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Rapid dissemination of Francisella tularensis and the effect of route of infection.

Ojeda SS, Wang ZJ, Mares CA, Chang TA, Li Q, Morris EG, Jerabek PA, Teale JM - BMC Microbiol. (2008)

Bottom Line: By 20 hours, there was significant tropism to the lung compared with other tissues.MicroPET images correlated with the biodistribution of isotope and bacterial burdens in analyzed tissues.Our findings suggest that Francisella has a differential tissue tropism depending on the route of entry and that the virulence of Francisella by the pulmonary route is associated with a rapid bacteremia and an early preferential tropism to the lung.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA. ojeda@uthscsa.edu

ABSTRACT

Background: Francisella tularensis subsp. tularensis is classified as a Category A bioweapon that is capable of establishing a lethal infection in humans upon inhalation of very few organisms. However, the virulence mechanisms of this organism are not well characterized. Francisella tularensis subsp. novicida, which is an equally virulent subspecies in mice, was used in concert with a microPET scanner to better understand its temporal dissemination in vivo upon intranasal infection and how such dissemination compares with other routes of infection. Adult mice were inoculated intranasally with F. tularensis subsp. novicida radiolabeled with 64Cu and imaged by microPET at 0.25, 2 and 20 hours post-infection.

Results: 64Cu labeled F. tularensis subsp. novicida administered intranasally or intratracheally were visualized in the respiratory tract and stomach at 0.25 hours post infection. By 20 hours, there was significant tropism to the lung compared with other tissues. In contrast, the images of radiolabeled F. tularensis subsp. novicida when administered intragastrically, intradermally, intraperitoneally and intravenouslly were more generally limited to the gastrointestinal system, site of inoculation, liver and spleen respectively. MicroPET images correlated with the biodistribution of isotope and bacterial burdens in analyzed tissues.

Conclusion: Our findings suggest that Francisella has a differential tissue tropism depending on the route of entry and that the virulence of Francisella by the pulmonary route is associated with a rapid bacteremia and an early preferential tropism to the lung. In addition, the use of the microPET device allowed us to identify the cecum as a novel site of colonization of Francisella tularensis subsp. novicida in mice.

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Presence of F. tularensis subsp. novicida in the GI tract at early time points. As a way to discard the possibility of swallowing of the inoculum during i.n and i.t infections we tested an endotracheal entubation of mice to directly deliver 35S labeled F. tularensis subsp. novicida into the trachea. Data shown represent an average of 3–4 mice and is given as cpm/g. Our biodistribution results showed that after 2 hrs p.i the majority of the labeled organisms were present in the lung and trachea; furthermore, a high proportion of labeled organisms were present in the stomach. In addition, after 20 hrs p.i the highest amount of labeled bacteria was present in the lung and further disseminated to other tissues including the cecum.
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Figure 4: Presence of F. tularensis subsp. novicida in the GI tract at early time points. As a way to discard the possibility of swallowing of the inoculum during i.n and i.t infections we tested an endotracheal entubation of mice to directly deliver 35S labeled F. tularensis subsp. novicida into the trachea. Data shown represent an average of 3–4 mice and is given as cpm/g. Our biodistribution results showed that after 2 hrs p.i the majority of the labeled organisms were present in the lung and trachea; furthermore, a high proportion of labeled organisms were present in the stomach. In addition, after 20 hrs p.i the highest amount of labeled bacteria was present in the lung and further disseminated to other tissues including the cecum.

Mentions: Francisella tularensis can establish infection in the host when entering through different routes. The severity of the disease differs according to the route of infection and the inhalation route is known to be the most dangerous form. Due to these facts and to the results obtained in our microPET studies following i.n infection, our laboratory decided to study F. tularensis subsp. novicida in vivo distribution following other routes of infection by which Francisella is also known to enter the host and cause disease. First, we included an important control for the i.n infection. When bacteria were administered i.n in just 20–25 μl, labeled organisms appeared in the GI tract by 0.25 hr (15 min) including the stomach, therefore it was important to control for swallowing. So in a set of experiments, mice were inoculated with the same dose of organisms (2 × 109 CFU) but by the i.t route (Fig. 1 and Additional File 4 left). The i.t inoculation was done by administering the bacterial dose at the back of the throat while holding the tongue in an extended position thus preventing swallowing as described previously [28,29]. This explains label in the head area at the site of inoculation. The results obtained were similar in that labeled organisms could be observed by microPET in the gastrointestinal tract by 0.25 hr (15 min). Similar to the i.n route, by 20 hrs the majority of label appeared in the lung and GI tract including the cecum although the relative amount of label in the lung was higher, especially at 0.25 and 2 hrs p.i. The results observed by microPET were confirmed by determining the biodistribution of label ex vivo in various tissues and the data represent an average of 3 animals after imaging (Fig. 2). To further discard the possibility of swallowing of the inoculum during i.n and i.t infections we decided to test an endotreacheal intubation of the mice to directly deliver 35S labeled F. tularensis subsp. novicida into the trachea. Our biodistribution data shows that after 2 hrs p.i the majority of the labeled bacteria are localized in the trachea and lung; interestingly, there is also a high proportion of labeled F. tularensis subsp. novicida present in the stomach (Fig. 4). In addition, after 20 hrs p.i the biodistribution data shows that the highest concentration of labeled bacteria appeared to be present in the lung and trachea followed by the spleen; furthermore, labeled F. tularensis subsp. novicida appeared to be present in the cecum as well (Fig. 4). These results correlate with the ones obtained by microPET, early after infection F. tularensis subsp. novicida localizes in the lung and stomach, while after 20 hrs p.i the bacterium remains in the lung and further disseminates to other tissues including the cecum. Also of relevance, F. tularensis is known to cause illness in humans by ingestion, through cuts in the skin, as well as systemically but with usually different clinical outcomes. Therefore, mice were also inoculated with the same dose of bacteria by the i.g, i.d, i.p and i.v routes. Following i.g administration, the majority of labeled organisms appeared in the GI tract and remained so for 20 hrs p.i (Fig. 1 and Additional File 4 right). Substantial tropism to the lung was not evident by 20 hrs. Mice inoculated by the i.d route exhibited a different pattern (Fig. 1 and Additional File 5 right and left). The vast majority of the bacteria injected appeared to remain at the site of inoculation for the initial hours of infection. Consistent with this, the distribution of label in isolated tissues indicated that the vast majority of the label was localized to the skin and muscle at 20 hrs (Fig. 2). Nonetheless, the isotope distribution still suggested that there were bacteria in the blood and several other tissues by 20 hrs although there was no preferential tropism to the lung at this early time point. In the case of mice infected by the i.p route, most of the labeled bacterium 20 hrs after infection appeared to be present in the liver as seen by microPET images with red-yellow color indicating the high concentration of bacteria in this tissue (Fig. 1), followed by the spleen and GI tract. In addition, our observations indicated that there was not a considerable amount of labeled bacteria present in the lung at this early time point (Fig. 1). Biodistribution data obtained for 64Cu labeled F. tularensis subsp. novicida at this time point correlates with the microPET data, showing a substantial tropism for the liver followed by the spleen that had a significantly higher %ID/g, than in the case of the i.n. infection (p < 0.05) (Fig. 2). In addition, statistical analysis showed that 20 hrs p.i, the lung, stomach and cecum showed a significantly higher %ID/g following i.n infection but not i.p infection (p < 0.005, p < 0.05 and p < 0.05 respectively). When the i.v infection was performed, we observed that 20 hrs p.i the majority of the labeled organisms were present in the liver, spleen and GI tract, seen by microPET image (Fig. 1). According to the biodistribution data obtained 20 hrs p.i, the majority of labeled F. tularensis subsp. novicida was present in the spleen and liver (Fig. 2), correlating with the microPET images (Fig. 1). Furthermore, statistical analyses showed that when comparing this infection route to the i.n route, there is a significantly higher tropism of F. tularensis subsp. novicida for spleen, liver and kidney (p < 0.05, p < 0.01 and p < 0.005 respectively) while there is a significantly lower tropism for the nasal cavity, trachea, lung, stomach and cecum (p < 0.05, p < 0.005, p < 0.005, p < 0.005 and p < 0.05 respectively) (Fig. 2).


Rapid dissemination of Francisella tularensis and the effect of route of infection.

Ojeda SS, Wang ZJ, Mares CA, Chang TA, Li Q, Morris EG, Jerabek PA, Teale JM - BMC Microbiol. (2008)

Presence of F. tularensis subsp. novicida in the GI tract at early time points. As a way to discard the possibility of swallowing of the inoculum during i.n and i.t infections we tested an endotracheal entubation of mice to directly deliver 35S labeled F. tularensis subsp. novicida into the trachea. Data shown represent an average of 3–4 mice and is given as cpm/g. Our biodistribution results showed that after 2 hrs p.i the majority of the labeled organisms were present in the lung and trachea; furthermore, a high proportion of labeled organisms were present in the stomach. In addition, after 20 hrs p.i the highest amount of labeled bacteria was present in the lung and further disseminated to other tissues including the cecum.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651876&req=5

Figure 4: Presence of F. tularensis subsp. novicida in the GI tract at early time points. As a way to discard the possibility of swallowing of the inoculum during i.n and i.t infections we tested an endotracheal entubation of mice to directly deliver 35S labeled F. tularensis subsp. novicida into the trachea. Data shown represent an average of 3–4 mice and is given as cpm/g. Our biodistribution results showed that after 2 hrs p.i the majority of the labeled organisms were present in the lung and trachea; furthermore, a high proportion of labeled organisms were present in the stomach. In addition, after 20 hrs p.i the highest amount of labeled bacteria was present in the lung and further disseminated to other tissues including the cecum.
Mentions: Francisella tularensis can establish infection in the host when entering through different routes. The severity of the disease differs according to the route of infection and the inhalation route is known to be the most dangerous form. Due to these facts and to the results obtained in our microPET studies following i.n infection, our laboratory decided to study F. tularensis subsp. novicida in vivo distribution following other routes of infection by which Francisella is also known to enter the host and cause disease. First, we included an important control for the i.n infection. When bacteria were administered i.n in just 20–25 μl, labeled organisms appeared in the GI tract by 0.25 hr (15 min) including the stomach, therefore it was important to control for swallowing. So in a set of experiments, mice were inoculated with the same dose of organisms (2 × 109 CFU) but by the i.t route (Fig. 1 and Additional File 4 left). The i.t inoculation was done by administering the bacterial dose at the back of the throat while holding the tongue in an extended position thus preventing swallowing as described previously [28,29]. This explains label in the head area at the site of inoculation. The results obtained were similar in that labeled organisms could be observed by microPET in the gastrointestinal tract by 0.25 hr (15 min). Similar to the i.n route, by 20 hrs the majority of label appeared in the lung and GI tract including the cecum although the relative amount of label in the lung was higher, especially at 0.25 and 2 hrs p.i. The results observed by microPET were confirmed by determining the biodistribution of label ex vivo in various tissues and the data represent an average of 3 animals after imaging (Fig. 2). To further discard the possibility of swallowing of the inoculum during i.n and i.t infections we decided to test an endotreacheal intubation of the mice to directly deliver 35S labeled F. tularensis subsp. novicida into the trachea. Our biodistribution data shows that after 2 hrs p.i the majority of the labeled bacteria are localized in the trachea and lung; interestingly, there is also a high proportion of labeled F. tularensis subsp. novicida present in the stomach (Fig. 4). In addition, after 20 hrs p.i the biodistribution data shows that the highest concentration of labeled bacteria appeared to be present in the lung and trachea followed by the spleen; furthermore, labeled F. tularensis subsp. novicida appeared to be present in the cecum as well (Fig. 4). These results correlate with the ones obtained by microPET, early after infection F. tularensis subsp. novicida localizes in the lung and stomach, while after 20 hrs p.i the bacterium remains in the lung and further disseminates to other tissues including the cecum. Also of relevance, F. tularensis is known to cause illness in humans by ingestion, through cuts in the skin, as well as systemically but with usually different clinical outcomes. Therefore, mice were also inoculated with the same dose of bacteria by the i.g, i.d, i.p and i.v routes. Following i.g administration, the majority of labeled organisms appeared in the GI tract and remained so for 20 hrs p.i (Fig. 1 and Additional File 4 right). Substantial tropism to the lung was not evident by 20 hrs. Mice inoculated by the i.d route exhibited a different pattern (Fig. 1 and Additional File 5 right and left). The vast majority of the bacteria injected appeared to remain at the site of inoculation for the initial hours of infection. Consistent with this, the distribution of label in isolated tissues indicated that the vast majority of the label was localized to the skin and muscle at 20 hrs (Fig. 2). Nonetheless, the isotope distribution still suggested that there were bacteria in the blood and several other tissues by 20 hrs although there was no preferential tropism to the lung at this early time point. In the case of mice infected by the i.p route, most of the labeled bacterium 20 hrs after infection appeared to be present in the liver as seen by microPET images with red-yellow color indicating the high concentration of bacteria in this tissue (Fig. 1), followed by the spleen and GI tract. In addition, our observations indicated that there was not a considerable amount of labeled bacteria present in the lung at this early time point (Fig. 1). Biodistribution data obtained for 64Cu labeled F. tularensis subsp. novicida at this time point correlates with the microPET data, showing a substantial tropism for the liver followed by the spleen that had a significantly higher %ID/g, than in the case of the i.n. infection (p < 0.05) (Fig. 2). In addition, statistical analysis showed that 20 hrs p.i, the lung, stomach and cecum showed a significantly higher %ID/g following i.n infection but not i.p infection (p < 0.005, p < 0.05 and p < 0.05 respectively). When the i.v infection was performed, we observed that 20 hrs p.i the majority of the labeled organisms were present in the liver, spleen and GI tract, seen by microPET image (Fig. 1). According to the biodistribution data obtained 20 hrs p.i, the majority of labeled F. tularensis subsp. novicida was present in the spleen and liver (Fig. 2), correlating with the microPET images (Fig. 1). Furthermore, statistical analyses showed that when comparing this infection route to the i.n route, there is a significantly higher tropism of F. tularensis subsp. novicida for spleen, liver and kidney (p < 0.05, p < 0.01 and p < 0.005 respectively) while there is a significantly lower tropism for the nasal cavity, trachea, lung, stomach and cecum (p < 0.05, p < 0.005, p < 0.005, p < 0.005 and p < 0.05 respectively) (Fig. 2).

Bottom Line: By 20 hours, there was significant tropism to the lung compared with other tissues.MicroPET images correlated with the biodistribution of isotope and bacterial burdens in analyzed tissues.Our findings suggest that Francisella has a differential tissue tropism depending on the route of entry and that the virulence of Francisella by the pulmonary route is associated with a rapid bacteremia and an early preferential tropism to the lung.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA. ojeda@uthscsa.edu

ABSTRACT

Background: Francisella tularensis subsp. tularensis is classified as a Category A bioweapon that is capable of establishing a lethal infection in humans upon inhalation of very few organisms. However, the virulence mechanisms of this organism are not well characterized. Francisella tularensis subsp. novicida, which is an equally virulent subspecies in mice, was used in concert with a microPET scanner to better understand its temporal dissemination in vivo upon intranasal infection and how such dissemination compares with other routes of infection. Adult mice were inoculated intranasally with F. tularensis subsp. novicida radiolabeled with 64Cu and imaged by microPET at 0.25, 2 and 20 hours post-infection.

Results: 64Cu labeled F. tularensis subsp. novicida administered intranasally or intratracheally were visualized in the respiratory tract and stomach at 0.25 hours post infection. By 20 hours, there was significant tropism to the lung compared with other tissues. In contrast, the images of radiolabeled F. tularensis subsp. novicida when administered intragastrically, intradermally, intraperitoneally and intravenouslly were more generally limited to the gastrointestinal system, site of inoculation, liver and spleen respectively. MicroPET images correlated with the biodistribution of isotope and bacterial burdens in analyzed tissues.

Conclusion: Our findings suggest that Francisella has a differential tissue tropism depending on the route of entry and that the virulence of Francisella by the pulmonary route is associated with a rapid bacteremia and an early preferential tropism to the lung. In addition, the use of the microPET device allowed us to identify the cecum as a novel site of colonization of Francisella tularensis subsp. novicida in mice.

Show MeSH
Related in: MedlinePlus