Summary
The extent that anaerobic digestate acts as a reservoir of antimicrobial resistance genes (ARGs) is likely underestimated as conventional metagenomics may underrepresent low-abundance determinants and lacks sufficient resolution to reliably link ARGs to mobile genetic elements (MGE). This study used hybrid assemblies to evaluate whether culture-enriched metagenomics (CEMG), with and without antimicrobial selectivity, improves detection of ARGs in digestate and characterization of ARG- MGE-host linkages. Culture enrichment substantially increased ARG recovery: mean ARG signal rose from 15.4 CPM in fresh digestate (FD; direct metagenomic samples) to 124 CPM in CEMG without antibiotics and 160.0 CPM in antibiotic-selective CEMG, corresponding to an approximately 10.4-fold increase over FD. While only 9 unique ARGs were detected in FD, enrichment recovered 112, including those of clinical importance such as those encoding for vancomycin resistance, extended-spectrum {beta}-lactamase, and linezolid resistance. Oxygen availability emerged as the strongest factor structuring enrichment, with aerobic and anaerobic samples forming distinct clusters and exhibiting shifts in dominant taxa and resistome composition. Antibiotic selection produced more targeted, class-specific shifts, with tetracycline resistance consistently enriched across treatments. Hybrid metagenomic assembly further resolved ARG-MGE-host linkages, revealing extensive co-localization of ARGs with MGEs and heavy metal resistance genes. Together, these findings demonstrate that antibiotic-selective culture enrichment enhances resistome surveillance by improving detection of low-abundance ARGs, while hybrid assembly provides critical genomic context to assess their mobility and host associations. IMPORTANCELivestock manure and its byproducts, such as anaerobic digestate, are recognized as important environmental reservoirs of antimicrobial resistance, yet current metagenomic approaches may underestimate this risk by failing to detect low abundance but clinically relevant resistance determinants. Here, we show that integrating culture enrichment with hybrid metagenomics improves the recovery of antimicrobial resistance genes and reveals their association with mobile genetic elements and bacterial hosts. This approach captures a cultivable and condition-responsive fraction of the resistome that is not readily accessible through direct metagenomic sequencing alone, providing a more informative framework for environmental AMR surveillance.
Outcomes reported
The extent that anaerobic digestate acts as a reservoir of antimicrobial resistance genes (ARGs) is likely underestimated as conventional metagenomics may underrepresent low-abundance determinants and lacks sufficient resolution to reliably link ARGs to mobile genetic elements (MGE). This study used hybrid assemblies to evaluate whether culture-enriched metagenomics (CEMG), with and without antimicrobial selectivity, improves detection of ARGs in digestate and characterization of ARG- MGE-host linkages. Culture enrichment substantially increased ARG recovery: mean ARG signal rose from 15.4 CPM in fresh digestate (FD; direct metagenomic samples) to 124 CPM in CEMG without antibiotics and 160.0 CPM in antibiotic-selective CEMG, corresponding to an approximately 10.4-fold increase over FD. While only 9 unique ARGs were detected in FD, enrichment recovered 112, including those of clinical importance such as those encoding for vancomycin resistance, extended-spectrum {beta}-lactamase, and linezolid resistance. Oxygen availability emerged as the strongest factor structuring enrichment, with aerobic and anaerobic samples forming distinct clusters and exhibiting shifts in dominant taxa and resistome composition. Antibiotic selection produced more targeted, class-specific shifts, with tetracycline resistance consistently enriched across treatments. Hybrid metagenomic assembly further resolved ARG-MGE-host linkages, revealing extensive co-localization of ARGs with MGEs and heavy metal resistance genes. Together, these findings demonstrate that antibiotic-selective culture enrichment enhances resistome surveillance by improving detection of low-abundance ARGs, while hybrid assembly provides critical genomic context to assess their mobility and host associations. IMPORTANCELivestock manure and its byproducts, such as anaerobic digestate, are recognized as important environmental reservoirs of antimicrobial resistance, yet current metagenomic approaches may underestimate this risk by failing to detect low abundance but clinically relevant resistance determinants. Here, we show that integrating culture enrichment with hybrid metagenomics improves the recovery of antimicrobial resistance genes and reveals their association with mobile genetic elements and bacterial hosts. This approach captures a cultivable and condition-responsive fraction of the resistome that is not readily accessible through direct metagenomic sequencing alone, providing a more informative framework for environmental AMR surveillance.
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