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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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MicroPET imaging of bacterial infection of mice. Two-dimensional images of representative mice (ventral view) are depicted in the figure. MicroPET images were recorded over time from mice infected with either 64Cu labeled F. tularensis subsp. novicida, 64Cu labeled K. pneumoniae or 64Cu labeled E. coli. Mice were infected i.n, i.t, i.g, i.p, i.v and i.d with F. tularensis subsp. novicida (2 × 109 CFU/20 μl) and i.n with K. pneumoniae and E. coli using the same infection dose. Images were obtained at 0.25 hrs, 2 hrs and 20 hrs p.i. The red punctuated line represents the liver. The color scale on the right hand side of the figure is a linear scale that indicates percentage with red indicating 100% of signal and purple 0–10% or the lowest amount detected by imaging. Abbreviations: N: Nasal cavity, T: Trachea, L: Lung, GI: Gastrointestinal Tract, Li: Liver, S: Spleen, Le: Leg, Ce: Cecum.
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Figure 1: MicroPET imaging of bacterial infection of mice. Two-dimensional images of representative mice (ventral view) are depicted in the figure. MicroPET images were recorded over time from mice infected with either 64Cu labeled F. tularensis subsp. novicida, 64Cu labeled K. pneumoniae or 64Cu labeled E. coli. Mice were infected i.n, i.t, i.g, i.p, i.v and i.d with F. tularensis subsp. novicida (2 × 109 CFU/20 μl) and i.n with K. pneumoniae and E. coli using the same infection dose. Images were obtained at 0.25 hrs, 2 hrs and 20 hrs p.i. The red punctuated line represents the liver. The color scale on the right hand side of the figure is a linear scale that indicates percentage with red indicating 100% of signal and purple 0–10% or the lowest amount detected by imaging. Abbreviations: N: Nasal cavity, T: Trachea, L: Lung, GI: Gastrointestinal Tract, Li: Liver, S: Spleen, Le: Leg, Ce: Cecum.

Mentions: F. tularensis subsp. novicida, a highly virulent strain in mice, was directly labeled with 64Cu-PTSM so that its route of dissemination and anatomical distribution after inoculation could be traced by repetitive microPET imaging of live animals. C57BL/6 mice were infected i.n with 2 × 109 CFU/20–25 μl of bacteria and accumulated positron emission measured for a 15 min period immediately after inoculation (labeled 0.25 hr), 2 hrs and 20 hrs post-infection (p.i) by using a microPET scanner. Still images are shown in Fig. 1, and images that can be rotated 360° can be found in Additional File 1. Surprisingly, by 0.25 hr (15 min) p.i we observed radiolabel not only in the nasal cavity at the site of inoculation but also in the lung and even in the digestive tract (Fig. 1). The inoculum volume of 20–25 μl has been shown to avoid introduction into the lung [26,27], but the rapid spread was found repeatedly in multiple animals. At 2 hrs p.i labeled organisms tracked to the same organs but the amount of 64Cu detected by the microPET increased in the lungs as well as the GI tract suggesting an increase in the number of bacteria present in these organs. Interestingly, the cecum was usually a hot spot for labeled organisms. The images obtained 20 hrs post-infection indicated a decrease in label intensity present in the nasal cavity, an additional increase in the lung and further spread through the GI tract (Fig. 1). The recent acquisition of a FLEX Pre-Clinical Platform allowed us to obtain an X-ray computed tomography (CT) image of the skeletal structure of mice that had been previously infected by the i.p and i.v routes prior to acquire the microPET images (Additional Files 2 and 3). The advantage is that a fused image of the skeletal structure with the microPET image is obtained and in this way a better indication of the localization of the labeled organisms in vivo is possible. Additional Files 1, 2 and 3 show fused images obtained at 0.25 hrs for i.n. i.p and i.v inoculations. To further analyze isotope distribution, inoculated animals were sacrificed, and several organs harvested to measure the amount of radioisotope using an automatic well-type γ counter. The combined ex-vivo data obtained from several experiments are expressed as % input dose/g of tissue (%ID/g) (Fig. 2). The results obtained with F. tularensis subsp. novicida i.n infected animals correlate with the microPET analyses indicating the highest concentration of 64Cu in the lung, stomach and GI tract. Importantly, essentially all organs and tissues tested contained radioactivity suggesting widespread dissemination of Francisella by 20 hrs p.i.


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)

MicroPET imaging of bacterial infection of mice. Two-dimensional images of representative mice (ventral view) are depicted in the figure. MicroPET images were recorded over time from mice infected with either 64Cu labeled F. tularensis subsp. novicida, 64Cu labeled K. pneumoniae or 64Cu labeled E. coli. Mice were infected i.n, i.t, i.g, i.p, i.v and i.d with F. tularensis subsp. novicida (2 × 109 CFU/20 μl) and i.n with K. pneumoniae and E. coli using the same infection dose. Images were obtained at 0.25 hrs, 2 hrs and 20 hrs p.i. The red punctuated line represents the liver. The color scale on the right hand side of the figure is a linear scale that indicates percentage with red indicating 100% of signal and purple 0–10% or the lowest amount detected by imaging. Abbreviations: N: Nasal cavity, T: Trachea, L: Lung, GI: Gastrointestinal Tract, Li: Liver, S: Spleen, Le: Leg, Ce: Cecum.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: MicroPET imaging of bacterial infection of mice. Two-dimensional images of representative mice (ventral view) are depicted in the figure. MicroPET images were recorded over time from mice infected with either 64Cu labeled F. tularensis subsp. novicida, 64Cu labeled K. pneumoniae or 64Cu labeled E. coli. Mice were infected i.n, i.t, i.g, i.p, i.v and i.d with F. tularensis subsp. novicida (2 × 109 CFU/20 μl) and i.n with K. pneumoniae and E. coli using the same infection dose. Images were obtained at 0.25 hrs, 2 hrs and 20 hrs p.i. The red punctuated line represents the liver. The color scale on the right hand side of the figure is a linear scale that indicates percentage with red indicating 100% of signal and purple 0–10% or the lowest amount detected by imaging. Abbreviations: N: Nasal cavity, T: Trachea, L: Lung, GI: Gastrointestinal Tract, Li: Liver, S: Spleen, Le: Leg, Ce: Cecum.
Mentions: F. tularensis subsp. novicida, a highly virulent strain in mice, was directly labeled with 64Cu-PTSM so that its route of dissemination and anatomical distribution after inoculation could be traced by repetitive microPET imaging of live animals. C57BL/6 mice were infected i.n with 2 × 109 CFU/20–25 μl of bacteria and accumulated positron emission measured for a 15 min period immediately after inoculation (labeled 0.25 hr), 2 hrs and 20 hrs post-infection (p.i) by using a microPET scanner. Still images are shown in Fig. 1, and images that can be rotated 360° can be found in Additional File 1. Surprisingly, by 0.25 hr (15 min) p.i we observed radiolabel not only in the nasal cavity at the site of inoculation but also in the lung and even in the digestive tract (Fig. 1). The inoculum volume of 20–25 μl has been shown to avoid introduction into the lung [26,27], but the rapid spread was found repeatedly in multiple animals. At 2 hrs p.i labeled organisms tracked to the same organs but the amount of 64Cu detected by the microPET increased in the lungs as well as the GI tract suggesting an increase in the number of bacteria present in these organs. Interestingly, the cecum was usually a hot spot for labeled organisms. The images obtained 20 hrs post-infection indicated a decrease in label intensity present in the nasal cavity, an additional increase in the lung and further spread through the GI tract (Fig. 1). The recent acquisition of a FLEX Pre-Clinical Platform allowed us to obtain an X-ray computed tomography (CT) image of the skeletal structure of mice that had been previously infected by the i.p and i.v routes prior to acquire the microPET images (Additional Files 2 and 3). The advantage is that a fused image of the skeletal structure with the microPET image is obtained and in this way a better indication of the localization of the labeled organisms in vivo is possible. Additional Files 1, 2 and 3 show fused images obtained at 0.25 hrs for i.n. i.p and i.v inoculations. To further analyze isotope distribution, inoculated animals were sacrificed, and several organs harvested to measure the amount of radioisotope using an automatic well-type γ counter. The combined ex-vivo data obtained from several experiments are expressed as % input dose/g of tissue (%ID/g) (Fig. 2). The results obtained with F. tularensis subsp. novicida i.n infected animals correlate with the microPET analyses indicating the highest concentration of 64Cu in the lung, stomach and GI tract. Importantly, essentially all organs and tissues tested contained radioactivity suggesting widespread dissemination of Francisella by 20 hrs p.i.

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