Preclinical Assessment of Bacteriophage Therapy against Experimental Acinetobacter baumannii Lung Infection

Abstract

Respiratory infections caused by multidrug-resistant Acinetobacter baumannii are difficult to treat and associated with high mortality among critically ill hospitalized patients. Bacteriophages (phages) eliminate pathogens with high host specificity and efficacy. However, the lack of appropriate preclinical experimental models hampers the progress of clinical development of phages as therapeutic agents. Therefore, we tested the efficacy of a purified lytic phage, vB_AbaM_Acibel004, against multidrug-resistant A. baumannii clinical isolate RUH 2037 infection in immunocompetent mice and a human lung tissue model. Sham- and A. baumannii-infected mice received a single-dose of phage or buffer via intratracheal aerosolization. Group-specific differences in bacterial burden, immune and clinical responses were compared. Phage-treated mice not only recovered faster from infection-associated hypothermia but also had lower pulmonary bacterial burden, lower lung permeability, and cytokine release. Histopathological examination revealed less inflammation with unaffected inflammatory cellular recruitment. No phage-specific adverse events were noted. Additionally, the bactericidal effect of the purified phage on A. baumannii was confirmed after single-dose treatment in an ex vivo human lung infection model. Taken together, our data suggest that the investigated phage has significant potential to treat multidrug-resistant A. baumannii infections and further support the development of appropriate methods for preclinical evaluation of antibacterial efficacy of phages.

Keywords: Acinetobacter baumannii; antibiotic resistance; bacteriophage; pneumonia; preclinical development.

Figure 1

Phage therapy mitigated murine A. baumannii pneumonia. Mice were transnasally infected with 5 × 10⁸ CFU A. baumannii RUH 2037 or sham-infected (DPBS) and intratracheally treated with specific phage, vB_AbaM_Acibel004, or control solution (buffer) 12 h post infection (p.i.). At defined time points mice were sacrificed for BAL and blood sampling. Separate groups were sacrificed for histological analyses. (A) Body temperature curves of all mice subjected to analysis at 24 h (n = 11–12; sham-infected groups or n = 16–17; A. baumannii infected groups) and 48 h (n = 12; sham-infected groups or n = 17; A. baumannii infected groups) are shown combined (mean ± SD). Representative images of H&E-stained whole lung sections from A. baumannii infected buffer or phage-treated mice are shown at 24 h and 48 h p.i. (B); n = 3–4 (sham-infected groups), n = 6 (A. baumannii infected groups). Murine clinical disease score, permeability index and VEGF levels in BAL were assessed at 24 h (C) and 48 h p.i. (D). Data are presented as box plots depicting median, quartiles, and range excluding outliers (open circles) and analyzed using 2-way ANOVA and Tukey’s multiple comparisons test. C and D (clinical score), n = 11–12 (sham-infected groups) or n = 16–17 (A. baumannii infected groups; including 3 mice euthanized due to humane endpoints); C and D (permeability index and VEGF levels), n = 8 (sham-infected groups) or n = 8–11 (A. baumannii infected groups). Grey box in A marks time point of phage application. §/#/* indicate significant differences between groups at corresponding time point; § sham + phages vs. Acinetobacter + phages (A), # infected vs. corresponding sham group, *Acinetobacter + buffer vs. Acinetobacter + phages, * p < 0.05, ##/** p < 0.01 and ###/***/§§§ p < 0.001.

Figure 2

Single intratracheal phage application reduced bacterial burden in mice. Colony forming units (CFU) of A. baumannii were quantified in BAL, homogenized half lungs and spleen at 24 h (A) and 48 h (C) post infection (p.i.). Representative images of immunohistochemically stained lung tissue sections to visualize A. baumannii at 24 h (B) and 48 h p.i. (D). Data are expressed as single values with mean ± SD. CFU data were logarithmized (Y = log(CFU+1)) and tested with unpaired student’s t-test; n = 9–11 (24 h p.i.); n = 8–10 (48 h p.i.) for each group. * p < 0.05 between indicated groups; n = 3–4 (sham-infected groups), n = 6 (A. baumannii infected groups) per time point for histology. #: neutrophils, arrowheads: bacteria, scale bar 50 μm.

Figure 3

Phage therapy did not increase lung inflammation in mice. Numbers of leukocytes and percentages of alveolar macrophages (AM) and polymorphonuclear leukocytes (PMN) in BAL of mice were analyzed at 24 h (A) and 48 h p.i. (B) by flow cytometry. Data are presented as box plots depicting median, quartiles and range excluding outliers (open circles) and analyzed using 2-way ANOVA and Tukey´s multiple comparisons test; n = 8 (sham-infected groups); n = 8–11 (A. baumannii infected groups). # indicates significant difference between infected vs. corresponding sham group. # p < 0.05, ## p < 0.01 and ### p < 0.001.

Figure 4

Phages lysed A. baumannii in human lung tissue without increasing proinflammatory cytokine release. Fresh human lung tissue was stimulated with 1 × 10³ CFU/mL A. baumannii RUH 2037. Thirty min after infection either phage vB_AbaM_Acibel004 or control solution (buffer) were injected. CFU and cytokine levels of IL-1β and IL-8 were determined 2 h (A) and 4 h (B) after treatment; n = 7, independent experiments. CFU data are expressed as single values with mean ± SD. CFU data were logarithmized (Y = log(CFU+1)) and analyzed with unpaired student’s t-test. *** p < 0.001 between indicated groups. Cytokine data are represented as box plots depicting median, quartiles and range excluding outliers (open circles) and analyzed using Mann-Whitney U Test.