Staphylococcal Biofilm Quantification and Removal

Staphylococcal biofilm formation and removal was performed as previously described using the RTCA technology (Gutiérrez et al., 2016 (link); Gutierrez et al., 2017 (link)). Briefly, 100 μl of S. aureus 15,981 were diluted in TSBg (TSB supplemented with 0.25% w/v D-(+)-glucose) and poured into 16-well E-plates (∼10⁶ CFU/well). E-plates were connected to the xCelligence RTCA-DP (ACEA Biosciences Inc., San Diego, CA, USA) holder which measure the impedance signal and represent cell index (CI) values. Biofilm were formed for 8 h at 37°C and then 100 μl of LysRODI and LysA72 were added to achieve a concentration gradient (0.14–9.15 μM and 0.21–13.42 μM, respectively) and incubated for an extra 16 h. Impedance values were further processed using the RTCA software 1.2.1 (ACEA Biosciences Inc.) as described previously (Gutierrez et al., 2017 (link)) in order to calculate: (i) the percentage of biofilm removal compared to control values after 16 h of treatment; (ii) the minimum biofilm eradicating concentration that removes 50% of the biofilm (MBEC₅₀), (iii) the lowest antibiofilm effect (LOABE; lowest concentration needed to observe an antibiofilm effect) and (iv) the specific antibiofilm activity expressed as Δbaseline normalized CI × min⁻¹ × mM⁻¹. All the experiments were performed in triplicate.