- Research article
- Open Access
Rapid dissemination of Francisella tularensisand the effect of route of infection
- Sandra S Ojeda†1,
- Zheng J Wang†2,
- Chris A Mares1,
- Tingtung A Chang3,
- Qun Li5,
- Elizabeth G Morris5,
- Paul A Jerabek4 and
- Judy M Teale1, 5Email author
© Ojeda et al; licensee BioMed Central Ltd. 2008
- Received: 21 May 2008
- Accepted: 09 December 2008
- Published: 09 December 2008
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.
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.
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.
- Colony Form Unit
- Infected Mouse
- Francisella Tularensis
- Biodistribution Data
Francisella tularensis is a facultative intracellular pathogen that is the causative agent of tularemia. Francisella has a broad host range as it is able to infect amoeba, arthropods, rodents and higher mammalian species [1–4]. The primary replication site in humans appears to be the macrophage although other cell types have been implicated [5–7]. Infection is established through contact with infected tissues, arthropod bites, inhalation or ingestion which can lead to various clinical manifestations [3, 8]. The route of infection is a key determining factor in the pathogenesis of this organism, and inhalation is the most dangerous [3, 9, 10]. There are two subspecies of F. tularensis, that are capable of causing disease in humans [9, 10]. F. tularensis (type A) is primarily found in North America. It is the most virulent strain and is capable of causing disease with as few as 10 organisms. The highly virulent nature of this microorganism combined with its ability to be aerosolized has led to its designation as a Category A biological warfare agent by the CDC [3, 11]. F. tularensis subsp. holarctica (type B) is less virulent than type A, but it is found more readily across the northern hemisphere. Type B is also the parent strain from which the important F. tularensis LVS was derived . F. tularensis subsp. novicida is another important laboratory strain as it remains highly virulent in mice but can be studied in a BSL-2 facility allowing access to new technology such as positron emission tomography (PET).
PET has been utilized to detect cells, proteins and gene expression in humans in vivo [13–15]. In humans, PET has been used as a research tool for whole body imaging and is now transitioning into diagnostic tools in oncology and in the detection of Alzheimer's disease . Recent advances in microPET design have led to the production of scanners that are capable of imaging rodents with high resolution [13, 17, 18]. Advancements in labeling techniques as well as different substrates [19, 20] have allowed for cell trafficking and distribution studies [15, 21, 22] as well as for tracing bacteria in infections [23, 24] and for tumor detection . The major advantage to utilizing microPET imaging is that it is a non-invasive process with high sensitivity that allows the investigator to perform longitudinal studies on one animal rather than sacrificing several animals at various time points .
We have exploited the capability of small animal microPET coupled with radionuclide labeling to help us investigate the spatial and temporal biodistribution of F. tularensis subsp. novicida in vivo. Mice were infected with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) ([64Cu] PTSM) labeled F. tularensis subsp. novicida by different inoculation routes including intranasal, intratracheal, intragastric, intraperitoneal, intravenous and intradermal. The trafficking of labeled F. tularensis subsp. novicida was compared with the control organisms E. coli and K. pneumoniae using both microPET analysis and radionuclide biodistribution analyses. Our results indicate that F. tularensis subsp. novicida has a differential tissue tropism depending on the route of entry and that when given by the intranasal or intratracheal route has a propensity to colonize the respiratory tract rapidly after infection tending to disseminate to other target organs within a very short period of time.
In vivo trafficking of bacteria in real time
To ensure that the radioactivity was indeed a measure of viable labeled bacteria, four tissues were analyzed for CFU/g of tissue at 20 hrs p.i (Additional File 6). Due to normal flora, intestinal tissues were not analyzed but Francisella is also known to colonize the liver. Consistent with radioisotope measurements in vivo and ex vivo, the lungs contained the highest bacterial burden with greater than 4–5 logs of bacteria/g of tissue compared with the bladder. The liver was also several logs lower reflective of a %ID/g that was visualized by microPET at 20 hrs with a blue/purple color indicative of lower levels of radioisotope/g. Taken together; these results indicate that levels of radioisotope reflect numbers of viable organisms. Because of the rapidity of dissemination, the presence of viable organisms in the blood was also tested and found to be present (Additional File 6).
Viable vs. non-viable bacteria
Several of the tissues from mice receiving viable organisms were also tested for CFU/g of tissue (data not shown). Similar to the results obtained with 64Cu labeling, the lung exhibited the highest bacterial burden from all the organs tested. Viable bacteria were also detected in the liver, spleen, superficial lymph nodes, Peyer's patches, and blood correlating with the presence of isotope but at lower levels in all of these tissues.
Effect of bacterial route of entry
Effect of bacterial strain
Isolation of Francisella tularensis subsp. novicidafrom gastrointestinal tract
Presence of Francisella in blood at a lower dose
Several studies have been published to examine small animal infection models using in vivo bioluminescence imaging (BLI) to track the course of infection or determine the efficacy of anti-microbial therapies [31–49]. As reviewed previously , there are many advantages to in vivo imaging which include the ability to perform longitudinal studies with the same animal, identifying novel sites of infection such as the cecum  and gall bladder , and increased information per animal allowing for the use of fewer animals. One of the disadvantages of BLI is the inability to accurately localize the focal point of bioluminescence within the living animal as bioluminescence produces only two dimensional data, and the spatial resolution is limited . In addition, the light is significantly attenuated per cm of tissue and scattered by fur and overlying tissues further complicating the data generated . The use of microPET overcomes these disadvantages providing high resolution data that are three dimensional . Thus, the entire depth of the body can be measured and specific organs visualized, particularly when combined with software that enables rotation of the image, and X-ray computed tomography (CT) that reveals the skeletal structure. Unfortunately, the CT capability only became available at the very end of our experiments. In addition, the high resolution of the microPET allows for accurate determination of radioactivity within user defined areas of interest. Using these measurements, both the CFU and the % of input bacteria can be calculated without sacrificing the animal and performing labor intensive bacterial burden assays in order to track infection processes or assess therapeutic modalities. Moreover, microPET is more easily transitioned to the clinic as compared with BLI. The studies described herein used microPET technology in addition to traditional labeling techniques to assess the progressive spread of F. tularensis subsp. novicida during the initial hours of infection to explore mechanisms associated with its extreme virulence in mice.
When multiple mice were infected with radiolabeled F. tularensis subsp. novicida by inhalation followed by imaging, there was rapid widespread dissemination of organisms from the upper respiratory tract to the lung despite low inoculum volumes known to avoid direct introduction into the lung . By 15 min, labeled organisms were found in the lung and stomach with further spread through the GI tract by 20 hrs. Similar results were obtained with mice infected by the i.t route using two different protocols indicating that swallowing was unlikely to be involved in the rapid dissemination to the GI tract. By 20 hrs after pulmonary infection, radioactivity substantially decreased in the upper respiratory tract. Activity in the lung was maintained and in several animals, accumulated to a greater degree suggesting an early tropism. This was confirmed by the distribution of radioisotope post necropsy where the highest levels of label were consistently found in the lung at 20 hrs. Comparable results were obtained when F. tularensis LVS was inoculated i.n. at the same dose suggesting similar dissemination patterns in Francisella. Similarly, bacterial burden assays have previously shown that mice intranasally infected with a low bacterial dose of Francisella show a relatively high bacterial burden in lung as early as 1 day post-infection [51, 52]. Therefore, regardless of the inoculum dose, Francisella has a high tropism for the lung when infected through the aerosol route.
Bacterial burden assays correlated with the imaging and biodistribution data. A Rif+ strain with equivalent virulence allowed assessment of viable bacteria in the GI tract. The results obtained show that there is a wide dissemination of bacteria as early as 2 hrs after i.n. infection, and CFU could be isolated from multiple organs including lung, stomach, small intestine, cecum and large intestine. It has been shown previously that Francisella can cause a typhoid-like disease  and survive the harsh environment of the GI tract in mice . However, the rapidity of spread to the gut after i.n infection was unexpected. One possible explanation of the rapid dissemination to the GI tract in our model could involve a hematogenous and/or lymphatogenous way through the Nasal Associated Lymphoid Tissue (NALT). It has been previously reported  that the NALT is composed of mainly B cells, to a lesser extent T cells and a small proportion of macrophages (CD11b+ cells), and that the percentages of these cell types observed in NALT are similar to the percentages observed in spleen . Since it is known that Francisella survives and replicates inside macrophages, a possible way of dissemination from the nasal passages could be by macrophages present in the NALT acting as 'Trojan horses', assuring the protection from the immune system and facilitating a rapid dissemination to other tissues. This has been observed for Salmonella typhimurium by trafficking through T, B cells and macrophages [55, 56].
One interesting aspect of working with microPET technology is the possibility to identify novel sites of infection. In our case, these experiments allowed us to identify the concentration of F. tularensis subsp. novicida in the cecum, which has not been described previously. Several studies using different organisms known to invade the gut including Citrobacter rodentium, Helicobacter pylori and Burkholderia cepacia appear to colonize the cecum [38, 48, 57]. In addition, it has been previously reported that intranasally delivered S. typhimurium in swine is present in cecum as early as 3 hrs p.i . The cecum, particularly the cecal patch, contain lymphoid tissue that could play an important role in immunity and possibly further bacterial dissemination. This aspect is currently under study in our laboratory.
K. pneumoniae, another pulmonary pathogen, was used for comparison as was a gut associated E. coli clinical isolate, and both were administered i.n. Interestingly, K. pneumoniae in contrast to F. tularensis subsp. novicida exhibited little or no dissemination to the stomach during the first two hours and was mainly confined to the nasal cavity and trachea. Even at 20 hrs, the largest % of label was still present in the nasal cavity although trafficking to the GI tract was apparent by then. Interestingly, at these early time points, the lung was not the major focus of infection. In the case of E. coli, organisms at 20 hrs were mainly confined to the GI tract. The results indicate distinct distribution patterns of pathogenic bacteria during the first hours of infection when mice are given the same number of organisms and by the identical route. Moreover, the early tropism to the lung in the case of Francisella was dependent upon viable organisms as non-viable, biosynthetically 35S labeled bacteria failed to preferentially traffic to the lung at 20 hrs compared to the viable (unfixed) labeled bacteria. More of the non-viable organisms appeared to traffic to the gall bladder as compared to the viable labeled bacteria in this tissue at the same time point. This suggests that the viable organisms begin colonizing the lung rapidly.
Initially, we thought that the extreme rapidity of dissemination was associated with the ability of Francisella to enter the bloodstream quickly as CFU could be detected in the blood at 20 hrs p.i. However, one of the disadvantages of in vivo imaging in general is the requirement to use large doses of organisms in order to visualize the images. Therefore, we questioned if more physiological doses of 103 or 104 organisms would result in a detectable bacteremia. And in fact, even at the lower doses, clear evidence of viable bacteria were found by 20 hrs supporting this contention, and likely explaining the presence of radioactivity in essentially all tissues analyzed by both labeling techniques. However, we then tested K. pneumoniae for its ability to establish a bacteremia at these early time points. Significantly, K. pneumoniae at both high and low doses entered the blood stream, and if anything, at increased levels with detection in some mice at earlier time points. Despite this, K. pneumoniae infected mice did not appear overtly ill whereas F. tularensis subsp. novicida mice did as judged by pilo-erection, lethargy, and hunched posture. Interestingly, K. pneumoniae maintained the bulk of the inoculum in the nasal cavity in contrast to F. tularensis subsp. novicida, and it is possible that this is an important site for entrance to the blood. In fact, bacteremia has been reported in tularemia [10, 59] and the presence of F. tularensis LVS as well as SchuS4 strains in blood early after i.d infection has been described . Nonetheless, the extreme virulence of F. tularensis subsp. novicida in mice may not be attributed solely to blood vasculature ingress.
Francisella tularensis is a bacterium that can adapt to many different environments including soil, ground water, and growth in amoeba . It also replicates intracellularly within humans and a variety of other species causing significant morbidity and mortality. It contains a number of genes that facilitate survival under a variety of conditions . Correlative to this, Francisella is known to cause infection in humans by inhalation, ingestion, eye contact, insect bites, and cuts in the skin [3, 9]. Interestingly, the pulmonary route causes the most severe infections with substantial mortality, although other routes of infection have been known to develop into severe infections but with less frequency . Therefore, other routes of infection were tested using the same number of bacteria. With oral infection (i.g), the majority of radiolabeled Francisella trafficked and remained in the GI tract through 20 hrs p.i. After an i.d injection during this same time period, most of the organisms remained in the skin and muscle tissue. Both oral and i.d (cuts in skin) routes cause illness, but usually less severe in patients, and it is possible that this alteration in trafficking is involved in the decreased severity. Mice were also infected by the i.p and i.v routes. In both cases, the majority of the bacteria were localized to the liver and spleen and to a lesser extent to the GI tract and the lung. Comparing the biodistribution data obtained at 20 hrs p.i among all of the infection routes, the results suggest that the numbers of F. tularensis subsp. novicida bacteria present in mice after i.n and i.t infection are higher in the lung than in mice infected through any of the other infection routes tested despite the same infection doses. Several studies have used LVS to examine various routes of inoculation, mostly at later time points of infection to study colonization [53, 62, 63]. However, KuoLee et al. , reported CFU in lung and GI tract at 1 day p.i following a dose of 108 CFU of LVS, consistent with our findings. In addition, Woolard et al.  used an i.d dose of 105 CFU of LVS and showed barely detectable CFU in the lung and spleen at 1 day consistent with our radiodistribution data at 20 hrs p.i. We speculate that the different trafficking patterns are due, in fact by differences in the innate immune response mounted depending on the initial tissue impacted as suggested by recent studies of organ specific immunity [64–69]. We and others have found evidence of a delay in the innate immune response following i.n infection, indicated by the absence of pro-inflammatory cytokines and chemokines early after infection (6 hrs p.i – 48 hrs p.i) and beginning to increase only after 72 hrs p.i [70, 71]. We propose that the acute virulence associated with inhaled Francisella is its extremely rapid colonization of the lung leading to pulmonary failure and eventual multiple organ failure before an effective immune response can be elicited. To test this, future studies will use microPET with CT capabilities together with radiolabeled antibodies to both bacteria and distinct immune cell subsets so that continued dissemination, increases in bacterial biomass, and the disease process can be evaluated at later time points.
By using various labeling techniques, imaging, and bacterial counting, we conclude that Francisella rapidly disseminates within hours to multiple tissues regardless of the route of infection with the possible exception of the intradermal route. However, the route of infection alters the trafficking patterns. In the case of the pulmonary route, which is the most dangerous, the bacteria rapidly traffic to the lung and throughout the GI tract but by 20 hrs appears to preferentially colonize the lung indicating an early tropism to this organ. We speculate that the mode of transmission alters the severity of the disease because of documented differences in organ specific immunity.
Bacterial strains and culture media
Francisella tularensis subsp. novicida strain U112 was obtained from Dr. Bernard Arulanandam (UTSA) through Dr. Fran Nano (University of Victoria). It was grown in Tripticase Soy Agar/Broth (Becton Dickinson), supplemented with 0.1% cysteine (TSAcys). Escherichia coli (clinical isolate) was obtained from Dr. Stephen Mattingly (UTHSCSA) and was grown in Luria Bertani Agar (LB medium). Klebsiella pneumoniae (ATCC # 43816) was obtained from Dr. Peter Dube (UTHSCSA) and was grown in LB. Bacteria were resuspended in broth and then taken to a titer of 2 × 109 colony forming units (CFU)/300 μl.
Radiolabeling of bacteria with [64Cu] PTSM
Resuspended bacteria were added to 400 μCi [64Cu] PTSM and incubated in a water bath at 42°C for 1 hr. [64Cu] was conjugated to PTSM as previously described . Radiolabeled bacteria were centrifuged at 15000 g for 4 min. Supernatants were removed, and both pellets and supernatants were measured for radioactivity to determine radiolabeling efficiency of the bacteria. Preliminary radiolabeling studies were performed using different bacterial doses ranging from 103 to 109 CFU/300 μl. It was determined that the concentration of 109/300 μl was most efficiently labeled and that doses of 109 labeled organisms were required for visualization by microPET. In addition, control experiments were performed to determine the effect of labeling on viability of the bacteria with time. It was found that there was no change in the viability of the organisms after 90 min of labeling indicating a lack of toxicity associated with the labeling method. In addition, the potential leakage of label was determined by incubating labeled and washed organisms for extended periods. After incubation, bacteria were washed again, and the amount of label in the supernatant and pellets determined at 24 hrs. 79.5 +/- 2.1% of the label remained in the pellet indicating the stability of the labeling process. In addition, bacteria viability post-labeling was checked at 90 min, 24 hrs and 48 hrs post-infection and was found to be 114.5%, 43.8% and 57.4% respectively. Labeled organisms were resuspended in sterile 1× phosphate-buffered saline (PBS) for inoculation of mice.
In vivobacterial biodistribution
Labeled bacteria pellets were resuspended in 30 μl or 50 μl of sterile 1× PBS to a final concentration of 2 × 109 CFU/20–25 μl intranasally (i.n) or 2 × 109 CFU/50 μl intradermal (i.d). C57BL/6 females 6–8 wk old were anesthetized with vaporized isofluorane for the initial experiments and later by intramuscular injection of 100 μl of ketamine-xylazine mixture containing (30 mg/mL ketamine, 4 mg/mL xylazine in 1× PBS). No apparent differences in images were observed between the two methods of anesthesia. Mice were then i.n inoculated with 10–12.5 μl of bacterial suspension in each nostril drop by drop thus allowing the mice to slowly inhale the inoculum. Intradermal inoculations were done by injecting 50 μl of 2 × 109 CFU in the right leg of mice. A total of fifteen mice were inoculated i.n, nine with F. tularensis subsp. novicida, three more with E. coli and three with K. penumoniae. Additional routes of inoculation included intratracheal (i.t), intragastric (i.g), intraperitoneal (i.p) and intravenous (i.v) inoculations of 64Cu labeled F. tularensis subsp. novicida using 2 × 109 CFU/50 μl for the i.t and i.v infections and 2 × 109 CFU/100 μl for the i.g and i.p infections; three mice were inoculated with F. tularensis subsp. novicida for each of the routes. The i.t infection was performed according to the procedure of Rubins et al. (40). Briefly, mice were anesthetized with vaporized isofluorane, suspended in an upright position, and the tongue was extended outwards and towards the side of the mouth with forceps. The inoculum was then delivered by pipette to the back of the throat while covering the nose. This procedure has been shown to prevent swallowing with the vast majority of the inoculum delivered to the trachea and lungs [28, 29]. The i.g infection was performed by using a gavage needle to directly deliver the inoculum into the stomach, while the intraperitoneal infection was accomplished by injecting 100 μl of the inoculum with a 23-gauge needle directly in the right bottom quadrant of the peritoneum. Finally, the intravenous infection was achieved by injecting 50 μl of the inoculum in the mice tail vein with a 28-gauge needle. All animals were imaged for 15–20 min, immediately after infection (labeled 0.25 hr) and at 2 and 20 hrs by using a microPET-R4 rodent scanner (Concorde Microsystems, Knoxville, TN, USA). MicroPET provides a 10 cm by 8 cm field of view with a reconstructed resolution of 2.25 mm in the central 40 mm of the field of view. Images are reconstructed using Fourier re-binning followed by two-dimensional filtered backprojection. Towards the end of our studies, a FLEX Pre-Clinical Platform (Gamma-Medica-Ideas, Northridge, CA) was acquired; this allowed the acquisition of an X-ray computed tomography (CT) image permitting the visualization of the skeletal structure of the mice being analyzed. The FLEX pre-Clinical Platform CT device provides an 8.7 cm field of view, fly acquisition, 2 × 2 binning, and 256 projections, with a spatial resolution of ~100 μm. This procedure was performed for 1 min prior to acquire the microPET image. We made use of this device to image mice that had been previously infected with 64Cu labeled F. tularensis subsp. novicida by the i.n, i.p and i.v routes of infection.
Once the mice had been scanned for the last time point, they were sacrificed and various tissues (as shown in Fig 2) were harvested in order to quantify the amount of 64Cu still present in them by using an automatic well-type gamma counter (γ 8000 Beckman Coulter Fullerton, CA). Finally, some of the tissues harvested from F. tularensis subsp. novicida infected animals were weighed, homogenized in 1× PBS, serially diluted, and plated on TSAcys (Becton Dickinson) to determine CFU/g of tissue in the i.n infected mice. All the experimental procedures were in compliance with federal guidelines and the institutional animal care and use committee.
Isolation of rifampicin resistant Francisella tularensis subsp. novicidafrom the gastrointestinal (GI) tract
In order to test for CFU in the GI tract, several rifampicin resistant F. tularensis subsp. novicida (F. tularensis subsp. novicida Rif+) isolates were generated and tested for virulence. F. tularensis subsp. novicida was grown in a liquid culture in tripticase soy broth (TSB) supplemented with 0.1% L-cysteine with agitation at 37°C overnight (ON). 1 mL of culture was then centrifuged at 13,000 rpm for 4 min; the cell pellet was resuspended in 100 μl of 1× sterile PBS and plated on TSAcys plates containing 200 μg/mL rifampicin. Rif+ bacteria were streaked again on TSAcys plates containing 200 μg/mL rifampicin. Colonies that grew better were then streaked again on TSAcys plates containing 30 μg/mL rifampicin. A Rif+ isolate that was shown to have equivalent virulence to the wt strain (LD50 10) was then used in subsequent studies. For analyses of the GI tract, nine C57BL/6 females 6–8 wk old were anesthetized and i.n inoculated with 2 × 109 CFU/20 μl of F. tularensis subsp. novicida Rif+ bacterial suspension. At serial time points, mice were sacrificed after anesthesia using a mixture of ketamine-xylazine. Lung, stomach, large intestine, small intestine and cecum were collected and organs homogenized; serial dilutions were made and plated on TSAcys plates containing 30 μg/mL rifampicin.
Metabolic labeling of bacteria with 35S
F. tularensis subsp. novicida cultures (50 mL) were incubated at 37°C for 2 hrs with agitation. Bacterial cultures were then centrifuged, supernatants were removed and cell pellets were washed twice with either TSB or LB liquid medium. Bacteria were re-suspended in 50 mL DMEM medium without L-glutamine, L-methionine and L-cysteine (MP Biomedicals) and incubated at 37°C for 15 min with agitation. Subsequently, 230 μCi of 35S were added to each bacterial culture and incubated for an additional 2 hrs. Bacterial cultures were then concentrated and the amount of 35S present in the cell pellets and supernatants were measured to determine efficiency of labeling. Once the bacteria were labeled, serial dilutions were prepared and plated on TSAcys to determine CFU.
Biodistribution of labeled bacteria with 35S
Labeled bacterial pellets were resuspended in 1× PBS to a final concentration of 2 × 109 CFU. Mice (C57BL/6 6–8 wk old) were anesthetized and inoculated i.n with 20–25 μl of the bacterial suspension and inoculated i.t with 50 μl of inoculum. In order to infect mice by the i.t route an endotracheal intubation was performed by using a BioLite small animal intubation system (BioTex, Inc., TX). Briefly, mice were anesthetized by intramuscular injection of ketamine-xylazine as previously described. Subsequently mice were suspended from the incisor wire on the intubation stand, followed by the tracheal intubation with a flexible intravenous catheter over a fiber-optic laser attached to an illuminator that allows a better visualization of the oralpharyngeal cavity. Once inside the trachea, the fiberoptic was drawn out and a 1 mL syringe was plugged to the catheter, and the inculum was delivered to the mouse. This method of inoculation allowed us to avoid any accidental swallowing of the inoculum.
After infection, mice were sacrificed at 20 hrs p.i in the case of i.n infected mice and at 1–2 hrs and 20 hrs post-inoculation in the case of i.t infected mice, various tissues were removed, and the amount of 35S in each tissue was determined by using a multi-purpose scintillation counter (LS6500 Beckman Coulter Fullerton, CA). In addition, several tissues were weighed, homogenized in 1× PBS, serially diluted, and plated to determine CFU/g of tissue in the case of the i.n infected mice.
This work was supported by a NIH AI 59703, PO1 AI057986, NS 35974 and in part by San Antonio Life Science Institute (10003177) and a start-up fund from Department of Radiology, University of Texas Health Science Center at San Antonio. The 64Cu was provided by Washington University Research Resource in Radionuclide Research (R24CA086307).
We thank David Lewis, Sergio Leal and Rick Perez for their assistance in the acquisition of images during the microPET studies. We are also grateful to Dr. Peter H. Dube for his advice and providing the strain of K. pneumoniae used in our studies, Dr. Stephen Mattingly for providing the strain of E. coli, Dr. Carlos J. Orihuela for instructing us on how to perform the intratracheal infection and Dr. Marcel Perret-Gentil DVM for his help with the endotracheal entubation of mice. The authors thank Dr. William Phillips for his helpful discussions regarding this project. We also thank Dr. Jorge Alvarez for his collaboration during the preparation of the figures for this manuscript.
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