Mitigating microbiological risks of potential pathogens carrying antibiotic resistance genes and virulence factors in receiving rivers: Benefits of wastewater treatment plant upgrade

● Abundance of MAGs carrying ARG-VF pairs unchanged in rivers after WWTP upgrade. ● Upgrade of WWTPs significantly reduced diversity of pathogenic genera in rivers. ● Upgrade of WWTPs reduced most VF (ARG) types carried by potential pathogens in rivers. ● Upgrade of WWTPs narrowed the pathogenic host ranges of ARGs and VFs in rivers. Wastewater treatment plants (WWTPs) with additional tertiary ultrafiltration membranes and ozonation treatment can improve water quality in receiving rivers. However, the impacts of WWTP upgrade (WWTP-UP) on pathogens carrying antibiotic resistance genes (ARGs) and virulence factors (VFs) in rivers remain poorly understood. In this study, ARGs, VFs, and their pathogenic hosts were investigated in three rivers impacted by large-scale WWTP-UP. A five-year sampling campaign covered the periods before and after WWTP-UP. Results showed that the abundance of total metagenome-assembled genomes (MAGs) containing both ARGs and VFs in receiving rivers did not decrease substantially after WWTP-UP, but the abundance of MAGs belonging to pathogenic genera that contain both ARGs and VFs (abbreviated as PAVs) declined markedly. Genome-resolved metagenomics further revealed that WWTP-UP not only reduced most types of VFs and ARGs in PAVs, but also effectively eliminated efflux pump and nutritional VFs carried by PAVs in receiving rivers. WWTP-UP narrowed the pathogenic host ranges of ARGs and VFs and mitigated the co-occurrence of ARGs and VFs in receiving rivers. These findings underline the importance of WWTP-UP for the alleviation of pathogens containing both ARGs and VFs in receiving rivers.