Free-floating extracellular DNA (exDNA) in different wastewaters: Status quo on exDNA-associated antimicrobial resistance genes*

Wastewater treatment plants (WWTPs) have been reported as major anthropogenic reservoirs for the spread of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) into the environment, worldwide. While most studies mainly focus on the intracellular DNA (iDNA), extracellular DNA (exDNA) accounting for a significant proportion of the total DNA in wastewater, was usually neglected. Following the One Health approach, this study focuses on wastewaters of municipal, clinical, and livestock origins (n = 45) that undergo different treatment processes (i.e., conventional activated sludge, ultrafiltration, and ozonation). Water samples were analysed for 12 ARGs as indicators of the different compartments associated with iDNA and exDNA by quantitative real-time PCR (qPCR). Taxonomic profiling of exDNA-fractions, obtained using nucleic acid adsorption particles, was conducted by sequencing the V3-V4 hypervariable regions of the 16S rRNA gene. Notified exDNA concentrations varied between on-site WWTPs and treatment stages, and ranged from 314.0 +/- 70.2 ng/mL in untreated livestock wastewater down to 0.7 +/- 0.1 ng/mL in effluents after ultrafiltration. In general, influents exhibited higher concentrations compared to effluents, while wastewater treated by advanced treatment processes (i.e., ultrafiltration and ozonation) showed the lowest exDNA concentrations. Despite the lower concentrations, free-floating exDNA accounted for up to 80.0 +/- 5.8% of the total DNA in effluents. Target ARGs were more common in the iDNA (100%, n = 45/45), compared to the exDNA-fractions (51.1%, n = 23/ 45), whereas exDNA-ARGs were mostly detected in clinical and slaughterhouse wastewaters as well as in the municipal influents. Compared to the iDNA-ARGs, the concentrations of exDNA-ARGs were in general lower. Nevertheless, significant higher concentrations for exDNA-associated genes were measured in clinical wastewaters for blaNDM (4.07 +/- 0.15 log gene copies (GC)/L) and blaVIM-2 (6.0 +/- 0.2 log GC/L). Overall, our results suggest that depending on the origin of wastewater and its treatment methods, exDNA represents an important reservoir for ARGs, particularly in clinical wastewater.

Automated summary

Wastewater treatment plants are major reservoirs for the spread of antibiotic-resistant bacteria and antibiotic resistance genes. This study highlights the importance of extracellular DNA in wastewater, which is often overlooked. The results suggest that depending on the origin and treatment methods of wastewater, extracellular DNA can serve as an important reservoir for antibiotic resistance genes.