Adenine imprinted beads as a novel selective extracellular DNA extraction method reveals underestimated prevalence of extracellular antibiotic resistance genes in various environments

Abstract

Despite severe threats of extracellular antibiotic resistance genes (eARGs) towards public health in various environments, advanced studies have been hindered mainly by ineffective extracellular DNA (exDNA) extraction methods, which is challenged by trace levels of exDNA and inference from abundant coexisting compounds. This study developed a highly selective exDNA extraction method based on molecular imprinting technology (MIT) by using adenine as the template for the first time. Results suggested that adenine imprinted beads were rough spheres at an average size of 0.39 ± 0.07 μm. They effectively adsorbed DNA in the absence of chaotropic agents, with superior capacity (796.2 mg/g), rate (0.0066/s) and regarding DNA of variable lengths, even the ultra-short DNA (<100 bp). They were also highly selective towards DNA, circumventing the interference of competitive compounds' interference. These properties contribute to efficient exDNA extraction (71 %-119 %) from various environmental samples. Specifically, adenine imprinted beads enabled significantly higher extraction rates of eARGs from river, air and vegetable samples (69 %-95 %) compared to that by commercial DNA extraction products (16 %-62 %). The adenine imprinted beads-based method reveals underestimated eARG levels in the environment and the corresponding risks, and thus will thus be a powerful tool for advanced exDNA research.

Keywords: Extended Derjaguin–Landau–Verwey–Overbeek (xDLVO); Extracellular DNA extraction; Extracellular antibiotic resistance genes (eARGs); Molecular imprinting; Selectivity.