Figure 4. Microbial taxa associated with choline diet-induced TMAO generation and a prothrombotic phenotype.
(A) Germ-free mice, conventionally reared mice, and germ-free mice subsequently conventionalized (all groups female, 4 week old, C57BL/6J), were fed the indicated sterile diets for 6 weeks and then both in vivo thrombosis potential (FeCl3 carotid artery injury model) and plasma TMAO levels were assessed. TMAO data represent mean ± SEM. (B) Correlation (Spearman) between plasma TMAO levels and time to cessation of blood flow among mice in all groups. (C) Microbial DNA encoding 16S rRNA was analyzed from cecum of mice in the indicated diet groups. Principal coordinates analyses demonstrate distinct cecal microbial composition between groups (p < 0.001 for Student’s t test with 1,000 Monte Carlo simulations). Each data point represents a sample from a distinct mouse projected onto the first three principal coordinates (percent variation explained by each PCo is shown in parentheses). (D) Linear Discriminant Analysis (LDA) Effect Size (LEfSe) identified taxa most characteristic (increased abundance) in chow (green) and choline (red) fed groups. (E) Example of choline diet group characteristic taxon (Allobaculum) and chow characteristic taxon (Candidatus Arthromitus) whose proportions are associated with both plasma TMAO levels and time to cessation of blood flow (occlusion time). A Kruskal Wallis test was used for multiple group comparison and a Mann Whitney test was used for two group comparison. See also Figure S3, Figure S4, Figure S5 and Tables S2–S4.