Comprehensive Study of Antibiotics and Antibiotic Resistance Genes in Wastewater and Impacted Mediterranean Water Environments

<b>Background:</b> The spread of antimicrobial resistance is a central public health problem. Wastewater treatment plants and impacted environments are well-known hotspots for antibiotic resistance. However, there is still limited knowledge regarding where antibiotic resistance genes (ARGs) acquire mobility. <b>Method:</b> In this study, we aimed to gather evidence on the seasonal patterns of ARG spread in two Mediterranean areas from NE and E of Spain (Ebro River and Ebro Delta, and Xúquer River and Albufera de València), correlating ARG presence, with special focus on the faecal bacteria <i>Escherichia coli</i>, with antibiotic residues and environmental conditions. The analytical methodology employed was based on a suspect screening approach, while a novel prioritisation approach for antibiotics was proposed to identify those areas more susceptible to the spread of ARG. <b>Results:</b> Our findings demonstrate that ARG levels in wastewater were similar across different seasons, although a greater diversity of ARGs was recorded in summer. We hypothesise that horizontal gene transfer among aquatic bacterial populations during the northeastern Mediterranean summer, when temperatures reach approximately 35~40 °C, could be a key driver of ARG dissemination. By contrast, the highest concentrations of antibiotics in winter samples, with temperatures around 5~10 °C, may promote the spread of microbial resistance. <b>Conclusions:</b> Our key findings highlight that water temperature and sunlight irradiation are crucial factors influencing antibiotic levels and microbial abundance, requiring further investigation in future studies.