Figure 4. Survival of B. melitensis 16 MΔotpR, 16 McΔotpR, and the wild-type strain under iron-limited conditions.

The survival of the strains was measured in the TSB medium containing 2.5 mM, 5.0 mM and 10.0 mM Fe²⁺ chelator 2,2`-dipyridyl. The measurements were taken at 24 h, 48 h and 72 h after inoculation. Experiments were performed in triplicates and a significant difference was observed. Statistical analysis was performed by comparing the CFUs of 16 M versus 16 MΔotpR bacteria at different time points using the Student’s t-test. p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) represents different levels of significant differences. It is noted that in the presence of 10.0 mM DIP, the otpR mutant could be detected at 24 and 48 h after inoculation, but not at 72 h. When the CFU/mL was determined, 200 μL of culture sample was used in each condition, which equals to a detection limit of 5 CFU/ml (or ~0.7 LOG CFU). It is possible that there were still some viable otpR mutant cells survived in the iron-limited medium at 72 h; however, the level of survival was below the laboratory’s detection limit.