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. 2011 Jun 16;2:133.
doi: 10.3389/fmicb.2011.00133. eCollection 2011.

Bioluminescent Diagnostic Imaging to Characterize Altered Respiratory Tract Colonization by the Burkholderia Pseudomallei Capsule Mutant

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Free PMC article

Bioluminescent Diagnostic Imaging to Characterize Altered Respiratory Tract Colonization by the Burkholderia Pseudomallei Capsule Mutant

Jonathan M Warawa et al. Front Microbiol. .
Free PMC article

Abstract

Pneumonia is a common manifestation of the potentially fatal disease melioidosis, caused by the select agent bacteria Burkholderia pseudomallei. In this study we describe a new model system to investigate pulmonary melioidosis in vivo using bioluminescent-engineered bacteria in a murine respiratory disease model. Studies were performed to validate that the stable, light producing B. pseudomallei strain JW280 constitutively produced light in biologically relevant host-pathogen interactions. Hairless outbred SKH1 mice were used to enhance the ability to monitor B. pseudomallei respiratory disease, and were found to be similarly susceptible to respiratory melioidosis as BALB/c mice. This represents the first demonstration of in vivo diagnostic imaging of pulmonary melioidosis permitting the detection of B. pseudomallei less than 24 h post-infection. Diagnostic imaging of pulmonary melioidosis revealed distinct temporal patterns of bacterial colonization unique to both BALB/c and SKH1 mice. Validation of these model systems included the use of the previously characterized capsule mutant, which was found to colonize the upper respiratory tract at significantly higher levels than the wild type strain. These model systems allow for high resolution detection of bacterial pulmonary disease which will facilitate studies of therapeutics and basic science evaluation of melioidosis.

Keywords: Burkholderia pseudomallei; bioluminescent diagnostic imaging; capsular polysaccharide; hairless mouse model; intranasal infection; melioidosis; pulmonary disease; upper respiratory tract infection.

Figures

Figure 1
Figure 1
Intracellular survival of B. pseudomallei in J774A.1 cell line. B. pseudomallei strains JW280 and JW280Δwcb were used to infect 7.5 × 104 J774A.1 murine macrophages in triplicate at an MOI of 0.5. After 1 h, extracellular bacteria were killed with gentamicin. At time points of 3, 5, 7, and 9 h post-inoculation, the light production of triplicate set of samples was measured, then samples were lysed with 0.1% Triton X-100 and enumerated by plate counting. The CFU/well was plotted as a function of time for both JW280 and JW280Δwcb (A). Similarly both CFU/well (left y-axis) and cps/well (right y-axis) were jointly plotted as a function of time for JW280 (B) and JW280Δwcb (C). The mean and standard deviation were plotted for each strain/time point. Asterisks indicate significant difference in bacterial colonization of J774A.1 cells at specific time points (*p < 0.05).
Figure 2
Figure 2
Virulence of Lux-producing B. pseudomallei strains. Groups of six mice were infected by the i.n. route with B. pseudomallei and monitored for 14 days. (A) Groups of BALB/c mice were infected with 105 CFU of wild type strains DD503 (lux−) or JW280 (lux+, left panel) or with 106 CFU of capsule mutant strains JW270 (lux−) or JW280Δwcb (lux+, right panel). (B) Groups of SKH1 or BALB/c mice were infected with 105 CFU of JW280 (left panel) or 106 CFU JW280Δwcb (right panel) to compare the susceptibility of each mouse strain to melioidosis. (C) SKH1 mice were infected with 3- to 10-fold serial dilutions of JW280 (103–105 CFU, left panel) or JW280Δwcb (105–107 CFU, right panel) to facilitate estimation of the ID50 for each strain. Mice were euthanized at the onset of moribund disease. Asterisks indicate significant differences between survival curves using the log-rank (Mantel–Cox) test.
Figure 3
Figure 3
Histological analysis of B. pseudomallei-infected tissues. Photomicrographs of representative tissue samples from SKH1 mice infected i.n. with JW280 (104 CFU), JW280Δwcb (106 CFU), or PBS mock-infected control animals. Samples were formalin-fixed, stained with hematoxylin and eosin (H&E), and imaged by bright field microscopy using either a ×10 (lung) or ×20 (liver) objectives.
Figure 4
Figure 4
Histopathological scoring of SKH1 infected tissue. Lung, liver, and spleen from SKH1 mice infected i.n. with JW280 (104 CFU) or JW280Δwcb (106 CFU) were fixed, stained with H&E, and scored in a blind study for varying degrees of pathology. The scoring system was defined as: 0 = within normal limits, 1 = minimal, 2 = mild, 3 = moderate, 4 = severe. Individual data points, the mean, and standard deviation are plotted, and statistically significant variation from JW280-infected tissue scoring is indicated by asterisks (*p < 0.05).
Figure 5
Figure 5
B. pseudomallei colonization of key sites of infection. SKH1 and BALB/c mice infected i.n. with JW280 (104 CFU) or JW280Δwcb (106 CFU) were euthanized at the onset of morbid disease symptoms, organs were harvested, homogenized in PBS and bacteria were enumerated. Data indicates the total number of bacteria detected per organ for lung (A), liver (B), and spleen (C). Significant difference between data sets indicated by asterisk (*p < 0.05).
Figure 6
Figure 6
In vivo detection of a developing B. pseudomallei infection. Overlay images captured with an IVIS Lumina system of representative SKH1 and BALB/c mice infected i.n. with JW280 (104 CFU) or JW280Δwcb (106 CFU). Infected mice were imaged twice daily and images from late infection (54–92 h) are presented until the time point at which mice were euthanized due to terminal disease. All images were normalized to the same photon saturation scale.
Figure 7
Figure 7
Dynamic assessment of lung colonization by B. pseudomallei. SKH1 and BALB/c mice were infected i.n. with JW280 (104 CFU), JW280Δwcb (106 CFU) or PBS mock-infected, and mice were imaged dorsally with an IVIS Lumina system beginning 24 h post-infection. Living Image 3.0 software was used to enumerate light emission from the lung. PBS mock-infected mice were used to establish baseline noise. At onset of morbid disease, mice were euthanized and bacteria enumerated from the lung. (A) Plot of emitted light immediately before euthanization of mice in morbid disease stage as a function of bacterial burden in the lung. The y-intercept of the trend line was constrained through the estimated noise determined from mock-infected mice. (B,C) Total flux plotted as a function of time for mice which developed morbid disease in BALB/c (B) and SKH1 (C) mice. The average noise level estimated from mock-infected mice is indicated with a horizontal bar (B) or the x-axis (C). (D) Total flux plotted as a function of time for mice which survived the pneumonia phase of disease in SKH1 mice. The average noise level estimated from mock-infected mice is set at the x-axis.
Figure 8
Figure 8
Preferential colonization of the nasal cavity by B. pseudomallei. SKH1 and BALB/c mice were infected i.n. with JW280 (104 CFU) or JW280Δwcb (106 CFU) and mice were imaged dorsally with an IVIS Lumina system beginning 24 h post-infection. The background corrected nasal cavity to thoracic cavity ratio was evaluated at all time points for mice that developed lethal pneumonia. The collective of all experimental ratios were plotted for each group. Significant differences between groups are indicated by asterisks (**p < 0.01, ***p < 0.001).

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