Context. Extracellular DNA (exDNA) is an emerging contaminant in wastewater which cannot be removed efficiently due to their small molecular size and poor settleability. Hence it can be disseminated into environment by water reuse.
Gap. The movement of exDNA across wastewater treatment is unknown, particularly for critical genes like antibiotic resistance genes (ARGs) and virulence factor genes (VFGs). Besides, current studies were limited to simple detection of these genes, which lacked investigation of their mobility by plasmids/phages across wastewater treatment.
Aim. Investigate ARGs/VFGs that are present at different stages of the wastewater treatment plant (WWTP), with emphasis on those ARGs/VFGs associated with plasmids/phages.
Methods. Samples of WWTP, namely influent, MBR effluent and chlorinated effluent, were collected monthly over a year. ExDNA were isolated and metagenomic sequencing was conducted to determine the presence of ARGs and VFGs in exDNA across the wastewater treatment. ARGs/VFGs associated with plasmid/phage from exDNA of MBR effluent were detected. To determine the mobility of these genes, their presence in intracellular DNA (inDNA) was also elucidated.
Findings. Alpha diversity of extracellular ARGs/VFGs were higher in MBR effluent compared to influent and chlorina effluent. 11 ARGs and 2 VFGs associated with plasmids/phages in exDNA of MBR effluent were also detected in inDNA of influent, suggesting activated sludge process lysed bacterial hosts to release these genes to extracellular environment. These genes were subsequently detected in inDNA and exDNA of chlorinated effluent, suggesting potential gene transfer to competent hosts. Two ARGs, blaTEM and blaOXA, were associated with exDNA of MBR effluent and chlorinated effluent, and exhibited novel variants after chlorination.
Utilization. This study offered a pipeline to include plasmid/phage into the investigation of exDNA in wastewater treatment. Detection of extracellular ARGs/VFGs with plasmids/phages elucidated the risk of exDNA as mediator for ARGs/VFGs across wastewater treatment.
