A chronic murine model of pulmonary Acinetobacter baumannii infection enabling the investigation of late virulence factors, long-term antibiotic treatments, and polymicrobial infections

Abstract

Acinetobacter baumannii can cause prolonged infections that disproportionately affect immunocompromised populations. Our understanding of A. baumannii respiratory pathogenesis relies on an acute murine infection model with limited clinical relevance that employs an unnaturally high number of bacteria and requires the assessment of bacterial load at 24-36 hours post-infection. Here, we demonstrate that low intranasal inoculums in immunocompromised mice with a tlr4 mutation leads to reduced inflammation, allowing for persistent infections lasting at least 3 weeks. Using this "chronic infection model," we determined the adhesin InvL is an imperative virulence factor required during later stages of infection, despite being dispensable in the early phase. We also demonstrate that the chronic model enables the distinction between antibiotics that, although initially reduce bacterial burden, either lead to complete clearance or result in the formation of bacterial persisters. To illustrate how our model can be applied to study polymicrobial infections, we inoculated mice with an active A. baumannii infection with Staphylococcus aureus or Klebsiella pneumoniae. We found that S. aureus exacerbates the infection, while K. pneumoniae enhances A. baumannii clearance. In all, the chronic model overcomes some limitations of the acute pulmonary model, expanding our capabilities to study of A. baumannii pathogenesis and lays the groundwork for the development of similar models for other important opportunistic pathogens.

Keywords: Acinetobacter baumannii; Antibiotic treatments; Murine model; Pathogenesis; Polymicrobial infections; Virulence factors.

Figure 1.

Low inoculums of modern respiratory A. baumannii clinical isolates result in chronic lung infection in tlr4 mutant mice. Groups of C3H/HeN (WT) or C3H/HeJ (tlr4 mutant) mice were intranasally inoculated with 10⁸ G636 (A), 10⁸ G654 (B), 10⁸ Ab19606 (C), 10⁵ G636 (D), 10⁵ G654 (E), or 10⁵ Ab19606 (F). Beginning at 24 hpi, groups of mice were sacrificed every three days, and bacteria in the lungs were quantified. Each data point indicates and individual mouse.

Figure 2.

Lower intranasal A. baumannii inoculums result in reduced lung neutrophil influx. Groups of C3H/HeN (WT) or C3H/HeJ (tlr4 mutant) mice were intranasally inoculated with 10⁵ G636, 10⁸ G636, or mock inoculated with PBS. At 4 h (A and D), 2 d (B and E), and 7 d (C and F) pi, alveolar macrophages (AMs) (A-C) and polymorphonuclear leukocytes (PMNs) (D-F) in the BALF were enumerated by flow cytometry. Shown are pooled results from at least two independent experiments, and each data point represents an individual mouse. The horizontal line represents the mean, and the standard error of the mean (SEM) is indicated by error bars. *P < 0.05; two-way analysis of variance (ANOVA), Tukey’s test for multiple comparisons.

Figure 3.

The chronic respiratory infection model results in lung pathology. Groups of C3H/HeJ (tlr4 mutant) mice were inoculated with 10⁵ G636 or mock-inoculated with PBS, and at 4 hpi, 2 dpi, 7 dpi, 14 dpi, and 21 dpi, lungs slices were prepared, H&E stained, and scored for alveolitis (A), peribronchiolitis (B), smooth muscle hypertrophy (C), squamous epithelium metaplasia (D), and BALT formation (E). The mean is shown on the graph, and the SEM is indicated by error bars. *P < 0.05, Unpaired Student’s t-test.

Figure 4.

InvL is a critical virulence factor for long-term respiratory infection, but dispensable in the acute infection model. C3H/HeJ (tlr4 mutant) mice were infected with 10⁵ G636, G636 ΔinvL, or G636 invL⁺. Groups of mice were then sacrificed at 1 dpi (A), 7 dpi (B), 14 dpi (C), and 21 dpi (D), and CFU in the lungs were quantified. Shown are the results from 3 independent experiments. For the acute infection model, groups of C57BL/6 mice were infected with 10⁹ G636, G636 ΔinvL, or G636 invL⁺. 24 hpi, mice were sacrificed, and CFU in the lungs (A), spleen (B), and kidneys (C) were enumerated. Each data point represents an individual mouse, the horizontal line represents the mean, and the SEM is indicated by error bars. Shown are the results of 2–3 independent experiments. *P < 0.05; Kruskal-Wallis H test with Dunn’s test for multiple comparisons; ns = not significant.

Figure 5.

The chronic respiratory infection model can be used to study outcomes of antibiotic treatment. Groups of C3H/HeJ (tlr4 mutant) mice were infected with 10⁵ G636 (A, C) or 10⁵ G654 (B, D) and sacrificed at 1, 3, and 5 dpi (long-term). Additionally, groups of C57Bl/6 mice were infected with 10⁹ G636 (A, C) or 10⁹ G654 (B, D) and sacrificed at 24 hpi (acute). Mice in both infection models were treated intraperitoneally treated with PBS or 100 mg/kg tigecycline (tig) every 12 h (A, B) or PBS or 500 mg/kg apramycin (apr) every 12 h (C, D) with all treatments beginning 4 hpi. At each timepoint, CFU were quantified in the lungs. Shown are the results from at least two independent experiments, each data point represents an individual mouse, the horizontal line represents the mean, and the SEM is represented by error bars. *P < 0.05; Mann-Whitney U test.

Figure 6.

Bacterial secondary infection alters the course of chronic A. baumannii pneumonia. C3H/HeJ (tlr4 mutant) mice were intranasally inoculated with 10⁵ G636, and groups of mice were sacrificed at 1, 7, and 14 dpi. At 14 days post-A. baumannii infection, groups of mice were either not inoculated (untreated), inoculated with PBS (mock-infected), infected with S. aureus, or infected on K. pneumoniae. Subsequently, on days 15 and 16 post-A. baumannii infection (1 and 2 days post-secondary infection), mice were sacrificed, and A. baumannii CFU were quantified in the lungs (A, D, E), spleen (B), and kidneys (C). In panels A, B, and C, each data point represents the mean, the SEM is represented by error bars, and the limit of detection is indicated by the dashed line. In panels D and E, each data point represents an individual mouse, the horizontal line represents the mean, and the SEM is indicated by error bars. Shown are results from at least 2 independent experiments. *P < 0.05; Kruskal-Wallis H test with Dunn’s test for multiple comparisons.