Figure 3

Chitosan and cloxacillin combination can eradicate preformed biofilms and increase bacterial killing inside them. CNS isolates from chronic intramammary infections were grown in 96-well plate for 24 h at 37 °C. Preformed biofilms were washed with PBS and incubated in the presence of medium or different concentration of cloxacillin [6, 48, 256 µg/mL], chitosan [200 µg/mL] or cloxacillin and chitosan combined for 24 h at 37 °C. Biofilm biomass and viable cells were evaluated by CV, plate count and CLSM. (A) Cristal Violet evaluation and biofilm generated in control condition (medium) was considered as 100% of biomass. (B) Bacterial Viability was evaluated in plate count (CFU/mL). (C) Bacterial viability and biofilm 3D structure were evaluated by staining with SYTO9 and PI dyes and analyzed by CLSM microscopy. CLSM images belong to CM-CNS7 isolation which was taken as representative. The other isolations showed similar efficiency results. These experiments were performed three independent times with two biological replicates and the data are represented by six independent replicates. Data are shown as mean ± SEM. P values were obtained using two-way ANOVA followed by Bonferroni post-hoc analysis. P values *< 0.05, **< 0.01 and ***< 0.001 were considered significant.