Biologically Relevant Murine Models of Chronic Pseudomonas aeruginosa Respiratory Infection

Abstract

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen and the leading cause of infection in patients with cystic fibrosis (CF). The ability of P. aeruginosa to evade host responses and develop into chronic infection causes significant morbidity and mortality. Several mouse models have been developed to study chronic respiratory infections induced by P. aeruginosa, with the bead agar model being the most widely used. However, this model has several limitations, including the requirement for surgical procedures and high mortality rates. Herein, we describe novel and adapted biologically relevant models of chronic lung infection caused by P. aeruginosa. Three methods are described: a clinical isolate infection model, utilising isolates obtained from patients with CF; an incomplete antibiotic clearance model, leading to bacterial bounce-back; and the establishment of chronic infection; and an adapted water bottle chronic infection model. These models circumvent the requirement for a surgical procedure and, importantly, can be induced with clinical isolates of P. aeruginosa and in wild-type mice. We also demonstrate successful induction of chronic infection in the transgenic βENaC murine model of CF. We envisage that the models described will facilitate the investigations of host and microbial factors, and the efficacy of novel antimicrobials, during chronic P. aeruginosa respiratory infections.

Keywords: Pseudomonas aeruginosa; S. aureus; chronic infection; in vivo; murine model; respiratory.

Figure 1

CD1 Mice were infected intranasally with clinical isolates of P. aeruginosa and compared to that of the common laboratory strain, PAO1 (1 × 10⁷). (A) Survival was monitored (n = 10 per strain). (B) In vitro biofilm production of the clinical isolates and PAO1 was measured in vitro (n = 8 replicates per strain) and (C) in vivo alginate production was measured. (n = 8 per group). ◯ represent individual data points, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 2

(A) Pre-treatment (at -10 days) of CD1 mice with S. aureus (OD5) prior to P. aeruginosa infection—clinical isolates of S. aureus all from sputum samples from patients with CF, control animals were pre-treated with PBS. (B) Comparison of inbred mouse strains infected with P. aeruginosa Q502 1 × 10⁷. (C) The bacterial CFU following pre-treatment of Biozzi mice with S. aureus Q154 OD5 and OD10 or control PBS prior to P. aeruginosa infection and (D) IL-6 levels pre and post P. aeruginosa infection. (E) Detection of Q154 within the lungs 10 days after inoculation. (F) In vitro co-culture of Q502 and Q154. (G) Infection of biozzi mice with high dose (1 × 10⁷) Q502—prior inoculation with Q154 significantly enhances survival and (H) there was detectable P. aeruginosa in the lungs 14 days post-infection. ◯ represent individual data points, * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

Figure 3

(A) Mice were infected intra-nasally with 1 × 10⁶ CFU of P. aeruginosa (PAO1). At 1 h post-infection, mice were administered inhaled tobramycin via nebulisation (10 mg/kg) for 7 days. At each time point, mice were sacrificed for enumeration of CFU in the lungs. (B) At 14 days post-infection, lung and faeces were collected; there was a significant correlation between bacterial levels in the lung and the amount being shed in faeces. Untreated n = 12, treated n = 6–12 per time point, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 4

C57BL6 mice infected with PAO1 in water bottle (1 × 10⁷ per ml) for 5 days. Mice were then transferred back onto fresh drinking water and culled at days 7, 14, 20, 30, and 50 and the levels of CFU in (A) the lungs (B) and NALT. (C) The levels of IL-6 in the lungs were quantified (n = 6–9 per time point). (D) Following treatment in this model, there is sustained bacterial clearance in water bottle infected mice (n = 6 per time point). βENaC mice were infected with PAO1, and the CFU in (E) the lungs was quantified and the (F) weights were measured (n = 6 per time point). ◯ represent individual data points, * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.