Resolving Host-Associated Antimicrobial Resistance in Municipal Wastewater Using Hi-C Metagenomics.

Authors

Abiola Senok, Subham Verma, Douha Shouqair, Rashed Alghafri, Danesh Moradigaravand, Rania Nassar, Dean Everett, Richard Goering, Mushtaq Khan

Published in

Microorganisms. Volume 14. Issue 10. Sep 24, 2026. Epub Sep 24, 2026.

Abstract

Conventional wastewater metagenomics enables population-level surveillance of antimicrobial resistance (AMR) but provides limited insight into the bacterial hosts, genomic context, and the mobility of resistance determinants. In this study, we applied high-throughput chromosome conformation capture (Hi-C) metagenomics, a proximity-ligation approach, to resolve host-associated AMR. Six influent wastewater samples collected from two municipal wastewater treatment plants over a four-month sampling period were investigated. Integrated shotgun metagenomic and Hi-C sequencing were used to reconstruct metagenome-assembled genomes (MAGs), plasmid bins, and viral bins, and to link AMR, stress-response, and virulence-associated genes to their bacterial hosts through Hi-C contacts. From 12,877 genome bins, 454 met quality thresholds (139 MAGs, 130 plasmid bins, 185 viral bins) spanning 70 bacterial and archaeal families. AMRFinderPlus identified 771 resistance and fitness-associated gene hits (517 AMR, 232 stress-response, 22 virulence-associated), dominated by macrolide-lincosamide-streptogramin B (n = 117), β-lactam (n = 114), and tetracycline (n = 96) determinants. Hi-C linked 490 gene hits to host families; Enterobacteriaceae had the highest number of host-assigned detections (n = 131), while Aeromonadaceae carried the broadest AMR repertoire (n = 102; 72% AMR). The host-plasmid network comprised 137 AMR-positive plasmid bins linked to 106 MAGs through 166 Hi-C-supported associations. Five plasmid contigs, including one harbouring bla_CMY-4, were linked to MAGs from two distinct bacterial classes, suggestive of cross-lineage plasmid carriage. Viral AMR attribution was limited, with erm(B) as the sole resistance determinant identified on a viral contig. Hi-C metagenomics extends wastewater resistome profiling beyond gene inventories by resolving bacterial hosts, plasmid associations, and genomic context, strengthening the ecological interpretation of wastewater-based AMR surveillance.

PMID:
42943393
Bibliographic data and abstract were imported from PubMed on 27 Oct 2026.

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