Comprehensive Shotgun Metagenomic Profiling of Antibiotic Resistance Genes in Sheep and Goat Farming Environments

Background: Antimicrobial resistance (AMR) is a growing global health concern, driven in part by antibiotic use in animal production systems. Despite its relevance, the microbiome and resistome of small ruminant farm environments remain largely underexplored. Methods: In this study, shotgun metagenomics was applied to environmental samples from 46 sheep, goat and mixed-species farms across 14 municipalities in central Portugal. Results: Microbial profiling revealed a well-preserved microbiome with Pseudomonadota, Actinomycetota, Bacteroidota and Bacillota (syn. Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes respectively) as the most dominant phylum across different farm types. Regarding AMR, a total of 706 unique antimicrobial resistance genes (ARGs), covering 15 antibiotic classes, were detected. Tetracycline, aminoglycoside and macrolide resistance genes dominated across all samples, forming a conserved core resistome. While overall resistome profiles were broadly similar among farm types, significant differences were observed in specific ARG classes, such as pleuromutilin and fosfomycin. Conclusions: These findings highlight small ruminant farm environments as potential reservoirs of clinically relevant ARGs, including WHO highest priority critically important antimicrobial (HPCIA) resistance genes for macrolides (mph(c), erm(f), erm(b)) and fluoroquinolones (qnrD1), as well as critically important antimicrobial (CIA) resistance genes for glycopeptides (vanR-SC, vanR-O) and aminoglycosides (str, aadA), supporting the need to incorporate these environments into surveillance strategies.