Inhalational Gentamicin Treatment Is Effective Against Pneumonic Plague in a Mouse Model

Abstract

Pneumonic plague is an infectious disease characterized by rapid and fulminant development of acute pneumonia and septicemia that results in death within days of exposure. The causative agent of pneumonic plague, Yersinia pestis (Y. pestis), is a Tier-1 bio-threat agent. Parenteral antibiotic treatment is effective when given within a narrow therapeutic window after symptom onset. However, the non-specific "flu-like" symptoms often lead to delayed diagnosis and therapy. In this study, we evaluated inhalational gentamicin therapy in an infected mouse model as a means to improve antibiotic treatment efficacy. Inhalation is an attractive route for treating lung infections. The advantages include directly dosing the main infection site, the relative accessibility for administration and the lack of extensive enzymatic drug degradation machinery. In this study, we show that inhalational gentamicin treatment administered 24 h post-infection, prior to the appearance of symptoms, protected against lethal intranasal challenge with the fully virulent Y. pestis Kimberley53 strain (Kim53). Similarly, a high survival rate was demonstrated in mice treated by inhalation with another aminoglycoside, tobramycin, for which an FDA-approved inhaled formulation is clinically available for cystic fibrosis patients. Inhalational treatment with gentamicin 48 h post-infection (to symptomatic mice) was also successful against a Y. pestis challenge dose of 10 i.n.LD₅₀. Whole-body imaging using IVIS technology demonstrated that adding inhalational gentamicin to parenteral therapy accelerated the clearance of Y. pestis from the lungs of infected animals. This may reduce disease severity and the risk of secondary infections. In conclusion, our data suggest that inhalational therapy with aerosolized gentamicin may be an effective prophylactic treatment against pneumonic plague. We also demonstrate the benefit of combining this treatment with a conventional parenteral treatment against this rapidly progressing infectious disease. We suggest the inhalational administration route as a clinically relevant treatment modality against pneumonic plague and other respiratory bacterial pathogens.

Keywords: Y. pestis; antibiotic treatment; gentamicin; infection; inhalation; mouse model; plague; tobramycin.

Figure 1

A schematic presentation of the aerosol treatment system. The exposure chamber holds up to 20 mice, and the two running wheels encourage activity and reduce huddling. The nebulizer (right-hand side) constantly generates aerosolized antibiotics, which are directed into the chamber. Other openings include a sampling port (lower left) connected to a filter and a pressure equilibration port (upper-left) connected to a low-resistance HEPA filter.

Figure 2

Concentration of gentamicin in the lungs and serum of mice following a single inhalation treatment with gentamicin solution. Average gentamicin concentration in the lungs (blue) and in the serum (red) after 60 min of inhalation of 100 mg/mL gentamicin solution delivered by a SideStream nebulizer (flow rate of 17 L/min). The time points represent min after the end of inhalation. Each data point depicts the mean and the standard error of the mean (SEM) of three individual lung extracts or sera.

Figure 3

Y. pestis loads in mouse organs at the time of antibiotic treatment initiation. Mice were exposed to 10 i.n.LD₅₀ (A) or 100 i.n.LD₅₀ (B) of the Y. pestis Kim53 strain and sacrificed at the indicated time points. Every point represents the bacterial burden in the lungs (full circles, total CFU/lung) or blood (clear circles, CFU/mL) of an individual mouse. Bacterial burden was quantified by plating the tissue homogenate/blood on BHIA plates in serial dilutions and counting the colonies. The horizontal continuous line represents the geometric mean value. The dashed line marks the level of detection-−5 CFU/mL.

Figure 4

Therapeutic efficacy of early or late initiation of gentamicin inhalation after exposure to 10 i.n. LD₅₀ or 100 i.n. LD₅₀ of Y. pestis. Groups of 8 CD-1 mice were intranasally exposed to 10LD₅₀ (A,B) or 100LD₅₀ (C) of Y. pestis. Antibiotic treatment with a comparable dose of gentamicin (3.5 mg/kg administered s.c.) was initiated either 24 h after i.n. infection (A,C) or 48 h after i.n. infection (B) and continued for 5 days as indicated: inhalation (green) and s.c. (purple). Control mice (red) were not treated.

Figure 5

Monitoring bacterial proliferation during pneumonic plague progression by bioluminescence imaging. Mice were treated with a low dose of gentamicin (3.5 mg/kg/q24 s.c. and in 3.5 mg/kg/q24 by inhalation) starting 24 h post-exposure to 25 i.n.LD₅₀ of Y. pestis Kim53-lux. (Upper) s.c. injections, and (Lower) inhalation. The mice were marked (black dye) and monitored dorsally in the prone position every 24 h.

Figure 6

Combining gentamicin inhalation with parenteral treatment against severe pneumonic plague. Mice were treated s.c. with the indicated antibiotics (gentamicin or ciprofloxacin) for 5 days starting 48 h after i.n. exposure to 25LD₅₀ of the bioluminescent Y. pestis Kim53-lux strain (20 mg/kg/q24 h). Mice in the right column were additionally treated with inhalational gentamicin starting at 48 h after i.n. infection, as described above (3.5 mg/kg/q24 h). The images present the condition of the mice 72 h post-exposure (24 h after the 1st treatment) (A). Light intensity around lungs of treated mice (Gentamicin-Genta., Ciprofloxacin-Cipro.), was quantified by ROI analysis (B). Statistical significance was determined using the non-parametric Mann-Whitney test.

Figure 7

Therapeutic efficacy of inhaled tobramycin. Groups of 8 CD-1 mice were exposed intranasally to 15LD₅₀ of Y. pestis Kim53. Inhalational treatment with tobramycin solution (50 mg/mL 1 h daily, (deposition of 1.7 mg/kg in the lungs) was initiated 24 h after i.n. infection and continued for 5 days. Control mice were not treated.