Saline solution

Bacterial colonies, grown overnight on blood agar plates, were inoculated into 3 ml of 0.45% saline solution (Air Life, Carefusion, CA, USA) to obtain a turbidity of 0.5 ± 0.1 McFarland (McF) corresponding approximately to 1 × 10⁸ colony-forming units (CFU)/ml. Samples were diluted 1:1000 and resuspended in 1 ml of brain heart infusion broth (BHI) in a μ-Slide, 8 well glass bottom chamber slides (Ibidi, Germany). The bacterial suspension was incubated at 37 °C for 24 h to allow biofilm formation. Afterwards, the medium was removed and biofilms were washed with 0.45% saline solution. The samples were stained using the LIVE/DEAD BacLight kit (Life Technologies, New York, NY, USA), according to supplier specifications. Biofilm samples were analyzed using a Zeiss LSM5 Pascal Laser Scan Microscope (Zeiss, Oberkochen, Germany) as described previously [36 (link)].

MICs were determined for each strain using the broth microdilution method described previously^{116 (link)} and adapted to the specific growth conditions of C. acnes. Specifically, C. acnes strains grown on Schaedler agar plates were inoculated into 2 mL of 0.45% saline solution (Air Life, Carefusion, CA, USA) to obtain turbidity of 0.5 ± 0.1 McFarland turbidity standard (approximately 10⁸ CFU/mL). Samples were diluted at 1:100 in BHI, and 100 μL of bacterial suspension, were seeded into a sterile 96-multiwell polystyrene plate containing different antibiotics at variable concentrations (Corning Inc., Corning, NY, USA). Serial two-fold dilutions of the amoxicillin/clavulanic acid, ampicillin, benzylpenicillin, clindamycin, doxycycline, ertapenem, imipenem, meropenem, moxifloxacin, piperacillin/tazobactam, tigecycline, vancomycin were prepared. After the antibiotic treatment, viable cells were determined by plate counting for the CFU/mL determination. The MIC was defined as the lowest concentration of an antibiotic preventing bacterial growth. Experiments were conducted in triplicate.