Evaluation of Peptide Effects on Bacterial Biofilm

Bacterial biofilm was grown on glass cover slips in 24-well plates in 0.5X MHB in static conditions. In particular, bacterial cells from an overnight culture were diluted to about 1 × 10⁸ CFU/mL and then seeded into wells for 4 or 24 h at 37 °C in the presence of the peptide under test, in order to evaluate biofilm attachment and formation, respectively. When effects on preformed biofilm were evaluated, bacterial biofilms were formed for 24 h at 37 °C, and then treated with peptides under test for further 24 h to evaluate their ability to eradicate preformed biofilm. Afterwards, non-adherent bacteria were removed by gently washing samples with sterile phosphate buffer and viability of cells embedded into biofilm structure was determined by sample staining with LIVE/DEAD^® BacLight™ Bacterial Viability kit (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA), while FilmTracer™ SYPRO^® Ruby biofilm matrix dye has been used to stain matrices of biofilms (Invitrogen, Carlsbad, CA, USA). Staining was performed accordingly to manufacturer instructions. Biofilm images were captured by using a confocal laser scanning microscopy (Zeiss LSM 710, Zeiss, Germany) and a 63X objective oil immersion system. Biofilm architecture was analyzed by using the Zen Lite 2.3 software package (Zeiss, Germany). Each experiment was performed in triplicate. All images were taken under identical conditions.