Animal type outweighs growth stage in shaping the active antibiotic resistome in livestock manure.

Animal manure is a major reservoir of antibiotic resistance genes (ARGs), yet the factors governing their composition and activity remain insufficiently understood. In this study, metagenomics, metatranscriptomics, and quantitative PCR (qPCR) were integrated to characterize microbial communities and resistome profiles in manure from five animal types across multiple growth stages. Results demonstrated that animal type was the dominant factor shaping both microbial community structure and ARG composition at DNA and RNA levels, whereas growth stage did not significantly influence overall community structure. Metagenomic analysis identified 944 ARG subtypes, predominantly associated with tetracycline, multidrug, and macrolide-lincosamide-streptogramin (MLS) resistance. Metatranscriptomic analysis further revealed 579 actively transcribed ARG subtypes, among which 29 [e.g., tet(W), sul1, and erm(B)] were consistently detected at both DNA and RNA levels, indicating their active nature. Among tested animal types, layer litter showed the highest ARG number at the DNA level, with qPCR results further revealing significantly higher abundances of specific ARGs, including bla TEM and erm(B). Although growth stage had limited influence on overall resistome composition, stage-dependent variations in ARG abundance were observed, with higher levels during early-life and juvenile stages. Procrustes analysis revealed strong correlations between microbial community composition and resistome profiles in both DNA and RNA levels, suggesting that microbiome structure plays a key role in shaping ARG dynamics and activity. Overall, animal type was the dominant measured correlate of resistome composition, whereas growth stage was associated mainly with variation in selected ARG abundances. These observational findings require validation in multi-farm longitudinal studies before management or environmental-risk conclusions are drawn.