A murine model of early Pseudomonas aeruginosa lung disease with transition to chronic infection

Abstract

Pseudomonas aeruginosa (PA) remains an important pathogen in patients with cystic fibrosis (CF) lung disease as well as non-CF bronchiectasis and chronic obstructive airways disease. Initial infections are cleared but chronic infection with mucoid strains ensues in the majority of CF patients and specific interventions to prevent this critical infection transition are lacking. The PA bead model has been widely used to study pulmonary P.aeruginosa infection but has limitations in animal husbandry and in accurately mimicking human disease. We have developed an adapted agar bead murine model using a clinical mucoid strain that demonstrates the key features of transition from transitory to chronic airways infection. Infected animals show very limited acute morbidity and mortality, but undergo infection-related weight loss and neutrophilic inflammation, development of anti-pseudomonal antibodies, variable bacterial clearance, endobronchial infection and microbial adaptation with PA small colony variants. We anticipate this model will allow research into the host and microbial factors governing this critical period in Pseudomonas aeruginosa pulmonary pathogenesis when transition to chronicity is occurring.

Figure 1. Technique for intra-tracheal instillation of agar beads using inhaled anaesthesia.

(a) Sterile surgical equipment set-up required for instillation. (b) Following anesthesia administered via isofluorane inhalation in an anesthesia box, animals were transferred to the surgical board and maintained under anaesthesia via nose cone. (c) Midline neck incision and careful division of soft tissues to expose trachea. (d) Insertion of 22G intravenous cannula into trachea, while extending neck using traction on lower jaw. (e) Injection of 50 μl of beads/PBS via cannula. (f) Elevation of anaesthetized animal on surgical board head-up to 45-degrees for a timed 1-minute in order to aid full inspiration of beads. (g) Closure of incision with surgical staples. (h) Completed closure of incision.

Figure 2. Weight loss and bacterial adaptation in mice infected with NH57388A-laden agar beads.

(a,b) Body weight measured daily in C57BL/6 mice treated with sterile or Pseudomonas aeruginosa-laden agar beads. Animals who were infected with NH57388A-laden beads and, at 2-weeks post-inoculation, remain infected are denoted as ‘Chronic infection’ (a), where as those who do not have pulmonary infection are denoted ‘Infection cleared’ (b). Percentage weight change from baseline for pooled experiments with animals treated with NH57388A-laden beads (N = 35) compared with controls treated with sterile beads (N = 30 mice). Each point represents mean of group and SEM. (a) Difference between groups significant at p < 0.0001 via repeated measures ANOVA. (b) Difference between groups significant at p = 0.0204 via repeated measures ANOVA. (c,d) Mucoid (white arrow) and small colony variants (black arrow) identified in lung homogenate of NH57388A-laden bead treated animal 2-weeks following inoculation. (e) Subcultures of mucoid NH75388A and small colony variant (SCV) phenotypes isolated from the lungs of animals treated with NH57388A-laden beads (grown on blood agar). (f) Transmission electron microscopy of biofilms of mucoid NH57388A and its small colony-variant. Mucoid NH57388A demonstrated low bacterial density with limited extracellular matrix. The related SCV produced a biofilm with a high density of highly adherent bacteria embedded in a dense network of extracellular material. Bacteria indicated by think white arrow and extracellular matrix by thin white arrow.

Figure 3. Neutrophilic inflammation, histological changes and Pseudomonas-binding immunoglobulin in mice two-weeks following NH57388A-laden agar bead inoculation.

C57BL/6 mice received sterile or NH57388A-laden agar beads. (a) Two-weeks post-instillation, bronchoalveolar lavage (BAL) leukocytes were strained for Gr-1 followed by flow cytometry. BAL neutrophils (percent of total leukocytes) in animals treated with sterile beads (N = 16) were compared mice inoculated with NH57388A-laden agar beads (N = 19) at 2-weeks. Line indicates median. P-value related to Mann-Whitney test. (b) Quantitative histological scores of animals treated with sterile agar beads (N = 9) or NH57388A (NH)-laden beads (N = 9). Line denotes median score. P-value related to Mann-Whitney test. (c–f) H&E staining of representative lung sections (all at x10 magnification) in C57BL/6 mice with no treatment (healthy control) (c), 2-weeks post-inoculation with sterile agar beads (d), 2-weeks post-inoculation with NH57388A-laden agar beads (e), or with overwhelming acute infection post-inoculation with PA-laden agar beads. Black arrows highlight intra-bronchial agar beads. (g) Mediastinal lymph node cell counts in mice treated with sterile and PA-laden agar beads two-weeks following bead instillation. Columns shown mean + SD. P-value relates to comparison by t-test and includes results from 3 experiments (each with N = 9–10 mice/group). (h) Serum PA-binding immunoglobulin M (IgM) and G (IgG) levels showing fold increase in IgM and IgG levels in NH57388A treated animals compared with sterile bead treated controls two-weeks post-instillation. Line represents median. *Denotes p < 0.05 compared with theoretical median of 1.0 via Wilcoxon Signed rank test. Combined results from two separate experiments.