Humanized in vivo model for streptococcal impetigo

Abstract

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.

FIG. 1

Gross pathology of infected grafts on hu-skin-SCID mice. Human skin grafts (arrows) were superficially wounded with a scalpel blade, inoculated with GAS, and occluded with a bandage for 48 h prior to observation. The grafts were inoculated with GAS strains D471 (A) and 29487 (B). Gross pathology scores are 0 and 3 for panels A and B, respectively. Note the glistening and whitish graft surface, and the adjacent wet and matted fur (double arrow) in panel B, characteristic of purulent exudate.

FIG. 2

Growth properties of GAS following inoculation of damaged human skin grafts. Human skin grafts were superficially damaged with a scalpel blade, inoculated with an overnight culture of bacteria over a wide range of doses, and occluded with a bandage. Biopsies were performed at 48 h (A), 96 h (B), or 168 h (C) postinoculation. Grafts inoculated with strain 29487 or D471 are indicated. The inoculating dose is shown on the x axis, whereas the y axis represents the log₁₀-fold change (increase or decrease) in CFU relative to the inoculum dose. Gross pathology scores at the time of biopsy are also indicated as 0 (open symbols), 1 or 2 (shaded symbols), and 3 (filled symbols).

FIG. 3

Histopathology of human skin grafts. Photomicrographs of formalin-fixed tissue sections of human skin grafts inoculated with strain D471 (A and B) or 29487 (C through H) and biopsied at 48 h (A through D), 96 h (E and F), or 7 days (G and H) are shown. Tissue sections are stained with hematoxylin-eosin (A, C, E, and G) or tissue Gram stain (B, D, F, and H). Panels A and B are from the same graft. The tissue section in panel F is adjacent to that shown in panel E and corresponds to the boxed area of panel E. The tissue section in panel H is adjacent to that shown in panel G and corresponds to the boxed area of panel G. Highlighted features are PMNs (black arrowheads), dermal blood vessels (green arrowheads), stratum corneum (red arrowhead), healthy granular keratinocyte layer (yellow arrowhead), gram-positive cocci (black arrows), parakeratosis (red arrow), damaged keratinocytes (green arrows), dermal ulcer (green asterisk), thrombotic vessel (black asterisk), cell nuclei (blue arrowheads). SC, stratum corneum; E, epidermis; D, dermis; P, purulent exudate. Bars, 35 μm (A, E, and G), 28 μm (C), 7 μm (B, F, and H), and 5.6 μm (D).

FIG. 3

Histopathology of human skin grafts. Photomicrographs of formalin-fixed tissue sections of human skin grafts inoculated with strain D471 (A and B) or 29487 (C through H) and biopsied at 48 h (A through D), 96 h (E and F), or 7 days (G and H) are shown. Tissue sections are stained with hematoxylin-eosin (A, C, E, and G) or tissue Gram stain (B, D, F, and H). Panels A and B are from the same graft. The tissue section in panel F is adjacent to that shown in panel E and corresponds to the boxed area of panel E. The tissue section in panel H is adjacent to that shown in panel G and corresponds to the boxed area of panel G. Highlighted features are PMNs (black arrowheads), dermal blood vessels (green arrowheads), stratum corneum (red arrowhead), healthy granular keratinocyte layer (yellow arrowhead), gram-positive cocci (black arrows), parakeratosis (red arrow), damaged keratinocytes (green arrows), dermal ulcer (green asterisk), thrombotic vessel (black asterisk), cell nuclei (blue arrowheads). SC, stratum corneum; E, epidermis; D, dermis; P, purulent exudate. Bars, 35 μm (A, E, and G), 28 μm (C), 7 μm (B, F, and H), and 5.6 μm (D).

FIG. 4

Western immunoblot of epidermal keratins. Damaged human skin grafts were infected with GAS strain D471 or 29487 for 96 h. The skin grafts were extracted without or with β-mercaptoethanol. The blot was first incubated with the anti-pan-keratin mixture of MAbs (AE1-AE3) (right panel); keratin 1 (ker 1) migrates at 68 kDa, whereas keratin 5 is observed at 58 kDa. The blot was then stripped and incubated with MAb directed to keratin 10/11, which reacts with the 56.5-kDa keratin 10 found in the skin (left panel).