4.9. Antibiofilm Assay

The inhibition of the peptides on biofilm attachment of S. aureus was detected. Overnight cultures of S. aureus were resuspended in TSB (containing 1% glucose) to a concentration of 2 × 10⁸ CFU/mL. Aliquots of 100 μL suspended bacteria were added to a 96-well plate containing 100 μL peptides in TSB by two-fold dilution, and the peptide-free were served as a negative control. The plate was incubated at 37 °C for 4 h without agitation to allow bacterial attachment and the biomass of S. aureus biofilm was examined with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay as below.

Briefly, S. aureus were grown to the logarithmic phase in TSB at 37 °C and diluted in TSB (with 1% glucose) to obtain a concentration of 2 × 10⁶ CFU/mL. S. aureus suspensions were inoculated into 96-well microplates in the absence (negative control) or in the presence of different concentrations of each peptide. Then the plates were incubated at 37 °C for 24 h and the biomass of S. aureus biofilm were examined with MTT assay as below.

To evaluate the biofilm-disrupting property, S. aureus (10⁶ CFU/mL) was made from exponentially growing bacteria in fresh TSB media. Two hundred microliters inoculum culture were seeded to a 96-well-flat-bottom plate and incubated at 37 °C for 24 h to obtain the mature biofilm. Afterwards, the plate was washed by sterile phosphate buffered saline (PBS) twice and treated with a series 200 μL peptide solution at 37 °C for 24 h. The ability of peptide disruption of biofilms was quantified with MTT assay as below.

The metabolic activity of the biofilms formed by the bacteria was assessed using a modified MTT assay [51]. Briefly, MTT was dissolved in PBS to 5 mg/mL. The culture medium in the plates treated as above was removed gently and the plates were air-dried. Five microliter of 5 mg/mL MTT solution and 95 µL of PBS (pH 7.2) was added to each well and incubated for 3 h at 37 °C. The insoluble purple formazan was further dissolved in 150 μL dimethyl sulfoxide (DMSO). The absorbance was measured at 570 nm using the microplate reader. The percentage inhibition of metabolic activity was calculated as: [(OD₅₇₀ control without peptide − OD₅₇₀ peptide)/OD₅₇₀ control without peptide] × 100%.