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. Author manuscript; available in PMC: 2023 May 16.
Published in final edited form as: Nature. 2022 Nov 16;611(7937):780–786. doi: 10.1038/s41586-022-05438-x

Extended Data Fig. 7. The enterococcal ADI pathway reshapes the metabolic environment in the gut during CDI.

Extended Data Fig. 7.

(a) C. difficile growth in presence of E. faecalis or E. faecalis arcD::Tn supernatants. (mean ± s.d., n = 8/group, two-way ANOVA, Time factor P<0.001). (b) Taxonomic distribution (at bacterial family level) of reads mapped to arc genes in adult patients (n=48) with symptomatic CDI. Each column is a subject and each row is a bacterial family. Each cell displays the percentage of reads mapped to arc genes of a specific family out of all arc mapped reads. (c) Relative abundance of the top 10 arc operon containing bacterial families in each adult patient symptomatically infected with C. difficile (n=48, lower and upper hinges correspond to the first (25%) and third (75%) quartiles. The upper and lower whiskers extend from the hinge to the largest value no further than 1.5*IQR. Data beyond the whiskers are plotted individually). (d) Toxin production of C. difficile following introduction of supernatants from E. faecalis and addition of exogenous L-ornithine measured by ELISA (mean ± s.d., n = 3, Tukey’s multiple comparisons test, C. difficile +/− ornithine P=0.705). (e) MALDI-IMS image of uninfected or infected mice (3d post-infection) (SPF) (representative of n = 5 mice) or (f) GF mice mono-infected with C. difficile CD196 or co-infected with E. faecalis OG1RF (2d post-infection) (representative of n = 4 mice). Individual heatmaps of arginine and ornithine. (g) Ornithine levels in stool measured by targeted metabolomics in GF mice infected with C. difficile only (mean ± s.d., n = 10) or C. difficile + E. faecalis OG1RF (mean ± s.d., n = 3, two-sided t-tests with Welch’s correction, P<0.001). Metabolomics were performed on GF mice prior to infection (GF group, n = 13). (h) Arginine levels in stool of GF mice infected with C. difficile only (mean ± s.d., n = 10, two-sided t-test with Welch’s correction, P=0.023) or C. difficile + E. faecalis OG1RF (mean ± s.d., n = 3, two-sided t-test with Welch’s correction, P<0.001). Stool metabolomics performed prior to infection (GF group, n = 13). (i) CFU of E. faecalis OG1RF (wild type) or E. faecalis arcD::Tn during CDI. Each strain introduced prior to CDI and naturally competed with endogenous enterococci (n = 5/group) (mean ± s.e.m. two-sided Mann-Whitney test with Bonferroni-Dunn method for correction for multiple comparisons, day 2 P=0.048, day 3 P=0.024). (j) Ornithine (P=0.014) and (k) arginine (P<0.001) levels in stool measured by targeted metabolomics in GF mice infected with C. difficile. Mice pre-colonized for 1 day with E. faecalis (n = 3) or E. faecalis arcD::Tn (n = 4) (mean ± s.d.. two-sided t-tests with Welch’s correction). Metabolomics performed on GF mice prior to infection (GF group, n = 5).