Penicillin

The antibiotic susceptibility of the all isolates was determined by the disk diffusion method on Mueller–Hinton agar (Becton–Dickinson, Sparks, MD, USA) according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI). The disk diffusion assay was run with 15 antibiotics: bacitracin (10 U), enrofloxacin (5 μg) (OXOID), streptomycin (10 μg), spiramycin (100 μg), sulfadiazine (25 μg), chloramphenicol (30 μg) (BD), doxycycline (30 μg), erythromycin (15 μg), gentamicin (10 μg), neomycin (30 μg), penicillin (10 U), tetracycline (30 μg), cefoxitin (30 μg), trimethoprim/sulfamethoxazole (1.25 μg/23.75 μg, respectively), and vancomycin (30 µg and 5 µg) (BIO-RAD, Hercules, CA, USA). S. aureus strain ATCC 25923 and Enterococcus faecalis strain ATCC 29212 were used as controls in the susceptibility test.

Susceptibility to 13 antibiotics was determined by the disc diffusion method and using the ADAGIO™ Automated System (Bio-Rad, Hercules, CA, USA) as described. The antibiotics tested included ampicillin (10 µg), penicillin (6 µg), ampicillin/sulbactam (20 µg), chloramphenicol (30 µg), vancomycin (5 µg), teicoplanin (30 µg), streptomycin (300 µg), gentamicin (120 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), quinupristin-dalfopristin (15 µg), linezolid (30 µg) and tigecycline (15 µg) (Bio-Rad, Hercules, CA, USA)]. Susceptibility to aminoglycosides, glycopeptides, quinolones and β-lactam antibiotics was also determined by an E-test (M.I.C. Evaluator™, OXOID, Basingstoke, UK). The methods and the interpretation of the results followed the CLSI guidelines [32 ]. Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 25923 were used as control strains.

FB and KC were separated from skins of nascent mice as previously mentioned [31 (link)]. Briefly, the newborn Kunming mice were executed with deep CO₂ anesthesia and sterilized. The skin were quickly removed, expanded, and floated on a cryogenic trypsin (with not EDTA, 0.25%, w/v, Sigma-Aldrich, Shanghai, China) overnight with the dermis side-down at 4 °C. The skins were treated with typsin, and then transferred to dry plates with the epidermis facing down and in contact with the plastic at various points on the edges. The dermis was directly raised higher than the epidermis. To prepare FB cells, dermis was transferred to plates containing Dulbecco’s Modified Eagle Media (DMEM, Solarbio) with 10% FBS, penicillin (200 U/mL), and streptomycin (200 μg/mL, Invitrogen, Carlsbad, CA, USA). To prepare KC cells, epidermis was transferred to plates containing S-MEM (Solarbio) chelexed with chelex®100 Resin (Bio-Rad, Hercules, CA, USA), 8% FBS, penicillin (200 U/mL) and streptomycin (200 μg/mL). After fully mincing and triturating, the suspension was filtered using cell strainer (1250 mesh, BD, Franklin Lakes, NJ, USA). After incubation for 24 h, the isolated cells were washed with PBS solution, while the suspension cells and fragments were discarded. The isolated cells were incubated in 5% CO₂ at 37 °C.