Fig 1.
The timeline of the C. difficile-infection mouse model is shown. C. difficile-infected mice developed diarrhea at D1-D3 and were recovered, or dead, after D4.
Fig 2.
The 18S rDNA analysis of mouse feces, with and without antibiotic administration (ATB and Control, respectively) was analyzed (combined feces from 3 mice in each group).
Fig 3.
Candida albicans dose determination.
To determine the appropriate dose of C. albicans, fecal fungi and fungemia were analyzed after the administration of C. albicans at several dose levels, without C. difficile gavage (n = 4/group) (A). The severity of the C. difficile-infection mouse model and control (non-C. difficile) with and without C. albicans administration (1x106 CFU) as determined by survival analysis (n = 8/group) (B) and weight loss (n = 6-8/time point, except for D7 of Candida + C. difficile group; n = 3) (C) are shown. 95%CI, 95% confidence interval.
Fig 4.
Impact of Candida gavage upon Clostridium difficile model.
To determine the impact of gavaged Candida in the C. difficile mouse model, fecal Candida (A), fecal C. difficile toxin B (B), gut leakage by FITC-dextran assay and serum (1→3)-β-D-glucan (BG) (C, D) and bacteremia (E), were measured (n = 6-8/group). *, p<0.05; #, p< 0.01; ##, p< 0.001.
Fig 5.
Inflammatory responses in the model.
The systemic and local inflammatory responses as determined by several inflammatory cytokines in serum and colon tissue, respectively, in C. difficile-infection mouse model and control (non-C. difficile) with and without C. albicans administration are presented (n = 6-8/group) (A-H). *, p<0.05; **, p<0.01; #, p<0.001; MIP-2, macrophage inflammatory protein 2; KC, keratinocyte chemoattractant; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β.
Fig 6.
Stimulations of HT-29 and Caco-2 cell-lines.
IL-8 cytokine levels in the culture supernatants of human intestinal epithelial cell lines (HT-29 and Caco-2 cell) after activation by C. difficile or phosphate buffer solution (PBS) as control, with and without the cell lysate of heat-killed C. albicans at the ratio of intestinal cells: C. albicans at 1:5 or 1:10 (see Method) are presented (A, B). In addition, lipopolysaccharide (LPS) alone or with heat-killed C. albicans or (1→3)-β-D-glucan (BG), with and without dectin-1 blocker, were used toward HT-29 and Caco-2 cells (C-F) (independent experiments were performed in triplicate) *, p<0.05; **, p<0.01; #, p<0.001.
Fig 7.
The in vitro effect of the probiotic.
IL-8 cytokine levels in the supernatants of human intestinal epithelial cell lines (HT-29 and Caco-2 cell) after activation by C. difficile or phosphate buffer solution (PBS) control, with and without Bifidobacterium adolescentis-B24 (BA-B24) and Bifidobacterium catenulatum-NB38 (BC-NB38), are presented (A, B) (independent experiments were done in triplicate); Survival analysis (C) (n = 12/ group); fecal fungal burdens (D); and blood bacterial count (E) were measured to determine the effect of Bifidobacterium cocktail (Bifido; see Methods) against C. difficile with and without C. albicans administration (n = 5-7/group for D-E). *, p<0.05; **, p< 0.02.
Fig 8.
The in vivo effect of the probiotic.
The systemic and local inflammatory responses as determined by several inflammatory cytokines in serum and colon, respectively, after the administration of Bifidobacterium cocktail (Bifido; see Method) in C. difficile-infection with and without C. albicans are presented (n = 6-8/group) (A-H). *, p<0.05; MIP-2, macrophage inflammatory protein 2; KC, keratinocyte chemoattractant; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β.