Systemic dissemination and cutaneous damage in a mouse model of
staphylococcal skin infections
Beth L. Hahna,b, Charles C. Onunkwoa,b, Christopher J. Wattsa,b, Peter G. Sohnlea,b,*
aDivision of Infectious Diseases, Department of Medicine, Medical College of Wisconsin, Milwaukee, WI 53226, USA
bConsultant Care Division and Research Service, VA Medical Center, Milwaukee, WI 53295, USA
a r t i c l e i n f o
Received 21 January 2009
Received in revised form
11 April 2009
Accepted 17 April 2009
Available online 3 May 2009
a b s t r a c t
Serious staphylococcal infections frequently begin in the skin. The present study used a mouse model of
such infections to evaluate the ability of Staphylococcus aureus to disseminate from the skin and to
determine if cutaneous damage from the infections was required for dissemination. The mice were
inoculated with S. aureus onto flank skin prepared by a tape-stripping method that caused minimal
disruption of the epidermal keratinocyte layers. After these inoculations the staphylococci were found to
disseminate to the spleen and kidneys of almost all animals within 6 h. Induction of leucopenia did not
affect this process. Cutaneous damage was prominent in these experimental infections and included loss
of the epidermis, neutrophil infiltration into the epidermis, and complete necrosis of the dermis. The
latter also occurred in cyclophosphamide-treated animals, indicating that the organisms themselves and
not the host inflammatory responses were responsible. Dermal necrosis did not develop until 48 h after
inoculation, a time by which dissemination had already occurred. Therefore, in this mouse model system
S. aureus is capable of penetrating the epidermal keratinocyte layers and disseminating rapidly after
inoculation; the experimental infections do produce significant dermal damage, but the latter develops
after dissemination has already taken place.
Published by Elsevier Ltd.
Staphylococcus aureus is a major human pathogen and is
particularlyadept at invading and infecting the skin. Staphylococcal
infections have assumed increased importance recently because of
the emergence of community associated methicillin resistant
S. aureus strains; of the infections caused by these organisms, the
majority involve skin and soft tissue , and some of these can
produce severe necrotizingfasciitis . Systemic dissemination and
metastatic infections are also common with staphylococcal infec-
tions. Nasal carriage of S. aureus probably represents the ultimate
source of the organisms causing bacteremia and it has been spec-
ulated that they may reach the bloodstream after colonizing
impaired skin . However, we know relatively little about the
relationship between staphylococcal skin infections and bacterial
dissemination from this site.
A number of experimental models of staphylococcal skin
infections have been described and these show that the organisms
can readily invade the epidermis and dermis to produce localized
infections [4–11]. These models have generally not been used to
study the relationship between staphylococcal skin infections and
systemic dissemination, although in one system positive blood
cultures were found after skin infection in leukopenic (but not
nonleukopenic) mice [9,10]. On the other hand, staphylococci can
cause a variety of pathologic changes in the skin, including impe-
tigo, furuncles, subcutaneous abscesses, scalded skin syndrome,
and necrotizing fasciitis [2,12]; it seems reasonable that blood
vessels in the damaged dermis of S. aureus infected skin might be
compromised, thereby allowing entry of the organisms into the
We have recently developed an experimental model of staphy-
lococcal skin infections in mice and in the present study have used
this system to investigate the relationship between cutaneous
damage and systemic dissemination of the organisms. The exper-
imental infection system employed skin preparation by a tape-
stripping method that caused minimal disruption of the epidermal
keratinocyte layers, along with cultures of spleen and kidney as
a sensitive method to demonstrate systemic dissemination of the
organisms. The goals of these studies were to determine the
incidence and timing of bacterial dissemination after inoculation of
* Correspondence at: Peter G. Sohnle, Research Service/151, VA Medical Center,
Milwaukee, WI 53295, USA. Tel.: þ1 414 384 2000x42878; fax: þ1 414 383 8010.
E-mail address: firstname.lastname@example.org (P.G. Sohnle).
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/micpath
0882-4010/$ – see front matter Published by Elsevier Ltd.
Microbial Pathogenesis 47 (2009) 16–23
S. aureus onto minimally damaged skin, and to relate dissemination
to the cutaneous changes caused by the local infection.
2.1. Dissemination after cutaneous inoculation
In order to determine if the inoculated organisms could pene-
trate a relatively intact epidermis and disseminate systemically,
cultures of spleen and kidneys were performed after the cutaneous
inoculations (Fig. 1). The spleen and kidney cultures were negative
after 1 h in all animals, indicating that that the inoculation and
retrieval procedures did not result in contamination of the organ
specimens from the inoculated skin. However, by 6 h almost all of
the spleen and kidney cultures had become positive, including 6 of
6 for each organwith S. aureus strain #25923 in C57BL/6 mice and 6
of 6 in spleen and 5 of 6 in kidney for this strain in Balb/c mice; in
addition, at 6 and 24 h with all the bacterial and mouse strain
combinations tested, 46 of 46 spleen cultures and 44 of 46 kidney
cultures were positive. The cultures generally remained positive for
the 96 h period, except for Balb/c mice, in which spleen and kidney
cultures both became negative at 96 h.
The number of CFU recovered from the skin increased between
1 and 48 h, indicating proliferation of the organisms in the skin
during this period. Also, studies were done with inoculations of
S. aureus strain #25923 onto C57BL/6 mouse skin that had been
shaved but not tape-stripped beforehand; the results of these
experiments showed a decrease in organisms retrieved from spleen
and kidney (log10values of 0.3?0.4 CFU in spleen and 1.0 ? 0.6 in
kidney; both significantly less compared to tape-stripped skin, at
P<.001 in each case by ANOVA and Tukey’s tests). In general,
treatment with cyclophosphamide to render the animals leuko-
penic did not significantly affect the results or produce increases in
the numbers of bacteria recovered from the skin, spleen, or kidney
sites. Similarly, studies with a second strain of S. aureus (Newman)
demonstrated results like those obtained using the standard strain
(ATCC #25923), and experiments using Balb/c instead of C57BL/6
mice also demonstrated equivalent findings, except as noted above.
2.2. Location of organisms in the skin
As early as 6 h after the epicutaneous inoculations onto tape-
stripped skin, bacteria were seen to have invaded from the stratum
corneum inoculation site into the epidermal keratinocytes and the
dermis (Figs. 2 and 3). Studies with the Newman strain also showed
penetration intothe epidermal keratinocyte layers and dermisat 24
and 48 h. In some cases bacteria were found within the epidermal
keratinocyte layers in the absence of an inflammatory cell infiltrate
(Fig. 3B). In addition, bacteria were sometimes found in the dermis
(Fig. 3C) and occasionally were associated with dermal blood
vessels there (Fig. 3d). The latterobservationwas infrequent (5 such
sites found in 35 sections from 11 animals inoculated 24 h before-
hand, with 4 of the 5 sites found in cyclophosphamide-treated
mice). In general, distribution of the organisms in infected skin was
approximately similar in Balb/c mice and in cyclophosphamide-
treated C57BL/6 mice as compared to untreated C57BL/6 mice. It
should be noted that loss of the epidermis at later time points
reduced the numbers of fields counted that listed organisms
present in this layer. Similarly, conversion of the dermis (and often
the overlying epidermis) into a separate crust by the dermal
necrosis process discussed below caused redistribution of the
organisms from infected epidermis and dermis into crusts located
above the skin surface.
Infections of hair follicle outlets were fairly frequent at 6 or 24 h
after inoculation in the experimental infections (Fig. 4). However,
relatively few of the hair follicle outlet infections resulted in
Fig. 1. S. aureus colony forming units (CFU) found in skin (checked bars), spleen (solid bars), and kidney (open bars) at various times (in hrs on the X-axis) after epicutaneous
inoculation. A. S. aureus ATCC strain #25923 in C57BL/6 mice; B. S. aureus ATCC strain #25923 in cyclophosphamide-treated C57BL/6 mice; C. S. aureus strain Newman in C57BL/6
mice; D. S. aureus strain ATCC #25923 in Balb/c mice. Note in panel A that no CFU were found in kidney and spleen at 1 h after inoculation, indicating lack of contamination during
the organ harvesting procedure; also, the difference in skin CFU between 1 and 48 h was significant (P<.001 by ANOVA and Tukey’s tests), indicating growth at the skin surface.
Data are from 4–13 animals per point, tested in 2–5 experiments.
B.L. Hahn et al. / Microbial Pathogenesis 47 (2009) 16–2317
Fig. 2. Location of bacteria in different skin layers, expressed as percent of microscopic fields with organisms at each site on the Y-axis, for locations in the stratum corneum or
crusts (checked bars), epidermal keratinocytes (solid bars), or dermis (open bars); times after epicutaneous inoculation are given in hrs on the X-axis. A. S. aureus ATCC strain
#25923 in C57BL/6 mice; B. S. aureus ATCC strain #25923 in cyclophosphamide-treated C57BL/6 mice; C. S. aureus strain Newman in C57BL/6 mice; D. S. aureus strain ATCC #25923
in Balb/c mice. Data are from 4–11 animals per point, studied in 3–5 experiments.
Fig. 3. Location of bacteria after epicutaneous inoculation of C57BL/6 mice with S. aureus ATCC strain #25923 24 h previously (photomicrographs of tissue gram-stained sections at
1000? original magnification under oil immersion): A. Section of normal uninoculated C57BL/6 mouse skin with the bracket marking the epidermal keratinocyte layers; the stratum
corneum lies above these layers and the dermis lies below; B. Bacteria located in epidermal keratinocytes (arrow); C. Bacteria located in the dermis (arrow); D. Bacteria associated
with a dermal blood vessel in a cyclophosphamide-treated mouse.
B.L. Hahn et al. / Microbial Pathogenesis 47 (2009) 16–2318
inflammatory cell infiltration into the follicular keratinocytes. Also,
deep hair follicle infections did occur at 6 or 24 h after inoculation,
but they were relatively infrequent. Generally, the deep hair follicle
infections observed were not associated with either infiltration of
inflammatory cells or disruption of follicular integrity.
Some experiments were done with heat-killed S. aureus strain
#25923 in C57BL/6 mice in order to determine if such organisms
could be found to penetrate into deeper layers of the skin. In 3 such
experiments with 6 mice in total, the percent of fields showing
organisms at 24 h were as follows: stratum corneum or crusts had
3.3%, epidermal keratinocytes had 0%, and dermis had 0%;
comparable values found with viable bacteria were 77.8%, 42.2%,
and 24.4%. Hair follicle outlets demonstrated organisms in 3.3%
with the heat-killed inoculum as compared to 60.5% with viable
bacteria; therewere nodeep hair follicle infections seen at this time
point with the heat-killed inoculum (compared to 8.8% with viable
bacteria). Therefore, it appears that viable bacteria are required for
entry into the skin.
2.3. Cutaneous damage from the infections
Damage to the inoculated skin from the experimental infections
was significant (Fig. 5). The major types of damage seenwere loss of
the epidermis (Fig. 5a), infiltration of neutrophils into the
epidermal keratinocyte layers (Fig. 5b), and complete necrosis of
the dermis (Fig. 5c). In general, the same types of cutaneous
damage developed after inoculation with S. aureus onto normal or
cyclophosphamide-treated C57BL/6 mice, and with Balb/c mice,
although the amounts of damage varied for the different experi-
mental conditions (Fig. 6a–e). Dermal necrosis resulted in conver-
sion of the dermis and overlying epidermis into a shrunken and
discolored layer overlying the remaining fat cells of the subcuta-
neous tissue underneath. This process did not occur until 48 h after
inoculation, but it was frequent in the infected skin; for example,
with the #25923 strain of S. aureus in C57BL/6 mice (Fig. 6a) it
affected approximately 67% of fields at 48 h after inoculation. Since
dermal necrosis alsooccurred
animals(Fig. 6b), it was apparently due to effects of the bacteriaand
not to those of the host inflammatory response. Studies using
saline-inoculated mice demonstrated that tape-stripping alone
produced only minimal amounts of cutaneous damage as
compared to the experimental infections (Fig. 6e).
2.4. Characteristics of dermal bacterial foci
Each site in the inoculated skin from either cyclophosphamide-
treated or untreated C57BL/6 mice at 6 h after inoculation was
examined for dermal bacterial foci (presence of bacteria located
below the dermal-epidermal junction), and then the characteristics
of each focus with respect to accompanying epidermal damage was
assessed (Table 1). This study was undertaken to determine if the
bacteria gained access to the dermis and the blood vessels located
Fig. 4. Infected hair follicle outlets (checked bars), infected hair follicle outlets with
inflammatory cell infiltration (solid bars), and deep (greater than 100 mm from the skin
surface) hair follicle infections (open bars) at 6 or 24 h after inoculation with S. aureus
ATCC strain #25923 in C57BL/6 mice.
Fig. 5. Types of cutaneous damage after epicutaneous inoculation of C57BL/6 mice with S. aureus 24 h previously (photomicrographs of tissue gram-stained sections at 1000?):
A. Loss of epidermis with organisms present in the superficial dermis; B. Infiltration of neutrophils into the epidermis, with associated bacteria (arrow); C. Dermal necrosis (arrows).
B.L. Hahn et al. / Microbial Pathogenesis 47 (2009) 16–2319
there because the epidermis had either been lost (absent
epidermis) or significantly damaged by the infection process.
However, most of the dermal foci were found either beneath intact
epidermis or associated with hair follicle outlet infections, indi-
cating that epidermal damage was not required for invasion into
In this model system it appears that S. aureus can rapidly (within
6 h) penetrate through intact keratinocyte layers of the epidermis
and into the dermis below; at some point within this 6 h the
invading organisms also distribute to distant organs. Although
severe damage to the dermis and dermal blood vessels does
develop in these infections, the visible effects would seem to occur
too late (between 24 and 48 h) after inoculation to really be
involved in the dissemination process. More subtle cutaneous
changes might perhaps be involved, as well as a number of other
routes into the bloodstream, as discussed below. The dermal
necrosis process is apparently due to the bacteria themselves and
not the host inflammatory cells in that it also occurs in cyclo-
phosphamide-treated animals. Infections of hair follicles, especially
at the infundibular outlets, do occur in this model system and could
be involved in the dissemination process by affording the bacteria
more rapid ingress to the deeper dermal blood vessels. However,
deep hair follicle infections (below 100 mm from the skin surface)
were relatively uncommon in the S. aureus-inoculated skin at early
time points and did not seem to disrupt the follicle integrity or
Fig. 6. Quantitation of cutaneous damage after experimental cutaneous S. aureus infections, including loss of epidermis (checked bars), neutrophil infiltration into the epidermal
keratinocyte layers (solid bars), and dermal necrosis (open bars) on the Y-axis at various times after epicutaneous inoculation (in hrs on the X-axis). A. S. aureus ATCC strain #25923
in C57BL/6 mice; B. S. aureus ATCC strain #25923 in cyclophosphamide-treated C57BL/6 mice; C. S. aureus strain Newman in C57BL/6 mice; D. S. aureus strain ATCC #25923 in Balb/c
mice; E. Inoculation with saline alone. Note the minimal damage that occurred after inoculation with saline alone; also, at 24 h loss of the epidermis was greater and neutrophil
infiltration into the epidermis was less in cyclophosphamide-treated than normal mice (P<.01 and .05 respectively by ANOVA and Tukey’s tests). Data represent mean ?SE of
percent of fields (of 10 counted per section) showing cutaneous damage; results are from 4–11 animals studied in 3–5 experiments per point.
Characteristics of dermal bacterial foci after epicutaneous Staphylococcus aureus
inoculation onto tape-stripped skin of mice.
Type of Dermal Focus
Epidermis Damaged with ICc
Epidermis Damaged w/o IC
CYa-Treated Mice Untreated Mice
aCY¼Mice treated with cyclophosphamide to render them leukopenic.
bHF-Associated¼Dermal foci associated with a hair follicle.
cEpidermis damaged with IC¼Epidermal keratinocyte layers disrupted with or
w/o host inflammatory cells (IC) being present. Data represent mean?SE of percent
of dermal bacterial foci found of the various types; dermal foci were counted as sites
where bacteria were found below the dermal-epidermal junction of infected skin,
with data from 4 CY and 10 normal animals studied in 3–5 experiments.
B.L. Hahn et al. / Microbial Pathogenesis 47 (2009) 16–2320
cause significant inflammatory responses. It should be noted that
the two strains of S. aureus used in these studies have previously
been found to be virulent in a variety of animal models and to
produce a number of important virulence factors; the organisms
are described more fully in the Methods section below.
Our staphylococcal skin infection model is based upon a number
of similar ones that have been described previously. In general, the
previously described experimental infections have used either
intracutaneous injections of the organisms or epicutaneous appli-
cation onto damaged skin with coverage by occlusive dressings in
a manner similar to that used here [4,8,11]. Resulting skin changes
were consistent with what we found and include hair follicle
infections , abscess formation [9–11], epidermal necrosis [4,8],
and cutaneous ulcer formation . These previous model systems
were developed primarily to assess local cutaneous changes from
the infections rather than bacterial dissemination from the skin
inoculation site. Blood cultures were performed in a model system
based on intracutaneous injections of S. aureus, and were found to
be negative in normal animals, but positive in some cyclophos-
phamide-treated mice (1 of 6 at 6 h and 5 of 6 at 48 h) . With
our experimental infections we wanted to assess the ability of the
inoculated organisms to penetrate a relatively intact epidermis and
disseminate systemically. Therefore, we used a mild tape-stripping
method to induce stratum corneum damage while leaving the
underlying keratinocyte layers relatively intact; in this model
system the organisms would have to traverse the keratinocytes in
order to gain access either to the bloodstream or the dermis below.
Also, we felt that organ cultures might be a more sensitive method
than blood cultures to determine bacterial dissemination in that
residence of organisms in the bloodstream would likely be tran-
sient. These studies did demonstrate a remarkable ability of
S. aureus organisms inoculated onto the skin surface to distribute to
distant organs within 6 h.
A number of routes for bacteria to pass through the epidermal
keratinocytes are possible. Small defects from shaving or tape-
stripping might allow direct entry into the dermis. We did not find
such defects in the 6-hr samples of saline-inoculated skin on
microscopic exam, although they still could have been present in
very small numbers. On the other hand, organisms were found in
the dermis at 6 h in our experimental infections, and these sites
were generally located beneath an intact keratinocyte layer.
Otherwise, there is evidence that S. aureus can either enter kera-
tinocytes, or bypass them by damaging their intercellular connec-
tions. This organism can be internalized by either immortalized
human keratinocytes [13–15] or primary keratinocytes [14–16].
Internalization appears to require the presence of fibronectin-
binding proteins on the bacteria in most cases . S. aureus ATCC
25923 does have the gene for fibronectin-binding protein A (Gen-
Bank accession number EU195388), as discussed in the Methods
section. Ingestion of S. aureus by keratinocytes may result in
increased expression of b-defensins and cathelicidins by the cells
and killingof the ingestedbacteria[15,16]. Alternatively, interaction
between keratinocytes and S. aureus may result in necrotic or
apoptotic keratinocyte death unrelated to staphylococcal hemoly-
sins , although staphylococcal a-toxin (hemolysin) is capable of
killing human keratinocytes in vitro by permeabilizing the plasma
membrane for monovalent ions . Direct killing of epidermal
cells by staphylococcal enzymes could produce cavities in the
epidermis that would allow entry of the bacteria to deeper sites in
Passage between epidermal keratinocytes is another route that
staphylococci might take to gain entrance to the dermis and the
blood vessels located there. S. aureus produces an impressive array
of enzymes and exotoxins that can either directly damage the skin
or subvert the host’s immune defenses against the invading
organisms [12,18,19]. Staphylococcal exfoliative toxins produce
subcorneal acantholytic cleavage that causes the bullous lesions
characteristic of staphylococcal scalded skin syndrome and bullous
impetigo. These enzymes have been shown to damage the inter-
cellular junctions between keratinocytes by cleaving desmoglein 1,
a desmosomal cadherin that is expressed in the upper epidermis
. Enzymes from Bacteroides fragilis and Helicobacter pylori act
similarly on intercellular junctions of endothelial cells, although
some of the proteins affected may be different [21–23]. As dis-
cussed below in the Methods section, the Newman strain of
S. aureus is positive for exfoliative toxin, whereas ATCC #25923 is
negative. We did not see acantholysis analogous to that of bullous
impetigo in our experimental infections, but in mice this process
generally requires use of exfoliative toxin in neonatal animals. Even
so, more subtle damage to keratinocyte intercellular junctions by
exfoliative toxins or other proteolytic enzymes could have allowed
the organisms to transverse the epidermis more readily.
Since staphylococci inoculated onto the skin surface could
rapidly make their way into the dermis, it seems likely that they
might enter the bloodstream through many blood vessels located
there. However, passage into the lymphatic system might be
another route for systemic dissemination. In inhalational anthrax,
the spores of Bacillus anthracis are thought to germinate within
alveolar macrophages, which then carry them to regional lymph
nodes  where they produce a destructive lymphadenitis and
eventually enter the systemic circulation. Epidermal Langerhans
cells are a type of dendritic antigen-presenting cell located in the
epidermis and capable of ingesting S. aureus . Epidermal
Langerhans cells have been shown to migrate to regional lymph
nodes  and could possiblycarry ingested microorganisms there.
In any event, distribution through the lymphatic system represents
an alternative to direct bloodstream invasion as a route to distant
organs for staphylococci on the skin surface.
In summary, S. aureus is capable of causing significant cutaneous
damage after being inoculated onto the skin surface, and also has
the ability to disseminate systemically from the skin within 6 h of
such inoculation. However, because the major dermal damage does
not develop until dissemination has already taken place, it would
seem that the two processes are not directly related.
4. Experimental/materials and methods
The inoculations werecarried out with 107CFU of S. aureus ATCC
strain 25923, with some parallel studies using the Newman strain
of S. aureus. The organisms were cultured overnight in tryptic soy
broth, then washed 3 times in sterile water before use. The inoc-
ulum of 107CFU was chosen from a review of previous studies
[4,7,9]. S. aureus strain ATCC #25923 has been shown to be virulent
in other animal models of staphylococcal infection [27,28]. In
addition to fibronectin-binding protein A, other virulence factors
and enzymes of this staphylococcal strain include the Panton–
Valentine leukocidin (PVL), coagulase, catalase, DNase, heat shock
protein 60, thermostable nuclease, a-toxin, and staphylococcal
enterotoxins G, I, M, N, and O, but not b-lactamase, exfoliative
toxins, or toxic shock syndrome toxin 1 (TSST) [27,29,30]. The
Newman strain has been sequenced and found positive for
enterotoxin A, exfoliative toxin A, and a variety of other exotoxins
and enzymes .
C57BL/6 and Balb/c mice were obtained from Charles Rivers
Laboratories (Wilmington, MA). C57BL/6 mice were used for most
B.L. Hahn et al. / Microbial Pathogenesis 47 (2009) 16–2321
of the studies, but some experiments were repeated with Balb/c
mice because they have previously been found to differ from
C57BL/6 mice in sensitivity to certain S. aureus infections . It
should also be noted that mouse leukocytes appear to be more
resistant to staphylococcal exotoxins such as PVL than are human
leukocytes , suggesting that inflammatory responses to the
infections may differ between species.
The animals were both male and female from 8–14 weeks of age
and were described by the supplier as being free of specific path-
ogens (including S. aureus). Some mice were treated with cyclo-
phosphamide (150 mg/kg intraperitoneally 3 days before and
100 mg/kg 1 day before the inoculations) to render them leuko-
penic, as previously described . The mice for these studies were
housed in a separate BSL 2 enhanced section of the Veterinary
Medical Unit at the Milwaukee VA Medical Center. The experi-
mental procedures were approved by the appropriate committees
at the Milwaukee Veterans Affairs Medical Center and the Medical
College of Wisconsin.
4.3. Epicutaneous inoculations
The skin was shaved with an electric razor, and then disinfected
with iodine, washed with alcohol followed bysaline, and then dried
with gauze. The skin surface was prepared by gentle tape-stripping
7? with Transpore tape (approximately 27 mm in width, from 3 M,
Minneapolis, MN). As shown in Fig. 6e, this technique was found to
cause only minimal damage to the epidermis or dermis. An inoc-
ulum of 107S. aureus CFU in 0.025 ml of saline was added to 4 mm
filter paper discs placed on prepared skin of the animal’s left flank,
with saline on the opposite side. Both sites were covered with
1.0 cm2pieces of plastic sheet and overwrapped with dressings of
Transpore tape and Nexcare waterproof tape (3 M). Photographs of
the tape-stripping and inoculation procedures are shown in Fig. 7.
4.4. Monitoring of infections
After 1–24 h the occlusive dressings were removed and the sites
washed 4 times with saline-soaked gauze pads. At various times
from 1–96 h after inoculation the animals were killed and skin
removed from the inoculated sites for histology. Paraffin sections
were prepared and stained with tissue gram stains, with analysis as
discussed below. In some cases when the mice were killed, skin
scrapings or homogenates of spleen or kidneys in 1 ml of saline
were cultured on tryptic soy agar, with results recorded as CFU per
ml. For skin scrapings the entire inoculation site was sampled by
abrasion with a scalpel blade until a glistening surface could be
seen; the material removed was vortexed in 1 ml of saline and then
cultured for CFU determinations.
Sections of either S. aureus or saline-inoculated skin were
examined in a blinded fashion for bacteria and cutaneous changes
at a 400? magnification under light microscopy. For the purposes
of this study, the epidermis was defined as the epidermal kera-
excluding the stratum corneum or crusts. In ten random fields in
each section the site of bacteria location was determined as being
in the stratum corneum or crusts, the epidermis, the dermis, or
combination thereof. Each hair follicle infundibular outlet across
the sections was also examined for the presence of bacteria, with
the data being recorded as percent of outlets infected and percent
of outlets with inflammatory cell infiltrates present. In addition,
the hair follicles located in the dermis were also examined for the
presence of bacteria located greater than 100 mm below the skin
surface; the data were recorded as the percent of hair follicles
with deep infections. It should be noted that mouse skin structure
is generally similar to that of humans except for a thinner
epidermis and grouping of hair follicles together in areas of ones
that are actively growing (anagen) or those that are resting (tel-
Cutaneous damage was also assessed for each linear high power
(400?) field across the section’s entire epidermis as absent
epidermis, neutrophil infiltration into the keratinocyte layers, or
complete dermal necrosis. The latter represented conversion of the
entire dermis into a discolored amorphous crust. Examples of
cutaneous damage are shown in Fig. 5.
Characteristics of dermal bacterial foci were assessed from
sections of inoculated skin taken at 6 h in order to determine if
epidermal damage was required for invasion of the organisms into
the dermis. Dermal bacterial foci were defined for the purposes of
this study as foci of bacteria located below the dermal–epidermal
junction of the inoculated skin. The foci were evaluated to deter-
mine if they were associated with a hair follicle or were located
beneath epidermis with an intact keratinocyte layer; otherwise,
epidermal damage associated with the dermal foci was assessed as
absent epidermis (with the epidermal keratinocyte layers missing
entirely) or as those with epidermis disrupted with or without host
inflammatory cells being present. Such data were generated from
cyclophosphamide-treated or untreated C57BL/6 mice inoculated
with the ATCC #25923 strainof S. aureus, and were expressed as the
percentages of each type of dermal focus found.
Fig. 7. Stages of the epicutaneous inoculation procedure. A. Appearance of skin after
shaving and tape-stripping; B. Arrangement of filter disc and plastic covering after
inoculation of S. aureus onto the disc.
B.L. Hahn et al. / Microbial Pathogenesis 47 (2009) 16–2322
4.5. Statistics Download full-text
Data were expressed as mean?SE, with statistics carried out in
the GraphPad Prism 4.0c statistical package using ANOVA and
Dunnett’s test for multiple comparisons. Generally 4–12 mice per
point were studied in 3–5 experiments (each consisting of animals
inoculated in a similar manner on a single day). Statistical signifi-
cance was taken as P<.05.
This work was supported by the United States Department of
Veterans Affairs and by the NIH/NIAID Regional Center of Excel-
lence for Bio-defense and Emerging Infectious Diseases Research
(RCE) Program. The authors wish to acknowledge membership
within and support from the Region V ‘Great Lakes’ RCE (NIH award
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