Dairy manure analysis reveals significant risk of Antibiotic resistance from Extracellular DNA in Manure storage Pit

AbstractDairy manure pit storage systems are significant reservoirs for antimicrobial resistance genes (ARGs). These genes occur in both intracellular DNA (iDNA) and extracellular DNA (exDNA), but their distribution across these categories in fresh (loafing pen surface) and pit-stored dairy manure has not been previously characterized. To address this gap, we quantified the abundance of six ARGs (tetG, tetM, tetX- tetracyline, sul1-sulfonamide, and ermB-macrolide) and three mobile genetic elements (MGEs) (intI1, intI2, and intI3) in iDNA and exDNA extracted from fresh and pit-stored manure collected at a dairy farm in Iowa. While total DNA yields were lower in pit-stored relative to fresh manure samples, exDNA-to-iDNA ratios were significantly elevated across all genes (p<0.001), indicating relative enrichment of exDNA during storage. Notably, tetM exhibited a higher free (unattached) to bound (surface-attached) exDNA ratio in pit samples, which suggested an increased potential for gene transfer in the pit. Correlation network analysis revealed similar numbers of strong ARG-MGE associations in pit and fresh exDNA, but lower interconnectivity in pit exDNA. Merging fresh and pit datasets showed wider ARG-MGE associations: intI1 and intI3 strongly co-occurred with tetracyclines and macrolide resistance in iDNA, while sul1 correlated with MGEs only in the exDNA network. Microbial community profiling showed similar taxa in exDNA across manure types, while iDNA communities diverged significantly. This result could support that exDNA is relatively stable over time and in varying environments, and that iDNA is relatively more reflective of selective pressures. Overall, our results highlight exDNA as a critical but overlooked reservoir of resistance determinants, warranting further investigation and targeted management strategies in dairy systems. ImportanceTo date, extracellular DNA (exDNA) has been shown to contribute to the spread of antibiotic resistance genes (ARGs) in the environment; however, few studies have evaluated its enrichment in dairy pit-stored manure systems. This study demonstrates that dairy manure pits concentrate exDNA during dairy manure storage and serve as a reservoir for ARGs, along with mobile genetic elements that can facilitate subsequent gene transfer. The results of this study are a strong rationale for further investigation and targeted management strategies of exDNA in manure pits.