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

was not statistically different from the placebo group. aspects, such as improving antibiofilm efficiency 22,23 and
Similar observations were reported by Tsonos et al. promoting contacts between phages and bacteria on
8
that an applied phage cocktail against avian pathogenic cell surfaces to achieve better bactericidal effects. 17,18,24-26
Escherichia coli (APEC) did not significantly improve the Furthermore, phages could also significantly reduce
cure rate in a chicken colibacillosis model when compared the rates of bacterial adhesion and invasion into cells,
with the untreated group. The mixed findings in translating possibly by killing the bacteria before they get close to
in vitro results to in vivo can be partly accounted for the the mammalian cells or competing with the receptors
incapability of the simple in vitro experimental setting at the cell surface. 27-29 Overall, results from these studies
to capture the complicated interactions of bacteria and suggested that their interactions are phage-, bacteria-, and
phages with the mammalian cells in vivo. These include: mammalian cell-specific. Further research to investigate
(i) bacterial invasion and survival within mammalian the dynamics between them would be critical in revealing
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cells; (ii) bacterial response to environmental stress by the roles of mammalian cells in phage therapy to provide
altering their capsule composition, 11,12 causing phage better prediction of the in vivo outcomes. In the present
receptor loss and subsequent loss of phage lytic activity; study, a three-component coculture system was established
and (iii) clearance of phages by the host immune system. 13 to study the dynamics of the MDR Acinetobacter baumannii
To improve the validity of in vitro results in predicting bacteria (MDR-AB2, isolated from the sputum samples of
the in vivo outcomes in animals or humans, it is beneficial a pneumonia patient), various anti-A. baumannii phages
to evaluate the in vitro antibacterial capability of phages (vB_AbaM-IME-AB2, vB_AbaM-IME-AB9, and vB_
in the presence of host mammalian cells. The dissected AbaM-IME-AB406), and a human bronchial epithelial cell
dynamic interactions between phages, bacteria, and tissue line (BEAS-2B).
cells at the sites of infections could then provide more 2. Methods
insights into the dosing regimen and predict the efficacy
of phage therapy. However, the inter-relationship between 2.1. Materials
each component in the coculturing system becomes Three distinct phages, vB_AbaM-IME-AB2 (AB2
more complicated. The bacteria secrete endotoxins and phage) , vB_AbaM-IME-AB9 (AB9 phage), and vB_
29
other toxic products to destroy the mammalian cells. AbaM-IME-AB406 (AB406 phage), active against A.
14
In response to the bacterial toxins, the host immune baumannii MDR-AB2 were gifted from the Beijing
system releases cytokines to potentiate the toxicity and Institute of Microbiology and Epidemiology. High
14
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generate antibacterial peptides to kill the bacteria. In titer phage lysates were produced and titered using well-
15
some cases, bacteria can invade host cells and multiply established protocols. Anion-exchange chromatography
31
intracellularly to establish an infection or reside within with a CIMmultus QA 1 mL Monolithic Column (BIA
™
cells for an extended period to escape the host’s immune Separations, Slovenia) was employed to purify the phage
16
response or antibiotic treatment. The intracellular lysates. Phage elutions were dialyzed with phosphate-
32
survival niche of bacteria has greatly increased the failure buffered saline (PBS, Sigma Aldrich, United States), and
rate of any therapeutic treatments as most antibacterial the obtained phage titers were 6.00 × 10 plaque forming
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agents are unable to penetrate, accumulate, and retain in unit (PFU)/mL for AB2 phage, 1.12 × 10 for AB406
11
mammalian cells. Previously, phage was also believed to phage, and 7.33 × 10 for AB9 phage. Morphologies of
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be incapable of penetrating mammalian cell membranes phage stocks were examined under the FEI Tecnai G2
™
to target pathogens residing within cells. However, recent Spirit Twin (Oregon, United States of America [USA])
findings revealed that phages can directly interact with transmission electron microscope (TEM) after staining
human cells and/or the mucin secreted by the epithelial with phosphotungstic acid for 30 s, confirming that the
cells, facilitating their internalization and transcytosis. 17-20 studied phages belonged to the Myoviridae family. Normal
Therefore, assessing the antibacterial efficiency of phages human lung epithelial cell line BEAS-2B was purchased
in the presence of mammalian cells would facilitate a from the American Type Culture Collection (USA). The
better understanding of their dynamics and the successful cell line was cultured in Dulbecco’s Modified Minimal
translation of phage therapy. Essential Medium (DMEM, Thermo Fisher Scientific,
A few studies have evaluated the antibacterial activity USA), supplemented with 10% fetal bovine serum, 100
of phages in the presence of mammalian cells. A detailed units/mL streptomycin sulfate, and 100 units/mL penicillin
review of these studies was provided in our previous G sulfate at 37°C and 5% CO . Nutrient broth (NB) and
2
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review, and a summary is highlighted here. Mammalian agar bacteriological (AGAR NO.1) were obtained from
cells may provide benefits to phage therapy from several OXOID (United Kingdom [UK]).

Volume 1 Issue 1 (2024) 82 doi: 10.36922/mi.3141