Removal of extracellular free DNA and antibiotic resistance genes from water and wastewater by membranes ranging from microfiltration to reverse osmosis

Free DNA in the effluent from wastewater treatment plants has recently been observed to contain antibiotic resistance genes (ARGs), which may contribute to the spread of antibiotic resistance via horizontal gene transfer in the receiving environment. Technical membrane systems applied in wastewater and drinking water treatment are situated at central nodes between the environmental and human related aspects of the “One Health” approach and are considered as effective barriers for antibiotic resistant bacteria. However, they are not evaluated for their permeability for ARGs encoded in free DNA, which may result, for example, from the release of free DNA after bacterial die-off during particular treatment processes. This study examined the potential and principle mechanisms for the removal of free DNA containing ARGs by technical membrane filtration. Ten different membranes, varied by the charge (neutral and negative) and the molecular weight cut off (in a range from microfiltration to reverse osmosis), were tested for the removal of free DNA (pure supercoiled and linearized plasmids encoding for ARGs and free linear chromosomal DNA with a broader fragment size spectrum) in different water matrices (distilled water and wastewater treatment plant effluent). Our results showed that membranes with a molecular weight cut off smaller than 5000 Da (ultrafiltration, nanofiltration and reverse osmosis) could retain ≥99.80% of free DNA, both pure plasmid and linear fragments of different sizes, whereas microfiltration commonly applied in wastewater treatment showed no retention. Size exclusion was identified as the main retention mechanism. Additionally, surface charging of the membrane and adsorption of free DNA on the membrane surface played a key role in prevention of free DNA permeation. Currently, majority of the applied membranes is negatively charged to prevent adsorption of natural organic matter. In our study, negatively charged membranes showed lower retention of free DNA compared to neutral ones due to repulsion of free DNA molecules, reduced adsorption and decreased blockage of the membrane surface. Therefore, the applied membrane may not be as an effective barrier for ARGs encoded in free DNA, as it would be predicted based only on the molecular weight cut off. Thus, careful considerations of membrane's specifications (molecular weight cut-off and charge) are required during design of a filtration system for retention of free DNA. [Display omitted] •Free