Furthermore, this study found

an association between geographical variation of the EAEC strains and their iron utilization genes with LY2835219 ic50 disease onset, indicating that most EAEC strains contain more than one iron transport system [15]. There is an urgent need to characterize additional virulence factors in E. coli O104:H4, besides the Shiga toxins, which might be associated with disease in the natural setting and not just in silico or in vitro. Therefore, we combined a murine model that mimics the enteropathogenicity of E. coli strains [16, 17] with bioluminescent imaging (BLI) technology, a method recently optimized in our laboratory [18]. We hypothesized that the murine model of experimental infection using E. coli O104:H4

bacteria not only is an appropriate way to visualize the site of intestinal colonization, but will also aid in rapid screening of putative virulence factors in vivo. This BLI infection method provided us with the advantage of quantitatively assessing the E. coli O104:H4 burden and facilitated the development of new insights into tissue tropism during infection. Furthermore, BLI application reduced the number of animals required for competition experiments, aided in the localization Sirolimus cost of E. coli O104:H4 infection sites, and enabled us to quickly screen the role of the aerobactin iron transport system (iut/iuc system) as a virulence factor in this pathogen. Results In vivo bioluminescence imaging The E. coli O104:H4 lux strain RJC001 was generated as described in Methods. We used the pCM17 plasmid containing the lux operon under the OmpC constitutive promoter. This plasmid was used for the following properties: to avoid the exogenous addition of luciferase substrate, it carries both a two-plasmid partitioning system and a post-segregational killing mechanism, and maintenance can be ensured for at least 7 days [19]. E. coli O104:H4 transformants were plated on the appropriate Thalidomide media, incubated

at 37 °C, and monitored for bioluminescence. Colonies that did not display any apparent difference in the bioluminescent signal after patching on plates containing the appropriate antibiotic were further evaluated for their resistance to multiple antibiotics (E. coli O104:H4 displayed an extended-spectrum β-lactamase phenotype [20]), presence of multiple plasmids, and growth phenotype similar to that of the wild-type strain (data not shown). E. coli strain RCJ001 was selected because it displayed wild-type characteristics and showed a strong bioluminescence signal. E. coli O104:H4 lux strain RJC001 was evaluated as a reporter strain in following intestinal infection of the ICR (CD-1) mouse model. A group of 10 ICR mice were infected intragastrically with 1 x 108 CFUs of E. coli strain RJC001 (Figure 1A).