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ARGs enhance bacterial survival against antibiotics, while VFs contribute to infection processes, and the microbiome composition influences host health. Understanding microbial exchange at the wildlife-livestock interface is essential for assessing risks to both animal and human health. This study addresses the gap in knowledge by investigating the microbial composition, ARGs, and VFs in fecal matter from livestock (<jats:italic>Bos taurus<\/jats:italic>, <jats:italic>Ovis aries<\/jats:italic>) and wildlife (<jats:italic>Microtus arvalis<\/jats:italic>) cohabiting grassland pastures. Sampling was conducted within the DFG Biodiversity Exploratories, which provides valuable and extensive long-term ecological datasets and enables the study of diverse environmental parameters. Using metagenomic sequencing and 16\u00a0S rRNA amplicon analysis, we compared bacterial diversity, antimicrobial resistance profiles, and virulence gene presence across the three host species. Metagenomic analysis revealed host-specific differences in bacterial community composition. Livestock samples exhibited higher microbial diversity than those from <jats:italic>M. arvalis<\/jats:italic>, likely due to greater environmental exposure and management practices. The most common VFs in livestock were associated with immune modulation, whereas motility-related VFs were prevalent in <jats:italic>M. arvalis<\/jats:italic>. ARG profiles differed among hosts, suggesting rare events rather due to environmental acquisition than direct transmission between the hosts. The limited numbers of ARGs and VFs shared between the species indicate that horizontal gene transfer events between wildlife and livestock are infrequent. Notably, <jats:italic>M. arvalis<\/jats:italic> harbored diverse ARGs, including resistance to tetracycline and vancomycin, which were likely acquired from the environment rather than from direct livestock contact. These findings highlight the significant role of environmental reservoirs in shaping microbial communities and the spread of resistance. This research underscores the need for enhanced surveillance and ecosystem management strategies to mitigate the risk associated with antimicrobial resistance and the potential impacts on both animal and human health.<\/jats:p>","DOI":"10.1186\/s42523-025-00448-2","type":"journal-article","created":{"date-parts":[[2025,8,6]],"date-time":"2025-08-06T12:17:54Z","timestamp":1754482674000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Microbial exchange at the wildlife-livestock interface: insights into microbial composition, antimicrobial resistance and virulence factor gene dynamics in grassland ecosystems"],"prefix":"10.1186","volume":"7","author":[{"given":"Lea","family":"Kauer","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Panagiotis","family":"Sapountzis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christian","family":"Imholt","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christian","family":"Berens","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ralph","family":"Kuehn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,8,6]]},"reference":[{"issue":"1","key":"448_CR1","doi-asserted-by":"publisher","first-page":"4413","DOI":"10.1038\/s41598-022-08230-z","volume":"12","author":"SA Bruce","year":"2022","unstructured":"Bruce SA, Smith JT, Mydosh JL, Ball J, Needle DB, Gibson R, et al. 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