Study overview and effects of ampicillin on the gut microbiome. (A) Study overview. Mice aged 3 weeks were weaned and then immediately commenced treatment with ampicillin, administered every 12 hours by gastric gavage for 7 days (n = 15) or not (n = 14). After a 2-week rest period, mice remaining in the study were challenged with 50 ng IV-administered FVIII. The mice were euthanized and samples collected (blue circles) for analyses at 6 weeks of age, immediately before the FVIII challenge began and at the study end point (10 weeks of age). (B) Differences in the gut microbiota between ampicillin-treated and control mice derived from a heat map of calculated z-scores. Bacterial 16s rRNA was sequenced, and samples were clustered based on study time point (6 weeks vs 10 weeks) and treatment (ampicillin vs control), using Pearson’s distance and Ward’s clustering methods. (C) Principal coordinate analysis based on weighted UniFrac distances of each microbiome sample. (D) α-Diversity was measured by using Faith’s phylogenetic diversity (PD) metric (means compared by unpaired, 2-tailed Student t test). Standard deviations are shown. (E) Relative abundance of bacterial phyla (left) and class (right). Each vertical bar corresponds to an individual sample. Sample size for all microbiome analyses remain consistent (n = 4-5). ****P < .0001.