Long-read metagenomics and methylation-based binning support the discovery of antibiotic resistance gene-host...

Antibiotic resistance genes (ARGs) circulating among clinically relevant bacteria pose serious challenges to public health. Given the ancient and environmental bacterial origins of ARGs, a better understanding of the carriers of ARGs beyond the clinically most relevant species is urgently needed for longer-term resistance monitoring and intervention measures. While the risks of emerging ARGs from environmental sources have been recognized, the identification bottlenecks stem from the limitations of shotgun metagenomic sequencing and bioinformatic methods.

We use long-read metagenomic sequencing and bacteria-specific methylation profiles to re-establish the links between established (well-described) or latent (absent in databases) ARGs and their bacterial and genetic contexts in wastewater. We analyze base modification data produced by PacBio SMRT sequencing using an in-house pipeline utilizing position weight matrices and UMAP visualizations, which we validate by a synthetic community with known bacterial composition. Our analysis reveals several previously unreported ARGs and ARG-host linkages in wastewater. For instance, we find that Arcobacter, a key wastewater associated taxon and emerging pathogen, carries a latent beta-lactamase gene with high predicted mobility potential. Of the other understudied beta-lactamases, we describe blaMCA within pdif-modules across highly varying contexts suggesting its recent acquisition events. Additionally, we uncover the wastewater resident taxa mediated carriage of clinically important ARGs.

By linking ARGs to their wider genetic contexts and hosts, our findings shed light on the previously unrecognized carriers of resistance genes in wastewater. The presented approach provides a valuable methodology for early identification of newly arising ARGs and their hosts.