Establishment of a Superficial Skin Infection Model in Mice by Using Staphylococcus aureus and Streptococcus pyogenes

Bacterial strains and growth media. Staphylococcus aureus FDA486 is a laboratory strain previously used to study wound infections in rats (25). Streptococcus pyogenes 301 is a clinical dermal infection isolate obtained from the University Hospital in Uppsala, Sweden. The Dry SPOT Streptococcal Grouping kit (DR0400 M; Oxoid Ltd., Basingstoke, United Kingdom) was used to determine the Lancefield type of the streptococcal strain. The streptococcal bacteria were grown overnight anaerobically at 37°C on defibrinated horse blood agar before they were tested according to the manufacturer's instructions. S. pyogenes 301 belongs to Lancefield serological group type A. The in vitro MICs of fusidic acid for these strains, as determined by Etest (AB BIODISK, Solna, Sweden), were as follows: S. aureus FDA486, 0.125 μg/ml and S. pyogenes 301, 4 μg/ml. S. aureus was grown in Luria broth and on Luria agar plates (Oxoid Ltd.). S. pyogenes was grown in Todd-Hewitt broth (Sigma Aldrich, Stockholm, Sweden) and on blood agar plates made by mixing 5% (wt/vol) defibrinated horse blood (National Veterinary Institute, Uppsala, Sweden) with Luria agar. S. aureus was grown aerobically at 37°C, and S. pyogenes was grown anaerobically (10% CO₂) at 37°C.

Tape-stripping infection model.Animal infection experiments were performed at the Microbiology and Tumor Biology Center, Karolinska Institute (Stockholm, Sweden), in accordance with institutional and national guidelines (ethical permit N154/02). Six- to 8-week-old female BALB/c mice (Taconic M&B, Ry, Denmark) were used for all experiments. The mice were anesthetized by intraperitoneal injection of 10 ml/kg of body weight of a 1:1:2 (vol/vol) mixture of Hypnorm (fentanyl/fluanisone; Janssen-Cilag Ltd., Saunderton, United Kingdom)-Dormicum (midazolam; Hoffman-La Roche AG, Basel, Switzerland)-distilled water. The fur was stripped from the mice with Tensoplast (Smith & Nephew Medical, Hull, United Kingdom), an elastic adhesive bandage. An area of ca. 2 cm² was tape stripped. In order to standardize the degree of barrier disruption elicited by the tape stripping, the transepidermal water loss (TEWL) was measured by using a DermaLab TEWL probe (Cortex Technology, Hadsund, Denmark). Measurements were made according to the guidelines of the Standardization Group of the European Dermatitis Society (27). TEWL is calculated automatically and is expressed in g/m² h. By tape stripping the back of the mice 7 to 10 times in succession, the TEWL reached approximately 70 g/m² h. Following this procedure, the skin became visibly damaged and was characterized by reddening and glistening but no regular bleeding. Microscopically, this procedure resulted in the controlled removal of most of the epidermal layer, with only a few basal epidermal cells remaining. After stripping of the skin, a bacterial infection was initiated by placing on the skin a 5-μl droplet containing 10⁷ cells concentrated from an overnight bacterial culture in stationary phase. In each experiment, a group of mice were killed 4 h after infection to control the infectious dose (Table 1). The mice were treated with FAO (LEO Pharma, Ballerup, Denmark) on a regular basis, as described here. This dose gave a significant reduction in the numbers of CFU in preliminary dose-finding studies (0.5%, 1%, and 2% fusidic acid) and is the dose recommended by the manufacturer for human use. The first application of antibiotic to the stripped skin of the mice was at 4 h postinfection. Thereafter, beginning at 16 h after the first treatment, additional antibiotic applications were made twice daily (in the morning and the evening, with an 8-h interval) for a period of 4 days. For each treatment 25 to 30 mg of ointment was applied (estimated by weighing the pellet of ointment on a spatula). After each day the ointment tube was weighed to determine the average amount of ointment used for each mouse. Two infection control groups were included for each experiment: one consisted of untreated mice and the other consisted of mice treated with placebo ointment. The placebo ointment was identical to FAO except for the lack of the 2% fusidic acid. For all experiments in which untreated or topically treated groups were included, the experiments were terminated 18 h after the last topical treatment in order to avoid carryover effects in vitro. The addition of fucidinase (2.5 units per sample) to the homogenized samples did not influence the numbers of CFU, showing that 18 h is sufficient to avoid a carryover effect. Immediately after the mice were killed, the wounds, approximately 2 cm², were excised and homogenized together with 1 ml of phosphate-buffered saline in stomacher lab system bags by using a Stomacher 80 machine (Seward Ltd., Thetford, United Kingdom) set at 260 strokes per min for 120 s. The homogenates were washed once in phosphate-buffered saline to decrease the concentration of ointment. Suitable dilutions of the homogenates were plated on Luria agar (S. aureus) or blood agar (S. pyogenes) plates to determine the number of living bacteria (CFU). In order to investigate the reproducibility of the infection with S. aureus and S. pyogenes, three independent experiments that included untreated and placebo-treated groups were performed. In each experiment the mean CFU was calculated by using log₁₀-transformed data. Based on the averages of these three experiments, the mean, range, and coefficient of variation were calculated.

Suture-wound model.The established skin suture-wound model was carried out as described previously (14).

Histological examinations.In order to characterize the histopathology of the model, biopsy specimens were taken after the following treatments: immediately after tape stripping, 4 days after tape stripping, and 4 days after inoculation with S. aureus and S. pyogenes with and without treatment with placebo ointment. Immediately after the animals were killed, 5-mm punch biopsy specimens of excised skin were taken and immediately fixed in phosphate-buffered (pH 7.4) formalin (4%). The formalin-fixed biopsy specimens were embedded in paraffin and stained with hematoxylin and eosin. For identification of the bacteria, the biopsy specimens were stained with Gram's crystal violet solution (94448; Sigma-Aldrich). The following parameters and semiquantitative scoring system were used to describe the inflammatory response: for scoring of the inflammation (in the dermis, subcutis, muscular tissue, and connective tissues), 0, no inflammation present; 1, little inflammation present; 2, moderate inflammation present; and 3, severe inflammation present; for scoring of the presence of neutrophils, 0, no neutrophils present; 1, a few neutrophils present; 2, moderate occurrence of neutrophils; and 3, abundant occurrence of neutrophils; for scoring of the presence of mononuclear leukocytes, 0, no mononuclear leukocytes present; 1, a few mononuclear leukocytes present; 2, moderate occurrence of mononuclear leukocytes; and 3, abundant occurrence of mononuclear leukocytes; for scoring of presence of bacteria, 0, no bacteria; 1, scattered bacteria; 2, moderate numbers of bacteria; and 3, many large collections of bacteria. The observer was blinded to the treatments for all biopsy specimens.

Behavioral responses of mice.The mice were observed at least twice each day for signs of fatigue, stress, and aggressiveness. The mice were weighed before and after each experiment.

Statistical analysis.Statistical analysis of the log₁₀-transformed data was performed to ensure variance homogeneity and normality. Thus, an analysis of variance (generalized linear models [8]) was applied, followed by four predefined pairwise treatment comparisons adjusted for multiplicity by the Bonferroni method (18), yielding a statistical ranking. Furthermore, to ensure robustness in the analysis performed, the nonparametric Kruskal-Wallis approach (21) was used. The method showed no conclusive dissimilarities to the generalized linear models approach. All testing was performed on an overall 5% significance level, meaning that P values less than 0.05 were considered a statistically significant difference. All calculations and analyses were performed with SAS version 8.2 (SAS OnlineDoc; SAS Institute Inc., Cary, NC). Log₁₀-transformed data are presented as the mean and standard deviation (SD) in Table 1, whereas actual values and corresponding median values are presented in the figures.

Establishment of staphylococcal infection.The number of CFU recoverable from the wound 4 h after application of 10⁷ CFU of S. aureus FDA486 was 7.03 ± 0.37 log₁₀ (Table 1). The different treatment regimens were begun after this initial 4-h period. There was no significant difference (P = 0.05) in the numbers of CFU per wound when 4 h versus 4 days of placebo treatment were compared (6.05 ± 0.31 log₁₀). This is evidence of the successful establishment of a staphylococcal infection in this model. There was a slightly greater reduction in the numbers of CFU per wound that was statistically significant (P = 0.01) when 4 h versus 4 days of no treatment (5.89 ± 0.64 log₁₀) were compared. The differences between the 4-day placebo treatment and 4 days of no treatment were not statistically significant (P = 1.00).

Effect of fusidic acid treatment on staphylococcal infection.Tape-stripped mice infected with S. aureus FDA486 were treated with FAO to test the efficacy of a topical antibiotic treatment in the new model (Fig. 1). Comparison of placebo (6.05 ± 0.31 log₁₀) and FAO (4.68 ± 0.78 log₁₀) treatment of the staphylococcal infection revealed a reduction in the numbers of CFU per wound that was statistically significant (P < 0.001).

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FIG. 1.

Tape-stripped mice infected with S. aureus FDA486. The number of bacteria (CFU) extracted from each mouse is represented by the symbol for the corresponding experimental group. The median value of the data for each group is shown as a horizontal bar. The number of mice in each experimental group was as follows: 4 h postinfection, n = 7; untreated, n = 8; placebo, n = 8; FAO, n = 12.

Establishment of streptococcal infection.The numbers of CFU recoverable from the wound 4 h after application of approximately 10⁷ CFU of S. pyogenes 301 was 6.48 ± 0.29 log₁₀ (Table 1). Comparison of the numbers of CFU after 4 h and 4 days of placebo treatment (6.15 ± 0.57 log₁₀) revealed no significant difference (P = 1.00). In contrast, comparison of the numbers of CFU at 4 h and 4 days without treatment (3.21 ± 1.62 log₁₀) revealed a significant reduction (P < 0.001). Comparison of the placebo and the untreated groups 4 days after infection confirmed the significance of the reduction in the numbers of CFU in the latter group (P < 0.001).

Effect of fusidic acid treatment on streptococcal infection.Comparison of placebo and fusidic acid treatments for the streptococcal infection (Fig. 2; Table 1) revealed a very significant effect (P < 0.001) of the antibiotic in clearing the infection. Thus, the mean CFU count after the 4-day FAO treatment (2.04 ± 1.31 log₁₀) was reduced more than 4 log₁₀ compared with that after the 4-day placebo treatment (6.15 ± 0.57 log₁₀). Statistical comparison of the 4-day FAO treatment group and the 4-day untreated group showed that there was no significant difference (P > 0.05).

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FIG. 2.

Tape-stripped mice infected with S. pyogenes 301. The number of bacteria (CFU) extracted from each mouse is represented by the symbol for the corresponding experimental group. The median value of the data for each group is shown as a horizontal bar. The number of mice in each experimental group was as follows: 4 h postinfection, n = 16; untreated, n = 16; placebo, n = 16; FAO, n = 8.

Reproducibility of the model.The reproducibility of the model was assessed in three independent experiments, each of which included both S. aureus and S. pyogenes, with untreated and placebo-treated mice. The coefficients of variance (CVs) for the untreated mice were 18% (mean, 6.09 log₁₀; SD, 1.10 log₁₀) for those infected with S. aureus and 36% (mean, 3.96 log₁₀; SD, 1.41 log₁₀) for those infected with S. pyogenes. The CVs for the placebo-treated mice were 30% (mean, 6.21 log₁₀; SD, 1.86) for those infected with S. aureus and 12% (mean, 6.62 log₁₀; SD, 0.81 log₁₀) for those infected with S. pyogenes.

Histological examination of infected and noninfected mice.The results for the scores of the inflammatory response, infiltrating neutrophils and mononuclear leukocytes, and the presence of bacteria are presented in Table 2. The tape-stripping procedure removed most of the epidermis, although a single layer of epidermal cells or scattered epidermal cells remained. Most of the remaining epidermal cells were necrotic. A minimal inflammatory response was evident immediately after tape stripping, and except for the missing epidermis, the skin appeared normal. Four days after tape stripping an acute subcutaneous phlegmonous inflammation with fibrin deposition and edema was observed with the infiltration of a few neutrophils. The inflammation was most pronounced in the subcutaneous and connective tissues. Inoculation of both S. aureus and S. pyogenes induced a late stage of subcutaneous fibrinoid necrosis after 4 days, with a few neutrophils mainly seen as nuclear dust deep in the subcutis (Fig. 3). In addition to neutrophils, the inflammatory cell infiltrate consisted of mononuclear cells, including lymphocytes and histiocytes. The inflammatory response was most intense in the subcutaneous tissues. For both mice infected with S. aureus and mice infected with S. pyogenes, the presence of bacteria (Fig. 4) was less evident in the untreated mice than in placebo-treated mice and the skin appeared dry compared to the skin of the placebo-treated mice. In some of the infected animals, fibrosis and remarkable regeneratory changes were observed in the subcutaneous tissue.

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FIG.3.

Histological appearance of normal dorsal skin of mice (A; magnification, ×100) and Staphylococcus aureus-infected skin lesion (B, magnification, ×100; C [boxed area in panel B], magnification, ×200; D [boxed area in panel C], magnification, ×1,000) on day 4. Biopsy specimens were taken immediately after the termination of the experiment, fixed in formalin, and embedded in paraffin. The biopsy specimens were stained with hematoxylin and eosin. The inflammatory cell infiltrate consists of mononuclear cells, including lymphocytes, histiocytes, and, to lesser extent, neutrophils. The inflammatory response is associated with marked fibrosis, edema, and fibrin deposition. Coccoid bacteria are present. The epidermal layer is absent in the infected lesions. Numbered arrows indicate the following: 1, epidermis; 2, dermis; 3, muscular layer; 4, bacteria; 5, fibrin deposition; 6, inflammatory cell infiltrate.

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FIG. 4.

Staining of gram-positive bacteria in Staphylococcus aureus-infected skin (magnification, ×1,000) on day 4. Biopsy specimens were taken immediately after the termination of the experiment, fixed in formalin, embedded in paraffin, and stained with Gram's crystal violet solution. Coccoid bacteria are present in the superficial layers of the dermis (arrows).

Behavioral responses of the mice.No abnormal behavioral patterns, such as fatigue, stress, or aggressiveness, were observed among the mice at any time during the course of these experiments. The mice gained, on average, 0.5 g of weight during the experiment, with no differences between the various test groups. A small fraction (2 of 48) of the mice displayed signs of deeper infections, with necrotized tissue and softening of the backbone skeletal structure (data not shown).