Selective extracellular DNA (exDNA) extraction method reveals underestimated associations between extracellular antibiotic resistance genes and bacteria in diverse environments

Extracellular antibiotic resistance genes (exARGs) constitute a significant type of antibiotic resistance genes. However, current exARGs studies are restricted due to the lack of efficient extraction methods for extracellular DNA (exDNA), which are laborious, dependent on extra experimental agents, and prone to interference by coexisting substances. In this study, we have developed an ultra-selective exDNA extraction method based on molecular imprinting technology. The resulting guanine imprinted beads are micron grade spheres with high capacity (384.6 mg/g) and speed (90% adsorption in 10 min) for DNA adsorption at variable lengths, including ultra-short DNA (<100 bp), without requiring extra agents. The guanine-imprinted beads demonstrated exclusive DNA adsorption capabilities and avoided interference by other competing substances. Furthermore, guanine imprinted beads effectively extracted DNA at ideal recovery efficiencies (81%similar to 109%) from diverse environments, such as soil, air, river water and vegetable. Higher abundance of exARGs was obtained by using guanine imprinted beads than currently used commercial magnetic beads. In addition to exDNA extraction quality, a higher diversity of exDNA-releasing bacteria was acquired by guanine imprinted beads. This contributes to 1.2-fold increase in ARG-genera (172 v.s. 150) and 3.6-fold increase in ARG-pathogen associations (96 v.s. 27), indicating a greater number of bacterial hosts of ARGs. This study provides a simple and robust method for advanced exDNA studies, such as discovering novel exARGs and identifying hosts of exARGs.