2.4. Biofilm Eradication Analysis on Nephrophane Membrane

The effectiveness of biofilm matrix degradation by PA5oct phage was evaluated by laser interferometry method [20]. PAO1 biofilm was formed on the surface of Nephrophane membrane during the incubation for 72 h at 37 °C in Trypticase-Soy broth (TSB, bioMerieux, Craponne, France). After supernatant removal, the level of membrane coverage was determined as around 93% (Figure 2A). Subsequently, the biofilm was treated for four hours at 37 °C with an intact or UV-inactivated phage suspensions (5 × 10⁸ pfu/mL). After treatment, the membrane was washed with saline and examined by interferometry technique. The degradation of biofilm was assessed as the increase of biofilm matrix permeability for low-weight molecular compounds, meaning the TSB medium itself. The quantitative measurements of TSB diffusion through biofilm structure treated with phage was obtained by laser interferometry method [20,23]. The data were analyzed using the Statistica software package (StatSoft, Tulsa, OK, USA). All values obtained at the end of 40 min measurements were expressed as mean ± SD and significant differences between variations (denoted p-values < 0.05) were assessed using the Snedecor–Fisher test using a one-way ANOVA.

Biofilm eradication was also examined by crystal violet (0.004%) staining for 15 min as previously described [20] and tested for pyocyanin and pyoverdin/pyochelin levels in the supernatants as described below.