Chronic Wound Biofilm in db/db Mice

Chronic wounds were made in db/db^−/− mice as previously described [24 (link)]. Six to seven-month-old db/db^−/− mice were treated intraperitoneally with a catalase inhibitor, 3-amino-1,2,4-triazole, at 1 g/kg body weight before wounding. Immediately after wounding, they were treated once topically with the glutathione peroxidase inhibitor, mercaptosuccinic acid, at 150 mg/kg body weight. Using our procedure, the wounds are fully chronic in 20 days and contain abundant biofilm. All bacteria forming the biofilm are from the mouse natural skin with no additional manipulation or introduction of laboratory-carried strains; therefore, the biofilm forms naturally from the microbiome in the skin. For these studies, the biofilms were collected using the Levine method with sterile cotton swabs and stored for bacteria identification both dry at -80°C and in Luria-Bertani (LB) media (10 g tryptone, 5 g yeast extract, and 10 g NaCl and Milli-Q water to 1 L; Teknova) supplemented with 20% glycerol. Swabs in LB media with 20% glycerol were then cultured in fresh LB media overnight at 37°C (150 rpm) and aliquots stored at -80°C.