Free-floating extracellular DNA: systematic profiling of mobile genetic elements and antibiotic resistance from wastewater

The free-floating extracellular DNA (exDNA) fraction of microbial ecosystems harbors antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). Natural transformation of these xenogenetic elements can generate microbial cells resistant to one or more antibiotics. Isolating and obtaining high yield exDNA is challenging due to its low concentration in wastewater environments. Profiling exDNA is crucial to unravel the ecology of free-floating ARGs and MGEs and their contribution to horizontal transfer. We developed a method using chromatography to isolate and enrich exDNA without causing cell lysis from complex wastewater matrices like influent (9 µg exDNA out of 1 L), activated sludge (5.6 µg), and treated effluent (4.3 µg). ARGs and MGEs were metagenomically profiled for both the exDNA and intracellular DNA (iDNA) of activated sludge, and quantified by qPCR in effluent water. qPCR revealed that ARGs and MGEs are more abundant in the iDNA fraction while still significant on exDNA (100-1000 gene copies mL−1) in effluent water. The metagenome highlighted that exDNA is mainly composed of MGEs (65%). According to their relatively low abundance in the resistome of exDNA, ARGs uptake by natural transformation is likely not the main transfer mechanism. Although ARGs are not highly abundant in exDNA, the prevalence of MGEs in the exDNA fraction can indirectly promote antibiotic resistance development. The combination of this method with functional metagenomics can help to elucidate the transfer and development of resistances in microbial communities. A systematic profiling of the different DNA fractions will foster microbial risk assessments across water systems, supporting water authorities to delineate measures to safeguard environmental and public health.