Use of a Primary Epithelial Cell Screening Tool to Investigate Phage Therapy in Cystic Fibrosis

Abstract

Antimicrobial-resistant microbes are an increasing threat to human health. In cystic fibrosis (CF), airway infections with Pseudomonas aeruginosa remain a key driver of lung damage. With few new antibiotics on the development horizon, alternative therapeutic approaches are needed against antimicrobial-resistant pathogens. Phage therapy, or the use of viruses that infect bacteria, is one proposed novel therapy to treat bacterial infections. However, the airways are complex microenvironments with unique characteristics that may affect the success of novel therapies. Here, three phages of P. aeruginosa (E79, F116, and one novel clinically derived isolate, designated P5) were screened for activity against 21 P. aeruginosa strains isolated from children with CF. Of these, phage E79 showed broad antibacterial activity (91% of tested strains sensitive) and was selected for further assessment. E79 genomic DNA was extracted, sequenced, and confirmed to contain no bacterial pathogenicity genes. High titre phage preparations were then purified using ion-exchange column chromatography and depleted of bacterial endotoxin. Primary airway epithelial cells derived from children with CF (n = 8, age range 0.2–5.5 years, 5 males) or healthy non-CF controls (n = 8, age range 2.5–4.0 years, 4 males) were then exposed to purified phage for 48 h. Levels of inflammatory IL-1β, IL-6, and IL-8 cytokine production were measured in culture supernatant by immunoassays and the extent of cellular apoptosis was measured using a ssDNA kit. Cytokine and apoptosis levels were compared between E79-stimulated and unstimulated controls, and, encouragingly, purified preparations of E79 did not stimulate any significant inflammatory cytokine responses or induce apoptosis in primary epithelial cells derived from children with or without CF. Collectively, this study demonstrates the feasibility of utilizing pre-clinical in vitro culture models to screen therapeutic candidates, and the potential of E79 as a therapeutic phage candidate in CF.

Aztreonam Lysine Increases the Activity of Phages E79 and phiKZ against Pseudomonas aeruginosa PA01

“…Therefore, the objective of this study was to determine whether a synergistic effect exists between the virulent phage E79 and the antibiotic aztreonam lysine (AzLys) [ 20 ] on P. aeruginosa strain PA01 in vitro. Trend et al [ 21 ] showed that phage E79 does not induce apoptosis or the production of inflammatory cytokines on airway epithelial cells isolated from children with or without CF. They also consider the potential of E79 as a phage therapy agent due to its wide host range against P. aeruginosa clinical isolates, stable high-titer stocks, and no known presence in bacterial pathogenicity islands [ 21 ].…”

“…Trend et al [ 21 ] showed that phage E79 does not induce apoptosis or the production of inflammatory cytokines on airway epithelial cells isolated from children with or without CF. They also consider the potential of E79 as a phage therapy agent due to its wide host range against P. aeruginosa clinical isolates, stable high-titer stocks, and no known presence in bacterial pathogenicity islands [ 21 ]. Our results extend these previous findings and identify a PAS effect of increased time to lysis, increased infection efficiency and plaque sizes by E79 in the presence of AzLys, as well as the increased destruction of biofilms.…”

Possible Role for Bacteriophages in the Treatment of SARS-CoV-2 Infection

“…Preincubation with T4 phage also showed protective activity [ 35 ]. It is known that SARS-CoV and SARS-CoV-2 induce apoptosis and result in lymphocytopenia [ 36 , 37 ]; however, when airway epithelial cells from the human bronchi harvested and cultured phages in vitro, it resulted in reduced apoptosis [ 38 ].…”

Phages in the Fight Against COVID-19?

“…We demonstrated protective activity of the T4 phage on the survival of human lung epithelial cells infected with human Adv when the phage was added before or during the entire time of Adv infection; preincubation with the phage was also protective [ 10 ]. Interestingly, while SARS-CoV and SARS-CoV-2 are known to induce apoptosis and resulting lymphocytopenia is a common laboratory finding (>60 of patients and >80% in the dead patients) [ 11 , 12 ], apoptosis is reduced in airway epithelial cells harvested from the human bronchi and cultured with phages in vitro [ 13 ]. Furthermore, both T4 and A3/R (staphylococcal phage) preparations significantly reduce the percentage of DEC-205+ human myeloid cells (lectin receptor recognizing ligands expressed during apoptosis and necrosis of different cell populations) [ 14 ].…”