Fig. 5: Prefrailty aging in mice is linked with altered gut biofilm and emergence of pathogenic behavior when transferred into recipient mice.

A–C Using 16S fluorescent in situ hybridization, we visualized tissue-associated microbiota in the distal colon of young (6 months, 6 M, n = 8, A), aged (24 months, 24 M) robust (n = 7, B), and aged prefrail (n = 7, C) mice. Representative images are presented with blue is DAPI staining for host nuclei, green is fluorescein-coupled wheat germ agglutinin for glycoproteins-rich content (e.g., mucus layer), and red is the 16S-Cyanine3 probe for all bacteria. Dashed lines represent the limits of mucosal tissue and the “inner mucus layer”. Arrows highlight areas of interest. Scale bars are 25 µm. D Biofilm-damage scores were calculated for the three groups of mice, with dots representing the mean score from at least 3 fields per animal. E–G C57Bl/6 animals (8 weeks old) were previously exposed to 10-day antibiotics and underwent oral gavage with either vehicle (PBS, n = 5), or with feces from young (6 M, n = 9), aged robust (24 M robust, n = 8), and aged prefrail (24 M prefrail, n = 7) mice. E Four days after inoculation, feces were collected for microbial taxonomy assessment using 16S rRNA V3-V4 gene sequencing. Colons were harvested for (F) parietal thickness measurement and (G) macroscopic damage score. D, F, G Bar plots correspond to mean value per group ±SEM. Statistical significance was determined by ANOVA followed by Dunnett’s test for multiple comparisons, where P < 0.05 was considered significant (* for P < 0.05, ** for P < 0.01).