Genetic diversity of dissolved free extracellular DNA compared to intracellular DNA in wastewater treatment plants

Dissolved free extracellular DNA (free-exDNA) coexists with intracellular DNA (inDNA) in aquatic environments. Free-exDNA can be taken up by bacteria through transformation, and wastewater treatment plants (WWTPs) are positioned as potential hot spots for genetic contamination. However, studies comparing the composition of free-exDNA and inDNA is limited. This study employed colloidal adsorption and foam concentration method to recover free-exDNA from different WWTP stages and compared its diversity with inDNA via metagenomic analysis. Free-exDNA concentrations were observed to increase after chlorination. Genetic analysis revealed a higher abundance of specific genes following chlorination, suggesting that free-exDNA in effluent originated from bacterial death in secondary treated water. This result indicates that free-exDNA, which increases due to chlorination, is subsequently released into the catchment. Additionally, several high-risk antibiotic-resistance genes (ARGs) were detected that colocalized with mobile genetic elements. These ARGs were expected to have a high potential for gene transfer via transformation, and the risk was highlighted. Overall, these findings deepen our understanding of horizontal gene transfer risks in WWTPs.