Extended Data Fig. 3. Enterococcal-mediated enhancement of C. difficile toxin gene expression and production.

(a) Fold change of the toxin-encoding genes in C. difficile in coculture with E. faecalis versus monoculture as measured by qPCR (mean ± s.d., n=3). (b) Toxin production by C. difficile when grown in co-culture with E. faecalis as measured by ELISA. Both C. difficile and E. faecalis were grown in the same culture and differential plating was used to measure C. difficile CFUs. Toxin levels (OD₄₅₀) were normalized to C. difficile CFUs in the culture to control for any difference in growth (mean ± s.d., n = 5, two-tailed t-tests with Welch’s correction, P=0.007). (c) C. difficile toxin levels measured from in vitro cultures by cytotoxicity with E. faecalis cell-free supernatants. (mean ± s.d., n = 5, Kruskal-Wallis test with Dunn’s correction for multiple comparisons, OG1RF P=0.014, V583 P=0.032). (d) C. difficile toxin production measured by cytotoxicity following introduction of cell-free supernatants from microbiota isolates cultured from human patients with CDI and IBD (mean ± s.d., n = 12 (C. difficile), 3 (Raoultella, Bifidobacterium, Enterobacter, Paeniclostridium, Lactobacillus), 5 (Klebsiella), 6 (Citrobacter, Clostridium, Shigella, Streptococcus), Kruskal-Wallis test with Dunn’s correction for multiple comparisons, Lactobacillus P=0.049). Isolates were selected to represent the spectrum of taxa cultured from these patients.