Fig. 6: Positive impact of grape pomace on prefrailty-associated human biofilm structure and virulence.

Polymicrobial anaerobic biofilms were generated from human feces of prefrails individuals aged >70 yrs, n = 4–5 per group) and exposed to grape pomace diluted in microbial broth at concentrations of 3–30 mg/ml for 24 hours. A Total biomass was evaluated using safranin-O stain, and the data are presented as the fold change of mean biomass from control prefrail biofilms (broth media). B The rate of dispersal for each biofilm was evaluated as a ratio between the amount of biofilm-dispersed bacteria and biofilm biomass previously assessed. C Normalized counts of biofilm-dispersed bacteria were cultured on the apical surface of the human Caco2/HT29MTX monolayer. After a 4-hour coculture, the number of bacteria adhered to the epithelium was quantified by plating and was represented as fold change of vehicle-treated biofilms (broth media). D–F Dispersed bacteria were collected from biofilms previously exposed to grape pomace. Human intestinal epithelial cell lines (Caco2 and HT29MTX, at a 3:1 ratio) were then exposed to these bacteria for 4 hours, and mRNA expression of IL6 (D), TNF (E), and IL8 (F) genes was assessed. A–C Bar plots corresponds to average value ± SEM of 12 biofilm replicates from human donors (represented as individual dots). D–F Bar plots corresponds to average value ± SEM of relative mRNA expression of vehicle control group (n = 4 human donors). Statistical significance was determined by ANOVA followed by Dunnett’s for multiple comparisons, where P < 0.05 was considered significant and * for P < 0.05 and *** for P < 0.001.