Pseudomonas aeruginosa Pneumonia Model

Pseudomonas aeruginosa D4 cultured on LB agar plates overnight at 37°C was adjusted to 2.5 × 10⁹ colony forming unit (CFU)/mL using sterile physiological saline. To produce the P. aeruginosa pneumonia model, 20 μl of bacterial suspension was inoculated through intranasal route in anesthetized mice. These mice were randomly divided in two groups and 10 μL of KPP10 bacteriophage (4 × 10¹¹/mL plaque forming unit, PFU) (MOI = 80) was administered in these mice intranasally after 2 h (group 1) and 8 h (group 2) of bacterial inoculation. Past study suggested that higher dose (MOI) of bacteriophage can promptly kill bacteria and help to minimize the chance of resistant development to phage (Taha et al., 2018). In addition, another study using in-vitro models showed that KPP10 became resistant to P. aeruginosa D4 strains at 210 min. of administration (Watanabe et al., 2007). Considering number and short therapeutic window of phage from these studies, we administered the bacteriophage in mice at a highest concentration (MOI) that we could harvest in our laboratory (MOI = 80). The mice in negative control (NC) group were inhaled 10 μL of phage buffer after 2 h of inoculation. We used imipenem/cilastatin sodium to prepare a positive control (PC) group. After 2 h post inoculation of P. aeruginosa, 25 mg/kg of imipenem/cilastatin sodium (MSD Co., Ltd.) was injected subcutaneously to a group of randomly selected mice at every 12 h for 3 days (Coopersmith et al., 2003). All groups were monitored daily for 6 days to calculate their survival rate.

Analysis of survival data showed that there was no significant difference in survival rates between the mice those inhaled KPP10 at 2 h and 8 h p.i. Therefore, in the bacteriology and histopathology experiments we only included the Pa pneumonia mice inhaled either phage KPP10 or phage buffer at 2 h p.i.