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Infection and Immunity, May 2000, p. 2880-2887, Vol. 68, No. 5
Departments of Epidemiology and Public
Health1 and
Dermatology,2 Yale University School
of Medicine, New Haven, Connecticut
Received 29 October 1999/Returned for modification 9 December
1999/Accepted 1 February 2000
An in vivo model for group A streptococcal (GAS) impetigo was
developed, whereby human neonatal foreskin engrafted onto SCID mice was
superficially damaged and bacteria were topically applied. Severe
infection, indicated by a purulent exudate, could be induced with as
few as 1,000 CFU of a virulent strain. Early findings (48 h) showed a
loss of stratum corneum and adherence of short chains of gram-positive
cocci to the external surface of granular keratinocytes. This was
followed by an increasing infiltration of polymorphonuclear leukocytes
(neutrophils) of mouse origin, until a thick layer of pus covered an
intact epidermis, with massive clumps of cocci accumulated at the outer
rim of the pus layer. By 7 days postinoculation, the epidermis was
heavily eroded; in some instances, the dermis contained pockets
(ulcers) filled with cocci, similar to that observed for ecthyma.
Importantly, virulent GAS underwent reproduction, resulting in a net
increase in CFU of 20- to 14,000-fold. The majority of emm
pattern D strains had a higher gross pathology score than
emm pattern A, B, or C (A-C) strains, consistent with
epidemiological findings that pattern D strains have a strong tendency
to cause impetigo, whereas pattern A-C strains are more likely to
cause pharyngitis.
Group A streptococci (GAS) are
highly prevalent bacterial pathogens that exclusively infect
humans. Primary infection and disease most often take place at the
mucosal epithelium of the upper respiratory tract (pharyngitis) or
epidermal layer of the skin (impetigo). Streptococcal impetigo is most
common in the tropical, developing world, and it can lead to serious
complications, such as acute glomerulonephritis. Impetigo can follow
minor trauma to the skin, and risk factors for disease include a warm
and humid climate. There is a wide array of cutaneous infection due to
GAS, ranging from a mild, superficial condition limited to the
epidermis (impetigo) to extensive dermal layer involvement (ecthyma) to deeper, subcutaneous spread (cellulitis) (8, 23). In all cases, a prominent feature is an acute inflammatory response, largely
attributed to infiltration of infected foci by polymorphonuclear leukocytes (PMNs).
Decades of epidemiological studies indicate that there are some
strains of GAS that often cause oropharyngeal infection but rarely
cause impetigo and, conversely, that there is a distinct subset of
strains that tend to cause infection at the skin but not at the throat.
This led to the concept of distinct throat and skin strains of GAS
(7, 18, 25). More recently, ecological markers for tissue
site preferences among GAS have been identified within phylogenetically
distant emm genes (6, 13). Strains bearing
emm patterns A, B, or C (A-C) have a high tendency to be
found in association with an upper respiratory tract infection, whereas
emm pattern D strains are most often recovered from impetigo lesions (5, 6). In order to establish the molecular basis for tissue tropisms among GAS, an experimental model for impetigo was developed.
(This work was presented in part at the XIVth Lancefield International
Symposium on Streptococci and Streptococcal Diseases, Auckland, New
Zealand, 11 to 15 October 1999.)
Bacteria.
Six emm pattern A-C strains and six
emm pattern D strains of GAS were emm typed, and
their sources (dates, places, and tissue sites of isolation) have been
described previously (5). Streptomycin-resistant variants of
strains D471 and 29487 were selected as previously described
(3).
Skin grafts.
Four- to six-week-old female C.B.-17
scid mice (Taconic or Charles River Labs) were engrafted
with human neonatal foreskins. Freshly isolated foreskins, obtained
from Yale-New Haven Hospital, were collected in RPMI medium containing
penicillin and streptomycin. The foreskins were aseptically freed of
subcutaneous tissue and trimmed to dimensions of approximately 1 by 1.5 cm. Mice were anesthetized by intraperitoneal injection of
Ketamine-Xylazine. Animals were shaved and wiped with alcohol, and a
section of mouse skin was surgically removed at each hind quarter,
exposing the underlying muscle fascia. The fascia was gently scored
with scissors, and human foreskins were stapled in place; the staples
were removed 1 week following transplantation. Because SCID mice lack
functional B and T lymphocytes, they fail to reject xenotransplants.
Engrafted mice (hu-skin-SCID mice) were ready for inoculation
approximately 4 weeks following surgery, after the scab had shed.
In vivo model for infection.
Frozen stocks of GAS were
streaked on Todd-Hewitt (TH) agar containing sheep blood and grown
overnight at 37°C. Five individual colonies were inoculated into 5 ml
of TH broth and grown for 22 to 26 h at 37°C; this constituted
the inoculum for hu-skin-SCID mouse virulence studies. For experiments
in which CFU measurements were made (i.e., those involving strains
29487 and D471), streptomycin was included in all bacterial growth
media and the broth culture inoculum was initially adjusted to an
optical density at 600 nm of approximately 0.600 and then diluted or
concentrated in order to approximate the intended doses with which to
inoculate mice. Mice inoculated with streptomycin-resistant GAS were
placed on streptomycin (5 mg/ml)-water 20 to 24 h prior to
inoculation and remained on streptomycin until biopsy.
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Humanized In Vivo Model for Streptococcal
Impetigo
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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7 in TH broth; 100 µl of each dilution was spread on
TH-blood agar plates, and bacterial colonies were counted using a grid
and dissection microscope. The calculated total number of CFU recovered
from the tissue was based on the total weight of the skin biopsy;
calculations based on skin surface area yielded comparable findings.
The CFU measurements for inoculation doses were performed in duplicate and averaged.
For analysis of keratin profiles, portions of tissue biopsy material
were placed in 150 to 200 µl of 20 mM Tris-HCl (pH 7.4) containing
1% sodium dodecyl sulfate (SDS) and 1× Complete protease inhibitor
cocktail (Boehringer GmbH, Mannheim, Germany) in 1.5-ml Microfuge
tubes. The tissues were minced with scissors, boiled for 10 min and
homogenized with a pellet disrupter tissue grinder (Kontes Glass
Company, Vineland, N.J.). Undissolved tissue was pelleted by
centrifugation at 16,000 × g for 30 s, and
supernatants containing protein extracts were removed, placed on ice,
and stored at 
80°C. Pellets were resuspended in 150 to 200 µl of
the Tris-SDS-protease inhibitor mixture, to which was added 150 mM
-mercaptoethanol; boiling, homogenization, centrifugation, and
collection of supernatant from the reduced extract were then carried
out as described above.
Western immunoblots.
The protein content of the tissue
extracts was quantified in microtiter plates using the Protein
Microassay (Bio-Rad Laboratories, Hercules, Calif.). Absorbance was
read at 595 nm in an MRX microplate reader (Dynex Technologies Inc.,
Chantilly, Va.). Equivalent amounts of protein extract were separated
by SDS-polyacrylamide gel electrophoresis, and gels were electroblotted
using standard methods (4). Electroblots were incubated with
monoclonal antibodies (MAbs) directed against high- and
low-molecular-weight keratins (AE1-AE3 MAb cocktail; ICN
Pharmaceuticals Inc., Costa Mesa, Calif.) or keratin 10/11 (
-cytokeratin 8.60; Sigma, St. Louis, Mo.). Immunodetection was performed by the ECL method (Amersham-Pharmacia Biotech, Piscataway, N.J.).
Statistics. Unpaired Student t test (two-tailed), arithmetic mean, and average deviation calculations were performed using Excel version 8.0 (Microsoft Corp.).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Requirements for skin infection.
Human skin-SCID mouse
chimeras were tested for their ability to support an impetigo-like
infection due to GAS. Strain 29487, originally derived from a human
impetigo lesion, was topically applied to human skin grafts that
had undergone superficial damage achieved by one of three
methods: a series of gentle cuts with a scalpel blade, sandpaper
treatment, or tape stripping. Inoculated grafts were occluded
with a bandage and visually examined for gross pathology at several
time intervals thereafter (Table 1). High
gross pathology scores were observed at both 24 and 48 h postinoculation with an undiluted, overnight bacterial culture. By
48 h, 82.4% of the human skin grafts inoculated with strain 29487 displayed a purulent exudate on the surface (Fig. 1B), whereas 5.9% of
the grafts failed to exhibit any obvious gross pathological changes.
Undamaged and/or nonoccluded grafts led to very low levels of gross
pathological alterations. The differences in gross pathology score
between GAS-inoculated skin grafts that had both damage and occlusion
and grafts lacking one or both treatments were highly significant. The
findings suggest that minor skin trauma and moisture are prerequisites
for the development of experimental impetigo, analogous to the risk
factors for naturally acquired disease in humans.
|
Correlation of emm pattern with gross pathology.
Several strains of GAS, defined for emm patterns A-C or D,
representing the so-called throat and skin strains, respectively, were
compared for virulence in the hu-skin-SCID mouse model. Undiluted, overnight bacterial broth cultures were topically applied to damaged skin grafts and occluded with bandages for 48 h. Findings for graft damage by scalpel blade cuts were comparable to those resulting from all wounding methods combined (Table
2). The majority of emm
pattern D strains exhibited a higher gross pathology score than the
pattern A-C strains tested. Overall, the difference in gross pathology
scores for emm pattern A-C versus D strains was highly
significant. The data suggest that the experimental hu-skin-SCID mouse
model for GAS impetigo is a sensitive measure for differences in strain
behavior as observed in human populations.
|
Growth properties of virulent and avirulent GAS strains.
The
highly virulent emm pattern D strain 29487 was compared to
the emm pattern A strain D471 for several virulence
measurements following inoculation on superficially damaged human skin
grafts. For inoculating doses of approximately 106 to
107 CFU, observations at 48 h revealed fulminant
infection for strain 29487, whereas strain D471 induced little or no
gross pathological alterations (Fig. 2A).
At 96 h, three of four grafts receiving strain 29487 at doses of
<106 CFU gave rise to purulent exudates,
whereas strain D471 inoculated at higher doses failed to induce
demonstrable infection by gross examination (Fig. 2B). At 7 days
postinoculation, low doses of strain 29487 induced infection
whereas high doses of D471 had no effect (Fig. 2C). In all instances in
which strain 29487 induced pathological changes at 96 h or 7 days,
there was also a net increase in the number of CFU recovered from the
tissue biopsy. In contrast, skin grafts which healed following
inoculation with strain D471 (or 29487) yielded a net decrease in
CFU. For the 96-h and 7-day time points, gross pathology was
directly proportional to the net growth of GAS, and thus, virulence and
reproductive capacity were positively coupled.
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Histopathological changes.
Human skin grafts underwent biopsy
at 48, 96, and 168 h following topical application of bacteria to
tissue damaged by gentle scalpel blade cuts. At 48 h following
inoculation of strain D471, an intact stratum corneum displaying a
basket-woven structure in tissue sections was observed over most of the
skin (Fig. 3A). Dermal
vessels were slightly dilated and a few PMNs in the dermis and
epidermis were evident, similar to observations for damaged skin
lacking GAS. This finding is typical of the normal host response to
wound healing, even in the absence of added bacteria. However, there
were occasional areas of inflammation which contained small foci of
subcorneal neutrophils (data not shown). A representative tissue Gram
stain revealed gram-positive cocci clinging to the outermost layers of
the sloughing stratum corneum (Fig. 3B). Also, premature shedding of
nucleated keratinocytes into the stratum corneum (parakeratosis), a
hallmark of epidermal injury, is evident.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Animal models for bacterial pathogens whose host is strictly humans often fail to mimic key aspects of the infectious process. The hu-skin-SCID mouse model for streptococcal impetigo satisfies several important criteria that are desirable for an in vivo model of a human disease. The inoculating dose of GAS which leads to infection, as well as its route of administration, closely parallels the physiological conditions encountered by its host under natural conditions. Doses as low as ~1,000 CFU of a virulent strain (29487) led to production of purulent exudate when mice were observed at 7 days. In all cases, the GAS are applied topically to the surface of slightly damaged skin, a delivery method that closely simulates the natural route of transmission for GAS which give rise to impetigo lesions. Furthermore, virulent organisms underwent expansive reproduction at this site, a hallmark of evolutionary success. GAS strains harboring different ecological markers for the preferred tissue site of infection (the emm patterns) also differ accordingly by their infectiousness in the in vivo model. emm pattern D strains (the so-called skin types) are significantly more virulent than pattern A-C strains (the so-called throat types). Thus, the hu-skin-SCID mouse model accurately reflects key events that occur within the human host population.
One key feature not prominent on gross examination is the honey-colored fibrin crust, although its microscopic equivalent, i.e., PMNs and coagulated serum, was readily observed (Fig. 3E). The relative absence of the fibrin crust may be the consequence of an expanding inflammatory response that fails to resolve. Future studies will aim towards defining the experimental conditions under which a fibrin crust is an obvious feature. Also, it should be emphasized that the inability to guarantee precise reproducibility in wounding of the skin, to a depth that is largely contained within the epidermal layer, is a potential shortcoming of this model.
Topical application of GAS onto the skin parallels the mode of administration encountered during natural infection. Both minor injury and moisture, maintained by occlusion of the wound, are required for induction of severe infection by virulent GAS in the hu-skin-SCID mouse model. Under experimental conditions using human subjects, minor abrasion and occlusion are also necessary for GAS infection (17).
Adherence to an epithelial cell surface is an important first step for many pathogenic bacteria that interact with their host at a nonsterile tissue site. Damage to the stratum corneum is a prerequisite for GAS infection in the hu-skin-SCID mouse model, presumably by providing the GAS access to subcorneal keratinocytes. Receptors for GAS adhesins, as measured by in vitro assays which employ keratinocyte cell lines or primary tissue cultures, include the CD46 membrane cofactor protein recognized by M proteins (19), the CD44 binding site for the hyaluronic acid comprising the GAS capsule (20), and binding sites for lipoteichoic acid, which possibly involve fibronectin (1, 10). The importance of differentiated keratinocytes for in vitro models of pathogenesis is underscored by studies which artificially induce a differentiated state that can distinguish between adherence of throat and skin isolates (11). Although during early infection, virulent strain 29487 is in tight association with the exposed surface of granular keratinocytes, rather than the spinous and basal cell layers, the identity of neither the tissue receptor nor the bacterial adhesin is obvious. However, adherence to granular keratinocytes may not even be a determinant of skin tropism among GAS. Strain differences in toxicity due to unsaturated fatty acids produced by highly differentiated keratinocytes could easily influence survival on the skin (21).
The stratum corneum is a key protective barrier of the human host. Loss of the stratum corneum is a prominent feature in natural disease, as well as in the hu-skin-SCID mouse infected with virulent GAS (Fig. 3C). Release of the stratum corneum could conceivably follow the detachment of underlying granular keratinocytes from the epidermis as a consequence of acantholysis (Fig. 3D). Local release of the hydrolytic enzymes present within the granules of infiltrating PMNs can conceivably lead to acantholysis by destroying the intercellular connections between the granular keratinocytes. Alternatively, blocking the maturation of granular keratinocytes into the cornfied form might lead to eventual loss of the stratum corneum due to normal turnover. Finally, GAS enzymes might exert direct action on the stratum corneum, leading to its breakdown and release. One or a combination of the proposed mechanisms may be responsible.
The presence of virulent strain 29487 on the granular cell layer led to a large influx of PMNs (Fig. 3E). If functional C5a peptidase (14) is produced by strain 29487, it appears to be overwhelmed. Alternatively, interleukin-8 production by keratinocytes might serve as the principal chemoattractant (24). Although it is reasonable to assume that M protein surface expression thwarts any attempts at opsonophagocytosis (12), it also seems possible that the massive tangles of streptococcal chains located at the outer pus layer impart a steric barrier to effective clearance. It also appears likely that GAS at the pus layer rim are in a logarithmic state of growth and are well positioned for transmission to a new host (or to a new site on the same host). The existence of high levels of replication of GAS at the outer rim of the pus layer is supported by both CFU measurements and tissue Gram-stained histology sections at 96 h postinoculation. The contents of the purulent exudate provide the bacteria with a rich source of nutrients. The data best support the notion that an intense inflammatory response (i.e., virulence) is positively coupled to the reproductive success of the organism.
At some point, a critical threshold is probably attained and the epidermis undergoes severe erosion. Conceivably, this is promoted by the breakdown of intercellular junctions due to spongiosis and/or movement of PMNs through the epidermis and, possibly, tissue damage resulting from their spilled granule contents. For some of the skin biopsies obtained at 7 days postinoculation, histopathological features closely resemble ecthyma, a severe form of impetigo (Fig. 3G). The dermal ulcers which are densely packed with gram-positive cocci point to bacterial replication occurring in situ. The cellular nuclei lying adjacent to the coccus-packed dermal pockets (Fig. 3H) suggest a diffusible bacterial cytolysin, perhaps streptolysin S or O, whose action results in lysis of mammalian cells. Potentially, the released cytoplasmic contents serve as a nutrient source for the replicating bacteria.
A critical issue is how well the inflammatory response in the SCID mouse resembles that of the immunocompetent human host. The histopathological features of the hu-skin-SCID mouse model for streptococcal impetigo show an infiltrate that appears to be exclusively neutrophilic; this finding parallels the observed human pathology (16). There is no evidence for a role of macrophages at the inflammatory site in the experimental model. However, SCID mice lack functional B and T lymphocytes, and if diffusible lymphokines are crucial to the human disease process, their effect may be absent in this model. In addition, the SCID mouse is deficient in immunoglobulin production, and therefore, protective or partially protective antibody is unavailable for modulating the development of the infected lesion. It is also possible that the low binding affinity of GAS for the mouse counterparts of several human plasma components, such as plasminogen (2, 9, 22), alters the natural course of infection in the hu-skin-SCID mouse model.
Innate host defenses prevented the development of septicemia in four of the five hu-skin-SCID mice tested for spleen cultures at 7 days. During natural infection of an immunocompetent naive host, antibody responses begin to rise within 7 days following exposure to antigen, and protective antibodies can reverse the progression of infection. The SCID mouse has no antibody response; however, its ability to contain the infection for at least 7 days, and the similar duration for an antibody response in a normal host, may not be mere coincidence. Using well-characterized antibodies directed to putative virulence factors, the hu-skin-SCID mouse model should prove useful for passive immunization studies that monitor neutralizing effects at specific stages of infection, in order to better understand disease pathogenesis and also to facilitate the development of a vaccine for streptococcal impetigo.
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ACKNOWLEDGMENTS |
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We thank Hong Wang and Marc Izzo for expert technical support, Ulf Sjöbring for comments on the manuscript, and Jeff Schechner and Bob Tigelaar for helpful discussions during the early stages of the study.
This work was supported by the National Institutes of Health (grant R01-AI-28944), the American Heart Association, and a Pilot/Feasibility award from the Yale Skin Diseases Research Center (supported by grant P30-AR-041942). D.E.B. is an Established Investigator of the American Heart Association.
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FOOTNOTES |
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* Corresponding author. Mailing address: Yale University School of Medicine, Dept. of Epidemiology & Public Health, 60 College St., Box 208034, New Haven, CT 06520. Phone: (203) 785-4480. Fax: (203) 737-4285. E-mail: debra.bessen{at}yale.edu.
Editor: E. I. Tuomanen
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Infection and Immunity, May 2000, p. 2880-2887, Vol. 68, No. 5
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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