Aims:The environment is increasingly recognized as an important reservoir of antimicrobial resistance genes (ARGs), which can be identified using molecular platforms. Yet, environmental surveillance remains an underutilised tool as there is no agreement on the best strategy for sample processing. We aim to develop a low-cost extraction method independent to commercial kits or reagents. Methods and Results:We present a novel, magnetic bead-based method for the isolation of ARGs from river water named MagnaExtract. We present this with analytic limit of detection as well as a case study in Southern Malawi. Here we compare the DNA yield from MagnaExtract with commercially available QIAGEN kits and the crude boil and spin method, using a high-resolution melt analysis PCR panel designed for the detection of third-generation cephalosporin and carbapenem-resistant genes from 98 water samples. Conclusion:The MagnaExtract method is comparable, and in some instance's superior to commercially available kits for the isolation of ARGs from river water samples. Significance and Impact of the Study: The MagnaExtract approach offers a simple, affordable, high yielding extraction method that could be used for the detection of ARGs from river water samples in surveillance campaigns in East Africa.

BackgroundEnvironmental water samples are increasingly recognised as an important reservoir of antimicrobial resistance (AMR) genes. Polymerase chain reaction (PCR) and next generation sequencing (NGS) offer a potentially inclusive surveillance platform for a wide range of AMR genes. However, molecular methods are dependent upon the extraction of DNA of high yield and quality. Current options for DNA extraction from complex environmental matrices for downstream molecular applications are either expensive or low yielding. We present here a novel magnetic bead-based DNA extraction method, for the detection of antimicrobial resistance genes (ARGs) from river water in Malawi, named MagnaExtract.MethodsMagnaExtract involves initial filtration of 250ml freshwater, followed by an overnight incubation of the filter in 15ml buffered peptone water (BPW), common procedure in microbiology laboratories. 200µl is then taken for a boil (95°C) and spin step and mixed with magnetic beads to bind DNA. Following washes with ethanol, the DNA is eluted in nuclease-free water. To determine the effectiveness of this method, 98 freshwater samples were collected from two rivers in Southern Malawi, and DNA was isolated using the MagnaExtract method, two commercial Qiagen (Germany) kits; PowerWater and DNeasy Blood and tissue, alongside a boil and spin of BPW, and a boil and spin from bacterial isolate grown on agar media. All samples were screened with a high-resolution melt (HRM) PCR panel previously validated for the detection of third generation cephalosporin and carbapenem ARGs. We compared the DNA yield obtained using all extraction methods, as well as the identification of each ARG.ResultsDNA yield using MagnaExtract was statistically greater than both boil and spin methods and DNeasy Blood & Tissue (Qiagen, Germany). DNA yield was slightly lower than using PowerWater (Qiagen) but the difference was not statistically significant. MagnaExtract was the only method to identify ARGs in all 98 water samples compared with PowerWater (n=82), DNeasy (n=95) boilate of BPW (n=75) and boilate of bacterial isolate (n=87). The most commonly detected ARG was OXA-48 (n=93). In addition, we found overnight incubation in non-selective enrichment broth (BPW) to promote the growth of bacteria harbouring extended spectrum beta lactamase (ESBL) genes and reduction in the detection of carbapenemase genes.ConclusionThe MagnaExtract approach offers a simple, affordable, high yielding DNA extraction method for the detection of ARGs isolated from river water samples.