Fig. 1. Enterococci promote C. difficile fitness and pathogenesis.

(a) Bacterial burdens (CFUs) for endogenous enterococci measured day of C. difficile inoculation. C. difficile burdens measured on first day post-inoculation to determine success of initial colonization. Burdens calculated following cefoperazone (cef) or cef + vancomycin (vanc) treatment (mean ± s.d., n = 10/group, two-sided Mann-Whitney for each comparison, P<0.001). (b) FISH image in lumen (left) or associated with the mucosa (right) during mouse infection. (c) CFUs from C. difficile CD196 (C.d.) monoculture or co-culture (E. faecalis = E.f., E. coli = E.c.) biofilms. Biofilms treated with vehicle (−) or vancomycin (+). Percent killing shown vs. untreated (mean ± s.d., n=5, two-sided Mann-Whitney test, C.d.: P = 0.007; C.d. + E.f:. P = 0.69; C.d. + E.c.: P = 0.008). (d) C. difficile toxin titers from mice infected with C. difficile or C. difficile + E. faecalis OG1RF following cef + vanc treatment (mean ± s.d., n = 15 for C. difficile only and n = 12 for C. difficile + E. faecalis, two-sided Mann-Whitney test, P=0.003). (e) Linear regression relating peripheral WBC to Log₁₀ Enterococcus 16S rRNA relative abundance (95% confidence interval shown, n = 41, P = 0.017). Two-sided Spearman correlation was also performed and showed a positive correlation between WBC and Enterococcus relative abundance (Spearman ρ = 0.252; n = 41). (f) C. difficile toxin levels measured by ELISA following growth with E. faecalis supernatants (mean ± s.d., n = 8 for C. difficile only and n = 4 for E. faecalis OG1RF and E. faecalis V583, one-way Welch ANOVA, P<0.001). (g) C. difficile toxin ELISA following introduction of cell-free supernatants from enterococci isolated from pediatric patients with IBD + CDI (mean ± s.d., n = 3/group, one-way Welch ANOVA, P<0.02, exact P values in Supplementary Table 5).