PCR for the S-layer RTX gene was conducted www.selleckchem.com/products/i-bet151-gsk1210151a.html using the primers FCCC13826_1838 and RFCCC13826_1838 (Table 5) for 30 cycles with an annealing temperature of 58°C. PCR for the zot gene was conducted using the primers FCCC13826_2075 and RFCCC13826_2075 for 30 cycles with an annealing temperature of 56°C. Intestinal epithelial cell culture and inoculation T84 human colonic epithelial cells (passages 7 to 20; ATCC, Manassas, VA) were grown in DMEM/Ham F-12 plus 10% fetal bovine serum, 200 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 80 μg/ml tylosin (all from Sigma, Oakville, ON), and incubated
at 37°C and 5% CO2. For cell culture assays, confluent T84 monolayers were washed twice and media was replaced with antibiotic-free DMEM/Ham F12. Monolayers were inoculated with sterile see more Columbia broth (= control) or Campylobacter to achieve a multiplicity of infection of 100 CFU per epithelial cell, and incubated for 3 h check details at 37°C. Due to the intensive nature of the assays for assessment of pathogenic potential (i.e., adherence, invasion, translocation, hemolytic ability, and cytotoxicity), representative isolates of C. concisus from diarrheic and healthy humans were examined for pathogenicity (n = 5 from AFLP cluster 1, n = 9 from AFLP cluster 2). Adherence and invasion T84 enterocyte monolayers were grown in
24-well plates and inoculated as described Rebamipide above. Following incubation, monolayers were washed three times with PBS. To assess adherence, monolayers were lysed with 0.1% Triton X-100 in PBS for 10 min at room temperature on an orbital shaker. Following lysis, bacteria were enumerated by plating ten-fold serial dilutions onto Karmali agar (Oxoid, Nepean, ON). Invasion was determined using a gentamicin protection assay. After incubation, monolayers were washed three times with PBS. Monolayers were then incubated for 2 h with fresh tissue culture medium containing gentamicin (500 μg/ml) to kill extracellular bacteria as previously described . Following incubation, monolayers were washed, lysed and
bacteria were enumerated as for the adherence assay. A preliminary experiment was conducted to ensure that a bactericidal concentration of gentamicin was used for the invasion assay. Translocation and epithelial permeability T84 cells were seeded onto Transwell filters at 4 × 105 cells/filter (5 μm pore size, 1.13 cm2; Costar, Corning Inc. Corning, NY) and cultured as described above. Transepithelial electrical resistance (TER) was monitored with an electrovoltohmeter (World Precision Instruments, Sarasota, FL), and monolayers were used at confluence (TER >1000 Ω × cm2). Monolayers were inoculated as described above. Following incubation, the basolateral medium was serially diluted, spread onto Karmali agar and incubated microaerobically at 37°C. Permeability was assayed as described previously .