Assessing resistome host range across water reclamation in three geographically distinct communities using Hi-C...

Antimicrobial resistance (AMR) is a growing problem, with annual deaths set to pass 10 million by 2050 if current trends continue. Sequencing untreated wastewater has been proposed as a strategy to understand population-level resistance, and water reclamation facilities (WRFs) have been identified as a control point for environmental dissemination of resistant bacteria. Understanding AMR dynamics across WRFs can provide insight into how WRFs may alter the resistome and requires advanced molecular tools that elucidate host bacteria, especially for mobile resistance carried on plasmids. To that end, influent, activated sludge, and effluent were collected from three WRFs in North Carolina, Texas, and California during 3 weeks of spring 2024. Samples were analyzed for chromosomal and, crucially, plasmid-based resistance using Hi-C proximity ligation sequencing to identify the AMR host range. A total of 1,868 hits for 244 unique resistance genes were observed, with seven resistance genes identified in all samples: bla_OXA, merE, P, R, and T, msr(E), and tet(C). Resistance genes within microbial hosts were more likely to be carried on a plasmid in influent, but more likely to be on a chromosome in activated sludge. Seventeen total microbial hosts for resistance genes were identified in effluent, with a Moraxellaceae sp. and Thiobacillaceae sp. carrying ARGs on plasmids. More than 50% of all identified host relationships were confined to just four bacterial families: Aeromonadaceae, Burkholderiaceae, Moraxellaceae, and Neisseriaceae. Hi-C contact mapping is a critical tool to more fully describe the AMR host range in complex matrices, particularly for plasmid-based resistance genes.IMPORTANCEWater reclamation facilities receive a complex mixture of antibiotics and rely on active microbial communities for treatment, thereby acting as critical systems to prevent environmental spread of resistance. However, antimicrobial resistance (AMR) dynamics are difficult to discern in complex wastewater environments due to antibiotic resistance genes (ARGs) being frequently carried on mobile pieces of DNA that are difficult to link to specific bacteria using conventional shotgun sequencing. In the current study, Hi-C sequencing was carried out on influent, activated sludge, and effluent collected from water reclamation facilities in California, Texas, and North Carolina to assess the resistome host range across treatment. The current study elucidated differences in the genomic context of ARGs across treatments, showing that ARGs are more likely to be plasmid-based in influent bacterial communities but located on chromosomes in activated sludge. Hi-C sequencing is uniquely able to provide untargeted linkage to plasmids with their microbial hosts in complex communities.