Preclinical Evaluation of Recombinant Microbial Glycoside Hydrolases as Antibiofilm Agents in Acute Pulmonary Pseudomonas aeruginosa Infection

Abstract

The bacterium Pseudomonas aeruginosa can colonize the airways of patients with chronic lung disease. Within the lung, P. aeruginosa forms biofilms that can enhance resistance to antibiotics and immune defenses. P. aeruginosa biofilm formation is dependent on the secretion of matrix exopolysaccharides, including Pel and Psl. In this study, recombinant glycoside hydrolases (GHs) that degrade Pel and Psl were evaluated alone and in combination with antibiotics in a mouse model of P. aeruginosa infection. Intratracheal GH administration was well tolerated by mice. Pharmacokinetic analysis revealed that, although GHs have short half-lives, administration of two GHs in combination resulted in increased GH persistence. Combining GH prophylaxis and treatment with the antibiotic ciprofloxacin resulted in greater reduction in pulmonary bacterial burden than that with either agent alone. This study lays the foundation for further exploration of GH therapy in bacterial infections.

Keywords: Pel; Pseudomonas aeruginosa; Psl; acute pulmonary infection; antibiotic; antimicrobial combinations; bacteria; biofilm; exopolysaccharide; glycoside hydrolase (GH).

FIG 1

GHs potentiate antibiotic activity against P. aeruginosa biofilms in vitro. Dose-response matrix analyses of P. aeruginosa biofilms cotreated with 2-fold serial dilutions of either PslG_h-PelA_h or PslG_h-Ega3_h and 4-fold serial dilutions of ceftazidime or ciprofloxacin in a MBEC high-throughput assay as indicated are shown. Biofilm biomass was quantified with crystal violet staining. Colored squares represent the ratio of treated to untreated biofilms of ≥2 independent experiments. Lighter yellow boxes represent high biofilm biomass and darker green boxes represent low biofilm biomass, compared with untreated control wells.

FIG 2

Intratracheal GH therapy does not induce pulmonary damage. Immunocompetent BALB/c mice were intratracheally administered a single dose of 250/250 μg PslG_h-PelA_h or PslG_h-Ega3_h. (A) Lactate dehydrogenase activity was quantified in BAL fluid. (B) Pulmonary leukocyte populations, including lymphocytes, macrophages, eosinophils, and neutrophils, were quantified by flow cytometry 7 days after GH treatment. Bars represent the means ± standard errors of 4 independent experiments with ≥13 mice per group. Asterisks indicate significant differences (P < 0.0020), relative to the buffer-treated group and where there are no asterisks no significant differences were observed as compared with the buffer-treated group (P > 0.7794), as determined by two-way ANOVA with Dunnett’s multiple-comparison test.

FIG 3

Pulmonary pharmacokinetics of intratracheally administered GHs. Mice were treated intratracheally with a single dose of 500 μg PelA_h, Ega3_h, or PslG_h (A) or 250/250 μg each of a combination of PslG_h and PelA_h (B) or PslG_h and Ega3_h (C) and then sacrificed at the indicated time points. Lung homogenates were assessed by Western blotting. Dots represent the means ± standard errors of band intensities normalized to total band intensity at 0 h from 2 independent experiments with ≥5 mice per time point.

FIG 4

Specific GH-antibiotic combinations reduce bacterial burden in an acute mouse model of pulmonary P. aeruginosa infection. Mice were intratracheally infected with 3 × 10⁷ P. aeruginosa CFU coadministered with or without PslG_h-PelA_h (A) or PslG_h-Ega3_h-HEK (B) and then treated as indicated with 10 mg/kg ciprofloxacin or 25 mg/kg ceftazidime every 8 h for 1 day. Pulmonary bacterial burden was determined by CFU quantification. Bars represent the medians with interquartile ranges of at least 2 independent experiments with ≥16 mice per group. *, significant difference (P = 0.0347) between combinations of GHs and ciprofloxacin and ciprofloxacin alone; ns, no significant difference (P > 0.9999), as determined by the Kruskal-Wallis test with Dunn’s multiple-comparison test. Cipro, ciprofloxacin; Cefta, ceftazidime.