Detection of extracellular antibiotic resistance genes in river water: Application of ultrafiltration-magnetic beads method

Antibiotic resistance genes (ARGs) are widespread contaminants that pose significant threats to public health. Rivers play a crucial role in the dissemination of ARGs within the aquatic environment. However, there are limitations in the current research on the differentiation of intracellular ARGs (iARGs) and extracellular ARGs (eARGs) in river water. In this study, we developed a method combining ultrafiltration and adsorption of silicahydroxy magnetic beads for efficient extraction of extracellular DNA (eDNA) from river water. The conditions of adsorption, washing, desorption, and pretreatment were optimized to enhance eDNA recovery. By using only 90 mL of water sample, our method could collect sufficient eDNA for subsequent detection of eARGs through qPCR analysis. The eDNA recovery rate ranged from 51.4% to 69.8%. The occurrence of five prevalent ARGs (tetC, sulI, blaTEM, ermB, qnrS) as well as integrase gene intl1 were investigated in both iDNA and eDNA extracted from river water samples collected from two tributaries of the Pearl River. Our results revealed that the absolute abundance levels of eARGs ranged from 10- 1 to 105 copies/mL, which were significantly higher than those observed for iARGs ranging from 10- 1 to 104 copies/mL. Moreover, there was a significant difference in contamination profiles for ARGs between two tributaries. The ultrafiltration-magnetic beads method overcomes challenges associated with low efficiency extraction when working with water samples containing low nucleic acid concentrations. This approach provides an improved technique for extracting eARGs from river water while also generating valuable data supporting assessments related to eARG contamination in such environments.