2.4. Biofilm Assay and Determination of Minimum Biofilm Eradication Concentration (MBEC)

The assay was carried out using the Calgary 96-well Biofilm Device (Innovotech Inc.: Edmonton, AB, Canada) as previously described in [21]. Briefly, bacterial suspension was adjusted to 1×10⁷ CFU/mL. The biofilm device was inoculated by adding 150 μL of the inoculum into the wells of the 96 peg-lids on which the biofilm cells could build up. Sterility wells were inoculated with 150 μL of PBS. The pegs were incubated in a humidified incubator for 18–24 h under a rotation of 110 rpm at 37 °C to allow biofilm formation on the purpose-designed pegs. Once the biofilms were allowed to form, the pegs were rinsed with phosphate-buffered saline (PBS) to remove planktonic cells. Each peg-lid was then transferred into a ‘‘challenge 96-well microtiter plate’’ containing 200 μL of serially diluted antibiotics. Growth control wells and sterile control wells were filled with 200 μL of mycorrhiza helper bacteria (MHB) and PBS, respectively. The peg lids in the challenge plate were then incubated for 18–24 h at 37 °C. Finally, the peg lids were removed from the challenge plate and rinsed with PBS, then transferred into the recovery plate containing 200 μL of MHB in each well. The plate was incubated overnight at 37 °C.

To determine the MBEC values, the recovery plate was assessed visually for turbidity, and by measuring the optical density at 630 nm using a microtiter plate reader. Any visible growth indicated the detachment and re-growth of bacterial cells from the treated biofilms. The MBEC value represents the minimum antibiotic concentration that eradicates the biofilm following a suitable period of incubation [22]. Thus, the lowest antibiotic concentration that showed wells with no growth—visually clear with measured optical density of less than 0.1—was considered the MBEC for the selected antibiotic.