Impact of human lifestyle on the pathogenic potential of urban wastewater

Domestic wastewater has been known for its pathogenic potential including the presence of pathogenic bacteria, virulence factor genes (VFGs) and antibiotic resistance genes (ARGs). While previous studies have investigated regional differences in microbial communities, the influence of population lifestyle factors on the pathogenic potential of wastewater microbiomes remains poorly understood. In this study, we analyzed ten Australian wastewater treatment plants (WWTPs) using Nanopore metagenomic sequencing to profile pathogens, ARGs, and VFGs, and examined their associations with health-related behaviors such as smoking, alcohol consumption, and obesity. A total of 196 pathogenic species, 951 ARG subtypes, and 380 VFGs were detected. Staphylococcus aureus was the most abundant pathogen, macB the dominant ARG, and lipooligosaccharides (LOS) the most prevalent VFG. While pathogen and ARG profiles were relatively stable across sites and showed limited association with lifestyle variables, VFG profiles demonstrated significant correlations with smoking rates, alcohol consumption, and nicotine levels in wastewater. Co-occurrence network analysis revealed strong interconnectivity among VFGs and between VFGs and ARGs, suggesting potential co-selection. Principal component analysis and diversity metrics further confirmed distinct patterns in VFG distribution across sites. These findings suggest VFGs as potentially sensitive indicators of behavioral health risks in wastewater-based epidemiology.