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

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 blaCMY-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.