888 • JID 2007:195 (15 March) • Bischof et al.
M A J O R A R T I C L E
Characteristics of Spore Germination in a Mouse
Model of Cutaneous Anthrax
Timothy S. Bischof, Beth L. Hahn, and Peter G. Sohnle
Division of Infectious Diseases, Department of Medicine, Medical College of Wisconsin, and Consultant Care Division and Research Service,
Milwaukee Veterans Affairs Medical Center, Milwaukee
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
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.
Cutaneous infection is the most common form of human anthrax, but little is known about
Anthrax is thought to begin when the endospores of
Bacillus anthracis enter the body, are ingested by mac-
rophages, and then germinate intracellularly into veg-
etative bacilli [1, 2]. In experimental inhalational an-
thrax, the alveolar macrophages are important not only
because they initiate germination but also because they
appear to carry the germinating organisms to the re-
gional lymph nodes in the lungs . Further studies
have shown that germination occurs in vesicles derived
from the phagosomal compartment and that the bac-
terial toxin genes and the atxA gene that regulatesthem
are expressed within macrophages after germination
Received 11 August 2006; accepted 29 October 2006; electronically published
2 February 2007.
Potential conflicts of interest: none reported.
Presented in part: 44th Annual Meeting of the Infectious Diseases Society of
America, Toronto, Canada, 12–15 October 2006 (abstract 347).
Financial support: Department of Veterans Affairs (VAMR 8033-17).
Reprints and correspondence: Dr. Peter G. Sohnle, Research Service/151, VA
Medical Center, Milwaukee, WI 53295 (firstname.lastname@example.org).
The Journal of Infectious Diseases
This article is in the public domain, and no copyright is claimed.
. Spore germination is a complicated process that
involves chemical germinants, germination operons of
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 . Therefore, macrophages appear to
be very important in the in vivo process of developing
vegetative bacilli from the initial inoculum of B. an-
thracis 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 in-
jury , others have not noted such a preexistent
lesion . We have recently developed a model of
cutaneous anthrax that uses epicutaneous spore inoc-
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894 • JID 2007:195 (15 March) • Bischof et al.
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 aminoacids
(particularly alanine) and certain purine nucleosides, that pro-
mote 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, thepresentinvestigationshaveshownthatabra-
sion of the skin is necessary to produce significant germination
of B. anthracis spores after epicutaneous application under oc-
clusive dressings. Ontheotherhand,sporegerminationbyitself
is apparently insufficient to initiate progressive infectionsinthe
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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