The routine preparation of the pig skin followed this procedure. Thawed pig skin (ex-abattoir, stored for weeks frozen) was epilated with a dry razor, stripped with a tape 10-times to remove the upper dead layers, and finally cut into 1 × 1 cm2 pieces. The pieces were sterilised firstly by immersing in 70% ethanol for 20 min, dried for 20 min in a biosafety cabinet, followed by soaking in a solution of antibiotics for 16 h (kanamycin sulfate 20 μg/mL and ampicillin 50 μg/mL). On the day of the experiment, a cut 0.9 cm long was created along the skin to reach the epidermal layer using a sharp knife. The cut pig skin squares were then placed on TSB agar plates prepared with antibiotics (kanamycin sulfate 20 μg/mL and ampicillin 50 μg/mL).
S. aureus MRSA252 or ATCC 25923 were cultured (15 h) in TSB-GN. Inoculation was with an aliquot (20 μL) of bacterial suspension applied to the epidermal side of the skin and spread uniformly. The inoculated skin pieces were incubated for 5 days at 37°C in a humidified chamber. Every day the pieces were transferred to a new agar plate. Four pig skin pieces were used in each repeat. On day 5, the pig skin pieces were covered with zein 3L, zein/PCL 3L (1 cm2), or a filter paper loaded with 30 μg Tet. A fourth pig skin piece was left untreated. Before covering the pig skin pieces with the 3L matrices, the surface of the biofilms was wetted with TSB-GN (10 μL) and, after coverage, another 20 μL of broth was applied on top of the matrices. The plates were then incubated again at 37°C for 24 h. Each skin piece was then transferred to a tube containing MH broth (5 mL) and kept cool on ice during the experiment. The tubes were vortexed extensively (~5 min) in order to detach mechanically the bacteria from the skin. Suspended cells were then serially diluted to 10−7 in broth, and aliquots (10 μL) of 10−4, 10−5, 10−6, and 10−7 dilutions were spotted on TSB-GN agar plates, to determine the colony forming units (CFU)/mL using the Miles-Misra method (23 (link)). The plates were incubated at 37°C for 24 h and the numbers of CFU were counted. The number of CFU/skin piece was determined using the above formula; this was then normalised by the number of CFU/untreated skin piece.
S. aureus MRSA252 or ATCC 25923 were cultured (15 h) in TSB-GN. Inoculation was with an aliquot (20 μL) of bacterial suspension applied to the epidermal side of the skin and spread uniformly. The inoculated skin pieces were incubated for 5 days at 37°C in a humidified chamber. Every day the pieces were transferred to a new agar plate. Four pig skin pieces were used in each repeat. On day 5, the pig skin pieces were covered with zein 3L, zein/PCL 3L (1 cm2), or a filter paper loaded with 30 μg Tet. A fourth pig skin piece was left untreated. Before covering the pig skin pieces with the 3L matrices, the surface of the biofilms was wetted with TSB-GN (10 μL) and, after coverage, another 20 μL of broth was applied on top of the matrices. The plates were then incubated again at 37°C for 24 h. Each skin piece was then transferred to a tube containing MH broth (5 mL) and kept cool on ice during the experiment. The tubes were vortexed extensively (~5 min) in order to detach mechanically the bacteria from the skin. Suspended cells were then serially diluted to 10−7 in broth, and aliquots (10 μL) of 10−4, 10−5, 10−6, and 10−7 dilutions were spotted on TSB-GN agar plates, to determine the colony forming units (CFU)/mL using the Miles-Misra method (23 (link)). The plates were incubated at 37°C for 24 h and the numbers of CFU were counted. The number of CFU/skin piece was determined using the above formula; this was then normalised by the number of CFU/untreated skin piece.
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