Fig. 4: Biofilm-associated microbiota can affect C. difficile biofilm formation.

A Biofilm formation of C. difficile when co-cultured with different microbial species, compared with the biofilm formed from a C. difficile monoculture. Blue or red bars indicate species that significantly (p ≤ 0.05) reduced or increased the biofilm formed and grey bars represent species that did not significantly affect biofilm formation. Results expressed as fold change of crystal violet absorption of the dual co-culture vs monoculture. B Antagonistic bacteria reduced C. difficile recoveries from biofilms. C. difficile recoveries from mono and dual culture biofilms and C the enhanced antagonistic effect of polymicrobial biofilms on C. difficile recoveries from biofilms. D Poly synergistic biofilms are greater than the sum of the individual monoculture biofilms. B–D Results expressed as box and whisker plots showing the median log₁₀ cfu/mL, lower and upper quartile ranges, and the minimum and maximum results from at least four technical replicates from three biological repeats. False-coloured SEM of C. difficile (E, ×10,000, scale bar is 2 µm) and C. parapsilosis (F, ×5000, scale bar 5 µm) monoculture biofilms. Polymicrobial biofilms of C. difficile (red cells), S. warneri (blue cells) and C. parapsilosis (green cells) showing close interaction between the microbial cells (G, ×2500, scale bar 20 µm). Insert, zoomed section highlighting the extracellular matrix-like substance (light green colour) (H, ×10,000, scale bar 2 µm). White arrows denote what appears to be extracellular matrix in all images. Asterix denotes significantly difference with a p value < 0.01.