Fig. 2: Prefrailty associated microb-aging is linked with physical alterations of human biofilms.

Feces from individuals aged 30–70 years, 70+ years robust, and 70+ years prefrail, were cultured in vitro as a polymicrobial anaerobic biofilm. A, B The total biofilm biomass and D, E the total sialic acid/N-acetylglucosamine glycoproteins content of extracellular biofilm matrix were measured with WGA stain. A–D Pearson correlation relationship between biofilm measures (biomass in A, WGA-matrix in D) and donor’s age is presented. Shaded area around the regression line represents the 95% confidence interval. B–E Scatter plots depict for each individual’s its corresponding value of biofilm biomass (B) and biofilm WGA-matrix (E) and were grouped for each three cohort of individuals. B, E each dot is the average value of an individual donor biofilm (at least 12 replicates). The gender of each donor is indicated using open(male)/filled(female) circles. Statistical significance was determined by ANOVA followed by Tukey’s for multiple comparisons, where P < 0.05 was considered significant. C–F Heatmaps represent Kendall (F) or Pearson (C) correlation coefficients between biofilm physical structure and clinical data (C) or biofilm taxa counts (F). Clinical parameters include discrete and continuous clinical features: household (1: solo 2: with a partner), gender (1: male, 2: female), MNA score, Charlson index, age, and Fried Frailty Index. The color gradient ranges from blue (negative) to red (positive) correlations, with statistically significant associations (P < 0.05) marked with *. For enhanced clarity, the heatmap in (F). was filtered to include only taxa rank with significantly positive or negative correlations (P < 0.05).