Effects of activated sludge processes on bacterial communities and antibiotic resistance assessed by intra- and extracellular DNA analysis

Wastewater treatment plants are widely recognized as hotspots for antibiotic resistance. Although activated sludge processes are not designed to lower the abundance of pathogens or antibiotic resistance genes (ARGs), their effects on the pathobiome and antibiotic resistome require in-depth investigation. To this end, we collected wastewater samples before and after activated sludge process from five municipal wastewater treatment plants, each characterized by different inlet composition and treatment capacity. We extracted both intracellular and extracellular DNA and performed shotgun sequencing to characterize the bacterial community, pathobiome, and antibiotic resistome. Our aim was to assess the effects of activated sludge processes on bacterial community composition, the abundance of potentially pathogenic bacteria and high-risk ARGs, and the potential horizontal mobility of detected ARGs. Our results showed that activated sludge processes significantly reduced the abundance of potentially pathogenic bacteria and several high-risk ARGs. Notably, while the prevalence of plasmid-associated ARGs decreased following treatment, ARG-carrying contigs assigned to bacteriophages increased, particularly in extracellular DNA samples. Overall, activated sludge processes demonstrated a beneficial microbiological effect by lowering potentially pathogenic bacteria. However, the enrichment of viral particles carrying ARGs highlights a potential risk for ARG spreading during the following processes. These findings underscore the importance of analyzing both intracellular and extracellular DNA to fully understand the role of activated sludge in mitigating antibiotic resistance and pathogens in wastewater.