Assessing Resistome Host Range Across Water Reclamation in Three Geographically Distinct Communities using Hi-C Sequencing

Antimicrobial resistance (AMR) is a growing problem, with annual deaths set to pass 10 million by 2050 if current trends continue. Wastewater surveillance 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 dynamics of AMR across WRFs 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 three weeks of Spring 2024. Samples were analyzed using Hi-C proximity ligation sequencing to identify the AMR host range for chromosomal and plasmid-based resistance. A total of 1,868 hits for 244 unique resistance genes were observed, with seven resistance genes identified in all samples. Resistance genes were more likely to be carried on a microbial plasmid in influent, but more likely to be in a chromosome in activated sludge. Seventeen total microbial hosts for resistance genes were identified in effluent, suggesting WRF effluents may be sources of resistant bacteria to receiving surface waters. A high proportion of all identified host relationships were confined to just four bacterial families. 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. ImportanceAntimicrobial resistance (AMR) threatens modern medicine. Water 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, 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. Novel proximity ligation sample preparation techniques like Hi-C physically link co-located sequences of DNA before shotgun sequencing. This allows sequencing to elucidate the bacterial hosts for both stable and mobile ARGs. 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. 5 Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/26346186v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1e4620eorg.highwire.dtl.DTLVardef@e1c3a7org.highwire.dtl.DTLVardef@1f40964org.highwire.dtl.DTLVardef@94b886_HPS_FORMAT_FIGEXP M_FIG C_FIG