Synthetic microbial consortia for the treatment of Clostridioides difficile infection in mice model

Abstract

Clostridioides difficile infection (CDI) as of recent has become a great concern to the impact on human health due to its high hazardous risk and rate of recurrence. Live bacterial therapeutics is a promising method to treat or prevent CDI. Here, a synthetic microbial consortia (SMC) B10 was constructed using probiotic strains with antibacterial and anti-quorum sensing activities, and the therapeutic effect of SMC B10 against C. difficile infection was evaluated in vitro. Compared to the model group, the treatment of SMC B10 significantly increased the survival rate. The clinical signs of mice were significantly ameliorated, especially the cecum injury, while the secretion of pro-inflammatory associated cytokines such as IL-1α, IL-6, IL-17A and TNF-α was reduced, the expression of TLR4 was inhibited, which alleviated the inflammatory response, and the expression of the tight junction protein Claudin-1 was increased, ultimately promoting the recovery of host health. The treatment of B10 restored gut microbiota dysbiosis and led to a healthy intestinal microbiota structure, significantly improved alpha diversity, suppressing potentially harmful bacteria and restoring other core bacterial species. In conclusion, SMC B10 can effectively treat CDI through modulate gut microbiota and attenuate the inflammatory response.

FIGURE 1

Selected strains can differentially inhibit the activity of Clostridioides difficile. (A) Growth curves of C. difficile when co‐cultured with a cell‐free supernatant of different strains, (i) MRS group, * represents Bifidobacterium longum vs. MRS, (ii) BHI group, * represents Clostridium butyricum vs. BHI, # represents Akkermansia muciniphila vs. BHI, (iii) NB group, n = 3. (B) The intensity of fluorescence expression of the reporter strain Vibrio harveyi BB170 at different times. (C) The expression of AI‐2 in the whole system when the live bacteria were co‐cultured with C. difficile, n = 6. (D) Expression of AI‐2 in the system when the living bacteria were co‐cultured and when they were monocultured, n = 3. (E) Concentration of TcdB in the system when the living bacteria were co‐cultured with C. difficile, n = 3. Significance calculated using unpaired t test (A, D) and ordinary one‐way ANOVA (C, E). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 2

Probiotic properties of B10 members, n = 3. (A) Survival in gastric acid environment. (B) Survival in bile salt environment. (C) DPPH radical scavenging ability, hydroxyl radical scavenging ability and superoxide anion scavenging ability.

FIGURE 3

B10 can exist stably in vitro environment and achieve the designed effects, n = 3. (A) Proportion of each member of B10 before co‐culture. (B) Proportion of each member of B10 after co‐culture. (C) The amount of biofilm formed by Clostridioides difficile monoculture, B10 co‐culture with C. difficile and B10 monoculture. (D) The amount of biofilm formed by C. difficile monoculture versus CFS of B10 co‐cultured with C. difficile. Significance calculated using unpaired t test (C, D). *p < 0.05 and **p < 0.01.

FIGURE 4

B10 ameliorates clinical signs caused by Clostridioides difficile infection (CDI). (A) Timeline of animal experiments. (B) Relative weight change after infection, animals were culled the next day after death. (C) Change in mortality after infection. (D) Highest Clinical Sickness Score for each animal within five days after infection. (E) Comparison of colorectal lengths of the three groups. (F) Representative colorectal photographs of the infected group versus the treated group. The number of independent samples used for each group was: Control group, n = 6; CDI group, n = 10; and B10 group, n = 10. Significance calculated using Mantel‐Cox test (C), Wilcoxon test (D) and ordinary one‐way ANOVA (F). *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 5

Treatment with B10 attenuated the inflammatory response and increased the expression of tight junction protein. (A) Representative H&E‐stained images of three groups of cecum and colon. (B) Histologic injury scores (HIS) of cecum and colon based on H&E staining results (n = 6 per group). (C) Cytokine concentrations in serum (n = 5 per group). (D) Relative expression levels of mRNA in colon tissue (n = 5 per group). Significance calculated using unpaired t test (B–E). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 6

B10 restores the alpha diversity of the intestinal microbiota while affecting the microbiota constitution. (A) ACE index. (B) Chao1 index. (C) PD whole tree index. (D) Venn diagram of all three groups at OTUs level. (E) NMDS based on Unweighted UniFrac algorithm. (F) Colony composition of the three groups at the phylum level. (G) Colony composition of the three groups at the family level. The number of samples per group is n = 5. Significance calculated using unpaired t test (A–C). *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 7

LEfSe analysis results. (A) LEfSe cladogram. (B) The taxa with Linear discriminant analysis scores ≥4.0 between all three groups, suggesting stronger discriminatory features that may have functional or taxonomic relevance, see Table S3 for details.

FIGURE 8

B10 recovered numerous species and exhibited a wide range of interactions. (A) Species with relative abundance greater than 0.01% and multigroup (p) less than 0.05 in the B10 group at the family level, significance calculated using ANOVA. (B) Random forest analysis of the B10 group versus the Clostridioides difficile infection (CDI) group, with yellow arrows labelling species endemic to the B10 group and green rectangles labelling species with higher relative abundance in the CDI group. (C) Correlation network within the CDI group. (D) Correlation network within the B10 group. (E) Correlation network constructed by joint analysis of both CDI and B10 groups.

FIGURE 9

Correlation of individual species with Clostridioides difficile infection‐related indicators at the genus level.