Figure 4.

Evaluation of the effect of modified CFSs from LJO02 produced in TIL on S. aureus growth and biofilm formation. (a) S. aureus planktonic cell and biofilm OD₆₀₀ after 24, 48, and 72 h of incubation with the CFSs from LJO02 in TIL at pH 6.6 or enzymatically-digested (considering T₀ = 100% viability in each experimental condition). OD = optical density. CFS = cell-free supernatant. TRY = trypsin; PK = proteinase K; PE = pepsin. (b) Planktonic cell and (c) biofilm S. aureus viability assessment after 24, 48, and 72 h of incubation with modified CFSs. Data are expressed as mean ± SD of 3 independent experiments (n = 4). Trypsin and proteinase K-treated CFSs significantly reduce the planktonic cell viability compared to basification and pepsin treatment, similarly to what occurs with the untreated CFS (p < 0.0001). Biofilm viability is significantly reduced by trypsin and proteinase K-treated CFSs with respect to basification and pepsin at all tested times (p < 0.0001), but undigested CFS still shows the best performance (p < 0.0001 at 24 and 48 h, and p < 0.01 at 72 h with respect to trypsin and proteinase K-digested CFS). Two-way ANOVA was applied with Tukey’s comparison test. **p < 0.01, ****p < 0.0001. RFU = relative fluorescence units. (d) CV-reading at 570 nm of S. aureus biofilm at the 3 tested time points. (e) Representative 2D and 3D images of CV-stained S. aureus biofilm after 72 h of treatment with the modified CFSs. 2D pictures magnification: 460 X. Scale bar: 100 μm.