Optimizing a Culture-Enriched Hybrid Metagenomics Pipeline to Assess the AMR Footprint of Livestock Manure in Anaerobic Digestate

The role of environmental samples from livestock production systems, including manure and anaerobic digestate, as reservoirs of antimicrobial resistance genes (ARGs) is likely underestimated because conventional metagenomic approaches can overlook low-abundance ARGs and often lack the resolution needed to reliably associate these genes with their microbial hosts and linked mobile genetic elements (MGEs). Here, we evaluated whether culture-enriched metagenomics (CEMG), with and without antibiotic selection, enhances ARG detection in anaerobic digestate and improves the resolution of ARG-MGE-host associations using hybrid short- and long-read metagenomic assembly. Culture enrichment substantially increased ARG recovery, mean ARG abundance rose from 15.4 counts per million (CPM) in culture-independent direct metagenomes from fresh digestate (FD) 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. In FD, only 9 unique ARGs were detected, whereas enrichment recovered 112, including ARGs of clinical importance such as glycopeptide resistance, {beta}-lactamase genes of the CTX-M, OXA, and TEM families, and the cfr 23S rRNA methyltransferase conferring cross- resistance to multiple antibiotic classes. Oxygen availability was the strongest factor structuring enriched community compositions and ARG profiles, with aerobic and anaerobic communities forming distinct clusters. Antibiotic selection induced targeted, class-specific shifts in ARG profiles, with ARGs associated with tetracycline resistance consistently enriched across treatments. Hybrid metagenomic assembly resolved the genomic context of 784 ARGs, of which 59.3% were co-localized with at least one class of mobile genetic element (MGE), predominantly plasmids, insertion sequences, and integrative and conjugative/mobilizable elements (ICEs/IMEs). Biocide and metal resistance genes frequently co-occurred with ARGs on the same contigs, highlighting the potential for co-selection. 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 for assessing their mobility and host associations. IMPORTANCELivestock manure and its byproducts, such as anaerobic digestate, are recognized as important environmental reservoirs of antimicrobial resistance genes and resistant bacteria, yet current metagenomic approaches may underestimate this risk by failing to detect low abundance but clinically relevant ARGs. Here, we show that integrating culture enrichment with hybrid metagenomics improves ARG recovery and reveals ARG co-localization with mobile genetic elements and putative 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.