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

stage by adding phages together with bacteria; and bactericidal effect was noted in the presence of BEAS-2B
(iii) prophylaxis of infection by pre-incubating phages with cells. The enhanced antibacterial effect followed the order,
BEAS-2B cells for 4 h. Figure 1 compares the efficiency of AB2 phage > AB406 phage > AB9 phage, suggesting the
AB406 phages in killing bacteria under various phage-to- interactions between the phage, bacteria, and epithelial
bacteria MOIs in the presence and absence of epithelial cells are phage-specific. Despite the different antibacterial
cells in the three specified treatment period. After 24 h, the efficiency among the three phages, all of them could
AB406 phage could effectively reduce the number of viable successfully protect the epithelial cells from bacterial
bacteria. In the absence of epithelial cells, the bacterial damage, reflected by the similar cell viability as that of
count was significantly higher (~7 log, corresponding to the non-infected epithelial cells (Figure 2C). This was also
2 log reduction from the control) than that in systems reflected in the integrity of the bacteria-infected BEAS-2B
containing BEAS-2B cells (3‒5 log, corresponding cell layer treated with the investigated phages (Figure 2D).
to 4‒6 log reduction), suggesting dependence on the The adsorption rate of the three studied phages and
time of phage addition to the co-culture (Figure 1B).
Among the three treatment periods, pre-incubating the the MDR-AB2 bacteria onto the BEAS-2B cell surface
epithelial cells with phages (Condition iii) resulted in the were investigated. Different adsorption rates were noted in
highest antibacterial efficiency, followed by simultaneous the two-component system (bacteria with BEAS-2B cells
co-incubation (Condition ii) and pre-incubation with or phages with BEAS-2B cells) and the three-component
bacteria (Condition i). system (phage, bacteria, and BEAS-2B cells). Figure 3A
indicates that approximately 6.2 × 10 CFU/mL bacteria
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Overall, the AB406 phage could propagate from the adhered to the BEAS-2B cells after 24-h incubation without
starting MOI to a peak value of ~10 PFU/mL after 24 h phage treatment. With phage treatment (Figure 3B),
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in the presence or absence of BEAS-2B cells, irrespective of the number of live bacteria adhered/internalized to the
the initial phage concentration, and the treatment period epithelial cell surface was below the detection limit,
(Figure 1C). Bacteria-induced damage to the BEAS-2B possibly due to the effective lytic effect of phages. In the
cells was also evaluated. Pre-incubating the epithelial cells absence of bacteria, the three studied phages were also
with bacteria 4 h before adding phages caused a significant found to be capable of adhering to the BEAS-2B cell surface
reduction in the cell viability in both the control (no phage) at a level of ~10 PFU/mL (Figure 3A). In the presence
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and phage-treated groups (Figure 1D-i). This reduction was of bacteria, the adhered phages were increased to ~10
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due to the toxicity of the endotoxin secreted by bacteria to PFU/mL (Figure 3B), suggesting that phage amplification
epithelial cells, and the damage was likely induced before occurred at the epithelial cell surface or that the presence
the phage treatment was initiated. The BEAS-2B cell of bacteria promoted phage adhesion to the cell surface.
viability was significantly improved when phage was added The adsorption of phage onto the non-mucus cell surface
simultaneously with bacteria (Figure 1D-ii) or 4 h before was also reported by Shan et al., but their study was
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the bacteria (Figure 1D-iii). The toxic effect of phages on the performed in an anaerobic environment instead of an
BEAS-2B cells was also assessed in the absence of bacteria. aerobic condition.
The BEAS-2B cell viability of the phage-alone group was
comparable to the cell-alone group (Figure 1D), suggesting The absorption of MDR-AB2 bacteria onto the BEAS-2B
the safe use of phage therapy. In assessing the effect of the epithelial cells was visualized with Giemsa staining.
phage-to-bacteria MOI on the antibacterial efficiency and Figure 3C displays visible attachments of bacterial clusters
protection of BEAS-2B cells, no significant difference was on the epithelial cell layer. Fluorescence microscopy was also
noted in the range of MOI studied. Therefore, the MOI employed to confirm phage adsorption onto the epithelial
value was fixed at 100:1 for further experiments. cell layer. The FITC-labelled AB2 phages (stained green)
interspersed over the BEAS-2B cell layer after the thorough
3.2. Phage-specific interactions washing and fixation steps, indicating that the phages
Next, we investigated whether our observations (from could effectively adhere to the cell surface during the 24-h
section 3.1.) were phage-specific using another two incubation period (Figure 3D). Similar findings were noted
A. baumannii phages, AB2 and AB9 phages, at a phage- for the AB9 and AB406 phages (Figures S1 and S2).
to-bacteria MOI of 100:1 and adding the phages
simultaneously with the bacteria (Figure 2). From the 3.3. Development of resistance toward phage
TEM images (Figure 2A), all three tested phages belonged treatment
to the Myovaridae family. Figure 2B displays that all three Figure 4A displays the change of OD of bacteria
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phages had comparable antibacterial capacity in the (~10 CFU/mL) under phage treatment in the presence
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absence of epithelial cells, and a 3 ‒ 5 log enhancement in and absence of BEAS-2B cells. The OD for all treatment
600
Volume 1 Issue 1 (2024) 85 doi: 10.36922/mi.3141