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 cyclophosphamide-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 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 application onto damaged skin with coverage by occlusive dressings in a manner similar to that used here [4
]. Resulting skin changes were consistent with what we found and include hair follicle infections [4
], abscess formation [9
], epidermal necrosis [4
], and cutaneous ulcer formation [10
]. 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 cyclophosphamide-treated mice (1 of 6 at 6 h and 5 of 6 at 48 h) [11
]. 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 transient. 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 keratinocytes, or bypass them by damaging their intercellular connections. This organism can be internalized by either immortalized human keratinocytes [13
] or primary keratinocytes [14
]. Internalization appears to require the presence of fibronectin-binding proteins on the bacteria in most cases [14
]. 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 β-defensins and cathelicidins by the cells and killing of the ingested bacteria [15
]. Alternatively, interaction between keratinocytes and S. aureus
may result in necrotic or apoptotic keratinocyte death unrelated to staphylococcal hemolysins [13
], although staphylococcal α-toxin (hemolysin) is capable of killing human keratinocytes in vitro by permeabilizing the plasma membrane for monovalent ions [17
]. 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 the skin.
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
]. 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 intercellular junctions between keratinocytes by cleaving desmoglein 1, a desmosomal cadherin that is expressed in the upper epidermis [20
]. 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
]. As discussed 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 [24
] 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 [26
] and could possibly carry 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.