Fecal microbiota transplantation and short-chain fatty acids reduce sepsis mortality by remodeling antibiotic-induced gut microbiota disturbances

Abstract

Objective: Sepsis is the leading cause of death in critically ill patients. The gastrointestinal tract has long been thought to play an important role in the pathophysiology of sepsis. Antibiotic therapy can reduce a patient's commensal bacterial population and raise their risk of developing subsequent illnesses, where gut microbiota dysbiosis may be a key factor.

Methods: In this study, we analyzed the 16S rRNA of fecal samples from both healthy people and patients with sepsis to determine if alterations in gut bacteria are associated with sepsis. Then, we developed a mouse model of sepsis using cecal ligation and puncture (CLP) in order to examine the effects of fecal microbiota transplantation (FMT) and short-chain fatty acids (SCFAs) on survival rate, systemic inflammatory response, gut microbiota, and mucosal barrier function.

Results: Sepsis patients' gut microbiota composition significantly differed from that of healthy people. At the phylum level, the amount of Proteobacteria in the intestinal flora of sepsis patients was much larger than that of the control group, whereas the number of Firmicutes was significantly lower. Mice with gut microbiota disorders (ANC group) were found to have an elevated risk of death, inflammation, and organ failure as compared to CLP mice. However, all of these could be reversed by FMT and SCFAs. FMT and SCFAs could regulate the abundance of bacteria such as Firmicutes, Proteobacteria, Escherichia Shigella, and Lactobacillus, restoring them to levels comparable to those of healthy mice. In addition, they increased the expression of the Occludin protein in the colon of mice with sepsis, downregulated the expression of the NLRP3 and GSDMD-N proteins, and reduced the release of the inflammatory factors IL-1β and IL-18 to inhibit cell pyroptosis, ultimately playing a protective role in sepsis.

Disccusion: FMT and SCFAs provide a microbe-related survival benefit in a mouse model of sepsis, suggesting that they may be a viable treatment for sepsis.

Keywords: antibiotic; fecal microbiota transplantation; gut microbiota; sepsis; short-chain fatty acids.

Figure 1

Human sepsis-related alterations in the gut microbiota. (A) The Ace, Chao, Shannon, and Simpson indices were used to compare the alpha diversity profiles of healthy individuals (n = 12) and patients with sepsis (n = 22). (B, C) NMDS analysis of beta diversity at the phylum and genus levels. Different hues symbolize different groups. The species in the two samples are more similar the closer the two sample points are to each other. (D) Comparison of serum SCFAs levels among two groups. (E) The distribution of the community at the phylum level. Different colors indicate distinct species, and the length of the columns indicates the relative abundance of those species in the group sample (represented by the Y-axis). (F, G) Variation in gut microbiota between the groups was statistically significant at the phylum and genus levels. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 2

Fecal microbiota transplantation (FMT) and supplementation with short-chain fatty acids (SCFAs) attenuate CLP-induced sepsis. (A) Illustration of the experimental design in schematic form. (B) FMT and SCFAs improve survival following cecal ligation and puncture (CLP). n = 14 mice per group. Mice in the AFC and ASC groups had a higher survival rate than those in the ANC group. (C) A representative cecum before and after four days of antibiotic treatment. (D) FMT and SCFAs increased bacterial clearance in sepsis mice. *P < 0.05; ***P < 0.001.

Figure 3

Fecal microbiota transplantation (FMT) and supplementation with short-chain fatty acids (SCFAs) prevent sepsis in mice against organ failure. (A-D) H&E staining revealed considerably higher levels of inflammatory infiltration, edema, and bleeding in the colon, lung, liver, and kidney tissue of the ANC group compared to the Sham group, but these abnormalities were drastically reduced in the AFC and ASC groups, originally magnification, ×100.

Figure 4

Fecal microbiota transplantation (FMT) and supplementation with short-chain fatty acids (SCFAs) protect the mouse intestinal epithelial tight junction from CLP-induced damage. (A) Electron micrograph of colonic segments taken from mice with different treatments. Groups are labeled as shown. Scale bar =500 nm. (B, C) Immunohistochemical staining of tight junction proteins Occludin, in colon tissues (X 100, scale bar, 100μm). Significant results are shown in the box plot. (D) Serum D-lactate and diamine oxidase (DAO) levels. *P < 0.05; ***P < 0.001; ****P < 0.0001.

Figure 5

Mouse feces microbiota diversity. (A) Alpha diversity in the A0 (blue), A4 (red), AF (green), and AS (purple) groups, as shown by the Ace, Chao, Shannon, and Simpson indices. (B) The beta diversity of gut microbiota was displayed by principal component analysis (PCoA) scatterplots. (C) Parallel and overlapping circles are seen in this Venn diagram. Species shared between groups are shown by the overlapping portion, while those unique to each group are shown by the non-overlapping portion. (D) Hierarchical clustering based on the Weighted Unifrac Distance of OTU profiles shows the community composition of each sample at the phylum level. (E) At the genus level, the abundance of species in the LEfSe for each group. The gradation of color represents the correlation value. n = 6 mice per group. (F, G) Relative abundance of SCFAs-producing bacteria in groups (F) Allobaculum (G) Bacteroides. A0 group (no antibiotics), A4 group (antibiotics for 4 days + normal saline for 3 days), AF group (antibiotics for 4 days + FMT for 3 days), AS group (antibiotics for 4 days + SCFAs for 3 days). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not significant.

Figure 6

Colonic protein expression of pyroptosis-associated genes after sepsis in mice: the role of Fecal microbiota transplantation (FMT) and short-chain fatty acids (SCFAs). (A) Western blotting for the detection of NLRP3, IL-18, GSDMD-fl, and GSDMD-N. (B–D) The quantitative examination of the ratio of NLRP3/β-actin, GSDMD-N/GSDMD-fl, and IL-18/β-actin expression was performed, with the Sham group serving as the reference value. (E) The serum level of IL-1β was measured using ELISA. n = 6 per group. Data are expressed as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns: not significant.

Figure 7

The role of fecal microbiota transplantation (FMT) and short-chain fatty acids (SCFAs) in fecal metabolism in mice with sepsis. (A–C) The Scatter plot of principal components analysis (PCA) for metabolites shows that the A0 group had a very different metabolic profile than the A4 group. (D–F) OPLS-DA analyses of the metabolite profile. OPLS-DA scores plot and OPLS-DA model test chart showed good discrimination between the groups. Volcano Plot showing differential metabolites between the A0 and A4 groups (G) and between the ANC and AFC groups (H), and between the ANC and ASC groups (I). Linear discriminate analysis (LDA) effect size analysis of the top 20 differential metabolites between the A0 and A4 groups (J) and between the ANC and AFC groups (K), and between the ANC and ASC groups (L). LDA scores > 1 and significance of p < 0.05 as determined by Wilcoxon’s signed-rank test.

Figure 8

(A–C) Comparison of the metabolic pathways of the A0 and A4 groups (A), the ANC and AFC groups (B), and the ANC and ASC groups (C).

Figure 9

Potential role of fecal microbiota transplantation (FMT) and short-chain fatty acids (SCFAs) in treating sepsis. In the pathogenesis of sepsis, there is an imbalance in the intestinal flora, which decreases the production of SCFAs from intestinal flora metabolites and decreases the ability of SCFAs to bind to GPR43. It also induces further NOD-like receptor protein-3 (NLRP3) inflammasomes to be made and more IL-1β and IL-18 to be released, exacerbating the inflammatory response of tissues and cells. In addition, NLRP3 triggers the activation of caspase-1, which in turn cleaves the gasdermin D (GSDMD) protein and triggers cell pyroptosis, hence exacerbating sepsis and perpetuating the vicious cycle. FMT and SCFAs can protect intestinal function, regulate the distribution and amount of intestinal flora, restore dominant intestinal flora, boost the production of SCFAs, limit inflammatory response and cell pyroptosis, and subsequently protect against sepsis. (Created with BioRender.com).