Methicillin-resistant Staphylococcus aureus strain MW2 was grown in Mueller Hinton broth at 37°C with shaking overnight. The culture was centrifuged, supernatant aspirated and the bacteria were gently washed once in sterile saline. The optical density was determined at 600 nm. The bacterial suspension was diluted to provide a challenge inoculum of approximately 500 CFU per wound in a volume of 0.05 ml in sterile 0.9% NaCl. The inoculum count was verified by viable counts on Mannitol Salt Agar plates spread with proper dilutions of the inoculum and incubated at 37 °C for 24–48 h.
For the wound infection model, 8-week-old male (~200 g) Sprague Dawley rats were given two wounds each. Two rats were used at each time point (day 1 and day 3) for each treatment group for a total of 4 rats (8 wounds) per drug. This sample size was statistically calculated on the basis of previous in-house wound burden studies comparing vehicle-treated groups with antibiotic controls. Rats were randomly selected into the treatment groups.
To generate wounds, the rats were anaesthetized by intraperitoneal injection of 100 mg kg−1 ketamine + 10 mg kg−1 xylazine and the dorsal side of the rats was shaved with electrical clippers and then depilated with Nair. The exposed skin was wiped with betadine. Two symmetrical wounds were made on the dorsum of each rat using a 0.8-cm-diameter disposable biopsy punch. Sterile polyurethane rings serving as wound chambers were placed over the fresh wounds and attached by surgical adhesive.
After the wound creation, rats from each group were infected with 0.05 ml of the bacterial suspension for a final infection dose of 500 CFU per wound. Wounds were covered with Tegaderm visible adhesive dressing, and the rats were rehydrated with physiological saline administered via intraperitoneal injection. The analgesic buprenorphine (0.05 mg kg−1) was administered to minimize pain during surgical recovery.
At 30 min post infection, rats were given single daily topical treatments of vehicle (25 mM CaCl2 in sterile water), or 0.5 mg malacidin A or daptomycin suspended in 25 mM CaCl2, and the wounds were covered in fresh Tegaderm dressing.
At 1 day and 3 days post infection, the rats were humanely euthanized and wounds were excised and assessed for bacterial burdens by plating on MSA. Rats were observed twice daily for morbidity and possible signs of acute toxicity. Abnormal clinical signs were recorded if observed. Rats were housed in the Public Health Research Institute’s Animal Biosafety Level-2 Research Animal Facility (ICPH RAF), a centre of the New Jersey Medical School, Rutgers University (NJMS-Rutgers).
The animal facility follows the Public Health Service and National Institute of Health Policy of Humane Care and Use of Laboratory Animals. All experimental protocols were approved by the Rutgers Institutional Animal Care and Use Committee (IACUC).
