Characteristics of Spore Germination in a Mouse Model of Cutaneous Anthrax

Division of Infectious Diseases, Department of Medicine, Medical College of Wisconsin, Milwaukee, WI, 53295, USA.
The Journal of Infectious Diseases (Impact Factor: 6). 04/2007; 195(6):888-94. DOI: 10.1086/511824
Source: PubMed


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

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    • "On the other hand, these organisms do not require an initial period of germination as does B. anthracis and therefore could possibly invade damaged skin before an initial neutrophilic response would clear them. Germination of B. anthracis spores is quite rapid in appropriate media and we have found that this process is well underway within 1–3 h after inoculation in this model system (Bischof et al. 2007b). However, it may be that even a short interval like this is enough to shift the kinetics of B. anthracis infection towards clearance by the host inflammatory cells, particularly for non-encapsulated strains. "
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    ABSTRACT: Skin window procedures in humans have shown rapid accumulation of neutrophils into the exuded fluids above abraded skin. The present study was undertaken to determine if similar epicutaneous neutrophil accumulation might explain the extreme resistance of HRS/J mice, both hairless (hr/hr) and haired (hr/+), to experimental cutaneous Bacillus anthracis Sterne infections on abraded skin. In this study, very early (6 h) biopsies demonstrated a lack of bacilli in skin from the HRS/J hr/hr mice, indicating that the organisms never did invade in these animals as opposed to early skin entry and then efficient clearance by host responses in the tissues. Touch preparations of either the inoculation filter or the skin surface revealed more inflammatory cells, fewer bacilli, and a higher percentage of cell-associated bacilli in the HRS/J hr/hr mice than in comparator strains. In the HRS/J mice, cyclophosphamide treatment or separation of inoculated spores from the inflammatory infiltrates by a second filter below both produced marked increases in the number of bacilli observed. Examination of inoculation filter specimens demonstrated ingestion of spores and bacilli by neutrophils inside the filter at 6 h after inoculation. These findings suggest that an early and vigorous inflammatory cell infiltrate in HRS/J mice attacks the inoculated organisms above the skin surface and does not allow them to invade the tissues below.
    International Journal of Experimental Pathology 07/2008; 89(3):180-7. DOI:10.1111/j.1365-2613.2008.00584.x · 2.17 Impact Factor
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    ABSTRACT: The Food and Drug Administration’s Bacteriological Analytical Manual recommends two enumeration methods for Bacillus spp.: 1) standard plating method using mannitol-egg yolk-polymyxin (MYP) agar and 2) most probable number (MPN) method with tryptic soy broth supplemented with 0.1% polymyxin sulfate. Preliminary research evaluated three inoculum preparation methods using EZ-Spore™ B. cereus pellets. Two methods involved EZ-Spore™ B. cereus pellets that were dissolved in deionized (DI) water, grown in brain heart infusion broth with manganese sulfate, and then heated to produce spores. The third inoculum preparation method of dissolving EZ-Spore™ pellets only in DI water was the most efficient due to 100% spores being present in the inoculum. Preliminary research also determined that MPN method recovered greater (p<0.05) B. cereus populations than MYP method in inoculated ultra-high temperature pasteurized skim and 2% milk. The objective of the main study was to compare the MYP and MPN method for detection and enumeration of B. cereus in raw and high-temperature-short-time pasteurized skim, 2%, and whole milk at 4 °C for 96 h. Milk samples were inoculated with B. cereus EZ-Spores™ dissolved in DI water and sampled at 0, 48, and 96 h after inoculation. No differences (p>0.05) were observed among sampling times so data was pooled for overall mean values for each treatment. The overall B. cereus population mean of pooled sampling times for MPN method (2.59 log CFU/mL) was greater (p<0.05) than MYP plating method (1.89 log CFU/mL). B. cereus populations ranged from 3.40 log CFU/mL to 2.40 log CFU/mL for inoculated milk treatments for MYP and MPN methods, which is well below the necessary level for toxin production. Even though MPN method enumerated more B. cereus, the MYP method should be used by industry for enumeration of B. cereus due to its ease of use and rapid turnover time (2 d compared to 5 d with MPN). However, MPN method should be used for validation research due to its greater populations recovered. EZ-Spore™ B. cereus pellets were found to be an acceptable spore inoculum for validation research because the inoculum consists of 100% spores and does not contain vegetative cells. USDA/CREES Master of Science Masters Food Science Institute- Animal Sciences and Industry Kelly J. K. Getty
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    ABSTRACT: We sought to visualize the site of Bacillus anthracis spore germination in vivo. For that purpose, we constructed a reporter plasmid with the lux operon under control of the spore small acid-soluble protein B (sspB) promoter. In B. subtilis, sspB-driven synthesis of luciferase during sporulation results in incorporation of the enzyme in spores. We observed that B. anthracis Sterne transformed with our sspBp::lux plasmid was only luminescent during germination. In contrast, Sterne transformed with a similarly constructed plasmid with lux expression under control of the protective antigen promoter displayed luminescence only during vegetative growth. We then infected A/J mice intranasally with spores that harbored the germination reporter. Mice were monitored for up to 14 days with the Xenogen In Vivo Imaging System. While luminescence only became evident in live animals at 18 h, dissection after sacrificing infected mice at earlier time points revealed luminescence in lung tissue at 30 min after intranasal infection. Microscopic histochemical and immunofluorescence studies on luminescent lung sections and imprints revealed that macrophages were the first cells in contact with the B. anthracis spores. By 6 h after infection, polymorphonuclear leukocytes with intracellular spores were evident in the alveolar spaces. After 24 h, few free spores were observed in the alveolar spaces; most of the spores detected by immunofluorescence were in the cytoplasm of interstitial macrophages. In contrast, mediastinal lymph nodes remained nonluminescent throughout the infection. We conclude that in this animal system, the primary site of B. anthracis spore germination is the lungs.
    Infection and immunity 04/2008; 76(3):1036-47. DOI:10.1128/IAI.00985-07 · 3.73 Impact Factor
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