Systemic dissemination and cutaneous damage in a mouse model of staphylococcal skin infections

Abstract

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 6h. 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 48h 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.

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.

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.

Fig. 4

Infected hair follicle outlets (checked bars), infected hair follicle outlets with inflammatory cell infiltration (solid bars), and deep (greater than 100 µm 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).

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.

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.