Murine Wound Infection Model

The procedure for mouse wound infections was modified from a previous study (15 (link)). Briefly, male C57BL/6 mice (6 to 8 weeks old, 22 to 25 g; NTU, Singapore) were anesthetized with 3% isoflurane. Following dorsal hair trimming, the skin was then disinfected with 70% ethanol before creating a 6-mm full-thickness wound using a biopsy punch (Integra Miltex). Bacteria (1 × 10⁷ CFU) were added to the wound site followed by addition of either 10 μl of PBS or 10 μl of MTX (0.515 μg/ml) immediately. Then, the wound site was sealed with a transparent dressing (Tegaderm 3M). When preventive treatment was tested, PBS or MTX was applied 24 hours before infection, and when cotreatment with vancomycin was performed, intraperitoneal injections of PBS or vancomycin (100 mg/kg in a maximum volume of 100 μl) were performed before the biopsy punch. When multiple treatments with MTX were performed, an 8-mm Finn Chamber on Scanpor was placed around the wound to facilitate removal of the transparent dressing for each treatment without disruption of the underlying bacterial biofilm. In total, five daily treatments of either 10 μl of PBS or 10 μl of MTX (0.515 μg/ml) were applied on the wound. After 24 hpi or 4 days post-infection, mice were euthanized and a 1 cm by 1 cm squared piece of skin surrounding the wound site was excised and collected in sterile PBS. Skin samples were homogenized, and the viable bacteria were enumerated by plating onto BHI plates.