McCoy's medium, phosphate buffer saline, trypsin-EDTA solution, T25 cell culture plates, T75 cell culture plates, de Man, Rogosa and Sharpe (MRS) agar, brain heart infusion broth, trypton-yeast broth, fetal bovine serum (Himedia, Mumbai, India), 5 mg streptomycin and 5000 units penicillin per ml, DMSO, L-cystine HCl, Giemsa stain, mucin and fibronectin (Sigma, Bangalore, India) 6 well plates (Corning, India), 12 well plates (Eppendroff, India), were procured for experimental studies until otherwise mentioned.
2.2 Bacterial strains and culture conditions
Bacillus licheniformis MCC 2514 isolated from goat milk (Shobharani and Halami 2014), a fecal isolate of Bifidobacterium breve NCIM 5671 (Achi and Halami 2019) and Kocuria rhizophila ATCC 9341 were used in the study. Potential probiotic cultures Bacillus licheniformis and Bifidobacterium breve are native laboratory isolates used to screen the adherence potential against cancerous epithelium HT-29 cell lines, which was procured from National Centre for Cell Sciences (NCCS), Pune, India. B. licheniformis were cultured in optimized trypton-yeast media, Bif. breve was grown in MRS medium with 0.05% L-cystine HCl and K. rhizophila was cultured in BHI (Brain Heart Infusion) medium. B. licheniformis and K. rhizophila were grown at 37° C for about 18 h in a shaker incubator at 150 rpm and Bif. breve was incubated on CO2 incubator at 37°C for 24 h. In the entire study, B. licheniformis and Bif. breve was used as an individual culture and as co-cultures.
2.3 Adhesion to mucin and fibronectin
The ability of B. licheniformis and Bif. breve to bind fibronectin and mucin was evaluated as described by Roos and Jonsson (Roos and Jonsson 2002; Archer and Halami 2015). Briefly, the 96 well microtiter plates were coated with fibronectin (50 µg/mL) and 150 µl/well of mucin (100 µg/ml) dissolved in 50 mM Na2CO3 buffer with a pH of 9.7. The 96 well plates were incubated overnight at 4° C with minimum shaking and later blocked with phosphate-buffered saline (PBS) of 0.2 mL supplemented with 1 % Tween20 for one h and it was thoroughly washed with PBST (0.05 % Tween 20 with PBS). Overnight grown bacterial cultures were washed using PBST, diluted to the OD600 of 1.0 in the same buffer and 150 µL was added to the wells and incubated at 37°C for one h. All the wells were thoroughly washed with PBST and cell adherence was witnessed under the inverted microscope. Besides, to remove the adhered bacteria, 0.2 mL of Triton X-100 (0.5 %) was used and incubated at 37°C for two h. From every well, 100 µL of cell suspension was diluted and plated in respective medium and CFU count was observed.
2.4 Maintenance of HT-29 Cell lines
The HT-29 Epithelium cells were grown in McCoy's medium with 10% (v/v) of heat-inactivated (56°C, 30 mins) fetal bovine serum, 5 mg streptomycin and 5000 U penicillin /ml in 25 cm2 culture flask at 37°C in 5 % CO2. The incubated cells were fed with fresh medium every alternate day. After cells reaching confluency around 80%, the cells were collected by incubating adhered cells with 0.25% Trypsin-EDTA solution at 37°C. The cells were centrifuged (1200 rpm, 2 mins at room condition) and the cells were re-suspended in McCoy's medium. Before incubating, the cells were counted using a hemocytometer (Rohem, India) and introduced into respective assay plates. For six-well plates, 3 x 105 cells/well, for 12 well plates 7.5 x 104 cells / well and T25 flask 1 x 106 cells / well were added as described earlier by Gagnon et al. 2013.
2.5 Binding efficiency on HT-29 cell line
Adhesion assay on HT-29 was carried out with the previously described procedure with slight changes (Duary et al., 2011). Adhesion of the B. licheniformis and Bif. breve cultures to the HT-29 cell line were measured. The cell suspension with 3 x 105 cells added in 3 ml McCoy's medium was transferred to each well into six-well adherent culture plates. The medium was replaced every alternate day. When these cells reached a confluency of 80%, the medium was replaced every day repeatedly for about 15–20 days. The exhausted medium was decanted three h prior to the adhesion assay and cells were introduced with McCoy's medium without antibiotics and incubated at 37° C for three h in 5% CO2. After the incubation period, cells were thoroughly washed with 3 ml phosphate-buffered saline (PBS) twice. The B. licheniformis and Bif. breve (at 1 x 108 CFU/ml) cells were added to 1.0 ml McCoy's medium (without antibiotics and FBS) and introduced into different wells. These treated plates were incubated at 37°C for four h in 5% CO2. The monolayers were washed five times thoroughly using sterile PBS.
2.6 Adhesion score, adhesion percentage and SEM analysis
By following the procedure described in Sect. 2.5, adhesion study experiments were carried out, 3 ml of methanol was introduced into each well containing the cells and incubated for 10 min at room condition. Methanol was aspirated completely and cells were stained using 1:20 diluted Giemsa for 90 min at ambient condition. The wells which were fixed are washed using pure ethanol to take out the unwanted stain. The air-dried plates were examined under 100x magnification (BX 5, Olympus, Japan). The bacteria were counted in 20 random microscopic fields and were labeled into different groups according to their counts, such as strongly adhesive (> 100 bacteria/field), adhesive (41–100 bacteria/field) and non-adhesive (≤ 40 bacteria/field). Continuing the adhesion assay procedure, the cell monolayer from the treated plate was separated using trypsinization. A 0.25% trypsin-EDTA solution was introduced into each well of six-well plates and incubated for 15 min at room condition. The detached cells were gently homogenized to make a uniform suspension. The cells were then plated on Tryptone-yeast agar for B. licheniformis and plated on MRS agar for Bif. breve using serial dilution. After the incubation at 37°C for 24–48 h, colonies were counted (B1 CFU/ml). Bacterial cells initially added to each well of six-well plates were also counted (B0 CFU/ml).
The adhesion percentage was calculated using the below formula:
% adhesion= (B1 / B0) * 100
For scanning electron Microscopy observation, the HT-29 cells were grown and maintained on glass coverslips until the monolayer is achieved. Later cells were fixed using 2.5% glutaraldehyde solution and incubated at 4°C for overnight. The ascending gradation of alcohol dehydration (30%, 50%, 70%, 90%, 95% and 100%) was carried on coverslips and finally, air-dried coverslips were subjected to gold plating and observed under SEM (LEO 435 VP, Carl Zeiss, Cambridge, UK) (Manjulata et al. 2018).
2.7 Inhibition of Kocuria rhizophila from colonizing HT-29 cell line
Inhibition of foodborne pathogen Kocuria rhizophila by B. licheniformis and Bif. breve from colonizing on HT-29 cell line was measured by the following assays as described before with slight modifications (Kumar et al. 2011).
For competition assay, both B. licheniformis and Bif. breve (1 x 108 CFU/ml) and K. rhizophila (1 x 108 CFU/ml) added in one ml Mc Coy's medium (without antibiotics and FBS) and suspended into HT-29 cells and incubated at 37°C for 90 min in 5% CO2 condition. After the incubation time, non-adhered bacterial cells were detached by washing the wells thoroughly with PBS trice and adhered bacterial cells were obtained by treating with 0.25% trypsin at 37°C for 10 min. The obtained bacterial cells were plated in respective medium (Optimized tryptone-yeast medium for B. lichenifomis, MRS agar for Bif. breve and BHI agar for K. rhizophila) for their culturing. The total bacterial count was denoted in log CFU/ml. Control or un-treated wells were maintained for both B. lichenifomis and Bif. breve and K. rhizophila, and they were standard for all of the assays. For exclusion assay, B. lichenifomis and Bif. breve (1 x 108 CFU/ml) cells were introduced to the HT-29 cell line and incubated at 37°C for 90 min in 5% CO2. Weakly attached cells were removed by thoroughly washing with PBS. After washing, K. rhizophila (1 x 108 CFU/ml) cells were added to the HT-29 cells, which are already colonized by B. lichenifomis and Bif. Breve was allowed for incubation at 37° C. At the end of the incubation, weakly attached cells were detached using PBS wash thoroughly, and bacterial cells adhered to were obtained using trypsinization and CFU/ml. For displacement assay, initially K. rhizophila in the concentration of 1 x 108 CFU/ml cells were introduced on HT-29 cells and incubated at 37° C for 90 min in 5% CO2.. Weakly attached cells were detached by washing thrice with PBS wash. After the wash, B. lichenifomis and Bif. breve (1 x 108 CFU/ml) were added to HT-29 cells which are already adhered with K. rhizophila. The treatment which is mentioned in the exclusion assay was followed. All three assays were carried out in triplicates, and results were interpreted statistically. SEM analysis was determined with glutaraldehyde and alcohol dehydration in ascending gradation (Manjulata et al., 2018).
2.8 Cytotoxicity studies using MTT assay and confocal microscope
To study the cytotoxic effects of bacteria on the epithelium cell line, MTT assay and confocal microscopy were performed (Xi et al. 2009). The B. licheniformis and Bif. breve cultures to the HT-29 cell line were measured. The HT-29 cell suspension with 4 x 104 cells /ml cell concentration prepared in 0.1 ml complete McCoy's medium and it was transferred to the individual well of 96-well culture plates. These plates were incubated at 37°C for 48 h in 5% CO2 for better adhesion and growth. After the incubation, the 96 wells were thoroughly washed using PBS and cells were fed with McCoy's medium lacking antibiotics of 1 X 108 CFU/ml of both B. licheniformis and Bif. breve and were incubated at 37°C for four h. Later, the incubated cells were washed 3 to 5 times thoroughly using PBS to remove bacteria from the wells and a 10 µl of MTT reagent was added to the cell containing fresh Mc Coy's medium and incubated at 37°C for four h in 5% CO2. The medium was aspirated from the well and 100 µl of DMSO reagent was added and incubated at 37°C for 30 mins. Reading taken at 570 nm and the percentage of the viability was calculated using the below equation
% Viability = Q1/Q0 x 100. (Q0 = control reading, Q1 = treated reading)
For confirmation of cytotoxicity on HT 29 cell line, confocal microscopy for the B. licheniformis and Bif. breve cultures were performed. The HT-29 cell with the concentration of 3 X 105 cells was prepared in 3 ml McCoy's medium and it was added to every individual well of 12-well culture plates. It was incubated at 37°C for 48 h for better adhesion and growth. After the incubation, wells were thoroughly washed using sterile PBS and cells were fed with McCoy's medium lacking antibiotics with the bacterial concentration of 1 x 108 of both B. licheniformis and Bif. breve and incubated at 37°C for four h. The incubated cells were then washed with sterile PBS several times to remove the bacterial cells and treated with 0.5 ml of absolute chilled alcohol and incubated for two h at -20°C. After incubation, the cells were suspended in 0.5 ml of PBS to avoid drying of the cell until the microscopy observation. Fluorescence dyes such as Acridine orange and ethidium bromide (EtBr) dye were added 5 mins before the observation under the confocal microscope (LSM 700, Carl Zeiss, Germany).
2.9 Statistical analysis
All the data was subjected to statistical analysis was performed using one-way ANOVA using the Graph Pad Prism 7. All data are denoted as mean ± standard deviation (SD). In all the experiments, significance was set at P < 0.05.