Journal: Communications Biology

Article Title: Defined microbial communities and their soluble products protect mice from Clostridioides difficile infection

doi: 10.1038/s42003-024-05778-6

Figure Lengend Snippet: CD + DMC-18 mice had significant reduction in relative concentrations of TcdA and TcdB in both CD027 ( a ) and CD078 ( b ) infected mice at 24 h and 48 h of infection (Mann–Whitney and a two-tailed test, n = 8 per group, ** p < 0.01 for all groups tested in triplicate). Fibroblast cell rounding assays revealed that CD + DMC-18 mice had reduced toxin-mediated cytotoxicity in their fecal pellets compared to CD-mice infected with both CD027 c and CD078 d at 48 h post infection (Kruskal–Wallis with Dunn’s test was used to analyze the data, n = 8, ** p < 0.01, *** p < 0.001). e 16S rRNA sequencing of murine stool from all groups demonstrated that DMC-18 led to a significant change in the relative abundance of bacteria in stool. f When comparing CD-infected mice treated with DMC-18 (Stool + CD + DMC-18) to CD-infected mice not exposed to DMC-18 we identified three bacterial genera only present in DMC-18, CD + DMC-18 but not CD-infected mice. Bifidobacterium, Parabacteroides, and Bacteroides were detected 24 h post-infection. These genera were absent prior to DMC-18 inoculation (stool) and in mice infected with CD but not treated with DMC-18 (Stool + CD). Operational taxonomic units were based on 97% identity; relative abundance is displayed. Error bars represent standard error of the mean.

Article Snippet: The polyvinylidene difluoride membrane (EMD Millipore, Germany) was incubated with either TcdA mouse monoclonal antibody (1:2500, Santa Cruz Biotechnology, Inc., USA, PCG4) or TcdB sheep polyclonal antibody (1:1,000 dilution, R&D Systems, USA, AF6246) overnight at 4 °C.

Techniques: Infection, MANN-WHITNEY, Two Tailed Test, Sequencing, Bacteria

Journal: Communications Biology

Article Title: Defined microbial communities and their soluble products protect mice from Clostridioides difficile infection

doi: 10.1038/s42003-024-05778-6

Figure Lengend Snippet: a Following the discovery that 4-members of DMC- 18 were found in the stool of mice protected from CDI, but not in the stool of CD-infected mice, a bacterial community of 4-bacteria (called DMC-4) was developed. DMC-4 treated mice were protected from significant weight loss following CD027 (two-way ANOVA with Tukey’s test, control animals n = 3 and infected animals n = 5, * p < 0.05). b Ceca from all mice were measured to compare sizes between uninfected, CD027-infected and DMC-4 + CD027 mice, similar to DMC-18; DMC-4 exposed mice were protected from ceca-shortening (one-way ANOVA with Dunnett’s correction, control animals n = 3 and infected animals n = 5, * p < 0.05, ** p < 0.01). c Representative images of the gross morphology of the cecum and colon from all groups of mice taken at the same magnification (20X). d Histological scores of all treatment groups based on the degree of edema, neutrophilic infiltration, and epithelial cell damage following CD027. DMC-4 exposure prior to CD infection reduced CD-mediated damage in mice infected with CD027 (Kruskal–Wallis with Dunn’s test, control animals n = 3 and infected animals n = 5, * p < 0.05). e DMC-4 exposure reduces toxin activity and cytotoxicity of fecal pellets from CD027-infected mice Representative H&E stained images of the colon of all mouse groups demonstrated that CD027infected mice had evidence of increased epithelial injury (black arrow), mucosal edema (orange arrow) and neutrophil infiltration (blue arrow). Scale bar, 100 μm. f Cell rounding assays also demonstrated significantly lower toxin activity in stool from mice infected with CD027 + DMC-4 infected mice 48 h after the infection compared to the CD027 group (Kruskal-Wallis with Dunn’s test, *** p < 0.001). Pooled supernatants from stool pellets, control animals ( n = 3) and infected animals ( n = 5). Error bars represent standard error of the mean. g TcdA and TcdB quantifications in DMC-4-treated mice 24 and 48 h after CD027 infection were significantly lower than in CD-infected mice ( t -Test with Mann–Whitney and two-tailed test, * p < 0.05. All groups were replicated in quadruplicate).

Article Snippet: The polyvinylidene difluoride membrane (EMD Millipore, Germany) was incubated with either TcdA mouse monoclonal antibody (1:2500, Santa Cruz Biotechnology, Inc., USA, PCG4) or TcdB sheep polyclonal antibody (1:1,000 dilution, R&D Systems, USA, AF6246) overnight at 4 °C.

Techniques: Infection, Bacteria, Control, Activity Assay, Staining, MANN-WHITNEY, Two Tailed Test

Journal: Communications Biology

Article Title: Defined microbial communities and their soluble products protect mice from Clostridioides difficile infection

doi: 10.1038/s42003-024-05778-6

Figure Lengend Snippet: a In addition to DMC-4 inhibiting toxin activity; conditioned media (CM) from DMC-4 also inhibited TcdA-mediated cell rounding in vitro (pooled results of assays run in triplicate, Kruskal-Wallis with Dunn’s test, p < 0.05). Silver staining ( b ) and western blot demonstrated the loss of purified TcdA (100 ng) ( c ) and TcdB (100 ng) ( d ) when incubated with DMC-4 (CM) for 1h. e Densitometry analysis was conducted to quantify glucosylation activity of Rac1 using DMC-4 (CM). Toxin-mediated glucosylation was inhibited ( e , f ) in cells treated with DMC-4 (CM) supernatants ( p < 0,05, One-way ANOVA with Dunnett’s test. Error bars represent standard error of the mean).

Article Snippet: The polyvinylidene difluoride membrane (EMD Millipore, Germany) was incubated with either TcdA mouse monoclonal antibody (1:2500, Santa Cruz Biotechnology, Inc., USA, PCG4) or TcdB sheep polyclonal antibody (1:1,000 dilution, R&D Systems, USA, AF6246) overnight at 4 °C.

Techniques: Activity Assay, In Vitro, Silver Staining, Western Blot, Purification, Incubation

Journal: Communications Biology

Article Title: Defined microbial communities and their soluble products protect mice from Clostridioides difficile infection

doi: 10.1038/s42003-024-05778-6

Figure Lengend Snippet: a DMC-4 (CM) treated mice were protected from significant weight loss following CD027 infection compared to CD-infected mice (two-way ANOVA with Tukey’s test, DMEM n = 3, CD027 n = 4, DMC-4 (CM) n = 4 and DMC-4 (CM) + CD027 n = 4, * p < 0.05). b DMC-4 CM + CD mice were protected from ceca shortening compared to CD-infected mice (one-way ANOVA with Dunnett’s correction, DMEM n = 3, CD027 n = 4, DMC-4 (CM) n = 4 and DMC-4 (CM) + CD027 n = 4, * p < 0.05). c Representative images of the gross morphology of the ceca and colons from all groups of mice taken at the same magnification (×20) demonstrated that uninfected mice and those exposed to DMC-4 (CM) prior to CD infection had large ceca filled with feces and fecal pellets in the colon, while CD infected mice had reduced cecum size and morphological evidence of colitis. d Stool from mice infected with CD027 resulted in significantly higher toxin-mediated cell rounding compared to DMC-4 CM + CD027 (Kruskal-Wallis with Dunn’s test, ** p < 0.01). Pooled supernatants from stool pellets, control animals n = 3 and infected animals n = 5. e TcdA and TcdB quantifications in DMC-4 (CM) treated mice after CD027 infection demonstrated that CD + DMC-4 CM mice had significantly lower levels of TcdA and TcdB at both 24 h and 48 h post-infection (Mann–Whitney test was used to analyze the data (* p < 0.05). All groups were carried out in triplicate). Error bars represent standard error of the mean.

Article Snippet: The polyvinylidene difluoride membrane (EMD Millipore, Germany) was incubated with either TcdA mouse monoclonal antibody (1:2500, Santa Cruz Biotechnology, Inc., USA, PCG4) or TcdB sheep polyclonal antibody (1:1,000 dilution, R&D Systems, USA, AF6246) overnight at 4 °C.

Techniques: Infection, Control, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Novel Clade C-I Clostridium difficile strains escape diagnostic tests, differ in pathogenicity potential and carry toxins on extrachromosomal elements

doi: 10.1038/s41598-018-32390-6

Figure Lengend Snippet: TcdB detection in cell-free supernatants of isolates HMX-149, HMX-152, and HSJD-312 ( a ) and RhoA glycosylation induced by them in HeLa cells ( b ). The TcdBs of HMX-149, HMX-152, and HSJD-312 were not detected by a monoclonal antibody that strongly reacted with the TcdB of the control NAP1 strain tested. Alike a control NAP1 strain, all three Clade C-I isolates glycosylated RhoA (the antibody used does not react with the glycosylated form of RhoA). The 7.5% SDS-PAGE gel in panel A was stained with Sypro Ruby. Only the portion of the gel containing TcdB is shown. Parts of the membranes not reacting with the corresponding antibodies were removed from the western blot (WB) images.

Article Snippet: These gels were stained with SYPRO ® Ruby (ThermoFischer Scientific) or used for electrotransfer of proteins to PVDF membranes (Macherey-Nagel) for Western Blotting with an anti-TcdB monoclonal antibody (2CV, tgcBIOMICS).

Techniques: SDS Page, Staining, Western Blot