bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.11.07.687174

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

Sakib, N.·Andersen, D. S.·Jarboe, L. R.·Howe, A.
View original source ↗Export metadata ↓

Abstract

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.

Source connections

Explore related subjects

microbiologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Sakib, N., Andersen, D. S., Jarboe, L. R., Howe, A.. 2025-11-07. Dairy manure analysis reveals significant risk of Antibiotic resistance from Extracellular DNA in Manure storage Pit. https://doi.org/10.1101/2025.11.07.687174

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Reverse Transcription Loop-Mediated Isothermal Amplification Combined with Lateral Flow Assay (RT-LAMP-LFA) for Rapid Detection of the 2026 Bundibugyo Virus (BDBV) Outbreak Strain

Making accurate diagnosis is a critical first step in patient management and control for emerging and re-emerging disease outbreaks. Bundibugyo virus (BDBV) is a filovirus that causes Ebola disease, and the 2026 outbreak highlights the need for rapid diagnostic approaches that can be deployed in settings with limited laboratory infrastructure. Here, we developed a reverse transcription loop-mediated isothermal amplification-lateral flow assay (RT-LAMP-LFA) for rapid detection of the 2026 BDBV outbreak strain. LAMP primers were designed based on the genome sequence of the 2026 outbreak strain, and the conservation of the primer binding sites among all available 2026 outbreak genomes was confirmed in silico. LAMP primers were evaluated for analytical specificity and sensitivity using in vitro-transcribed RNA (ivtRNA) from BDBV and other filoviruses, as well as RNA derived from arboviruses and mammalian cells. Amplification products were detected using a Chromatography-Printed Array Strip (C-PAS) lateral flow platform. The newly designed primers specifically detected the presence of BDBV RNA without nonspecific amplification of the other viral or cellular RNA samples tested. Consistent amplification was observed down to 103 copies per reaction. In addition, BDBV viral RNA was successfully amplified, and the amplification products were detected using C-PAS, with the total workflow being completed within 25 min. These findings demonstrate the analytical specificity and sensitivity of the developed RT-LAMP-LFA assay and its compatibility with rapid visual lateral flow detection, supporting its potential application as a point-of-care approach for BDBV detection.

microbiology↗
bioRxiv2026

Divergent methane production and microbial community responses to carbon enrichment in freshwater wetland sediments

Methane (CH4) accumulation in wetland sediments reflects interactions among organic matter, sediment chemistry, and microbial communities. We combined depth-resolved field characterization across four wetland habitats in northern Israel with complementary incubations to examine methane accumulation and responses to labile-carbon enrichment. Sediment cores were characterized using Rock-Eval analysis, porewater chemistry, oxygen profiling at three sites, and mcrA amplicon sequencing. High CH4 concentrations occurred under contrasting conditions: Ein Nimfit (EN) contained approximately 12% total organic carbon and reached 2.1 mM CH4, whereas Ein Afek site EA2 contained approximately 5% carbon and accumulated 0.7-1.7 mM CH4. Both showed sulfate depletion, but EN exhibited pronounced sulfide accumulation, whereas EA2 showed greater dissolved iron and nutrient enrichment. Their classified methanogenic assemblages also differed. EA1 remained methane poor despite increasing carbon content with depth, while EA3 combined low carbon content and CH4 accumulation with detectable methanogens and lower methanogenic diversity. In a 36-day bottle experiments using sediments from two of the four wetland habitats, sucrose-amended EA3 exhibited a transient maximum of 18.8 mmol collected CH4 kg-1 wet sediment during Days 22-29, whereas EN reached its highest collected amount during Days 29-36. Methane proportions increased before the large output increase in EA3 but remained comparatively stable and high in EN. Soluble chemical oxygen demand trajectories and microbial community changes also differed. These findings identify distinct biogeochemical settings supporting methane accumulation and demonstrate methanogenic potential in low-CH4 sediment, emphasizing the complementary information provided by field profiles, microbial characterization, and exploratory enrichment experiments.

microbiology↗
bioRxiv2026

The absence of microbiome in germ-free mice exacerbates the pathogenicity of mouse-adapted SARS-CoV-2 MA10

Germ-free mice, which are devoid of microbiota, provide a powerful system to define the contribution of the microbiome to host responses during infection. Severe COVID-19 outcomes are associated with multiple comorbidities that are themselves characterized by microbiome dysbiosis; however, the causal role of the microbiota in SARS-CoV-2 pathogenesis remains poorly understood. Here, we use germ-free mouse models to evaluate the impact of the microbiome on disease severity following infection with mouse-adapted SARS-CoV-2 MA10. Comparison with conventionally housed mice revealed that the absence of the microbiome markedly exacerbates SARS-CoV-2-induced disease in both C57BL/6 and Swiss Webster backgrounds. Germ-free mice exhibited greater weight loss, impaired lung function, worsened pulmonary pathology, and pronounced alterations in circulating cytokine and chemokine profiles compared with conventional controls. These findings show that resident microbiota limit SARS-CoV-2 pathogenicity and establish germ-free COVID-19 models as a powerful platform to identify protective microbial taxa and microbiota-based interventions to reduce severe disease.

microbiology↗