Fig. 7: Nystatin reduces C. difficile spore internalization and reduces R-CDI rates.

a Internalization and b adherence of undifferentiated Caco-2 cells were pretreated nystatin for 1 h, and subsequently infected with C. difficile spores R20291 preincubated for 1 h with FBS. c Colony-forming units of spores on BHIS-CC with 0.1% of sodium taurocholate of a lysate of undifferentiated Caco-2 were pretreated with 30 µM of nystatin (shown as NYS), or DMEM alone as control and infected with C. difficile spores, and treated with taurocholate and ethanol (EtOH). Cells were lysed and plated, so only spores that remain dormant after taurocholate treatment (dormant) germinate in the plates. The number of CFU mL⁻¹ was determined, and the percentage of adherence relative to the control. a–c In bars, each dot represents one independent well representative of three independent experiments. Loops of approximately of the ileum and colon of C57BL/6 were injected with 3 × 10⁸ C. difficile R20291 with RGD peptide (n = 4), or nystatin (n = 4) and saline as control (n = 4). d–f Representative confocal micrographs C. difficile spore is shown in red, F-actin is shown in green, and nuclei in blue (fluorophores colors were digitally reassigned for a better representation). The white arrow and empty arrow denote internalized and adhered C. difficile spores. Scale bar, 20 µm. Percentage of internalized spots (spores) and the number of adhered per 10⁵ µm² in the g, h colonic mucosa or in the i, j ileum mucosa. k Schematics of the experimental design of a mouse model of R-CDI. ATB cocktail treated mice were infected with 6 × 10⁷ R20291 spores. The CDI symptoms were treated from days 3 to 7 with nystatin and vancomycin (shown as VAN + NYS; n = 18 mice) or vancomycin alone as control (shown as VAN; n = 23 mice) and were monitored daily for l relative weight during the R-CDI. Weight loss comparison for animals treated with m vancomycin or n vancomycin and nystatin, and o onset to diarrhea during the R-CDI. Error bars indicate the mean ± S.E.M. Statistical analysis was performed by a–c two-tailed unpaired Student’s t test; g–j, l two-tailed Mann–Whitney test; post-Dunn’s; m, n one-tailed Wilcoxon matched-pairs signed-rank test; o log-rank (Mantel–Cox) test; ns, p > 0.05.