Experimental protocol.

Animals were randomized and assigned to five groups. Animals were further assigned to subgroups according to the time of observation (7, 14, and 21 days; 10 animals for each subgroup) for a total of 120 rats. The study included two uninfected control groups treated and not treated with daptomycin (7 mg/kg every 24 h [q24h]), an infected group that did not receive any treatment, and two infected groups treated, respectively, with intraperitoneal daptomycin (7 mg/kg q24h) and teicoplanin (7 mg/kg every 12 h) for 21 days. Drug dosages were determined as described in our previous studies and according to pharmacokinetic and pharmacodynamic information from other experimental studies (39,–42).

The rats were anesthetized by intramuscular injection of ketamine-xylazine (40 mg/kg and 13 mg/kg, respectively), and the back surface of their bodies was shaved and washed with povidone iodine-propanol solution. A copper bar (12 by 12 mm) heated in boiling water (100°C for 10 min) was placed on the paraspinal site of each animal for 40 s without pressure. Only the weight of the block was used to create the burns. After reheating of the probe in boiling water, a second burn was made as symmetrically as possible on the other side of the back, resulting in two full-thickness burns. In order to avoid variations in the creation of the burns, one person (F.O.) created all burns. A small gauze was placed over each burn and then inoculated with 5 × 10⁷ CFU of S. aureus ATCC 43300. The pocket was closed by means of skin clips (43). This procedure resulted in a local infection at 24 h. After 24 h, in control animals the burn was opened, the gauze was removed for quantitative bacterial culture, and treatment was initiated. Antibiotics were administered intraperitoneally daily for 21 days.

During surgery, the body temperature (36 or 37°C) of the animals were maintained and controlled using a homeothermic blanket (Harvard Apparatus). The rats were then resuscitated with 1 ml of Ringer's lactate solution administered by intraperitoneal injection. The injury was protected with a strip of cloth. The animals were then returned to their individual cages and thoroughly examined daily.

To mimic the clinical situation in burned patients, surgical debridement was performed 48 h after the injury. The skin and subcutis of the animals, which underwent general anesthesia with isoflurane, were excised in the dorsal area near the edge of the burn, avoiding the panniculus carnosum and the muscle layer. This was done to reduce the high risk of wound colonization and minimize the high risk of hypertrophic scarring. The wounds were left to heal by secondary intention (i.e., the wound edges were not closed by sutures). To protect the wounds from outside contamination and infection, a sterile hydrated gauze was used. The animals were returned to their individual cages and thoroughly examined daily. Each wound was gauged every 3 or 4 days according to the method described by Morton and Malone (44). Each dressing change was made while the animals were under general anesthesia with isoflurane. After each measurement, the dressing was removed and the wound was flushed with sterile solution. When the healing was completed, tissue specimens were collected from each treated area and samples were processed for morphological analysis. At the end of the experiment, all the rats were sacrificed with excess anesthetic. Skin samples were divided into two pieces. One piece was used for histological examination (see below), and the other was homogenized in 1 ml phosphate-buffered saline (PBS) using a stomacher. Quantitation of viable bacteria was performed by culturing serial dilutions (0.1 ml) of the bacterial suspension on blood agar plates. All plates were incubated at 37°C for 48 h and evaluated for the presence of bacteria. The organisms were quantitated by counting the number of CFU per plate. The limit of detection for this method was approximately 10 CFU/g. Toxicity was evaluated on the basis of the presence of any drug-related adverse effects, behavioral alterations, and local signs of inflammation. In particular, all the animals were evaluated for the correct consumption of food and drinking water, general locomotor activity, and the display of kyphosis.

Body weights were measured once a day during the experimental period. The amounts of drinking water and food supplied and the residual amounts of drinking water and food were weighed daily in order to calculate the average daily water and food consumption through the entire treatment period. Those animals who were sick, did not adequately eat or drink, or were crouching in the corner were excluded from the study.