The fold variation of gene expression was obtained by the comparative cycle

threshold (∆∆CT) method. The iutA expression expressed as a value of 1 represented bacteria grown in LB, and variations in expression in other media conditions are related to this value. The expression of iutA resulted in 2.15- (*, P = 0.01), AZD9668 4.9- (*, P = 0.001) and 12.13-folds (*, P = 0.01), increase in bacteria grown on MacConkey, LB/DIP and MacConkey/DIP respectively. Student’s T-test was used for the statistical analysis. Quantitative real-time PCR was performed to support the results obtained with the heat-extracted proteins and to quantify the expression of iutA in the E. coli O104:H4 wild-type strain, while grown in LB or MacConkey media with and without DP. Basal expression

of iutA in the wild-type strain was set at a value of 1, and all other values of expression were related to this baseline. The expression of iutA was 2.1-fold higher in the wild-type strain grown in MacConkey as compared to LB (Figure 3B, P = 0.01). In the presence of DP, the iutA expression level in the wild-type strain increased (4.9-fold, P = 0.001) when grown in LB + DP and reached 12.1-fold when the wild-type strain was grown on MacConkey agar supplemented with DP (Figure 3B, P = 0.01). Overall, data confirmed that the aerobactin receptor is expressed on the surface of E. Regorafenib in vivo coli O104:H4 wild-type strain, while grown on MacConkey agar, and that expression 3-mercaptopyruvate sulfurtransferase increased in response to iron depletion. Contribution of aerobactin to intestinal colonization Given that

the aerobactin transport system has been proposed as a contributor to the strong intestinal colonizing capability of some strains [24], the influence of the mutation of this iron transport system in E. coli O104:H4 intestinal colonization in mice was assessed. In a wild-type background, deletion of iutA aerobactin receptor gene had a significant effect upon colonization of the cecum (Figure 4). Starting at 24 h post-infection, the wild-type strain outcompeted the iutA mutant [geometric mean (95% confidence interval)]; [0.042 (0.01-0.178)]), suggesting that aerobactin production makes a contribution to colonization early during infection. Consistent with the results at 24 h, the CIs of the iutA mutant at 48 h [0.047 (0.01-0.183)], 72 h [0.01 (0.01-0.137)], 96 h [0.030 (0.01-0.177)], and 168 h [0.005 (0.01-0.140)], were drastically diminished as compared to the wild-type strain. Data suggested that the in vivo intestinal colonization of the E. coli O104:H4 strain required the aerobactin transport system, and the defects observed were due to the inability of the strain to acquire iron. Figure 4 The iutA mutant is outcompeted by E. coli O104:H4 strain C3493 in the murine intestine. Female ICR mice were intragastrically inoculated with 1:1 mixtures of (A) E.