coli O104:H4 lux; 1 × 108 CFUs) and, for the competition experiments, with a mixture of E. coli O104:H4 wild-type strain and CSS001 (E. coli O104:H4 iutA::cat; 5 × 107 CFUs per strain) in a final volume of 0.4 ml delivered by gavage (20-gauge needle), thereby using the mouse intestinal model to study enteropathogenicity of E. coli strains previously described by our group [16, 17]. Briefly, animals received streptomycin (5 g/L in drinking water) for 48 h prior to
oral inoculation with the E. coli strains and were food restricted for 12 h check details before oral inoculation. The concentration of the initial inoculum was determined by plating on selective antibiotic LB media by using the dot plate method [42]. Groups of mice (n = 10) were maintained for 7 days, and at different time points (24 h, 48 h, 96 h, and 169 h post-inoculation), groups of two or four animals were euthanized, and the cecum of each animal was collected, weighed, and homogenized for bacterial load enumeration. After homogenization, centrifugation at 3,000 xg for 30 seconds was done in order to sediment the cell debris, allowing for collection of accurate volumes
needed to make serial dilutions. Samples were plated on LB agar, LB + streptomycin (100 mg/mL), EPZ-6438 in vivo and LB + streptomycin + kanamycin (50 mg/mL) to determine total bacterial cell counts from those of E. coli O104:H4 or the iutA mutant strain. The vast majority of bacteria recovered from the cecum corresponded to the O104:H4 isogenic strains (data not shown). The replicates plated for each mouse were averaged, and competitive indices were calculated as previously described [43]. Groups were compared by using the Mann Whitney non-parametric test. Bioluminescent quantification For in-vivo imaging, mice were anesthetized with 2-3% isofluorane in an oxygen-filled induction chamber and then transferred to an isolation chamber placed inside the imaging chamber. Bioluminescent images Y-27632 2HCl were acquired by using an IVIS Spectrum (Caliper Corp., Alameda, CA) as we previously described [18]. The ex vivo images of the intestine were acquired at each time point immediately after
euthanasia. Bioluminescent signal is represented in the images with a pseudocolor scale ranging from red (most intense) to violet (least intense) indicating the intensity of the signal. Scales were manually set to the same values for every comparable image (in-vivo and ex-vivo) to facilitate comparison of intensity of the bioluminescence at each time point. Electron microscopy analysis and histopathology Segments of the mouse cecum infected with the wild-type E. coli O104:H4 strain were collected, washed gently with PBS, and fixed in a mixture of 2.5% formaldehyde, 0.1% glutaraldehyde, 0.03% trinitrophenol, and 0.03% CaCl2 in 0.05 M cacodylate buffer (pH 7.2) as previously described [16]. Samples were processed further by postfixing in 1% OsO4, stained en bloc in 2% aqueous uranyl acetate (in 0.