Urban wastewater and sewage sludge as environmental sources of antimicrobial resistance, potential pathogens, and selective agents: insights from three European wastewater treatment plants.

Antimicrobial resistance (AMR) is a growing global human health threat, and it is increasingly recognized as a One Health issue, complicated by the complex interactions between humans, animals, and the environment. Wastewater treatment plants (WWTPs) link human activities with the environment and may act as a reservoir of AMR, facilitating environmental dissemination. This study investigated bacterial communities, antibiotic resistance genes (ARG), and antibiotic susceptibility in wastewater and digested sludge at three WWTPs in Norway and Greece, representing regions with low and high antibiotic consumption and clinical AMR prevalence. Antibiotic residues and potentially toxic metals were also quantified. Of 21 antibiotics analyzed, 13 were detected in at least one sample, with ciprofloxacin, cefotaxime, and meropenem occasionally exceeding predicted no-effect concentrations. Removal efficiencies varied, with limited removal at the Norwegian WWTPs compared with the Greek facility. Potentially toxic metals were detected at all sites, with exceeded minimum co-selective concentrations for Cu, Ni, Pb, and Zn. Bacterial community composition was influenced by location, sample type, and sampling time point (p<0.05), and was associated with antibiotic concentrations. Removal of fecal and environmental indicator bacteria was 3.1 ± 1.4 log CFU/100 mL at the Greek WWTP but negligible at the Norwegian WWTPs. Most ARGs (79%) were detected across all WWTPs, and clinically important resistance genes were also prevalent in outlet and digested sludge samples, suggesting considerable environmental spread. Antibiotic susceptibility testing revealed reduced susceptibility to all tested antibiotics, including 3rd generation cephalosporins, meropenem, and last-resort antibiotics, although enumeration and susceptibility testing of environmental indicator bacteria was limited by methodological constraints. These findings demonstrate that WWTPs can contribute to the environmental dissemination of ARB and ARG in both high and low antibiotic consumption and clinical AMR regions, highlighting the need for effective wastewater treatment, improved environmental surveillance and integrated One Health approaches linking human and environmental AMR compartments.