An identical reaction without reverse transcriptase was performed to assess DNA contamination. Regions corresponding to fim2A, fim2H and fim2K were PCR amplified using primers pairs PR1607-PR1608, PR1609-PR1610, and PR1611-PR1612, respectively. Regions linking 116met56-10

to fim2A and fim2H to fim2K were detected using primer pairs PR1626-PR1627 and PR16268-PR1629, respectively. Amplicons were visualised on 1.5% agarose gels. Transmission electron microscopy Five μl of sample was applied to a hydrophilic Formvar-carbon coated copper grid (Agar Scientific) and allowed to adsorb for 5 min. After wicking excess liquid, the grid was washed once using distilled deionised water and then negative-stained for 15 s with a droplet of 1% uranyl acetate (pH 4.5). Electron microscopy was performed on a JEOL JEM-1400 microscope at 80 kV. Biofilm, GDC973 growth curve and epithelial adhesion assays Biofilm assays selleck compound were performed using a modified microtiter plate-based method [63]. Briefly, strains were grown

for 16 h (37°C, 200 rpm) in LB broth with antibiotics if necessary and subcultured 1:100 into 100 μl LB medium with 0.05 mM IPTG and ampicillin, when required, in 96-well microtiter plates (Nunc). Plates were incubated statically for 48 h at 37°C and OD595 (optical density at 595 nm) readings obtained at the end of incubation. Following incubation the medium was removed and the plate washed once with distilled water. 125 μl of 0.1% (v/v) crystal violet was added to each well and left to stain for 10 min. The plate was then washed twice with distilled water, dried thoroughly and the stain eluted with 200 μl of 95% ethanol check details per well and the absorbance measured at 595 nm (BioRad Model

680 Microplate reader). Each was strain tested in eight wells and three replicate experiments were performed. Growth curves were performed similarly to biofilm assays with a few minor modifications. Plates were incubated statically for 24 h at 37°C in a Varioskan (Thermo Scientific) instrument. The plates were subjected to a brief vigorous shake HSP90 every 10 min immediately prior to the absorbance being measured at 600 nm (OD600). Each strain was tested in seven wells and two duplicate experiments were performed. Quantitative assessment of bacterial adhesion to epithelial cells was performed using human HCT-8 ileocaecal and 5637 bladder cells. HCT-8 cells were subcultivated (1:10) twice a week in RPMI 1640 medium containing 25 mM HEPES, 2 mM glutamine, 1 mM pyruvate, 10% fetal calf serum, 0.002% neomycin and 0.01% streptomycin. 5637 cells were cultivated similarly but no pyruvate was added to the medium. Epithelial cells were seeded into two 24-well tissue culture plates (Nunc) and grown to confluent monolayers. After carefully washing each well three times with warm PBS, 1 ml of fresh supplement-free RPMI 1640 was added and inoculated with ~2 × 106 CFU from an overnight culture. Plates were incubated for 3 h at 37°C.