2.7. Minimum Biofilm Eradication Concentration (MBEC)

Biofilms of PA01 and PA11 adjusted to 0.5 McFarland standard (1.5 × 10⁸ CFU/mL) were grown for 72–96 h in MHB, in an MBEC^TM plate (CBD-Innovotech, Edmonton, AB, Canada) as recommended [55]. After incubation, the peg lid with biofilms was rinsed in a fresh 96-well plate filled with PBS, transferred to another plate containing serial dilutions of antibiotics combined with PABN at 25 mg/L and incubated for 24 h, at 37 °C, 110 rpm in the shaking incubator. Control wells were filled with MHB instead. After incubation, the peg lid was rinsed with PBS for a few seconds and placed in a recovery plate, a fresh 96-well plate with 200 µL of MHB per well and biofilms were sonicated for 30 min in an ultrasonic dismembrator bath. The recovery plate was incubated for 24 h at 37 °C, 110 rpm and the MBEC was determined as the smallest concentration of antibiotics to eradicate biofilms. Reductions of 2 fold or more with PABN were considered significant [44].