What samples are suitable for monitoring antimicrobial-resistant genes? Using NGS technology, a comparison between eDNA and mrDNA analysis from environmental water.

Abstract

The rise in antimicrobial resistance (AMR) that is affecting humans, animals, and the environment, compromises the human immune system and represents a significant threat to public health. Regarding the impact on water sanitation, the risk that antimicrobial-resistant genes (ARGs) and antimicrobial-resistant bacteria in surface water in cities pose to human health remains unclear. To determine the prevalence of AMR in environmental surface water in Japan, we used DNA sequencing techniques on environmental water DNA (eDNA) and the DNA of multidrug-resistant bacteria (mrDNA). The eDNA was extracted from four surface water samples obtained from the Tokyo area and subjected to high- throughput next-generation DNA sequencing using Illumina-derived shotgun metagenome analysis. The sequence data were analyzed using the AmrPlusPlus pipeline and the MEGARes database. Multidrug-resistant bacteria were isolated using a culture-based method from water samples and were screened by antimicrobial susceptibility testing (for tetracycline, ampicillin-sulbactam, amikacin, levofloxacin, imipenem, and clarithromycin). Of the 284 isolates, 22 were identified as multidrug-resistant bacteria. The mrDNA was sequenced using the Oxford nanopore MinION system and analyzed by NanoARG, a web service for detecting and contextualizing ARGs. The results from eDNA and mrDNA revealed that ARGs encoding beta-lactams and multidrug resistance, including multidrug efflux pump genes, were frequently detected in surface water samples. However, mrDNA also revealed many sequence reads from multidrug-resistant bacteria, as well as nonspecific ARGs, whereas eDNA revealed specific ARGs such as pathogenic OXA-type and New Delhi metallo (NDM)-beta-lactamase ARGs. To estimate potential AMR pollution, our findings suggested that eDNA is preferable for detecting pathogen ARGs.