Fig 2: Biofilm formation on Candida species isolated from patients with persistent and non-persistent candidemia. Candida bloodstream strains isolated from all included patients were evaluated for biofilm formation with standardized methods. The first Candida spp. isolate was conducted for biofilm formation evaluation when multiple isolates were collected from some of these patients within the study period. Candida isolates standardized (1 × 106 cells/mL) in RPMI-1640 were grown in flat-bottomed 96-well microtiter plates for 24 h at 37°C. The biomass quantified by staining with 0.05% w/v crystal violet solution. Biomass was quantified spectrophotometrically by reading the optical density (OD) at 550 nm in a microtiter plate reader (FluoStar Omega BMG Labtech, Aylesbury, United Kingdom). Three replicates were used for each isolate. The outlier was abandoned and the average of the 2 remaining values was represented. Higher biofilm formation in C. albicans, C. tropicalis, and C. glabrata were found among isolates in the persistent candidemia group compared with those in the non-persistent candidemia group.

Fig 3: Fluorescence signal intensity of individual transformants and correlation with expression of Cel7A by western blotting. a Transformants for Cel7A-2A-eGFP were analyzed for fluorescence intensity and Cel7A expression. b Transformants for eGFP-2A-Cel7A were analyzed for fluorescence intensity and Cel7A expression. Fluorescence intensity was measured using FLUOstar Omega plate reader (BMG Labtech GmbH) and is represented using arbitrary fluorescence units. Western blotting was performed on cell-free media as shown in Figs. 4 and 5. Each bar represents data obtained for a single transformant colony. Blue bars indicate transformants showing no Cel7A expression. Yellow bars indicate transformants showing Cel7a expression. Red bars represent control strains. Controls: JLT102A (AST1116 expressing native Cel7A under the eno promoter), AST1116 (Cel7A-deleted T. reesei QM6a strain), and SV004 (AST1116 expressing Cel7A-2A-eGFP; colony A5-4 in Fig. 3a)

Fig 4: Monospecies cariogenic biofilm biomass is influenced by carbohydrate source. Biomass was quantified spectrophotometrically by reading absorbance at 570 nm in a microtiter plate reader (FluoStar Omega, BMG Labtech). Four replicates were used for each isolate and was carried out on triplicate separate occasions, with the mean of each represented. Data represents mean with significance *p < 0.05. (A) Ten isolates with caries and caries free were standardized to 1 × 107 cells/mL in BHI with different carbohydrate and grown as biofilms in flat-bottomed 96 well microtiter plates for 24 h. Different concentrations of sucrose, mannose, fructose, glucose, galactose were added into BHI media (1.0% w/v). Biomass quantified by crystal violet staining. Biomass from caries group and caries free group were compared. (B) Supernatant of the mature biofilm were collected and the pH values of them were detected by pH meter. pH values from caries group and caries free group were compared. (C) Biofilms were also analyzed by SEM at both 1000× and 5000×. Samples were processed and viewed on a JEOL JSM-6400 scanning electron microscope and images assembled using Photoshop software. Strains from caries free group were grown in 1% galactose and 1% sucrose were compared with caries group separately.

Fig 5: Growth inhibition of N. crassa by increasing concentrations of PAF and PAFB. The antifungal activity was tested in a microdilution broth assay by inoculating conidia (104/mL) with increasing concentrations of PAF or PAFB in undiluted Vogel's medium and incubation for 30 h at 25 °C without shaking. Growth was determined spectrophotometrically (FLUOstar Omega, BMG Labtech) measuring the optical density at ? = 620 nm. Values represent the mean ± SD (n = 3, technical triplicates) growth (%) in the presence of AMPs in comparison to the untreated control which was set to be 100%. The result of one representative experiment of two biological repeats is shown.