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Characteristics of Spore Germination in a Mouse Model of Cutaneous Anthrax

  1. Timothy S. Bischof,
  2. Beth L. Hahn and
  3. Peter G. Sohnle
  1. Division of Infectious Diseases, Department of Medicine, Medical College of Wisconsin, and Consultant Care Division and Research Service, Milwaukee Veterans Affairs Medical Center, Milwaukee
  1. Reprints and correspondence: Dr. Peter G. Sohnle, Research Service/151, VA Medical Center, Milwaukee, WI 53295 (psohnle{at}mcw.edu).

  1. Presented in part: 44th Annual Meeting of the Infectious Diseases Society of America, Toronto, Canada, 12–15 October 2006 (abstract 347).

 
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Abstract

Background. Cutaneous infection is the most common form of human anthrax, but little is known about Bacillus anthracis spore germination in these infections.

Methods. We used experimental inoculations of B. anthracis Sterne spores or vegetative bacilli onto intact or abraded mouse flank skin, followed by evaluation of the infections and enumeration of germinating spores and vegetative bacilli.

Results. Bacilli developed from a spore inoculum after application onto abraded, but not intact, skin of the mice. Germination appeared to occur extracellularly at the skin surface before the development of a phagocytic response; in fact, vegetative bacilli were seen after inoculation of the spores on top of a filter that separated them from the host phagocytic cells below. Malachite green staining demonstrated that spores began germinating 1–3 h after inoculation onto abraded skin. Vegetative bacilli were found not to be capable of initiating infection in the absence of cutaneous abrasion.

Conclusions. The results indicate that epidermal damage is required for germination of B. anthracis spores in these infections; even so, spore germination by itself is not sufficient to produce infection of undamaged skin. In contrast to events in experimental inhalational anthrax, spore germination in these cutaneous infections appears to occur extracellularly.

Anthrax is thought to begin when the endospores of Bacillus anthracis enter the body, are ingested by macrophages, and then germinate intracellularly into vegetative bacilli [1, 2]. In experimental inhalational anthrax, the alveolar macrophages are important not only because they initiate germination but also because they appear to carry the germinating organisms to the regional lymph nodes in the lungs [3]. Further studies have shown that germination occurs in vesicles derived from the phagosomal compartment and that the bacterial toxin genes and the atxA gene that regulates them are expressed within macrophages after germination [4]. Spore germination is a complicated process that involves chemical germinants, germination operons of the organism, membrane-permeability changes, and the activation of enzymes that degrade the outer layers of the spore [5–7]. Germination of B. anthracis spores is very rapid in complex media, being virtually complete within 1 h; however, in serum alone this process is inefficient, although greatly enhanced by the presence of macrophages [8]. Therefore, macrophages appear to be very important in the in vivo process of developing vegetative bacilli from the initial inoculum of B. anthracis spores introduced into the host tissues.

The very early events occurring at the skin surface during cutaneous anthrax have been more difficult to study because of a lack of animal models of this type of infection. Cutaneous anthrax is thought to begin with inoculation of spores into a cut or abrasion in the skin [9–11] or, in some cases, through the bite of an insect [11, 12]. Although some reports of cutaneous anthrax have clearly identified the initial cutaneous injury [13], others have not noted such a preexistent lesion [14]. We have recently developed a model of cutaneous anthrax that uses epicutaneous spore inoculation in mice and have shown that skin abrasion markedly promotes the infections in this model system [15]. The present experiments were designed to use the experimental infections to answer the following questions about spore germination at the skin surface: (1) Does germination occur after inoculation onto intact skin? (2) Will inoculation of previously germinated vegetative bacilli produce infections of intact skin? (3) Does germination in these cutaneous infections require ingestion of the spores by macrophages?

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Methods

Organism. The Sterne strain of B. anthracis was used for these experiments. The organism was obtained from the Colorado Serum Company and was cultured on brain-heart infusion agar plates. Sporulation was induced by maintaining the plates at room temperature for 4–7 days after confluent growth at 37°C. When the cultures were found to consist of >90% spores by microscopic examination, the organisms were removed from the plates, washed with distilled water, and treated by heating to 60°C for 30 min to kill remaining vegetative forms. The preparations were then layered onto 58% renografin (Bracco Diagnostics), centrifuged at 3000 g for 30 min, and washed 3 times in saline to remove remaining vegetative forms. The spores were quantitated by both microscopic counts and colony counts to assure a viability of >90%. Spores were stored in saline with 10% glycerin at −20°C. Preparations of vegetative bacilli were produced by culturing the spores in brain-heart infusion broth for 24 h at 37°C. Vegetative bacilli were quantitated microscopically, with bacterial chains in the bacillus inoculum counted as individual organisms. Germination of spores was quantitated using a malachite green stain [16] and was expressed as the percentage of germinated (red) spores over the total counted.

Animals. Mice of the C57BL/6 strain were used for these experiments; these animals are more resistant to B. anthracis than complement-deficient strains such as DBA/2 but are less resistant than strains such as BALB/c [17]. In preliminary experiments, we found that, using the inoculation method discussed below, C57BL/6 mice generate a minimal inflammatory cell response at the skin surface 6 h after inoculation with B. anthracis spores. The mice were obtained from Charles Rivers Laboratories, were of either sex, and were used at 8–14 weeks of age. They were housed in a separate biosafety level 2/3 section of the Veterinary Medical Unit of the Milwaukee Veterans Affairs Medical Center. Some mice were treated with cyclophosphamide (150 mg/kg intraperitoneally 3 days before and 100 mg/kg 1 day before the inoculations) to render them leukopenic, as described elsewhere [18]. In some cases, the skin was abraded by scraping with a scalpel blade until formation of a (nonbloody) glistening layer, representing damage to the epidermal water barrier [15]. The experimental procedures were approved by the appropriate committees at the Milwaukee Veterans Affairs Medical Center and the Medical College of Wisconsin.

Epicutaneous inoculations. On the day before the inoculations, the mice were carefully shaved over their flanks with an electric razor; the next day, the sites were examined for cutaneous defects, and only mice with clear skin were used. An inoculum of 1×107 B. anthracis spores or vegetative bacilli was added in 0.025 mL of saline to 4-mm paper filter discs (GB002; Schleicher & Schuell) placed onto a shaved or abraded area on the left flank of a mouse, with a similar quantity of saline alone added to a disc on the opposite side. Both sites were covered with 1.0-cm2 pieces of plastic sheet (Handi-Wrap; Dow Chemical), which were then taped with Transpore tape and over-wrapped with Nexcare waterproof tape (both from 3M). In some cases, a 1.2-cm piece of 0.45-µm filter (Millipore) was put on top of the abraded skin before placement of the 4-mm spore-containing filter above it, to separate the spore inoculum from the host's phagocytic cells entering the inoculation site from below.

Monitoring of infections. After 1, 3, 6, or 24 h, the occlusive dressings were removed, and filter-touch preparations were made by touching a glass slide with the skin side of the 4-mm filter disc; the slides were then stained using the LeukoStat method (Fisher). Afterward, the inoculation site was washed 3 times with saline-soaked gauze pads. Some of the mice were followed for death or the development of a moribund state (in which case they were killed and recorded as having died on that day). Mice were considered to be moribund if they appeared relatively inactive, had ruffled fur, or seemed to be unable to eat or drink. In other cases, the mice were killed 24 h after removal of the dressings, and skin samples were obtained for routine histological analysis, as discussed below.

Examination of filter-touch preparations. After staining, the slides were examined at ×400 by light microscopy using a 20×20 square ocular micrometer grid to enumerate spores, vegetative bacilli, and host inflammatory cells. A total of 10 random fields were examined for each slide. Each chain of bacilli was counted as a single organism, and from each field the first 2 bacillus chains seen were measured to determine their length in micrometers. Germination of spores was determined by malachite green staining and was expressed as percentage of germinated (red) spores.

Histological analysis. Skin from inoculated or control areas was taken for histological analysis, with paraffin sections being prepared and stained with tissue Gram stains. Each of 10 sections was examined in a blinded fashion for depth of organism penetration (expressed in micrometers) into the dermis by use of the 20×20 grid system discussed above. In addition, the outlet of each hair follicle infundibulum seen on the entire section was examined for the presence of vegetative bacilli, with the data expressed as percentage of hair follicle outlets infected.

Statistics. Results were expressed as the number of mice dying or becoming moribund per the total number tested; comparisons were made between groups using the x2 test. Data were also expressed as the number of days until death or a moribund state, with the values given as means ± SDs. Spore germination was expressed as the numbers of vegetative bacilli or spores seen per square micron on the slides. Ratios of bacilli to remaining spores were determined; therefore, the data reflect both spore germination and proliferation of the resulting vegetative bacilli in the epicutaneous fluids during these periods, rather than quantitative spore germination alone. The latter value was assessed at early time points using malachite green stains and was expressed as the percentage of spores counted. Values were given as means ± SDs, and comparisons were made using the unpaired t test. Significance was taken as P=.05.

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Results

Generation of vegetative bacilli from the spore inoculum. Examination of filter-touch preparations from the inoculated, abraded skin of nonleukopenic mice demonstrated an increase in vegetative bacilli that occurred primarily between 3 and 6 h after inoculation, as shown in table 1. Although the total numbers of spores seen decreased over this period, the numbers and percentages of bacilli increased, reaching 21.8% of the bacterial forms seen at 6 h. The length of the bacillus chains increased only modestly during this time. The numbers of germinated spores indicated by malachite green staining increased between 1 and 3 h. Very few host inflammatory cells were seen on the preparations, with a ratio of 8583 bacteria to each host cell at 6 h. In contrast, after inoculation of spores onto intact skin, little evidence of germination could be seen; percentages of bacilli remained about the same as in the spore inoculum, and no host cells were seen. As shown in figure 1A and 1B, significant numbers of vegetative bacilli were seen 6 h after inoculation of the spore preparations onto abraded skin but not intact skin. Germination of the spore inoculum was also seen by malachite staining after inoculation onto abraded skin at 1 h, as shown in figure 1C.

Figure 1.
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Figure 1.

Bacillus anthracis organisms in filter-touch preparations from inoculated skin of C57BL/6 mice at 6 h after inoculation of spores onto unabraded skin (A), 6 h after inoculation onto abraded skin (B), 1 h after inoculation onto abraded skin (C), and 24 h after inoculation above a 0.45-µm filter on abraded skin (D). Also shown are the spore inoculum (E) and the inoculum after germination in brain-heart infusion broth (F). Touch preparations were made from the underside of the filter disc onto which the organisms had been inoculated and were then stained with malachite green stain (photomicrographs were taken at an original magnification of ×1000 under oil immersion; bars represent 20 µm). Note that there are germinated bacilli in the 6-h preparations from abraded skin but not in those from unabraded skin. Also, there are significant numbers of germinating spores (stained red) from abraded skin at 1 h and of germinated bacilli from preparations with the inoculation done above a 0.45-µm filter.

Table 1.
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Table 1.

Generation of vegetative bacilli of Bacillus anthracis after epicutaneous inoculation of spores onto abraded or intact skin in nonleukopenic mice.

Leukopenic mice were studied in this system to further decrease the likelihood that germination was occurring after ingestion of the spores by host phagocytic cells (table 2). Again, very few host cells were seen on the preparations, with similar ratios of bacteria to host cells as in the nonleukopenic mice. Generation of vegetative bacilli from the spore inoculum was again seen in preparations from the leukopenic mice at 6 h, with ratios of vegetative bacilli to spores of 25.1%. After inoculation of spores onto intact skin in these mice, germination was not evident even after 24 h. To further reduce the likelihood that the spores were germinating after being ingested by host phagocytic cells, some inoculations in leukopenic mice were done with a 0.45-µm filter separating the spores from the skin surface; in this case, the spores would be germinating in the cell-free fluids that diffused upward through the second filter. Again, significant generation of bacilli was seen, at 24 h in this case, reaching values of ∼56% bacillus forms. Figure 1D shows the vegetative bacilli that had developed 24 h after inoculation of spores on top of the second filter.

Table 2.
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Table 2.

Generation of vegetative bacilli of Bacillus anthracis after epicutaneous inoculation of spores onto abraded or intact skin in leukopenic (treated with cyclophosphamide before inoculation) mice.

Progression of infection after inoculation of spores or vegetative bacilli. Previous studies in this system have shown that skin abrasion is usually required for rapid progression of infection, even in leukopenic animals. As shown in table 3, all of the cyclophosphamide-treated mice that received inoculations onto abraded skin with either spores or vegetative bacilli died, with a mean time to death of ∼2.5 days. On the other hand, most survived after inoculation onto intact skin, and, for those that did die, the time to death was longer (∼8 days). Histological examination revealed striking differences as well, with organisms not being seen after inoculation of either spores or vegetative bacilli onto intact skin, compared with prominent invasion of dermis and hair follicle outlets after inoculation onto abraded skin.

Table 3.
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Table 3.

Progression of infection in cyclophosphamide-treated C57BL/6 mice after epicutaneous inoculation of Bacillus anthracis spores or bacilli onto intact or abraded skin.

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Discussion

The present series of experiments was performed to determine (1) whether B. anthracis spore germination could occur at the surface of intact skin or if abrasion was required; (2) whether inoculation of vegetative bacilli could produce progressive infections of intact skin; and (3) whether ingestion by host phagocytic cells was required for germination of spores in this system. The data clearly show that significant germination of inoculated spores required that the skin be abraded beforehand. With unabraded skin, there appeared to be minimal progression of the infection or germination of the spores into vegetative bacilli. Inoculation of the latter onto intact skin also did not result in progressive infections, indicating that germination of spores is necessary, but apparently not sufficient, to produce infections of intact skin. Finally, the results also demonstrate that, in contrast to findings of previous investigations of other experimental B. anthracis infections, germination of spores inoculated onto abraded skin did not require ingestion by macrophages.

The present investigations used the Sterne strain of B. anthracis, which is toxigenic but unencapsulated. Although this strain is less infectious than encapsulated ones, it appears to germinate [19] and to survive within peritoneal macrophages in a similar fashion [20]. There is a germination operon called gerX present on one of the B. anthracis virulence plasmids, but it is contained in the 40-kb toxin-encoding region of pX01 that is present in the Sterne strain [21]. The capsule of fully virulent B. anthracis strains appears to be very important in protecting the bacilli from phagocytic cell attack, but it probably does not affect spore germination significantly.

The findings of the present study contrast with those reported for experimental inhalational anthrax, in which spore germination has clearly been shown to require ingestion by macrophages [2–4]. In early studies, large numbers of bacilli were seen in infected tissues after intradermal injections in monkeys [22] and subcutaneous injections in mice [23]; however, in these infections the bacilli could have resulted from spore ingestion by macrophages, proliferation within the cells, and later escape into the extracellular fluids. In our system, we were able to isolate the spores in cell-free fluids by use of a double-filter system and then clearly show that extracellular germination did occur. The rate may be slower than that observed in in vitro germination experiments but is likely rapid enough to produce progressive infections. Also, access to macrophages is probably different for inhalational and cutaneous inoculation routes. Spores or other small particles entering the lower respiratory tract are likely to encounter alveolar macrophages lining the mucous membranes at that site; however, neutrophils constitute most of the cells present in the tissue fluids of abraded skin, and they take some time to accumulate there [24, 25]. Therefore, contact with macrophages is less likely to occur in abraded skin, and the spores appear to germinate without them. There is apparently an adequate supply of germinants present in the extracellular fluids of the damaged skin.

It is not clear that the bacilli we see in these surface preparations are the ones that eventually produce systemic infections in this model system. Small numbers of ungerminated spores could enter damaged skin, be ingested by macrophages, and then initiate distant and/or systemic infections through the sequence of events envisioned for inhalational anthrax. In our previous studies using this model system, we found that very susceptible mice of the DBA/2 strain did die after receiving an inoculation on intact skin, although the time to death was longer and organisms could rarely be located in inoculated, unabraded skin by histological examination [15]. In any event, our present results show that extracellular spore germination does occur and likely produces the large number of bacilli seen at the surface of the infected skin. However, these findings do not prove that the organisms seen are important in producing fatal infections after epicutaneous inoculation.

It is interesting that incubation of B. anthracis spores on intact skin under occlusive dressings does not result in spore germination. Occlusion has been shown to promote cutaneous infections with some organisms, such as Candida albicans and Staphylococcus aureus [26, 27]. Therefore, the occluded skin is capable of being infected, indicating that its moisture content and other characteristics allow for microbial growth. It is likely that the fluids present at the skin surface under occlusion are lacking the necessary chemical germinants, such as amino acids (particularly alanine) and certain purine nucleosides, that promote germination of this organism [5, 6]. It is also possible that occlusion causes some physical change at the skin surface that interferes with germination.

In summary, the present investigations have shown that abrasion of the skin is necessary to produce significant germination of B. anthracis spores after epicutaneous application under occlusive dressings. On the other hand, spore germination by itself is apparently insufficient to initiate progressive infections in the model system used. The data also show that, in contrast to results obtained for experimental inhalational anthrax, most of the spore germination seen in these experimental cutaneous infections occurs extracellularly.

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Footnotes

  • Potential conflicts of interest: none reported.

  • Financial support: Department of Veterans Affairs (VAMR 8033-17).

  • Received August 11, 2006.
  • Accepted October 29, 2006.
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