Nylon foam swab

Bacteria were cultured in tryptic soy broth (TSB) at 37°C overnight, washed with PBS, and re-suspended in PBS at 1 × 10⁶ CFU/5μL. To determine a capacity of antibiotic-producing CoNS, non-antibiotic-producing CoNS or S. aureus strain to colonize on the inflamed or non-inflamed skin, an 8mm TSB-agar disk containing each bacterial strain (1 × 10⁶ CFU) was applied on the skin after disinfecting the skin with alcohol swabs twice. The entire dorsal skin was then covered with wound dressing film for quantitative bacterial application and to avoid contamination.^{18 (link)} Tegaderm and agar disc were removed 24 h after bacterial application and mice were kept additional 24 or 48 h in clean cage. After bacterial application, up to 2 mice with the same bacterial strain were housed. An agar disc without bacteria was used as negative control. The entire skin sheet was rubbed 50 times with nylon foam swab (Puritan) pre-moisturized with PBS to collect bacteria on the skin surface. Swab sample was suspended in 1mL of 85% TSB/15% glycerol and frozen until analysis.

Fresh-frozen pigskin sheets were obtained from Loretta Tomlin Animal Technologies (Livermore, CA) and sanitized by surgical brush with 3% chloroxylenol. The skin sheet was cut into 2.5 cm × 2.5 cm and rinsed with sterile PBS more than 20 times to remove chloroxylenol residue. Synthetic LL-37 (2.5nanomol/50μL H₂O) or equal volume of vehicle was applied to the surface of sanitized pig skin. Antibiotic-producing or non-antibiotic-producing strain of CoNS (1 × 10⁶ CFU/10μL PBS) was applied on the pigskin. Antimicrobial activity of CoNS strains against S. aureus (ATCC35556) is shown in Figure S5. Pigskin sheet was placed on a 6-well plate and incubated at 30°C for 24 h. The entire skin sheet was rubbed 50 times with nylon foam swab (Puritan) pre-moisturized with PBS to collect bacteria on the skin surface. Swab sample was suspended in 1mL of 85% TSB/15% glycerol and frozen until analysis.