy-Based Structural Analysis.

Plastispheres, microbial biofilms formed on plastic surfaces, are increasingly recognised as ecological niches capable of transporting pollutants and antibiotic-resistant microorganisms. However, mechanistic insights into antimicrobial resistance (AMR) dynamics in natural plastispheres remain limited, particularly for priority pathogens such as carbapenem-resistant Enterobacterales (CRE). Here, we evaluated plastispheres as environmental reservoirs and vectors of carbapenem-resistant bacteria, comparing wastewater (secondary settling tanks, representing the final stage before environmental discharge) and riverine environments. Using a combined SEM-CFM approach, we resolved plastic surface topography and the spatial organisation of biofilm-associated bacteria. Although CRE were not detected, carbapenem-resistant bacteria constituted a stable fraction of heterotrophic communities in both environments and were primarily associated with intrinsic resistance mechanisms. Carbapenem-resistant isolates included Aeromonas spp. (bla), Stenotrophomonas maltophilia (bla), and Pseudomonas putida (efflux-based resistance). Microscopy revealed dense bacterial clusters on plastic surfaces, suggesting microenvironments that may facilitate cell-cell interactions, including horizontal gene transfer. These findings highlight plastispheres not only as vectors of AMR but also as potential evolutionary hotspots shaping resistance persistence and dissemination in aquatic systems. Future integrating metagenomic and genomic data on resistance gene mobility with spatially resolved microbial community structure will provide critical insights into the mechanisms and risks of AMR dissemination in plastisphere environments.