Novel mouse model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis

Abstract

Pseudomonas aeruginosa causes a chronic infection in the lungs of cystic fibrosis (CF) patients by establishing an alginate-containing biofilm. The infection has been studied in several animal models; however, most of the models required artificial embedding of the bacteria. We present here a new pulmonary mouse model without artificial embedding. The model is based on a stable mucoid CF sputum isolate (NH57388A) with hyperproduction of alginate due to a deletion in mucA and functional N-acylhomoserine lactone (AHL)-based quorum-sensing systems. Chronic lung infection could be established in both CF mice (Cftr(tmlUnc-/-)) and BALB/c mice, as reflected by the detection of a high number of P. aeruginosa organisms in the lung homogenates at 7 days postinfection and alginate biofilms, surrounded by polymorphonuclear leukocytes in the alveoli. In comparison, both an AHL-producing nonmucoid revertant (NH57388C) from the mucoid isolate (NH57388A) and a nonmucoid isolate (NH57388B) deficient in AHL were almost cleared from the lungs of the mice. This model, in which P. aeruginosa is protected against the defense system of the lung by alginate, is similar to the clinical situation. Therefore, the mouse model provides an improved method for evaluating the interaction between mucoid P. aeruginosa, the host, and antibacterial therapy.

FIG. 1.

Photos of a stable, mucoid P. aeruginosa NH57388A colonies after serial subculture on BAP (a), a stationary-shaken ox broth flask culture of nonmucoid P. aeruginosa NH57388B after 2 weeks (b), alcohol precipitation of alginate (white arrow) from stationary-shaken ox broth flask cultures (30 h) of mucoid P. aeruginosa NH57388A (left) and nonmucoid NH57388B (right) (d), and mucoid P. aeruginosa NH57388A after 14 days stationary-shaken (left) or static (right) aerobic culture in a tube (c). Three phenotypes are indicated by the black arrows in panel c: M, mucoid; NM, nonmucoid; and SCV.

FIG. 2.

(a and b) Bacterial CFU in the lung of mice after intratracheal challenge of P. aeruginosa resuspended in culture supernatant (crude alginate) with mucoid strain NH57388A at 5 × 10⁶ CFU/lung in CF mice or 5 × 10⁷ CFU/lung in BALB/c mice (a) or mucoid strain NH57388A or nonmucoid strain NH57388B at 5 × 10⁷ CFU/lung in BALB/c mice (b). (c and d) Intratracheal challenge of P. aeruginosa resuspended in purified alginate with mucoid strain NH57388A at 5 × 10⁷ CFU/lung in BALB/c mice (c) or mucoid strain NH57388A, nonmucoid strain NH57388C, or nonmucoid strain NH57388B at 5 × 10⁶ CFU/lung in CF mice (d). Abbreviations: d, day; PA, P. aeruginosa; +QS, functional QS system; −QS, nonfunctional QS system.

FIG. 3.

Photographs showing mucoid P. aeruginosa NH57388A isolated from a CF mouse lung 7 days postinfection. (a) Sections of mouse lung tissue at 7 days postinfection stained with HE and Alcian blue for detection of alginate. Mucoid P. aeruginosa NH57388A-infected CF mouse lung showing large P. aeruginosa biofilms (in black circles) encapsulated in alginate (blue color) in the alveolar space surrounded by PMN (white arrow) at ×40 (b) and ×100 (c) magnifications; scanning confocal photomicrographs of freeze microtomy sections of CF mouse lung tissue (40 μm) showing alveolus sacs filled with mucoid P. aeruginosa NH57388A biofilms (d) and lung tissues from a CF mouse challenged with saline (control) (e) and from a BALB/c mouse challenged with nonmucoid P. aeruginosa NH57388B (f). No alginate is seen. HE and Alcian blue stain were used in panel f. Mucoid P. aeruginosa microcolonies from bronchoalveolar lavage from a CF patient with chronic P. aeruginosa lung infection (g) and autopsy of a female CF patient who died from chronic P. aeruginosa lung infection showing alveoli sacs filled with P. aeruginosa biofilms (black arrow) at magnifications of ×100 (h), ×1,000 (i), and ×250 (j) with HE and Gram stain. Alveoli without infection (magnification, ×250; HE stain) (k) and sputum with a mucoid biofilm of P. aeruginosa (in black circle) from a CF patient (magnification, ×1,000; Gram stain) (l) are also shown.