Mouse pneumonia model by Acinetobacter baumannii multidrug resistant strains: Comparison between intranasal inoculation, intratracheal instillation and oropharyngeal aspiration techniques

Abstract

Infectious pneumonia induced by multidrug resistant (MDR) Acinetobacter baumannii strains is among the most common and deadly forms of healthcare acquired infections. Over the years, different strategies have been put in place to increase host susceptibility to MDR A. baumannii, since only a self-limiting pneumonia with no or limited local bacterial replication was frequently obtained in mouse models. Direct instillation into the trachea or intranasal inoculation of the bacterial suspension are the techniques used to induce the infection in most of the preclinical models of pneumonia developed to date. More recently, the oropharyngeal aspiration procedure has been widely described in the literature for a variety of purposes including pathogens administration. Aim of this study was to compare the oropharyngeal aspiration technique to the intranasal inoculation and intratracheal instillation in the ability of inducing a consistent lung infection with two MDR A. baumannii clinical isolates in immunocompromised mice. Moreover, pneumonia obtained by bacteria administration with two out of three techniques, intratracheal and oropharyngeal, was characterised in terms of histopathology of pulmonary lesions, biomarkers of inflammation level and leukocytes cells infiltration extent after mice treatment with either vehicle or the antibiotic tigecycline. The data generated clearly showed that both strains were not able to colonize the lungs when inoculated by intranasal route. By contrast, the bacterial load in lungs of mice intratracheally or oropharyngeally infected significantly increased during 26 hours of monitoring, thus highlighting the ability of these strains to generate the infection when directly instilled into the lower respiratory airways. Furthermore, the intragroup variability of mice was significantly reduced with respect to those intranasally administered. Tigecycline was efficacious in lung bacterial load and cytokines release reduction. Findings were supported by semi-quantitative histopathological evaluation of the pulmonary lesions and by inflammatory biomarkers analysis. To conclude, both intratracheal instillation and oropharyngeal aspiration techniques showed to be suitable methods for inducing a robust and consistent pneumonia infection in mice when difficult MDR A. baumannii clinical isolates were used. Noteworthy, oropharyngeal aspiration not requiring specific technical skills and dedicated equipment, was proven to be a safer, easier and faster technique in comparison to the intratracheal instillation.

Fig 1. Bacterial load in lungs and trachea of immunocompromised mice infected with A. baumanii ACC001 (A) and ACC002 (B).

Bacterial counts in lungs and trachea of animals infected with MDR A. baumannii strains ACC001 (challenge 5.92 Log10 CFU/mouse) and ACC002 (challenge 6.10 Log10 CFU/mouse) with intranasal (IN), intratracheal (IT) or oropharyngeal (OP) techniques. Data are expressed as scatterplot distribution (included mean). Statistical analysis was performed by a One Way ANOVA followed by Tukey’s Multiple Comparison Test, ****p<0.0001, **p<0.01 (26 hours vs 2 hours post infection for each infection technique).

Fig 2. Bacterial load in lung of immunocompromised mice infected with A. baumanii ACC001 during therapy with TGC.

Bacterial counts in lungs of animals intratracheally (IT) and oropharyngeally (OP) infected with MDR A. baumannii ACC001 (challenge 5.77 Log10 CFU/mouse) untreated (T0) and treated with vehicle (VEH) or tigecycline (TGC) 120 mg/kg/day (T24). Data expressed as scatterplot distribution (included mean). Statistical analysis was performed by a Two Way ANOVA followed by Tukey’s Multiple Comparison Test, ****p<0.0001 (VEH and TGC vs start of therapy).

Fig 3. Lung infiltrating leukocytes in immunocompromised mice, upon IT or OP infection with A. baumannii ACC001.

Lung BALF Leukocyte sub-population distribution, in intratracheally (IT) or oropharyngeally (OP) infected mice, treated with vehicle (VEH) and tigecycline (TGC) 120 mg/day. Data expressed as percentage (%) ±SD of total Leukocytes. Abbreviations. LYM: Lymphocytes; NEU: Neutrophils; MONO: Monocytes. Statistical analysis was performed by by two tailed Student’s T test considering significant p≤ 0.05.

Fig 4. Cytokine quantification in BALF of A. baumannii ACC001 infected mice.

Lung BALF cytokine quantification in vehicle (VEH) treated infected mice to assess the role played by the route of the infection intratracheal vs oropharyngeal (IT vs OP) (A) and to compare the effect of the tigecycline vs vehicle (TGC vs VEH) on the establishment of the innate immune response in intratracheally (IT) and oropharyngeally (OP) infected mice (B and C). Data expressed as mean ±SD. Statistical analysis was performed by two tailed Student’s T test considering significant p≤ 0.05.

Fig 5. Sections of pulmonary parenchyma after IT and OP infection with A. baumannii ACC001 of VEH and TGC treated mice.

Extracellular and intracellular bacteria were detected in untreated mice infected with both intratracheal (IT) (A) and oropharyngeal (OP) (C) techniques, with concurrent inflammation (hematoxylin and eosin staining, 600x magnification). Arrows indicate presence of bacteria in the cytoplasm of macrophages (black arrows) and in the extracellular space (white arrows). No bacteria were noted in mice treated with tigecycline (TGC) 120 mg/day and challenged by both intratracheal (IT) (B) and oropharyngeal (OP) (D) routes, with inflammatory changes similar in severity and distribution to untreated animals (hematoxylin and eosin staining, 600x magnification). All scale bars represent 30μm.