Impact of differential dietary concentrations of cobalt, manganese and zinc on gastrointestinal microbiome and resistome of lactating dairy cattle

Dietary trace mineral (TM) concentrations for lactating cows often exceed national recommendations under commercial feeding practices. Excess TM supplementation may exert selection pressure on the gut microbiota, promoting metal resistance and potentially co-selecting for important antimicrobial resistance genes (ARGs). This study used a cross-over design to investigate the impact of overfeeding a commericially representative TM premix on the gut microbiome and resistome of lactating dairy cattle. Cows were fed either recommended or surplus TM levels for 31 days followed by sample collection from rumen papillae, whole rumen content, and feces. Targeted amplicon, shotgun metagenomic sequencing, and droplet digital PCR (ddPCR) were used to assess microbial community compositions and the associated resistome. While a surplus TM did not significantly affect the overall microbial diversity, specific taxa differed between the matrices and to a lesser extent by diet. Spirochaetota were more abundant in papillae of cows fed the recommended TM diet, whereas Bacillota were more prevalent in the rumen and feces from cows fed the surplus TM. Phosphorus had the greatest impact on prokaryotic taxa in rumen content. Cows fed surplus levels of TM showed increased abundances of Ruminococcus in their rumen, but decreased Campylobacter, Desulfovibrio, and Treponema adhered to their rumen papillae, Methanosphaera in their rumen and feces, as well as Treponema in their feces. Despite these changes, no significant differences in the presence of key ARGs or metal resistance genes were detected by metagenomics, and ddPCR showed no significant impact of TM supplementation on blaCTX−M, pcoA and zntA gene levels. Overfeeding TM in a commercial premix resulted in modest matrix specific shifts I the microbial composition without detectable enrichment of selected ARGs or metal resistance genes. These findings suggest short-term resilience of the rumen ecosystem to surplus TM supplementation. Additionally this work provides foundational knowledge to further guide mechanistic and long-term investigations.