Minimal bactericidal concentration of biofilms (MBC-B) assay

The MBC-B was determined as described previously (26). Briefly, cells were grown overnight at 37°C at 220 rpm, collected by centrifugation, washed twice, and resuspended in BM2. The bacterial suspension was then diluted in BM2 biofilm medium to an optical density at 600 nm (OD_600nm) of 0.1 (18) to obtain approximately 1 × 10⁸ CFU/mL. An aliquot of 100 µL was placed into 96-well microtiter plates, and the plates were incubated for 24 h at 37°C under static conditions to allow biofilm formation. The supernatant was removed, and 200 µL of NaOCl (2–1,024 µg/mL) or H₂O₂ (1.95–100 mg/mL) diluted in BM2 was added to the pre-formed biofilms. The plates were incubated for 24 h at 37°C, unless otherwise stated, the supernatant was removed, and 100 µL of LB media was added to the wells, followed by incubation for 24 h at 37°C. Finally, the 96-well microplates were stamped and transferred on a 2% (wt/vol) LB agar plate and incubated for 24 h at 37°C. The MBC-B was considered the lowest concentration of oxidizing agent where no bacterial growth was observed.