Widespread antimicrobial resistance in the middle Tagus River Basin: Environmental drivers and seasonal dynamics

Antimicrobial resistance (AMR) is an emerging environmental threat intensified by the continuous release of antibiotics and resistant microorganisms into aquatic systems, with Mediterranean rivers-subject to strong anthropogenic pressure and climatic stress-expected to act as important reservoirs of antibiotic-resistant bacteria (ARB) and resistance genes (ARG). Despite its ecological and socio-economic importance, AMR dynamics in the Tagus River, the longest river in the Iberian Peninsula, remain poorly characterized. This study examined microbiological water quality, fecal indicator bacteria (FIB), Acinetobacter spp., culturable ARB to four antibiotics, and the presence of ARG across 19 sites along the middle Tagus River over four seasons (2022-2023), including Natura 2000 protected areas. High and widespread levels of total bacteria, FIB, and Acinetobacter spp. were observed, with marked seasonal variability linked to hydrological conditions. β-lactam and sulfonamide-resistant bacteria were the most abundant ARB, whereas ciprofloxacin and doxycycline ARB were less prevalent and more spatially variable. Of the 13 genes screened, only blaTEM, sul2, and intI1 were detected, with blaTEM at the highest concentrations. No consistent evidence indicated that wastewater treatment plants (WWTP) acted as AMR hotspots along the studied river section. Seasonal patterns showed reduced ARB in winter, likely due to increased flows, while ARG levels peaked in spring. Temperature, pH, and conductivity significantly shaped microbial abundance and resistance trends. Overall, the widespread dissemination of ARB and ARG underscores the inadequacy of current monitoring frameworks and highlights the urgent need for strengthened surveillance and mitigation strategies to curb AMR propagation in Mediterranean river systems.