Characterizing Aeromonas spp. as a Potential Sentinel Organism for Antimicrobial Resistance Dissemination in Wastewater and Drinking Water Treatment Systems: A Case Study in the Barcelona Metropolitan Area, Spain

<b>Background:</b> Wastewater treatment plants (WWTPs) are hotspots of antimicrobial resistance (AMR) due to inputs from diverse anthropogenic sources. <i>Aeromonas</i> spp., ubiquitous in aquatic environments, often carry clinically relevant antibiotic resistance genes (ARGs) and can persist beyond fecal contamination indicators, making them promising sentinel organisms for AMR dissemination. The aim of this study was to assess the suitability of <i>Aeromonas</i> spp. in this role by characterizing resistance profiles, associated virulence factor genes (VFGs), genetic mobility, and persistence across wastewater and drinking water treatment processes in the Barcelona metropolitan area, Spain. <b>Methods</b>: Isolates were phenotypically characterized and screened for ARGs, VFGs, integrons, and heavy metal tolerance genes, followed by whole-genome sequencing (WGS). Biofilm formation was assessed in vitro. Conjugation assays with <i>Escherichia coli</i> evaluated horizontal gene transfer (HGT) potential. <b>Results:</b> A total of 428 antibiotic-resistant <i>Aeromonas</i> spp., the most abundant antibiotic-resistant bacteria isolated during the 2023 sampling campaigns from two WWTPs and one drinking water treatment plant (DWTP), were characterized. Trimethoprim/sulfamethoxazole (SXT) non-susceptibility was most frequent (72%), followed by cefoxitin resistance (65.4%). The <i>sul1</i> (57.5%) and <i>bla</i>_MOX (78.6%) genes predominated among SXT- and β-lactam-resistant isolates. The <i>merA</i> gene was detected in 23.6%; 97.9% harbored at least one VFG (<i>aerA</i>, <i>act</i>, <i>fla</i>, <i>alt</i>, or <i>hlyA</i>), and 70.3% carried <i>intI1</i>. Half formed biofilm. Conjugation confirmed bi-directional HGT, and WGS revealed persistent ST3458 clones across treatment stages. <b>Conclusions:</b> WWTPs and DWTPs act as reservoirs of antibiotic-resistant <i>Aeromonas</i> spp., demonstrating persistence and HGT potential. Findings support their use as sentinel organisms for AMR surveillance in aquatic environments and for assessing treatment efficacy, highlighting variability across treatment types and locations, and reinforcing their relevance for urban water reclamation monitoring.