Fig. 6.

Colibactin drives changes in the gut microbiome in mice and humans and specifically kills B. fragilis. (A) PCoA of mouse microbial communities from fecal samples based on weighted UniFrac distance. Ellipses show 95% confidence intervals. Statistical tests by permutational multivariate ANOVA indicate significantly different microbiome structures between the ΔclbN mutant and EMS1; ΔclbN, n = 4; EMS1, n = 5. *P < 0.05; **P < 0.01. ns, no significance. (B) Relative abundance of B. fragilis in EMS1- and ΔclbN-treated animals day 5 after inoculation. Mann–Whitney U test; *P < 0.05; **P < 0.01. (C) Extrapolated B. fragilis relative abundances by MetaPhlAn3 analysis in presymptomatic fecal samples of humans exposed to V. cholerae by presence of colibactin biosynthetic genes; **P < 0.01 edgeR. (D) Colibactin effects on B. fragilis growth in vitro. Approximately 10⁶ CFU/mL overnight cultures of B. fragilis tested were inoculated into M9 plus 1% BHI (Brain Heart Infusion) with 10⁷ CFU/mL of EMS1, ΔclbN, or ΔclbL and incubated at 37 °C anaerobically. After 24 h, CFU values of assayed strains were determined, and the survival rate was calculated by dividing CFU with E. coli by CFU without E. coli. The mean of three independent assays is shown, with error bars representing SDs; **P < 0.01; ***P < 0.0005; ****P < 0.0001 (one-way ANOVA). (E) Effects of coinoculation of EMS1 and ΔclbN in suckling mice. Levels of B. fragilis in small intestine determined by plating after 16 h of colonization. Mann–Whitney U test; *P < 0.05. (F) Colibactin does not kill related Bacteroides commensals. Commensal Bacteroides strains were incubated with EMS1, ΔclbN, or ΔclbL at 37 °C anaerobically. After 24 h, CFU values of assayed strains were determined, and the survival rate was calculated by dividing CFU with E. coli by CFU with Bacteroides grown alone. The mean of three independent assays is shown, with error bars representing SD. (G) Distribution of the species in genera exhibiting highest colibactin-dependent changes in mice from household contacts of confirmed cholera patients in Bangladesh. Species with relative abundance >1% in at least one sample across the whole dataset (Dataset S3) are shown; edgeR: *P < 0.05; **P < 0.01; ***P < 0.001.