Bacteriophages φMR299-2 and φNH-4 can eliminate Pseudomonas aeruginosa in the murine lung and on cystic fibrosis lung airway cells

Abstract

Pseudomonas aeruginosa is a common cause of infection in the lungs of patients with cystic fibrosis (CF). In addition, biofilm formation and antibiotic resistance of Pseudomonas are major problems that can complicate antibiotic therapy. We evaluated the efficacy of using bacteriophages to kill the pathogen in both biofilms and in the murine lung. We isolated and characterized two phages from a local wastewater treatment plant, a myovirus (φNH-4) and a podovirus (φMR299-2). Both phages were active against clinical isolates of P. aeruginosa. Together, the two phages killed all 9 clinical isolate strains tested, including both mucoid and nonmucoid strains. An equal mixture of the two phages was effective in killing P. aeruginosa NH57388A (mucoid) and P. aeruginosa MR299 (nonmucoid) strains when growing as a biofilm on a cystic fibrosis bronchial epithelial CFBE41o- cell line. Phage titers increased almost 100-fold over a 24-h period, confirming replication of the phage. Furthermore, the phage mix was also effective in killing the pathogen in murine lungs containing 1 × 10(7) to 2 × 10(7) P. aeruginosa. Pseudomonas was effectively cleared (reduced by a magnitude of at least 3 to 4 log units) from murine lungs in 6 h. Our study demonstrates the efficacy of these two phages in killing clinical Pseudomonas isolates in the murine lung or as a biofilm on a pulmonary cell line and supports the growing interest in using phage therapy for the control and treatment of multidrug-resistant Pseudomonas lung infections in CF patients.

Importance: Given the rise in antibiotic resistance, nonantibiotic therapies are required for the treatment of infection. This is particularly true for the treatment of Pseudomonas infection in patients with cystic fibrosis. We have identified two bacterial viruses (bacteriophages) that can kill Pseudomonas growing on human lung cells and in an animal model of lung infection. The use of bacteriophages is particularly appropriate because the killing agent can replicate on the target cell, generating fresh copies of the bacteriophage. Thus, in the presence of a target, the killing agent multiplies. By using two bacteriophages we can reduce the risk of resistant colonies developing at the site of infection. Bacteriophage therapy is an exciting field, and this study represents an important demonstration of efficacy in validated infection models.

FIG 1

Scanning electron microscopy images of phage ϕ229-2, a podophage (A) and phage ϕNH-4, a myophage (B), stained with 0.2% phosphotungstic acid. The arrows point to the short tail (10 to 20 nm long) of the podophage in panel A and to the contracted tail sheath of the myovirus in panel B.

FIG 2

Genome organization of phage ϕMR299-2 (top) and ϕNH-4 (bottom). The predicted open reading frames are indicated by the thick arrows, which are shaded to show the level of protein identity to the corresponding regions of the closest P. aeruginosa (phage PaP3 for ϕMR299-2) or phage LMA2 (for ϕNH-4).

FIG 3

(A) Growth of lux-tagged Pseudomonas biofilms on the surface of the CFBE410- cell monolayer. Light was measured 1, 5, and 24 h (before and after the monolayer was washed with MEM). (B) Readings from 6 wells are shown. Values are shown as means ± standard deviations (SD) (error bars).

FIG 4

Fluorescent image of 24-h-old culture of P. aeruginosa cells grown on a CFBE41o- cell monolayer after Calcofluor white (fluorescent enhancer) staining. Staining confirms that P. aeruginosa NH57388A (A) and MR299 (B) are embedded in an exopolysaccharide structure prior to phage exposure. After 24-h incubation in the presence of mixed phages, staining indicates open and weak matrices with reduced numbers of cells for both NH57388A (C) and MR299 (D). An increase in phage titer was observed over the 24-h incubation period for both MR299 and NH57388A strains (E).

FIG 5

(A) Light emitted from nonmucoid P. aeruginosa MR299 strain and mucoid NH57388A strain grown on a CFBE410- cell monolayer for 24 h in the presence (+) and absence (−) of phage mix. (B) The RLU values are mean ± SD readings from 3 wells.

FIG 6

Mice (n = 8) were infected with nonmucoid P. aeruginosa MR299 (A) and mucoid NH57388A (mucoid strain) (B). Test mice (+) were treated with the phage mix (ϕMR299-2 and ϕNH-4B). Phage was given 2 h after the mice were infected with Pseudomonas. Control mice (−) did not receive the phage mix.