Synergistic activity of phage PEV20-ciprofloxacin combination powder formulation-A proof-of-principle study in a P. aeruginosa lung infection model

Abstract

Combination treatment using bacteriophage and antibiotics is potentially an advanced approach to combatting antimicrobial-resistant bacterial infections. We have recently developed an inhalable powder by co-spray drying Pseudomonas phage PEV20 with ciprofloxacin. The purpose of this study was to assess the in vivo effect of the powder using a neutropenic mouse model of acute lung infection. The synergistic activity of PEV20 and ciprofloxacin was investigated by infecting mice with P. aeruginosa, then administering freshly spray-dried single PEV20 (10⁶ PFU/mg), single ciprofloxacin (0.33 mg/mg) or combined PEV20-ciprofloxacin treatment using a dry powder insufflator. Lung tissues were then harvested for colony counting and flow cytometry analysis at 24 h post-treatment. PEV20 and ciprofloxacin combination powder significantly reduced the bacterial load of clinical P. aeruginosa strain in mouse lungs by 5.9 log₁₀ (p < 0.005). No obvious reduction in the bacterial load was observed when the animals were treated only with PEV20 or ciprofloxacin. Assessment of immunological responses in the lungs showed reduced inflammation associating with the bactericidal effect of the PEV20-ciprofloxacin powder. In conclusion, this study has demonstrated the synergistic potential of using the combination PEV20-ciprofloxacin powder for P. aeruginosa respiratory infections.

Keywords: Antibiotic aerosols; Bacteriophage therapy; Combination therapy; Dry powder inhalation (DPI); Inhaled phage therapy; Mouse lung infection model; Phage and antibiotic combination.

Figure 1.

Bactericidal activity of PEV20 only, ciprofloxacin only and PEV20-ciprofloxacin combination powders (PEV20: 10⁶ PFU; Ciprofloxacin: 0.33 mg) in mouse lung infection model (initially 10⁶ bacterial cells) 24 h after treatment (n=4). Statistical significance between groups was determined by ANOVA (*p < 0.005).

Figure 2.

Leukocyte recruitment to lungs post-infection. BALB/c mice (n = 4) were culled via CO₂ overdose 24 hours post treatment. Lungs were harvested 24h later and cell composition determined by flow cytometry. (A) Neutrophil (Ly6G⁺ CD11b⁺), (B) B cell (B220⁺), (C) CD8+ T cell (CD8⁺), and (D) monocyte/macrophage (Mo/Mϕ, Ly6G⁻ CD11b⁺) proportions were calculated. Data (average ± SEM) is representative of three independent experiments. Statistical significance between groups was determined by ANOVA (*p < 0.05; **p < 0.01).