Competition assays

Each of the ivi gene deletion strains was tested in vivo during competition assays in mice to evaluate if gene inactivation affects the bacterial fitness of EDL933 as previously described [²⁷]. Briefly, individual EDL933 mutant and the wild-type (WT) strain were grown to mid-exponential phase in LB, washed once with PBS and mixed 1:1. Approximately 10⁷ CFU of both strains were administered intragastrically into 10 to 15 BALBc mice, 6 weeks of age, that were previously given drinking water containing Sm (5 g/L) for 24 h in order to selectively remove facultative anaerobic bacteria and open the niche for E. coli [²⁷]. Eight days post-infection, faecal pellets were resuspended in PBS and plated on LB agar plates + Sm and LB agar plates + Sm + Kan to count WT + ivi mutant and ivi mutant alone, respectively. The WT population was obtained by subtracting CFU from LB + Sm + Kan plates to CFU from LB + Sm plates. Results are expressed as competitive indices obtained by dividing output ratio (WT/mutant day 8 post-infection) by the corresponding input ratio (WT/mutant from mouse inoculum).