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Port d'entry of Bacillus anthracis

The symptoms in anthrax depend on the type of infection and can take anywhere from 1 day to more than 2 months to appear. All types of anthrax have the potential, if untreated, to spread throughout the body and cause severe illness and even death.

Four forms of human anthrax disease are recognized based on their portal of entry.

•Cutaneous, the most common form (95%), causes a localized, inflammatory, black, necrotic lesion.

•Inhalation, a rare but highly fatal form, is characterized by flu like symptoms, chest discomfort, diaphoresis, and body aches.

•Gastrointestinal, a rare but also fatal (causes death to 25%) type, results from ingestion of spores. Symptoms include: fever and chills, swelling of neck, painful swallowing, hoarseness, nausea and vomiting (especially bloody vomiting), diarrhea, flushing and red eyes, and swelling of abdomen.

•Injection, symptoms are similar to those of cutaneous anthrax, but injection anthrax can spread throughout the body faster and can be harder to recognize and treat compared to cutaneous anthrax.

Bacillus anthracis Has Two Independent Bottlenecks That Are Dependent on the Portal of Entry in an Intranasal Model of Inhalational Infection

David E. Lowe, Stephen M. C. Ernst, Christine Zito, Jason Ya, and Ian J. Glomski

S. R. Blanke, Editor

Author information ► Article notes ► Copyright and License information ►

This article has been cited by other articles in PMC.

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ABSTRACT

Bacillus anthracis can cause inhalational anthrax. Murine inhalational B. anthracis infections have two portals of entry, the nasal mucosa-associated lymphoid tissue (NALT) and the lumen of the lungs. Analysis of the dissemination from these sites is hindered because infections are asynchronous and asymptomatic until the hosts near death. To further understand and compare how B. anthracis disseminates from these two different environments, clonal analysis was employed using a library of equally virulent DNA-tagged clones of a luminescent Sterne strain. Luminescence was used to determine the origin of the infection and monitor the dissemination in vivo. The number of clones and their proportions in the portals of entry, lymph nodes draining the portals, and kidneys were analyzed. Clonal analysis indicated a bottleneck for both portals of entry, yet the extent and location of the reduction in represented clones differed between the routes. In NALT-based infections, all clones were found to germinate in the NALT, but they underwent a bottleneck as the infection spread to the cervical lymph node. However, lung-based infections underwent a bottleneck in a focal region of growth within the lung lumen and did not need to spread through the mediastinal lymph nodes to cause a systemic infection. Further, the average number of clones found in the kidney and the rate at which genetic drift was affecting the disseminated populations were significantly higher in lung-based infections. Collectively, the data suggested that differences in the host environment alter dissemination of B. anthracis depending on the site of initial colonization and growth.

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INTRODUCTION

Bacillus anthracis is a Gram-positive sporulating bacterium that is the causative agent of anthrax. Anthrax can initiate via cutaneous, gastrointestinal, and inhalation inoculation, with the last two causing significant mortality. The spore is the infectious particle and is notable for being metabolically dormant and resistant to host-mediated killing and common disinfectants (1). Once within the host, spores germinate and become vegetative bacilli, which cause the disease (2–4). The bacilli are then able to replicate to high levels and disseminate through the host with the aid of an antiphagocytic capsule and production of lethal toxin and edema toxin (5–8). The combination of bacteremia and toxicemia leads to host death. After the disease kills the host, the bacilli sense the decreasing nutrient levels, sporulate within the carcass, and return to the soil, where they reside until they infect the next host (1).

The high mortality rate associated with inhalational anthrax is in part due to the bacterium's success in dissemination through the host and a lack of diagnostic clinical signs of an infection until late in infection. Early research with B. anthracis suggested that inhalational infections disseminate via resident phagocytes acting as a “Trojan horse” (9, 10). In the Trojan horse model, inhaled spores are deposited in the alveoli and phagocytosed by resident lung phagocytes. Either in transit or within the mediastinal lymph nodes (MLN), the spore germinates, escapes from the phagocyte, and replicates in the MLN; the bacteria eventually disseminate to the bloodstream via efferent lymphatics (10–12). However, experimentally testing this model for early events in pathogenesis is challenging in animal models due to the fulminant nature of the infection and its asynchronous dissemination (13, 14).

Bioluminescent imaging (BLI) of B. anthracis strains with luminescent reporters enabled researchers to dissect early events in pathogenesis that previously were difficult to study. These BLI studies identified two novel sites of establishment and dissemination of infection (15–18). In the upper respiratory passage, bacteria were first observed to replicate and disseminate from the nasal mucosa-associated lymphoid tissue (NALT) (13, 15, 16, 19). A second portal of entry was identified in the lower respiratory passage in mice, where germination and outgrowth occurred directly in the alveolar lumen (15, 17). The finding of germination and dissemination from the lung was a surprising result, since B. anthracis does not generally provoke a classic bronchopneumonia and there is evidence that the lung is refractory to germination (10, 14–17, 20). However, induction of germination in the lungs or preexisting damage to the lungs has been previously shown to permit spore germination and outgrowth in the lungs (16, 21, 22). While several studies have sought to understand the Trojan horse model of dissemination, fewer studies have investigated the dissemination from NALT- and lung-based portals of entry.

The dissemination patterns of several bacterial pathogens have been analyzed through clonal analysis to clarify portals of entry and understand how the host alters the pathogen's population dynamics (23–27). Clonal analysis consists of creating a library of isogenic clones that vary only by a signature tag, e.g., an antibiotic marker or recombinant DNA sequence. The host is then infected with an equally proportioned mixture of clones that are isolated from tissues of interest at particular time points to determine if and how the proportions of clones vary during dissemination. These analyses have been used to determine the sequence of dissemination as well as to identify population bottlenecks for several pathogens (23–25, 27). Such bottlenecks increase the rate of genetic drift and allelic fixation, leading to a drastic reduction of genomic diversity at the population level, which can be measured by effective population (28). Effective population differs from population size because it measures the intensity or rate of genetic drift on a population, rather than the number of individuals. While previous research has demonstrated that NALT-based bottlenecks occur as the bacilli disseminate, it is unknown whether a bottleneck occurs in lung-based infections (26). If bottlenecks were to occur in both sites, they could be compared to inform researchers on how the site of establishment influences the ensuing infection.

The research described here compared dissemination of B. anthracis from the lungs and dissemination from the NALT using a library of luminescent DNA-tagged clones of the B. anthracis Sterne strain. The simultaneous use of BLI with clonal analysis was essential to dissect how spatial and temporal events alter the bacterial population dynamics in vivo. Lung-based infections initiate when a small number of spores germinate in a focal region and spread rapidly through the host. Since lung infections initiate from a small, distinct focal area and rapidly disseminate through the host, it is difficult to envision such an event being captured by classic pathological or diagnostic methods with significant frequency. Compared to NALT-based infections, lung-based infections undergo bottlenecks at the site of germination and have a significantly higher effective population. This suggests that B. anthracis is able to establish infections in the unique microenvironments of the NALT or lung lumen within the host respiratory system and that these locations impart distinct characteristics to the resultant disseminated bacterial population that reflect how the infection unfolds within the host.

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MATERIALS AND METHODS

Bacterial strains, culture procedures, and media.

Bacillus anthracis Sterne strain 7702 (pX01⁺, pX02⁻) was used for construction of clonal mutants (see below) and was obtained from BEI (Manassas, VA). A luminescent B. anthracis 7702 strain, BIG23, was constructed by integrating the plasmid pIG6-19 into pX01, a kind gift from Michele Mock (18). The pIG6-19 construct is a derivative of the pAT113 conjugative suicide plasmid, which allowed for insertion of the plasmid upstream of the endogenous paglocus without interrupting the gene. This strain contains the luxABCDE operon from Photorhabdus luminescens under the control of the protective antigen (PA) promoter, leading to light expression in vivo. BIG23 was used as the base strain for all mouse infections. B. anthracis was grown in brain heart infusion (BHI) broth (Becton Dickson, MD) supplemented with 0.5% glycerol at 37°C unless otherwise noted. Spores were produced by plating on nutrient broth-yeast extract (NBY) medium with antibiotics when necessary, incubated at 30°C for 10 days, and then harvested and purified on an Omnipaq gradient (350 mg of iodine/ml) (GE Healthcare, NJ), similar to previously described methods (16, 18, 29). Escherichia colistrains were either α-select (Bioline, MA) or dcm- and dam-deficient GM119 (dcm-6 dam-3) (30) and were grown in LB broth with appropriate antibiotics when necessary (Becton Dickson, MD). All strains and plasmids are described in Table 1.

Table 1

Strains and plasmids used in this study

Construction of B. anthracis clonal mutants.

Unique DNA-tagged clones were constructed by inserting recombinant DNA from a progressive 100-bp DNA step ladder (Promega, WI) into the chromosomal B. anthracis eag gene. DNA tags were inserted into B. anthracis by allelic exchange with pSE02 and pCZ03, which are modified from the pHY304 temperature-sensitive allelic exchange vector, kindly provided by S. Leppla (Table 1) (31). To facilitate allelic exchange in B. anthracis, a 2-kb section of the eag gene was constructed by splice-by-overlap (SOE) PCR using primers FIG4, RIG4, FIG5, and RIG5, which also introduced a BamHI site (see Table S1 in the supplemental material). The modified eagfragment was then inserted into pHY304 to generate pIG13. pIG13 then had two different multiple-cloning sites (MCS) inserted into the recombinant BamHI site in eag to allow for greater flexibility for cloning in DNA tags. The pSE02 vector contains a short DNA linker with an EcoRV site and a SalI site flanked by BamHI sites, whereas pCZ03 has the linker flanked by EcoRI, SpeI, and NotI sites. The pSE02 vector was constructed by SOE PCR where the overlap region contained an EcoRV, SpeI, SalI, and BamHI sites within the eag gene. The two SOE regions were amplified from pIG13 using RDL22 and F SOE linker and FIG8 and R SOE linker and were spliced together by PCR using the FIG8 and RDL22 primers. The pCZ03 vector was generated by inserting the linker into the MCS of pGEM-T Easy (Promega, WI) and then isolating the MCS containing pGEM-T Easy plasmid by EagI digestion and blunting using the NEB Quick Blunt kit (NEB, MA). Next, pIG13 was digested with BamHI blunted using the NEB Quick Blunt kit, and the MCS was inserted into pIG13. The fragments from the DNA ladder were blunted with the NEB Quick Blunt kit and ligated into the EcoRV site in the eag region of homology of the MCS of either pCZ03 or pSE02 using T4 ligase (NEB, MA).

After pSE02 and pCZ03 were ligated with fragments from a progressive 100-bp step ladder, they were transformed into α-select chemically competent E. coli (Bioline, MA). Transformants were then screened with M13 primers to ensure that the fragments were ligated into the plasmid. The verified plasmid was then passaged through GM119 to remove DNA methylation and purified before electroporation into 7702 as previously described (32). Mutants were passaged at 42°C twice to select for merodiploid insertion of the temperature-sensitive plasmid carrying erythromycin (Erm) resistance into eag and then repeatedly passaged at 37°C until bacteria became Erm sensitive, indicating that allelic exchange had occurred. PCR was used to identify bacteria with the DNA ladder insert and confirm the presence of pX01. Lastly, to make the tagged strains luminescent, pIG6-19 was conjugated into tagged 7702 strains and selected on BHI plates supplemented with 5 μg/ml Erm and 60 μg/ml polymyxin B (33). Strains were sporulated and purified as described above.

Determination of growth rates of B. anthracis clones.

Strains were grown overnight in BHI medium at 37°C and back diluted to an optical density at 600 nm of ∼0.005 in 50 ml of fresh BHI medium. The optical density was then measured every hour for 8 h and then at 12 h and 24 h. This was repeated three times independently.

Infection and dissection of mice.

Mouse manipulations and husbandry were performed in accordance with the guidelines set forth in the Guide for the Care and Use of Laboratory Animals of the National Research Council (34). All techniques and animal husbandry were approved by the University of Virginia Animal Care and Use Committee (protocol 3671) and were designed to minimize distress and pain for the animals. All mice were 6- to 12-week-old female A/J mice (Jackson Laboratory, ME) and were housed and/or bred using specific-pathogen-free rearing procedures at the University of Virginia. Infections were performed by anesthetizing mice with methoxyflurane (Metafane; Matrix Scientific, SC) in a nose cone and then delivering spores by intranasal instillation as previously described (16). The spore inoculum varied from 5 × 10⁶ CFU/mouse to 5 × 10⁷ CFU/mouse. In preliminary studies, some mice would be infected with 1.4 × 10⁸ CFU/mouse to develop lung infections; however, lung infections were found to occur at lower doses. The infections were monitored by anesthetizing mice with a 2.5% (vol/vol) isoflurane-oxygen mix with an XGI-8 gas anesthesia system (Perkin-Elmer, MA), and light production was followed in the host with either the IVIS100 or the IVIS Spectrum (Perkin-Elmer, MA) until light was detected in the NALT or ventral thoracic cavity after 1 min of exposure with large binning. Light production was measured and quantified using LivingImage software (version 3.2; Perkin-Elmer, MA). Any mouse with light in the kidneys was considered to have a disseminated infection and was euthanized.

After euthanasia, the NALT, cervical lymph nodes (CLN), mediastinal lymph nodes, lungs, and kidney were removed and homogenized on ice using Dounce homogenizers and ice-cold 1× phosphate-buffered saline (PBS) as previously described (35). The homogenates were divided in half, with one half heated to 65°C for 20 min to kill vegetative bacilli such that the remaining CFU represent spores. Both fractions were serially diluted and spread on LB plates with 5 μg/ml Erm to enumerate CFU. A portion of each sample was also used to inoculate 3 ml BHI broth with 5 μg/ml Erm and grown overnight at 37°C. If any bacterial growth occurred in overnight cultures, an aliquot was brought to 10% (vol/vol) glycerol and stored as frozen samples in order to confirm clonal proportions or increase the number of colonies analyzed at a later date, if necessary.

Determination of clonal identity in organs.

Clonal proportions were established for each organ that contained at least 30 CFU. Organs that contained fewer than 30 CFU were not used for further study, as small numbers of bacteria are subject to greater skewing of clonal proportions due to chance. For each organ, 48 colonies were individually lysed using the HotSHOT technique to extract DNA (36). Using 5 μl of the lysed colony DNA sample, clonal identity was established by PCR amplification of the eag region flanking the DNA tag using Agilent Paq5000 polymerase (Agilent, CA) and primers FDL26 and RDL26 to yield unique fragment sizes for each clone.

Probability modeling to determine the size of the clonal library.

The probability of two exact clones passing through a bottleneck was determined by using the hypergeometric formula h(x; N, n, k) = (_kC_x)(_N−kC_n−x)/(_NC_n), where N is the inoculum delivered in CFU, x is the number of identical clones found in the kidney, k is the number of CFU from a particular clone in the inoculum, and n is the number of clones that pass through the bottleneck (bottleneck size). The probability was then calculated for a given bottleneck size for a given number of clones in the library.

Statistical analysis.

Student t tests, one-way and two-way analyses of variance (ANOVAs), Sidak's multiple-comparison posttest, and Tukey multiple-comparison posttests were performed using GraphPad Prism (version 5; GraphPad Software, CA). Growth rates were statistically compared by nonlinear regression and comparing F values from each strain. Fisher's exact tests were performed using the R project for statistical programming version 2.15.0 (The R Foundation for Statistical Programming, Vienna, Austria).

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RESULTS

Bacterial burdens are similar when B. anthracis disseminates from NALT and lung portals of entry.

BLI analysis of infections identified the NALT and the lung lumen as sites of initial germination and growth for B. anthracis (15–17). Because BLI analysis indicated that the bacteria had different dissemination patterns in NALT- and lung-based infections, it was hypothesized that these routes of dissemination may have different levels of bacterial burden in similar lymph nodes. To test this hypothesis, A/J mice were infected intranasally with BIG23, a luminescent derivative of Sterne 7702. BIG23 has the genes necessary for light production under the control of the protective antigen promoter and produces light when vegetative bacteria reside under the toxin-inducing conditions found throughout the host (18). This construct is capable of having luminescence detected both in vitro and in vivo as early as 2 h after germination and has no differences in its ability to produce PA relative to that of the parental 7702 (see Fig. S1 in the supplemental material) (16, 17). Intranasally infected mice developed either NALT- or lung-based infections (Fig. 1). Concurrent dual-portal infections were observed in only 2 mice out of all 30 of our infections; therefore, mice were analyzed only if there was luminescence exclusively in the NALT or the lungs to avoid confounding each location's contribution to the infectious process. After either a NALT- or lung-based infection was detected, the cervical lymph nodes (CLN), lungs, MLN, and kidneys were homogenized and the total bacteria and spore populations were analyzed. Since spores are more resistant to heat than bacilli, any CFU obtained from the heated homogenate is considered to have been derived from an ungerminated spore. Thus, the proportion of the bacterial population consisting of spores can be isolated by heating an aliquot of the tissue homogenate and comparing it to an unheated aliquot.

Fig 1

Inhalational B. anthracis infections cause similar bacterial burdens in most organs. (A) Dorsal and ventral black-and-white photographs of an A/J mouse with pseudocolor overlay of luminescence intensity. The mouse was infected with 4.6 × 10⁶ spores ...

A NALT infection was indicated when the mouse had measurable luminescence above background solely in the nasal passages (Fig. 1A). Luminescence from NALT-based infections were detected at between 2 and 7 days postinfection (dpi), with the median at 3 dpi (see Fig. S2A in the supplemental material). In agreement with previous publications, these mice had luminescence detected in the NALT, in the CLN, and, if the infections were allowed to progress, in the kidney (data not shown). The kidneys were chosen as a proxy to indicate systemically disseminated bacilli since the kidneys reach a high bacterial burden, lack spores, and are the first major organs to become luminescent after dissemination (15). Mice that had infections disseminate from the NALT did not show luminescent foci in their lungs when dissected (data not shown) (15, 17). Bacteria in the lymph nodes and kidneys were almost exclusively heat sensitive, as all CFU from heat-resistant spores were below the limit of detection (Fig. 1B). Enumeration of CFU in the lungs demonstrated that spores were present in the lungs even when a NALT-based infection occurred. However, the heat-resistant bacteria comprised only ∼10% of the bacterial population when mice with luminescence in the CLN were dissected (1.1 × 10⁶ CFU total bacteria [standard deviation {SD}, ±9.9 × 10⁵ CFU] versus 1.1 × 10⁵ heat-resistant CFU [SD, ±1.6 × 10⁵ CFU]).

A lung infection was defined as when light was detected solely through the ventral thorax, as previously described (Fig. 1C) (16, 17). Luminescence from lung-based infections were detected at between 3 and 8 days dpi, with the median at 5 dpi (see Fig. S2A in the supplemental material). A correlation between the CFU in the organs and the luminescence in NALT- and lung-based infection indicated that a higher bacterial burden in the lungs was needed to be detected (see Fig. S2B in the supplemental material). Upon closer examination after dissection, the luminescence in lung infections was found to be confined to a single focal region in the lungs and was regularly detected in the draining MLN. If the lung infection was allowed to progress to later stages, the kidneys also displayed measurable luminescence, in agreement with previous publications (data not shown) (15–17). Similarly, in lung-based infections the MLN and the kidneys contained high numbers of heat-sensitive CFU, and all heat-resistant CFU were below the limit of detection (Fig. 1D). Additionally, the lungs in lung-based infections contained 12-fold more heat-sensitive bacteria than heat-resistant bacteria. The NALT was not analyzed, since luminescence was not detected when a lung infection occurred, suggesting that it lacked vegetative bacilli. The CLN, however, typically had very small amounts of CFU present. In some cases, there was bacterial growth in the LB broth that was inoculated with the CLN homogenates. This suggested that bacteria were present but were too few to enumerate directly from the tissue.

The lung-based bottleneck is distinct from the NALT-based bottleneck.

B. anthracis undergoes a bottleneck as it disseminates from the NALT to the draining CLN (26). A population bottleneck occurs when there is a dramatic population decrease that leads to a nonbiased reduction in genetic variability relative to that of the original population. BLI analysis has revealed that B. anthracis has two portals of entry for inhalational anthrax (15). Given that the respiratory mucosa in the alveoli is distinct from the NALT and that there were differences in the bacterial burden in the CLN, we tested to see if lung-based infections also passed through a bottleneck. Probability modeling determined that 5 clones was the optimum number of DNA-tagged clones to allow for the greatest resolution of the bottleneck without causing unnecessary experimental burden (see Fig. S3 in the supplemental material). DNA-tagged strains were then tested for growth rate defects and in vivo virulence to ensure that B. anthracis was undergoing a bottleneck and not selection against a less virulent strain. A comparison of the in vitro growth rates showed no significant growth defects between any of the tagged strains and the parent BIG23 strain (see Fig. S4A in the supplemental material) (nonlinear regression and comparison of F values, P = 0.9991). Additionally, there were no statistically significant differences between the frequencies of clones in the kidneys and those delivered to the mouse, supporting the conclusion that no single clone had a competitive advantage over the others and that passage through a bottleneck is random (see Fig. S4B in the supplemental material) (Fisher's exact test, P = 0.521).

In agreement with previous publications, NALT-based infections were found to have no difference in the clonal numbers or proportions between the NALT and the initial inoculum early in infection (see Fig. S5 in the supplemental material). However, NALT-based infections undergo a significant reduction in the number of clones between the NALT and CLN, as all organs other than the NALT contained an average of 1 clone (Fig. 2A) (one-way ANOVA with Tukey multiple-comparison posttest, P < 0.0001) (26). Mice with lung-based infections had a less pronounced decrease in the number of clones represented as the bacilli disseminated from the lung lumen (Fig. 2B). Organs were dissected, and clonal identities were determined when light was detected in the ventral thoracic cavity but before luminescence was detected in the kidneys. At this stage of infection, there were too few CFU in the CLN for clonal analysis, and the CLN therefore were not analyzed. This allowed for the determination of the average number of clones in each organ before the bacteria returned to the lungs in large numbers from the circulatory system, which would confound analysis. The number of clones represented in the whole lungs was not significantly different from the number of clones delivered in the initial inoculum (Fig. 2B) (one-way ANOVA with Tukey multiple-comparison posttest, P > 0.05). The average number of clones in the MLN, which drain bacteria from the lungs, ranged from 1 to 4 clones, with a significantly lower average of 2 clones represented, compared to the initial inoculum's 5 clones (Fig. 2B) (one-way ANOVA with Tukey multiple-comparison posttest, P < 0.0001). In areas where heat-resistant spores were never found, i.e., the kidneys, there was an average of 2 different clones, which was significantly lower than in the inoculum (Fig. 2B) (one-way ANOVA with Tukey multiple-comparison posttest, P < 0.0001).

Fig 2

Mice with lung-based bottlenecks have a higher number of clones in the lungs and MLN. (A) Number of different clones represented in each organ of mice dissected after a NALT-based infection had progressed to the draining CLN or kidneys. Each symbol represents ...

To test if there were differences between the bottlenecks in NALT-based infections and lung-based infections, the numbers of different clones found in the lungs, MLN, and kidneys were compared. Mice with lung infections did not have bacteria present in their CLN until they were moribund and were thus not compared. In contrast to those with NALT-based infections, mice with lung-based infections had a significantly higher number of clones represented in the lungs and MLN (Fig. 2C) (two-way ANOVA with Sidak's multiple-comparison test, P < 0.01 and P < 0.0001).

Lung-based infections have disseminated clonal populations that do not match populations in the MLN and the whole lung, whereas NALT-based infections have disseminated populations that resemble those in the CLN.

In order to determine whether the clone or clones that pass through the bottleneck are the founders of the disseminated population in the host, the clonal proportions in each organ were analyzed. BLI was used to synchronize the infections to when light was first detected in the kidneys. Organs were then removed from the infected mice, and the proportions and identities of clones were compared between the organs to determine dissemination patterns. NALT-based infections were found to be almost completely comprised of 1 clone in all organs, and the clone found in the CLN had the same identity as that in the kidneys (Fig. 3A) (Fisher's exact test, P > 0.05). The lungs in mouse 1 had all clones present; however, the lung homogenates were further tested to determine if this population represented ungerminated spores in the lungs. The percent germination was calculated by comparing heated and unheated lung tissue homogenates. There was a small difference in CFU between the heated (spores only) and unheated (bacilli and spores) lung homogenates (1.2 × 10⁵ spore CFU/lung and 1.8 × 10⁵ total bacterial CFU/lung), implying that most bacteria in the lungs remained as spores. Additionally, there was no statistical difference between the clonal proportions from the heat-resistant bacteria in the lung homogenates and total bacterial lung homogenates for mouse 1, further suggesting that the lungs contained only spores (Fig. 3A) (Fisher's exact test, P > 0.05). This suggests that the increased number of different clones in the lungs in mouse 1 is due to it being dissected before the disseminated bacteria returned to the lungs after dissemination from the NALT and CLN.

Fig 3

Clonal proportions in NALT-based infections are similar between organs, whereas lung-based infections show greater diversity. (A) Clonal proportions in the CLN, lungs, and kidneys of mice that had a NALT infection that disseminated at least to the draining ...

In agreement with the data in Fig. 2, the clonal proportions in lungs and MLN present in lung-based infections were more diverse than those in NALT-based infections. Out of six mice, only mice E and F had a single clone constitute the majority of the whole lung, i.e., 82% and 52%, respectively (Fig. 3B) (Fisher's exact test, P > 0.05). Importantly, mice E and F were dissected later than the other four mice, meaning that the bacilli had a longer time to replicate and disseminate through the mouse, which could allow recolonization of the lung from the periphery. The other 4 mice had no more than 35% of the clonal proportions containing a single clone. Therefore, if a bottleneck occurred, clonal analysis was not sufficient to detect lung bottlenecks when the whole lungs were analyzed in a single homogenate. Similarly, only one mouse out of six had no statistical difference between the kidneys and MLN, potentially suggesting the bacilli disseminated without using the lung lymphatics (Fig. 3C) (Fisher's exact test, P < 0.0001).

The focal regions of bacterial growth arise from a few clones that replicate in a small area, and these founders go on to disseminate through the host.

The population structure of the disseminated bacteria from lung-based infections suggested that a bottleneck occurred by the time the bacteria were systemically disseminated, but whole-lung analysis was potentially hindered by the high spore burden in the lungs (Fig. 1D and ​and2B).2B). Since previous publications have noted that lung germination and outgrowth occur in distinct focal regions in the lung, it was investigated whether these regions contained a higher proportion of bacilli than spores (15, 17). Moreover, the high degree of luminescence within this region suggests a higher proportion of bacilli, which would reduce the likelihood of sampling spores and allow for a greater ability to determine if a bottleneck occurred in the lungs. After luminescence was detected in the ventral thoracic cavity, the lungs were removed and separated into luminescent and nonluminescent regions (Fig. 4A). The focal luminescent region had a greater percentage of bacilli than the nonluminescent regions as determined by heat sensitivity (Fig. 4B) (Student's t test, P < 0.0001). Germination in the nonluminescent region averaged 39% (SD, 15.9%), indicating that the majority of the bacteria were still in spore form. However, an average of 86% (SD, 18.9%) of the bacteria in the luminescent focal region were heat sensitive in addition to expressing a luminescent reporter that produces light only in metabolically active vegetative bacilli. This is in contrast to the NALT, where luminescence is detected throughout the anterior portion of the palate (see Fig. S6 in the supplemental material). If a bottleneck occurred in the focal region, this could suggest that a few spores in the focal region germinated, replicated, and eventually disseminated. Alternatively, it is possible that all 5 clones in the library germinated in a small area, but only the first few clones that reached the draining lymph node or bloodstream were the founders of the disseminated population. To differentiate between these two scenarios, the clonal diversities of the focal regions were analyzed separately from the rest of the lungs and compared to the clonal diversity of a similarly sized nonluminescent area of the lung. Mice were first dissected when there was clearly luminescence present in the thoracic cavity, which corresponded to >3.5 × 10⁶ CFU/g in the focal region. In these mice, all 5 mice had significant differences in clonal diversity between the initial inoculum and the focal region (Fig. 5A) (Fisher's exact test, P < 0.0001). Two mice, mouse 4 and mouse 5, had the same clonal population comprise a majority in the focal region as well as in the nonluminescent region. These mice had among the greatest amount of luminescence and bacterial burden (>9.5 × 10⁶ CFU/g) of the group. A second group of mice was dissected at the earliest sign of luminescence, which corresponded to <3.5 × 10⁶ CFU/g, to determine how early in infection the bottleneck could be detected. In this group, there were no significant differences between the initial inoculum and the focal region in 3 out of 4 mice (Fig. 5B). Most nonluminescent regions were significantly different from the focal region, but no single subpopulation was greater than 42%. This suggests that slight shifts in clonal proportions occur as spores transit from the nasal passage to the lungs. Germination varied in the focal regions of these mice, with mouse 6 having only 38% germination, mouse 9 having 91% germination, and mouse 7 and mouse 8 having >95% germination. To ensure that the clonality in the focal region is not an artifact due to the intranasal delivery of the spores to the lungs, the heated homogenates from the focal regions were analyzed. All clonal populations were present in the focal regions as spores, but most mice showed statistically significant shifts from the focal regions (Fig. 5C). However, averaging the clonal proportions in all heated homogenates of the focal regions showed no difference from the initial inoculum.

Fig 4

Lung-based infections occur in distinct focal regions that are composed mainly of vegetative bacilli. (A) Photograph of a dissected mouse lung with luminescent focal region with a pseudocolor overlay representing luminescence intensity as indicated on ...

Fig 5

The luminescent focal region is the site of the bottleneck in lung-based B. anthracis infections. (A and B) The percentages of clones in the focal region were compared to the initial inoculum and a nonluminescent region outside the focal region when there ...

The dominant clone in the lung focal region is the founder of the disseminated population.

To test whether the dominant clone in the focal region goes on to disseminate through the host, the clones in the kidneys were compared to the clones in the focal region. A comparison between the subpopulations in the focal regions and kidneys in five out of eight mice did not show a significant difference, suggesting that the dominant population in the lung went on to disseminate through the mouse (Fig. 6A) (Fisher's exact test, P > 0.05). The focal regions in mice 6 and 7 had a significant difference compared to the kidneys, but these were dissected early after luminescence was detected in the lungs. A bottleneck, however, occurred between the focal region and the kidneys. Mouse 3 had two populations comprise 76% of the focal region and the same two populations comprise 98% of the kidney, but the two populations were found to be significantly different from each other (Fig. 6A). The MLN, however, had unique clonal proportions compared to those in the kidneys and focal regions of five out of six mice, suggesting that these populations did not contribute to the disseminated population (Fig. 6B).

Fig 6

Clonal proportions in the lung focal regions resemble those in the kidney but not the MLN. The percentages of clones in the focal region (A) or the MLN (B) were compared to the percentages of clones in the kidneys. Each bar represents the indicated organ ...

The lung-based bottleneck allows more clones to pass through and has a lower effective population.

In order to determine if the lung-based bottleneck allowed more clones to pass through the bottleneck, additional mice were infected to increase the number of lung infections analyzed. The average number of clones represented in the disseminated population was measured by analyzing the average number of different clones in the kidneys from NALT-based and lung-based infections. The average number of different clones in the kidneys was significantly greater in lung-based infections than in NALT-based infections (Fig. 7) (Student's t test, P = 0.014). The effective population (N_e) could then be calculated from the number of clones in the disseminated population and used to compare the sizes of the bottlenecks that the bacilli pass through from different portals of entries. The effective population (N_e) size is an idealized population that has the same rate of genetic drift as the actual population size and allows for a quantitative comparison of bottlenecks between portals of entry. One method of determining N_e is to analyze the number of offspring between nonoverlapping generations. If there is a decrease in population due to a bottleneck, then the effective population will decrease in proportion to the stringency of the bottleneck. In a B. anthracis infection, the generations occur between the inoculation with spores and the death of the host. As such, one can calculate the effective population by determining the number of clones in the disseminated population, i.e., the kidney clonal population. The effective population was significantly higher in lung-based infections (N_e, 1.65) than in NALT-based infections (N_e, 0.72) (Student's t test, P = 0.014) (the N_e for no bottleneck was 5).

Fig 7

Mice with lung-based bottlenecks have a higher number of clones in the kidneys. The mean numbers of different clones represented in the kidneys of mice with NALT-based (closed circles; n = 12) and lung-based (closed squares; n = 16) were compared. Each ...

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DISCUSSION

Our work provides insight on the ability of B. anthracis to colonize and disseminate from unique microenvironments in the host respiratory tract by combining BLI technology with clonal analysis. Utilization of the luminescent reporter under the control of the PA promoter allowed for detection of the vegetative bacilli early and throughout infection (15, 16). Potential drawbacks to this reporter are that (i) the bacteria would not be detected if there were areas where toxin production does not occur and (ii) there is a slight delay of approximately 2 h between luminescent detection and germination (16). All mice had B. anthracis pass through a bottleneck regardless of where the infection initiated. Clonal analysis of lung-based infections suggested that a bottleneck occurred in the lumen of the lung, possibly as a few bacteria germinated in the lungs. These bacteria then rapidly disseminate from the lungs to the bloodstream and other organs. Further, the clone population in most MLN was statistically different from those in the respective kidneys and focal growth in the lung. NALT-based infections, however, passed through a bottleneck between the NALT and the CLN. In contrast to the lung-based infections, the CLN always had the same tagged clone as the NALT and kidneys. Analysis of the kidneys from mice with lung-based infections showed a greater mean number of clones and a higher effective population than in mice with NALT-based infections. Overall, these data suggest that B. anthracis disseminates differently from the two inhalational portals of entry and that some routes of inhalational dissemination allow greater numbers of bacilli to disseminate; thus, the portal of entry defines the characteristics of the subsequent disseminated infection.

The experiments performed in this study were done using the BSL-2 Sterne strain of B. anthracis in the A/J mouse model, which has advantages and disadvantages. Since the Sterne strain lacks the pX02 plasmid encoding the antiphagocytic capsule, the experiments can be performed without the difficulties of biosafety level 3 (BSL3) facilities, providing a more economical model of study. Additionally, several studies have noted key similarities between the A/J mouse model of anthrax and larger animal models (13, 15, 37–39). These include a similar dissemination pattern and a high level of bacteremia within the host (13, 15, 37). As such, mice are useful models to analyze colonization and early steps in pathogenesis (37, 40). A disadvantage, however, is the lack of capsule, which is thought to account for pathological differences found during the bacteremic stages of infection. Acapsular strains have limited splenic colonization and do not cause vasculitis and mediastinitis (40). Therefore, comparisons between the Sterne-A/J model and human anthrax must be considered carefully. It is important to note that the focus of this study was to investigate the dissemination of B. anthracis in inhalational infections, not to model the effects of late-stage anthrax.

In our studies, A/J mice were given spores by intranasal instillation to cause inhalational infections. A concern with the intranasal model is effective delivery of spores to the lungs (38). In our hands, mouse lungs would contain about 5% of the inoculum at the time of dissection. However, when the clonal proportions were analyzed both within the focal region and in the nonluminescent regions, all clonal populations were present, and when averaged they were not significantly different from the inoculum. This suggests that while our mice had fewer spores reach the lungs than previously published numbers reported in studies using aerosol models, all clonal identities were capable of reaching the focal regions (37). As such, the bottlenecks detected in the focal region are unlikely to occur due to uneven delivery of the spores to the lungs due to intranasal instillation.

The high spore burden relative to vegetative bacteria within the lung from the initial inoculum was likely the factor that obscured the detection of a bottleneck at early time points. If an organ has a large and random population of spores, there is a greater chance of sampling ungerminated spores of a dissimilar clonal subpopulation. While heat treatment of homogenates can differentiate spores from total bacteria, there is no current methodology for isolating only the vegetative bacilli for clonal analysis, which, if possible, would permit earlier analysis of the bottleneck.

To circumvent the high spore burden in the lungs, luminescent focal regions and nonluminescent regions were separated to (i) attempt to isolate a region with a higher proportion of bacilli to spores and (ii) determine the extent to which the bacilli can spread through lung tissue without hematogenous dissemination. The luminescent focal areas were found to comprise mainly vegetative bacilli, whereas the rest of the lung tissue had the majority of bacteria in spore form (Fig. 4A and ​andB).B). While previous studies found that germination and outgrowth occur in the lungs, it is interesting to note that the bacilli do not migrate far from the focal region without prior systemic dissemination through the host. This suggests that the site of lung germination as previously reported by Sanz et al. may represent the earliest moments of the focal infection formation in the lung (17).

The luminescent focal regions were examined at various levels of bacterial burdens to determine when the bottlenecks could be detected. When focal regions were analyzed with ≥3.5 × 10⁶ CFU/g, there were large increases in only 1 or 2 clones. However, a bottleneck could not be detected when mice were dissected at the earliest observation of luminescence. This suggests that detection of the lung bottleneck is dependent on both germination and bacterial replication and that the disseminated population arises from a small number of spores. Interestingly, bacilli could be found in the kidneys before the bottleneck could be detected in the lungs; this suggests rapid dissemination from the lungs after germination. Mice were also dissected when there was a high level of luminescence (>9.5 × 10⁶ CFU/g in the focal regions) and were found to have increased amounts of the disseminated population in the nonluminescent regions. This suggests that the nonluminescent areas of the lung have the disseminated population return secondarily, rather than primarily spreading through the lung.

In lung-based infections, bacterial dissemination from the portal of entry to the draining lymph nodes is sufficient but not necessary for dissemination. A comparison between the MLN and the kidneys found that six out of 12 mice had the greatest clonal proportion in the MLN in common with the kidney. Lethal toxin is also capable of increasing epithelial and endothelial permeability, from which one could speculate that bacilli are able to bypass the lymphatic routes in lung-based infection and access the vasculature directly (41, 42). However, these experiments alone do not provide sufficient evidence to definitively support this hypothesis, since the earliest events in the lung-based infections cannot be analyzed by these methods. It is also possible that the MLN comprise a population of bacteria distinct from that in the focal region. If this is the case, either these bacteria do not disseminate through the host or they contribute to the disseminated population to a lesser extent, since the focal regions with dominant clones do not statistically vary from those in the kidneys. Interestingly, other routes of dissemination in murine anthrax, such as subcutaneous and gastrointestinal infections, have also shown dissemination through the lymph node to be required (16, 18). We speculate that such a route could be possible, given that both exotoxin components, edema toxin and lethal toxin, are capable of increasing vascular and alveolar epithelial permeability (41, 42). However, future studies will need to be performed in order to determine if and to what degree the mouse lung epithelium becomes permeabilized. Epithelial layers have also been identified as being capable of spore uptake and to allow transcytosis of spores (43). Therefore, another possibility is that spores are directly internalized by the lung epithelium to then transit directly to the circulatory system and thereby bypass the lymph nodes. In contrast to the case for mice, pulmonary infections of rabbits have shown that germination occurs not in the lungs but in the lung-associated lymph nodes (44). The difference in germination and dissemination may be due to differences in the animal models, method of spore instillation, and/or bacterial strains. Given the paucity of human cases of inhalation anthrax and the difficulty of early detection of infection, it is not clear how early events in these animal models resemble anthrax infection in humans. Nonetheless, it is clear that the site of germination and replication plays a role in how the bacteria disseminate.

Germination and outgrowth from the lungs is not without controversy. The potential of lungs to act as primary sites of infection was first suggested by preliminary work which found bacterial replication and damage in the lung and bronchi (45, 46). It is worth noting that several of these studies used experimental animals in which the lungs were compromised by an unrelated previous infection or experimental manipulation (22, 47, 48). Similarly, a majority of male victims in the Sverdlosk anthrax outbreak were moderate to heavy smokers and their most common occupation was welder, factors which increase the risk of lung damage and disease (49). Therefore, previous damage to the lungs may skew infections to germinate and disseminate from the lungs rather than to disseminate from the NALT or lymphatics. Conversely, there is support for lymphatic dissemination from the lungs, since large-scale germination does not occur in the lungs and there is no sign of bronchopneumonia (9, 10, 14, 20, 37, 50). A central caveat to previous pathological analyses and determination of bacterial burden is that the infection was analyzed either at defined time points or after death and in particular organs. Mice in the current study had similar bacterial burdens in most organs at the time of dissection regardless of where the infection initiated. This suggests that determining dissemination patterns by quantifying bacterial loads in organs may not be conclusive by itself. In a number of animal models, lung lesions have been observed upon postmortem analysis, but there were no real-time assays to track the infection in the host (22, 47, 48, 51–55). Therefore, caution should be used when concluding where an infection began, given that there is still little known about the early events in pathogenesis in vivo or how differences in delivery, host, and environment could alter the portal of entry.

Our results provide a possible reconciliation between conflicting reports on B. anthracis infections that initiate and later disseminate from the lung and previous observations of initiation and dissemination from the MLN. This is not to say that MLN-based infections do not occur; rather, it is to argue that lung-based infections can occur and may not be easily detected. Clonal analysis suggests that a small number of spores germinate in a small, discrete location in the lung. The lungs of these mice contained bacteria in spore form throughout most of the lungs, except for the focal area where a small number of spores germinated and replicated. Rather than spreading primarily through the lungs, the bacilli rapidly spread either through the lymphatics or directly to the bloodstream. Therefore, even in our mice with a lung infection, the lungs on the whole were mostly refractory to germination, and overt lung damage was limited to a portion of a lobe that could have easily gone undetected in past studies that did not utilize BLI. Therefore, when the lungs were studied as a complete unit, rather than focusing on an important subregion that contained the growth as we have achieved, it would have been difficult to detect vegetative growth from this small area above the background of CFU from ungerminated spores within the rest of the lung tissues. Moreover, a postmortem dissection of the animal without the BLI data would have greater difficulty discerning if the lung lesions occurred prior to dissemination.

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SUPPLEMENTARY MATERIAL

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ACKNOWLEDGMENTS

We acknowledge Doug Taylor for thoughtful discussions and comments on population genetics. Kevin Janes assisted with concepts related to probability modeling. We also thank Heath Damron, Zach P. Weiner, and Margot K. Williams for thoughtful discussion and comments on the manuscript.

This work was supported by grant 5R03AI090205 from the NIAID, and David Lowe was supported by grant 5T35AI060528.

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FOOTNOTES

Published ahead of print 16 September 2013

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00484-13.

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REFERENCES

1. Mock M, Fouet A. 2001. Anthrax. Annu. Rev. Microbiol. 55:647–671 [PubMed]

2. Ireland JA, Hanna PC. 2002. Amino acid- and purine ribonucleoside-induced germination of Bacillus anthracis DeltaSterne endospores: gerS mediates responses to aromatic ring structures. J. Bacteriol.184:1296–1303 [PMC free article] [PubMed]

3. Titball RW, Manchee RJ. 1987. Factors affecting the germination of spores of Bacillus anthracis. J. Appl. Bacteriol. 62:269–273 [PubMed]

4. Corre JP, Piris-Gimenez A, Moya-Nilges M, Jouvion G, Glomski IJ, Mock M, Sirard JC, Goossens PL. 2013. In vivo germination of Bacillus anthracis spores during murine cutaneous infection. J. Infect. Dis.207:450–457 [PubMed]

5. Green BD, Battisti L, Koehler TM, Thorne CB, Ivins BE. 1985. Demonstration of a capsule plasmid in Bacillus anthracis. Infect. Immun. 49:291–297 [PMC free article] [PubMed]

6. Makino S, Uchida I, Terakado N, Sasakawa C, Yoshikawa M. 1989. Molecular characterization and protein analysis of the cap region, which is essential for encapsulation in Bacillus anthracis. J. Bacteriol.171:722–730 [PMC free article] [PubMed]

7. Smith H, Stanley JL. 1962. Purification of the third factor of anthrax toxin. J. Gen. Microbiol. 29:517–521 [PubMed]

8. Stanley JL, Sargeant K, Smith H. 1960. Purification of factors I and II of the anthrax toxin produced in vivo. J. Gen. Microbiol. 22:206–218 [PubMed]

9. Barnes JM. 1947. The development of anthrax following the administration of spores by inhalation. Br. J. Exp. Pathol. 28:385–394

10. Ross JM. 1957. The pathogenesis of anthrax following the administration of spores by the respiratory route. J. Pathol. Bacteriol. 73:485–494

11. Guidi-Rontani C. 2002. The alveolar macrophage: the Trojan horse of Bacillus anthracis. Trends Microbiol. 10:405–409 [PubMed]

12. Lincoln RE, Hodges DR, Klein F, Mahlandt BG, Jones WI, Jr, Haines BW, Rhian MA, Walker JS. 1965. Role of the lymphatics in the pathogenesis of anthrax. J. Infect. Dis. 115:481–494 [PubMed]

13. Loving CL, Khurana T, Osorio M, Lee GM, Kelly VK, Stibitz S, Merkel TJ. 2009. Role of anthrax toxins in dissemination, disease progression, and induction of protective adaptive immunity in the mouse aerosol challenge model. Infect. Immun. 77:255–265 [PMC free article] [PubMed]

14. Swartz MN. 2001. Recognition and management of anthrax—an update. N. Engl. J. Med. 345:1621–1626 [PubMed]

15. Glomski IJ, Dumetz F, Jouvion G, Huerre MR, Mock M, Goossens PL. 2008. Inhaled non-capsulated Bacillus anthracis in A/J. mice: nasopharynx and alveolar space as dual portals of entry, delayed dissemination, and specific organ targeting. Microbes Infect. 10:1398–1404 [PubMed]

16. Glomski IJ, Piris-Gimenez A, Huerre M, Mock M, Goossens PL. 2007. Primary involvement of pharynx and peyer's patch in inhalational and intestinal anthrax. PLoS Pathog.3:e76.10.1371/journal.ppat.0030076 [PMC free article] [PubMed] [Cross Ref]

17. Sanz P, Teel LD, Alem F, Carvalho HM, Darnell SC, O'Brien AD. 2008. Detection of Bacillus anthracis spore germination in vivo by bioluminescence imaging. Infect. Immun. 76:1036–1047 [PMC free article][PubMed]

18. Weiner ZP, Boyer AE, Gallegos-Candela M, Cardani AN, Barr JR, Glomski IJ. 2012. Debridement increases survival in a mouse model of subcutaneous anthrax. PLoS One7:e30201.10.1371/journal.pone.0030201 [PMC free article] [PubMed] [Cross Ref]

19. Dumetz F, Jouvion G, Khun H, Glomski IJ, Corre JP, Rougeaux C, Tang WJ, Mock M, Huerre M, Goossens PL. 2011. Noninvasive imaging technologies reveal edema toxin as a key virulence factor in anthrax. Am. J. Pathol. 178:2523–2535 [PMC free article] [PubMed]

20. Guidi-Rontani C, Weber-Levy M, Labruyere E, Mock M. 1999. Germination of Bacillus anthracis spores within alveolar macrophages. Mol. Microbiol. 31:9–17 [PubMed]

21. Cote CK, Bozue J, Twenhafel N, Welkos SL. 2009. Effects of altering the germination potential of Bacillus anthracis spores by exogenous means in a mouse model. J. Med. Microbiol. 58:816–825[PubMed]

22. Gleiser CA, Berdjis CC, Hartman HA, Gochenour WS. 1963. Pathology of experimental respiratory anthrax in Macaca mulatta. Br. J. Exp. Pathol. 44:416–426 [PMC free article] [PubMed]

23. Bakardjiev AI, Stacy BA, Portnoy DA. 2005. Growth of Listeria monocytogenes in the guinea pig placenta and role of cell-to-cell spread in fetal infection. J. Infect. Dis. 191:1889–1897 [PubMed]

24. Bakardjiev AI, Theriot JA, Portnoy DA. 2006. Listeria monocytogenes traffics from maternal organs to the placenta and back. PLoS Pathog. 2:e66.10.1371/journal.ppat.0020066 [PMC free article] [PubMed][Cross Ref]

25. Barnes PD, Bergman MA, Mecsas J, Isberg RR. 2006. Yersinia pseudotuberculosis disseminates directly from a replicating bacterial pool in the intestine. J. Exp. Med. 203:1591–1601 [PMC free article][PubMed]

26. Plaut RD, Kelly VK, Lee GM, Stibitz S, Merkel TJ. 2012. Dissemination bottleneck in a murine model of inhalational anthrax. Infect. Immun. 80:3189–3193 [PMC free article] [PubMed]

27. Schwartz DJ, Chen SL, Hultgren SJ, Seed PC. 2011. Population dynamics and niche distribution of uropathogenic Escherichia coli during acute and chronic urinary tract infection. Infect. Immun. 79:4250–4259 [PMC free article] [PubMed]

28. Charlesworth B. 2009. Fundamental concepts in genetics: effective population size and patterns of molecular evolution and variation. Nat. Rev. Genet. 10:195–205 [PubMed]

29. Sylvestre P, Couture-Tosi E, Mock M. 2005. Contribution of ExsFA and ExsFB proteins to the localization of BclA on the spore surface and to the stability of the bacillus anthracis exosporium. J. Bacteriol. 187:5122–5128 [PMC free article] [PubMed]

30. Arraj JA, Marinus MG. 1983. Phenotypic reversal in dam mutants of Escherichia coli K-12 by a recombinant plasmid containing the dam+ gene. J. Bacteriol. 153:562–565 [PMC free article] [PubMed]

31. Chaffin DO, Beres SB, Yim HH, Rubens CE. 2000. The serotype of type Ia and III group B streptococci is determined by the polymerase gene within the polycistronic capsule operon. J. Bacteriol.182:4466–4477 [PMC free article] [PubMed]

32. Koehler TM, Dai Z, Kaufman-Yarbray M. 1994. Regulation of the Bacillus anthracis protective antigen gene: CO₂ and a trans-acting element activate transcription from one of two promoters. J. Bacteriol.176:586–595 [PMC free article] [PubMed]

33. Janes BK, Stibitz S. 2006. Routine markerless gene replacement in Bacillus anthracis. Infect. Immun.74:1949–1953 [PMC free article] [PubMed]

34. National Research Council 2011. Guide for the care and use of laboratory animals, 8th ed. National Academies Press, Washington, DC

35. Cote CK, Van Rooijen N, Welkos SL. 2006. Roles of macrophages and neutrophils in the early host response to Bacillus anthracis spores in a mouse model of infection. Infect. Immun. 74:469–480 [PMC free article] [PubMed]

36. Truett GE, Heeger P, Mynatt RL, Truett AA, Walker JA, Warman ML. 2000. Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). Biotechniques 29:52–54[PubMed]

37. Loving CL, Kennett M, Lee GM, Grippe VK, Merkel TJ. 2007. Murine aerosol challenge model of anthrax. Infect. Immun. 75:2689–2698 [PMC free article] [PubMed]

38. Lyons CR, Lovchik J, Hutt J, Lipscomb MF, Wang E, Heninger S, Berliba L, Garrison K. 2004. Murine model of pulmonary anthrax: kinetics of dissemination, histopathology, and mouse strain susceptibility. Infect. Immun. 72:4801–4809 [PMC free article] [PubMed]

39. Welkos SL, Keener TJ, Gibbs PH. 1986. Differences in susceptibility of inbred mice to Bacillus anthracis. Infect. Immun. 51:795–800 [PMC free article] [PubMed]

40. Twenhafel NA. 2010. Pathology of inhalational anthrax animal models. Vet. Pathol. 47:819–830[PubMed]

41. Langer M, Duggan ES, Booth JL, Patel VI, Zander RA, Silasi-Mansat R, Ramani V, Veres TZ, Prenzler F, Sewald K, Williams DM, Coggeshall KM, Awasthi S, Lupu F, Burian D, Ballard JD, Braun A, Metcalf JP. 2012. Bacillus anthracis lethal toxin reduces human alveolar epithelial barrier function. Infect. Immun.80:4374–4387 [PMC free article] [PubMed]

42. Warfel JM, Steele AD, D'Agnillo F. 2005. Anthrax lethal toxin induces endothelial barrier dysfunction. Am. J. Pathol. 166:1871–1881 [PMC free article] [PubMed]

43. Russell BH, Vasan R, Keene DR, Koehler TM, Xu Y. 2008. Potential dissemination of Bacillus anthracis utilizing human lung epithelial cells. Cell. Microbiol. 10:945–957 [PubMed]

44. Lovchik J, Drysdale M, Koehler TM, Hutt JA, Lyons CR. 2012. Expression of either lethal toxin or edema toxin by Bacillus anthracis is sufficient for virulence in a rabbit model of inhalational anthrax. Infect. Immun. 80:2414–2425 [PMC free article] [PubMed]

45. Buchner H. 1888. Special conditions for the penetration of infective pathogens through the intact pulmonary surface. Arch. Hyg. 8:217–245

46. Fraenkel E. 1925. Inhalation anthrax. Virchow's Archiv. 254:363–378

47. Velu H, Soulie P, Bellocq B. 1943. Pulmonary anthrax and chlorine poisoning: minimum infectious dose of spores. C. R. Soc. Biol. 137:160–161

48. Velu H, Soulie P, Bellocq B. 1943. Experimental pulmonary anthrax of mice: initial bacteremia or septicemia. C. R. Soc. Biol. 137:159–160

49. Meselson M, Guillemin J, Hugh-Jones M, Langmuir A, Popova I, Shelokov A, Yampolskaya O. 1994.The Sverdlovsk anthrax outbreak of 1979. Science 266:1202–1208 [PubMed]

50. Young GA, Jr, Zelle MR, Lincoln RE. 1946. Respiratory pathogenicity of Bacillus anthracis spores; methods of study and observations on pathogenesis. J. Infect. Dis. 79:233–246 [PubMed]

51. Twenhafel NA, Leffel E, Pitt ML. 2007. Pathology of inhalational anthrax infection in the African green monkey. Vet. Pathol. 44:716–721 [PubMed]

52. Gleiser CA, Gochenour WS, Jr, Ward MK. 1968. Pulmonary lesions in dogs and pigs exposed to a cloud of anthrax spores. J. Comp. Pathol. 78:445–448 [PubMed]

53. Zaucha GM, Pitt LM, Estep J, Ivins BE, Friedlander AM. 1998. The pathology of experimental anthrax in rabbits exposed by inhalation and subcutaneous inoculation. Arch. Pathol. Lab. Med. 122:982–992[PubMed]

54. Fritz DL, Jaax NK, Lawrence WB, Davis KJ, Pitt ML, Ezzell JW, Friedlander AM. 1995. Pathology of experimental inhalation anthrax in the rhesus monkey. Lab. Invest. 73:691–702 [PubMed]

55. Abramova FA, Grinberg LM, Yampolskaya OV, Walker DH. 1993. Pathology of inhalational anthrax in 42 cases from the Sverdlovsk outbreak of 1979. Proc. Natl. Acad. Sci. U. S. A. 90:2291–2294 [PMC free article] [PubMed]