Early-life development of the microbiome and resistome in antibiotic-naïve dairy calves

This study aimed to characterize early-life changes in the fecal microbiome and resistome of calves. Fecal samples were collected from 49 Holstein heifers born and raised at a large organic dairy in Texas without antimicrobial drug exposures. Samples were collected from five age groups: early pre-weaning at 2-3 days old (Pre 1), late pre-weaning at 5 weeks old (Pre 2), prior to weaning at 12-13 weeks old (Pre 3), post-weaning in group hutches at 12-13 weeks old (Post 1), and later post-weaning at 13-14 weeks old (Post 2). Fecal samples were analyzed using 16S rRNA gene sequencing to characterize microbial communities and target-enriched shotgun sequencing to characterize antimicrobial resistance genes in the resistome. Richness of microbial communities increased as calves aged through the Pre 1, 2, and 3 samplings, before plateauing in the Post 1 and 2 groups. Diversity also increased in the Pre 1 and 2 groups, remaining similar thereafter. In contrast, resistome richness and diversity decreased during early life and then stabilized at around 5 weeks of age (Pre 2). Changes in microbial community structures were dramatic during the first 12 weeks, largely due to a significant decrease in the relative abundance (RA) of Pseudomonadota (Proteobacteria) and an increase in the RA of Bacillota (Firmicutes) and Bacteroidota. The resistome changed with an increased RA of tetracycline resistance genes, while drug and biocide resistance genes decreased. The apparent stabilization of microbial community features after 12 weeks of age may reflect a period when gut microbiome structure begins to establish greater stability.IMPORTANCEEarly-life development of the gut microbiome can have lasting effects on animal health, immune maturation, and productivity. Using 16S rRNA gene sequencing together with target-enriched metagenomic sequencing, we provide an in-depth characterization of the fecal microbiome and resistome of antibiotic-naïve dairy calves during early life. We demonstrate that microbiome diversity increased with age while resistome diversity decreased, revealing distinct temporal trajectories and suggesting ecological succession as a potential driver of resistance gene dynamics independent of antimicrobial drug exposure. Major resistome features appeared to stabilize earlier than overall microbiome structure, highlighting critical windows in early development when resistance gene composition may be most dynamic. These findings establish an important baseline for interpreting microbiome-resistome interactions and for evaluating how management practices and antimicrobial exposures may influence calf health and antimicrobial resistance ecology in dairy production systems.