2.8. Determination of Minimal Biofilm Inhibitory Concentration (MBIC) and Minimum Biofilm Eradication Concentration (MBEC) Assay

The antibiotic susceptibility test for biofilm was performed according to the procedures described by Moskowitz et al. [23]. After the biofilm was formed on the CBD pegs, the pegs were rinsed three times in a 96-well plate containing 150 µL of Maximum Recovery Diluent. The lid with pegs was then transferred to the new a standard 96-well plate with wells containing 150 µL Mueller–Hinton broth with antibiotics diluted two-fold serially (ranged from 2 to 1024 µg/mL). The 96-well plate was incubated overnight at 37 °C. Following incubation, turbidity in each well was examined visually. MBIC was defined as the minimal antibiotic concentration at which no bacterial growth was observed, which meant the minimal antibiotic concentration that inhibited the release of planktonic bacteria from the biofilm.

Following the MBIC examination, MBEC was determined. The lid with pegs was removed and rinsed three times in a 96-well plate containing 150 µL of Maximum Recovery Diluent to remove the planktonic cells. The lid was then placed in a second 96-well plate containing 150 µL Mueller–Hinton broth. The plate was shaken at a speed of 180 rpm for 10 min to detach the biofilm cells from the pegs into the Mueller–Hinton broth. The viability of the biofilm was determined by plate count after 24 h of incubation at 37 °C. MBEC was defined as the minimal antibiotic concentration required to eradicate the biofilm.