Anti-Biofilm Activity of CMB001

Anti-biofilm activity of CMB001 was evaluated using the method of Cruz et al. (2018) (link) with minor modifications. Briefly, aliquots of S. aureus ATCC 29213 suspended in TSB supplemented with 2.5 g/L glucose were incubated in Nunclon polystyrene 96-well plates (Thermo Fisher Scientific), for 24 h, after which the wells were washed to remove planktonic cells. Fresh media was added, and the plates were further incubated overnight at 32°C to allow for biofilm formation. CMB001 was added to the final concentration 1.6-100 μg/mL and incubated with the biofilm for 4 h. To assess the viability of cells in biofilm plates were washed and stained with the cell permeable resazurin-based PrestoBlue Cell Viability Reagent (Thermo Fisher Scientific) and fluorescence measured using SpectraMax M microplate reader (Molecular Devices). To test in a “pre-treatment” format, the surfaces of 96-well plates were coated with CMB001, then washed to remove unbound peptide. S. aureus ATCC 29213 suspended at an OD_600nm of 1 was added to peptide-coated plates and incubated for 1 or 24 h at 37°C. At the end of the incubation period, non-adherent cells were removed, wells were washed 3× in PBS and cells were stained with PrestoBlue, as described above. Percent cell viability was calculated based on fluorescence after treatment relative to control fluorescence, measured in the presence of media.