Dairy manure reception pits as reservoirs of extracellular DNA-associated antibiotic resistance genes

ABSTRACT Dairy manure reception pits receive fresh manure and milkhouse wash water, and have short residence times, yet their role as reservoirs of extracellular antibiotic resistance genes (exARGs) remains poorly characterized. Here, we quantified six ARGs ( tetG , tetM , tetX- tetracycline, sul1 -sulfonamide, and ermB- macrolide) and three integron-associated mobile genetic elements (MGEs: intI1 , intI2 , and intI3 ) in intracellular DNA (iDNA) and extracellular DNA (exDNA) from fresh manure and from a dairy reception pit with an estimated residence time of <6 h. While total DNA yields were lower in the pit relative to fresh manure samples, exDNA-to-iDNA ratios were significantly higher for all genes ( P < 0.001), indicating rapid enrichment of exDNA after manure entered the pit and mixed with the wash water. Notably, tetM exhibited a higher free (unattached) to bound (surface-attached) exDNA ratio in pit samples, which suggested greater accessibility to this gene within the extracellular pool in the pit. Exploratory correlation analysis identified recurring ARG-MGE associations, whereas 16S rRNA gene profiling showed iDNA-associated taxonomic composition diverged between fresh and pit samples, while exDNA-associated taxonomic profiles remained more similar. This result could support that exDNA is relatively stable over time and in varying environments, and that iDNA is more reflective of selective pressures. Together, these results indicate that even short-term reception pits can act as reservoirs of exDNA-associated resistance determinants and justify further work on their persistence, mobility, and management in dairy systems. IMPORTANCE To 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 short-term dairy pit-stored manure systems. This study demonstrates that dairy manure pits concentrate exDNA during short-term storage and serve as reservoirs 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.