Bacterial strains and growth conditions
Lactobacillus strains used in this study were Lactobacillus plantarum PCS20, PCS22, PCS25 (cheese isolates, from the collection of the Department of Biochemistry and Nutrition, Faculty of Medicine, University of Maribor, Slovenia), Lactobacillus rhamnosus LGG (from American Type Culture Collection; ATCC53103) and Lactobacillus plantarum PCK9 (from a cheese isolate, obtained during the European research project PathogenCombat; FP6-007081) . The strains applied on the intestinal epithelial cell lines in our study were selected on the basis of previous in vitro and in vivo research. All Lactobacillus strains were previously determined for their probiotic characteristics and showed good adhesive properties to intestinal epithelial cells [56-59]. The tested strains were grown in De Man Rogosa, Sharpe (MRS) broth (Merck, Darmstadt, Germany) for 24 h at 37 °C, and under anaerobic conditions. The final bacterial suspensions for the competition assays contained approximately 1×108 CFU/mL.
c. jejuni K49/4, a poultry meat isolate was grown at 42 °C microaerophilically (5% O2, 10% CO2, 85% N2) on Columbia agar (Oxoid, Hampshire, UK) supplemented with 5% defibrinated horse blood (Oxoid, Hampshire, UK). C. jejuni were transferred to Preston broth (Oxoid) at 42 °C, and grown microaerophilically for 9 h. These C. jejuni cultures in exponential phase were diluted in cell culture media containing no antibiotics to approximately 2 ×108 CFU/mL and were used for cell culture assays.
PSI cl.1, B1OXI and CLAB cell monolayers
The normal PSI cl.1 epithelial-derived cell line (partially differentiated cryptic enterocyte-like) was obtained from an adult pig at slaughter, as previously described . These cells represent the closest model to humans in terms of genome, organ development, anatomy, physiology and metabolism of the intestinal tract, and for disease progression,d intestine–microbe interactions [60,61]. The B1OXI cells represent normal enterocytes of the chicken small intestine. The B1OXI cell line was developed and characterised at the University of Maribor, Slovenia, in the framework of the EU funded ‘PathogenCombat’ project, to build a three-dimensional functional epithelial cell model of the chicken intestine . The CLAB cells are enterocytes that were obtained from an adult pig at slaughter in Slovenia and represent adult mucin secreting enterocyte-like cell line . Although CLAB cells are epithelial in origin, they do not polarise in vitro . These cells are of non-tumorigenic origin, which makes them more suitable as the in-vitro model to study pathogen–host interactions than tumorigenic cell lines. The phenotypical and functional characterization of the cells was performed with immunocytochemistry and the search for the presence of key epithelial markers. In addition, all cell lines were tested for mycoplasma contamination prior to the experiments.
The PSI cl.1 and B1OXI cell lines can form a tightly packed epithelial barrier when grown on microporous inserts, and for this reason, they were chosen for further studies to evaluate the efficacy of the probiotics for prevention of C. jejuni K49/4 adhesion, invasion and translocation across polarised cell monolayers. PSI cl.1, B1OXI and CLAB cells are of non-tumorigenic origin; instead, they were isolated from dissected animal tissue using the limiting dilution technique. The functional PSI cl.1 and B1OXI intestinal cell models were developed for studies into probiotic/pathogen/gut epithelial interactions in more detail following initial screening for the efficacy of the probiotics for prevention of C. jejuni K49/4 adhesion and invasion on PSI cl.1 and CLAB cell monolayers.
Cultivation and propagation of cell lines
PSI cl.1, B1OXI and CLAB cell lines were grown in advanced Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, Grand Island, USA), supplemented with 5% foetal calf serum (Lonza, Basel, Switzerland), 2 mmol/L L-glutamine (Sigma), 100 U/mL penicillin (Sigma) and 1 mg/mL streptomycin (Fluka, Buchs, Switzerland), at 37 °C in a humidified atmosphere of 5% CO2. To form monolayers, 96-well microplates were seeded with approximately 5.0 ×105 cells/mL PSI cl.1 and CLAB, and incubated for 24 h to reach confluence. Thereafter, the cultures were washed three times with phosphate-buffered saline (PBS) and cultivated in antibiotic free DMEM for the cell adhesion and invasion assays.
PSI cl.1 and B1OXI polarised cell model
To obtain polarised monolayers, PSI cl.1 or B1OXI cells were seeded on Transwell filter inserts (pore size, 0.4 μm; 12 mm; Corning) that were placed into 12-well plates (22.1 mm; Corning), at a density of 1 ×105 cells/cm2. The TEER was measured using an electrical resistance system (Millicell-ERS; Millipore, Bedford, MA, USA). The net TEER was corrected for background resistance by subtraction of the resistance of the microporous membranes with the cell cultures (108 Ω/cm2) from the resistances measured with the system. Functional polarity was established when the TEER between the apical and basolateral surfaces of the monolayers exceeded 1600 Ω/cm2 for PSI cl.1 cells and 240 Ω/cm2 for B1OXI cells. TEER was measured before the cells reached confluence, after the addition of the bacteria to the medium (with or without gentamicin), and 24 h post-infection. The TEER of cell monolayers without bacteria was used as the control for each experiment.
Effects of probiotic bacteria and C. jejuni K49/4 on viability of cell cultures
To test the effects of the bacteria on the viability of the PSI cl.1, B1OXI and CLAB cell monolayers, the cells were seeded separately in microplates at a density of 6 ×105 cells/well. Later, each strain of viable probiotic bacteria (1 ×108 CFU/mL) and C. jejuni K49/4 (2 ×108 CFU/mL) were added to pre-washed monolayers of the PSI cl.1, B1OXI and CLAB cells, and the cell monolayers were incubated for 24 h at 37 °C in a humidified atmosphere of 5% CO2. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was used to determine cell viability, as described previously . For each probiotic strain and for co-incubation of C. jejuni with probiotic bacteria three wells were used for each replicate. The mean absorbance of the control wells containing only confluent cell culture without any bacteria was taken as 100%. The percentage of metabolically active cells treated with probiotic bacteria and Campylobacter jejuni was then calculated.
Cell culture Assays
Adhesion, Invasion, and Intracellular survival of C. jejuni K49/4 with PSI cl.1 and CLAB cells
Bacterial adhesion tests on PSI cl.1 and CLAB cell monolayers were carried out in 96-well tissue culture plates. After washing the cell monolayers with PBS, each of the probiotic bacteria (approximately 1 × 108 CFU/mL) were added simultaneously with C. jejuni K49/4 (2 × 108 CFU/mL) to each well. The control wells were prepared by adding C. jejuni K49/4 to the cell monolayers. The cells were incubated at 37 °C in 5% CO2 for 2 h, to allow adhesion and invasion. After washing with DMEM, DMEM containing 100 μg/mL gentamicin was added, to determine the number of invaded C. jejuni. After a 1-h incubation, the monolayers were lysed with 1 mL/L (v/v) Triton X-100, for 5 min and were serially diluted. The intracellular bacteria were determined by plate counting at 3, 9, 24 and 48 h post-infection. The total numbers of adherent and internalised bacteria were determined simultaneously by performing the invasion assay, but without gentamicin treatment. The differences between the numbers of total and intracellular bacteria were calculated as the number of adherent C. jejuni cells.
Adhesion, invasion, and translocation of C. jejuni K49/4 using the PSI cl.1 and B1OXI polarised cell model
A functional cell model using PSI cl.1 and B1OXI cells was developed to determine inhibitory effect of the probiotic bacteria on the C. jejuni K49/4 adhesion, invasion and translocation of C. jejuni to the basolateral compartment of the well. When cells were confirmed to have reached confluence using TEER (>1600 Ω/cm2 for PSI cl.1 cells; >240 Ω/cm2 for B1OXI cells), the monolayers were washed twice with 100 μL DMEM without antibiotic/supplements, and infection assays were performed by seeding the bacterial inoculum of each probiotic culture (approximately 1 ×108 CFU/mL) together with C. jejuni K49/4 (approximately 2 ×108 CFU/mL) in the apical chamber. The total numbers of adhered and invaded C. jejuni K49/4 were determined at 3, 17 and 24 h post-infection after lysing the cells by addition of 500 μL Triton X-100 and plating on Columbia agar plates. Bacterial count for infection of cell lines and chosen time intervals were similar as in our previous studies [32,33]. In addition, the numbers of translocated C. jejuni K49/4 were determined at the same time intervals. To investigate the effects of C. jejuni K49/4 on PSI cl.1 and B1OXI cell monolayer integrity following the infection of the cell lines, the TEER was measured at 0, 3, 17, and 24 h post-infection. The TEER of the infected cells was compared to non-infected cells. Furthermore, the effects of co-incubation of probiotics with C. jejuni K49/4 were also assessed.
To define the effects of lactobacilli on C. jejuni K49/4 adhesion, data from triplicate samples from at least three independent experiments were analysed statistically with the Predictive Analytics (PASW) statistics 202 software, version 18.0 (IBM Corp., Armonk, NY, USA), for the significance of any changes in bacterial numbers. Statistical analyses were performed with unpaired Student’s t-tests to estimate the statistical significance. All data are presented as means ±standard deviations (error bars) of the replicate experiments. All statistical values were considered significant at P ≤0.01.