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Characterization of Bacillus anthracis persistence in vivo.

Jenkins SA, Xu Y - PLoS ONE (2013)

Bottom Line: Immunofluorescence staining of lung sections showed that spores associated with the alveolar and airway epithelium.We also showed that the anthrax toxins did not play a role in persistence.Together, the results suggest that B. anthracis spores have special properties that promote their persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence.

View Article: PubMed Central - PubMed

Affiliation: Center for Infectious and Inflammatory Diseases, Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, Texas, USA.

ABSTRACT
Pulmonary exposure to Bacillus anthracis spores initiates inhalational anthrax, a life-threatening infection. It is known that dormant spores can be recovered from the lungs of infected animals months after the initial spore exposure. Consequently, a 60-day course antibiotic treatment is recommended for exposed individuals. However, there has been little information regarding details or mechanisms of spore persistence in vivo. In this study, we investigated spore persistence in a mouse model. The results indicated that weeks after intranasal inoculation with B. anthracis spores, substantial amounts of spores could be recovered from the mouse lung. Moreover, spores of B. anthracis were significantly better at persisting in the lung than spores of a non-pathogenic Bacillus subtilis strain. The majority of B. anthracis spores in the lung were tightly associated with the lung tissue, as they could not be readily removed by lavage. Immunofluorescence staining of lung sections showed that spores associated with the alveolar and airway epithelium. Confocal analysis indicated that some of the spores were inside epithelial cells. This was further confirmed by differential immunofluorescence staining of lung cells harvested from the infected lungs, suggesting that association with lung epithelial cells may provide an advantage to spore persistence in the lung. There was no or very mild inflammation in the infected lungs. Furthermore, spores were present in the lung tissue as single spores rather than in clusters. We also showed that the anthrax toxins did not play a role in persistence. Together, the results suggest that B. anthracis spores have special properties that promote their persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence.

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Related in: MedlinePlus

Representative images of immunofluorescently stained lung sections from mice challenged with B. anthracis spores.Mice were infected i.n. with ∼108 spores/mouse. Lungs were harvested at 2 and 4 weeks, fixed, sectioned and stained with anti-BclA antibodies and secondary antibodies conjugated to Alexa Fluor 594 (red), Alexa Fluor 488-conjugated phalliodin (green) and DAPI (blue), as described in the Materials and Methods section. Representative images are shown to indicate spore association with the small airway epithelium (A and B) and the alveolar epithelium (C–E). Arrows indicate spores. The areas around those spores were enlarged and shown in boxed insets. Scale bars represent 10 µm.
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pone-0066177-g006: Representative images of immunofluorescently stained lung sections from mice challenged with B. anthracis spores.Mice were infected i.n. with ∼108 spores/mouse. Lungs were harvested at 2 and 4 weeks, fixed, sectioned and stained with anti-BclA antibodies and secondary antibodies conjugated to Alexa Fluor 594 (red), Alexa Fluor 488-conjugated phalliodin (green) and DAPI (blue), as described in the Materials and Methods section. Representative images are shown to indicate spore association with the small airway epithelium (A and B) and the alveolar epithelium (C–E). Arrows indicate spores. The areas around those spores were enlarged and shown in boxed insets. Scale bars represent 10 µm.

Mentions: To detect spores in the lung, immunohistochemistry was performed. Microscopic examination of the stained sections revealed that spores were present in the lung at all three time points. Spores were observed associated with the alveolar and the small airway epithelium as single spores rather than in clusters (Fig. 6, A–E), indicating that they did not form biofilms in the lung. Additionally, spores were seen distributed relatively evenly throughout the lung tissue similar to what was reported previously [13], [14]. Some of the spores that associated with the airway and alveolar epithelium appeared intracellular. To determine if the latter group of spores were indeed intracellular, we analyzed the Z-stacks of confocal images of 124 potential intracellular spores from 2 and 4 weeks. Of these, 101 (81%) were surrounded by F-actin (Fig. 7, A) or enclosed within the plasma membrane (Fig. 7, B) and therefore were likely to be intracellular.


Characterization of Bacillus anthracis persistence in vivo.

Jenkins SA, Xu Y - PLoS ONE (2013)

Representative images of immunofluorescently stained lung sections from mice challenged with B. anthracis spores.Mice were infected i.n. with ∼108 spores/mouse. Lungs were harvested at 2 and 4 weeks, fixed, sectioned and stained with anti-BclA antibodies and secondary antibodies conjugated to Alexa Fluor 594 (red), Alexa Fluor 488-conjugated phalliodin (green) and DAPI (blue), as described in the Materials and Methods section. Representative images are shown to indicate spore association with the small airway epithelium (A and B) and the alveolar epithelium (C–E). Arrows indicate spores. The areas around those spores were enlarged and shown in boxed insets. Scale bars represent 10 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672131&req=5

pone-0066177-g006: Representative images of immunofluorescently stained lung sections from mice challenged with B. anthracis spores.Mice were infected i.n. with ∼108 spores/mouse. Lungs were harvested at 2 and 4 weeks, fixed, sectioned and stained with anti-BclA antibodies and secondary antibodies conjugated to Alexa Fluor 594 (red), Alexa Fluor 488-conjugated phalliodin (green) and DAPI (blue), as described in the Materials and Methods section. Representative images are shown to indicate spore association with the small airway epithelium (A and B) and the alveolar epithelium (C–E). Arrows indicate spores. The areas around those spores were enlarged and shown in boxed insets. Scale bars represent 10 µm.
Mentions: To detect spores in the lung, immunohistochemistry was performed. Microscopic examination of the stained sections revealed that spores were present in the lung at all three time points. Spores were observed associated with the alveolar and the small airway epithelium as single spores rather than in clusters (Fig. 6, A–E), indicating that they did not form biofilms in the lung. Additionally, spores were seen distributed relatively evenly throughout the lung tissue similar to what was reported previously [13], [14]. Some of the spores that associated with the airway and alveolar epithelium appeared intracellular. To determine if the latter group of spores were indeed intracellular, we analyzed the Z-stacks of confocal images of 124 potential intracellular spores from 2 and 4 weeks. Of these, 101 (81%) were surrounded by F-actin (Fig. 7, A) or enclosed within the plasma membrane (Fig. 7, B) and therefore were likely to be intracellular.

Bottom Line: Immunofluorescence staining of lung sections showed that spores associated with the alveolar and airway epithelium.We also showed that the anthrax toxins did not play a role in persistence.Together, the results suggest that B. anthracis spores have special properties that promote their persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence.

View Article: PubMed Central - PubMed

Affiliation: Center for Infectious and Inflammatory Diseases, Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, Texas, USA.

ABSTRACT
Pulmonary exposure to Bacillus anthracis spores initiates inhalational anthrax, a life-threatening infection. It is known that dormant spores can be recovered from the lungs of infected animals months after the initial spore exposure. Consequently, a 60-day course antibiotic treatment is recommended for exposed individuals. However, there has been little information regarding details or mechanisms of spore persistence in vivo. In this study, we investigated spore persistence in a mouse model. The results indicated that weeks after intranasal inoculation with B. anthracis spores, substantial amounts of spores could be recovered from the mouse lung. Moreover, spores of B. anthracis were significantly better at persisting in the lung than spores of a non-pathogenic Bacillus subtilis strain. The majority of B. anthracis spores in the lung were tightly associated with the lung tissue, as they could not be readily removed by lavage. Immunofluorescence staining of lung sections showed that spores associated with the alveolar and airway epithelium. Confocal analysis indicated that some of the spores were inside epithelial cells. This was further confirmed by differential immunofluorescence staining of lung cells harvested from the infected lungs, suggesting that association with lung epithelial cells may provide an advantage to spore persistence in the lung. There was no or very mild inflammation in the infected lungs. Furthermore, spores were present in the lung tissue as single spores rather than in clusters. We also showed that the anthrax toxins did not play a role in persistence. Together, the results suggest that B. anthracis spores have special properties that promote their persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence.

Show MeSH
Related in: MedlinePlus