Tracking antibiotic resistance and respiratory viruses in municipal wastewater: Site-specific dynamics from two wastewater treatment plants and an upstream Sewershed community

The rise of antimicrobial resistance (AMR) and the ever-increasing prevalence of respiratory viruses pose serious public health threats, necessitating innovative wastewater-based epidemiology (WBE) strategies. We monitored and quantified three β-lactamase resistance genes (bla_GES, bla_IMP-27, & bla_OXA-48), one integrase gene (intI1), and two respiratory viruses (influenza A and SARS-CoV-2) in raw wastewater samples from two wastewater treatment plants (WWTPs) and a university campus in Ontario over five months. Using real-time qPCR, we detected absolute abundances of the antibiotic-resistance genes (ARGs) and intI in the range of 10⁴ to 10⁷ copies/mL, while influenza A and SARS-CoV-2 reached up to 10² and 10³ copies/mL, respectively. Statistical analyses revealed significant site-specific differences in gene abundances (p < 0.001 to p < 0.05), with bla_GES and intI1 detected in 100% of samples compared to bla_IMP-27 with lower detection (12.1-27.6%). Microbial community profiling via 16S rRNA analysis revealed distinct profiles across sites, with the university campus showing the highest diversity. Notably, a transient increase in gene relative abundance was observed following the influenza A wave in late 2022, suggesting a link between viral activity and resistance dynamics. These findings advance our understanding of the environmental dimension of AMR and respiratory virus interactions, supporting the integration of wastewater surveillance into public health strategies to manage infectious disease outbreaks and curb resistance proliferation.