The occurrence and diversity of antibiotic resistance and virulence factor genes in wastewater from four North American treatment plants

The spread of antibiotic resistance in aquatic environments is an emerging environmental issue due to potential threats to public health. Wastewater treatment plants (WWTPs) could serve as a sink for removal of antibiotic-resistant bacteria (ARB), antibiotic resistance genes (ARGs), and virulence factor genes (VFGs) from wastewater before discharge to the environment, or an amplifier because the stress conditions prevailing in sewage treatment processes may favor the proliferation of ARB, ARGs, and VFGs. In this study, we performed metagenomic sequencing and analyses to examine the diversity of microbial communities and the fate of ARGs, antibiotic biosynthesis genes (ABSGs) and VFGs in sixteen (16) sewage samples collected from four large WWTPs located at two major metropolitan centers on the east and west coast of the United States. Multivariate analysis revealed that the diversity and composition of microbial communities and ARGs in sewage samples were primarily associated with the wastewater treatment processes. The overall relative abundances of ARGs and VFGs declined by over 20% after treatments. However, the activated sludge process (ASP) selectively enriched ARGs encoding multidrug resistance and certain VFGs as indicated by the increase of their relative abundance in mixed liquor suspended solids. The relative abundances of sub-groups of ABSGs were also substantially amplified during ASP. These findings provide insights into the impact of conventional wastewater treatment processes on the prevalence of ARGs and VFGs to better understand the dissemination of ARGs and VFGs through human sewage. Comparison of different treatment processes in relation to ARGs removal also helps to identify strategies to reduce the spread of antibiotic resistance through sewage discharge. The total ARGs and VFGs declined after treatments. Antibiotic biosynthesis genes were amplified during activated sludge processes. Multidrug ARGs dominate in wastewater.