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Microbes & Immunity Dynamics between phage, bacteria, and mammalian cells

stocks were re-suspended in 200 μL Na CO /NaHCO and 5% CO2 in the absence and presence of BEAS-2B
3
2
3
buffer (pH 9.2) and 5 μL FITC solution (5 mg/mL) for cells for 24 h. The cultures were plated to obtain isolated
incubation in a dark environment at room temperature bacterial colonies. These plated colonies were sub-cultured
with gentle shaking (100 rpm) for 2 h to allow conjugation 3 times to avoid residual free phages, and each sub-culture
of FITC to the protein coat of the phages. Thereafter, 200 generation had three replicates. Thereafter, ten colonies
μL solution of 20% PEG8000 with 2.5 M NaCl was added were selected for the spot test to measure sensitivity
to the samples and incubated overnight at 4°C. The samples toward phages. Phage-resistance development was defined
were subsequently centrifuged at 15000 ×g at 4°C for 1 h. as no observable plaque in the spot test assay. The phage-
The phage pellets were re-suspended in 100 μL PBS and resistance rate was determined as:
stored at 4°C before use. Approximately 1 × 10 BEAS-2B
5
cells were seeded in a glass-bottom cell culture dish Number of colonies
(diameter of 20 mm) with 1 mL DMEM. After overnight form no plaque
incubation, the cells were cultured with 100 μL FITC- Phage resistance rate− = 10 × 100% (II)
labeled phages. After 24-h co-culturing in the incubator
at 37°C and 5% CO , 4% paraformaldehyde in PBS was The adsorption rate of phages onto the phage-treated
2
added to fix the cells for 30 min before the dishes were bacteria was also examined using incubating phages
washed 3 times with PBS to remove planktonic or loosely with the sub-cultured bacteria for 10 min followed by
adhered phages. The samples were then stained with immediate centrifugation at 12000 ×g and 4°C for 5 min.
300 nM DAPI staining solution (Thermo Fisher Scientific, The supernatant was then collected, and serial-diluted for
USA) at room temperature for 5 min, followed by washing the plaque assay. Adsorbed phages were evaluated as the
with PBS 3 times. A fluorescence microscope (TI-DH, difference between the initial phage titer and phage titer in
Nikon, Japan) was used to visualize the phage adhesion at the supernatant.
×40 magnification. The NIS-Elements imaging software 2.7. Cytokine secretion by BEAS-2B cells
(version 5.01) was employed for image processing.
The level of cytokines (tumor necrosis factor-alpha
2.5.2. Bacteria-epithelial cell adsorption assay [TNF-α], interleukin 1-beta [IL-1β], and interleukin-6
6
4
BEAS-2B cells were seeded in a 96-well plate at 3 × 10 cells [IL- ), secreted by BEAS-2B cells toward the bacterial
per well in 100 μL DMEM culture medium. After overnight challenge, was evaluated after incubating with the bacteria
incubation, the spent medium was removed, and the cells with and without phage treatment for 24 h. Analysis was
were further incubated with 100 μL bacteria (~10 CFU/ performed with commercially available inflammatory
7
mL) at 37°C and 5% CO for 24 h. The wells were then biomarker ELISA kits (eBioscience, Affymetrix Inc., USA)
2
washed 3 times with PBS to remove any unadhered bacteria. according to the manufacturer’s protocols. The experiment
A volume of 100 μL PBS containing 0.01 mM EDTA was was performed in three biological replicates (n = 3) and
then added to wells for 10 min at 37°C, following vigorous repeated two independent times.
pipetting. The plate counting method was used to determine 2.8. Statistical analysis
the number of viable bacteria adhered to the epithelial cells.
The experiment was performed in biological replicates (n = All experiments were repeated in two independent
3) and repeated two independent times. experiments. The number of biological replicates in each
experiment and individual assays was used to determine
Bacterial adhesion onto the BEAS-2B cells was
visualized with Giemsa staining. Briefly, the bacteria and the statistically significant differences between groups.
One-way analysis of variance (ANOVA) at a confidence
BEAS-2B co-culture were fixed with 4% paraformaldehyde level of 95% was employed to identify any statistically
in PBS for 30 min and subsequently stained with Giemsa significant differences in cell and bacteria viability. p-value
solution (1:20 dilution of stock solution) for 45 min. The < 0.05 was considered statistically significant.
stained co-culture was then washed with PBS 3 times for
visualization using a fluorescence microscope (TI-DH, 3. Results
Nikon, Japan) under the bright field visualization mode at
×20 magnification. 3.1. Effect of treatment period on antibacterial
efficiency by phage
2.6. Phage-resistance development assay The effects of phage addition on the antibacterial efficiency
The frequency of bacterial variants emerging with phage in the co-culture system were studied to mimic the scenario
resistance was determined using incubating phages (MOI of (i) after an infection initiated by pre-incubating bacteria
100) with ~10 CFU/mL MDR-AB2 bacteria at 37°C with BEAS-2B cells for 4 h; (ii) at the bacterial colonization
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Volume 1 Issue 1 (2024) 84 doi: 10.36922/mi.3141