Fig. 1.

Anti-Vibrio activities exhibited by gut commensal E. coli. (A) Screening. Mouse-isolated E. coli strains were grown to saturation in 96-well plates and then inoculated into minimal medium containing V. cholerae. The cultures were incubated at standing 37 °C for 24 h, and viable V. cholerae was determined by plating a four-log dilution of the mixed culture on selective LB agar. (B) Confirmation of selected mouse E. coli isolates on V. cholerae growth. E. coli isolates identified by the screen and controls (MG1655 and MP1) were coincubated with V. cholerae at a 10:1 ratio in minimal medium and incubated at standing 37 °C for 24 h. V. cholerae CFU values were determined by serial dilution and plating on selective LB agar plates. The mean of three independent assays is shown. (C and D) EMS1 kills V. cholerae in a dose-dependent manner. V. cholerae alone or V. cholerae with different ratios of EMS1 was inoculated into M9 medium and incubated without shaking at 37 °C for 24 h. V. cholerae CFU values were then determined. The mean of three independent assays is shown, and error bars represent the SD. *P < 0.05; ***P < 0.001; ****P < 0.0001 (one-way ANOVA). (E) Contact-dependent killing. EMS1 and V. cholerae were separately inoculated into 10-kDa–cutoff dialysis bags in minimal medium or coinoculated into the medium and incubated for 24 h before V. cholerae CFU determination. The survival rate was calculated by normalizing CFU of V. cholerae cocultured with EMS1 against that of V. cholerae cultured alone. The mean of four independent assays is shown; ***P < 0.001 (t test). (F) EMS1 inhibitory effects on various Vibrio strains. Overnight cultures (10⁶ CFU/mL) of Vibrio strains were inoculated into M9 medium without and with 10⁷ CFU/mL EMS1 and incubated for 24 h. CFU of Vibrio were then determined. The mean of three independent assays is shown, and error bars represent the SD.