Protective effect of gut microbiota restored by fecal microbiota transplantation in a sepsis model in juvenile mice

Abstract

Introduction: Restoring a balanced, healthy gut microbiota through fecal microbiota transplantation (FMT) has the potential to be a treatment option for sepsis, despite the current lack of evidence. This study aimed to investigate the effect of FMT on sepsis in relation to the gut microbiota through a sepsis model in juvenile mice.

Methods: Three-week-old male mice were divided into three groups: the antibiotic treatment (ABX), ABX-FMT, and control groups. The ABX and ABX-FMT groups received antibiotics for seven days. FMT was performed through oral gavage in the ABX-FMT group over the subsequent seven days. On day 14, all mice underwent cecal ligation and puncture (CLP) to induce abdominal sepsis. Blood cytokine levels and the composition of fecal microbiota were analyzed, and survival was monitored for seven days post-CLP.

Results: Initially, the fecal microbiota was predominantly composed of the phyla Bacteroidetes and Firmicutes. After antibiotic intake, an extreme predominance of the class Bacilli emerged. FMT successfully restored antibiotic-induced fecal dysbiosis. After CLP, the phylum Bacteroidetes became extremely dominant in the ABX-FMT and control groups. Alpha diversity of the microbiota decreased after antibiotic intake, was restored after FMT, and decreased again following CLP. In the ABX group, the concentrations of interleukin-1β (IL-1β), IL-2, IL-6, IL-10, granulocyte macrophage colony-stimulating factor, tumor necrosis factor-α, and C-X-C motif chemokine ligand 1 increased more rapidly and to a higher degree compared to other groups. The survival rate in the ABX group was significantly lower (20.0%) compared to other groups (85.7%).

Conclusion: FMT-induced microbiota restoration demonstrated a protective effect against sepsis. This study uniquely validates the effectiveness of FMT in a juvenile mouse sepsis model, offering potential implications for clinical research in critically ill children.

Keywords: antibiotics; fecal microbiota transplantation; microbiota; mouse model; pediatric; sepsis.

Figure 1

Study design. ABX, antibiotic treatment; CLP, cecal ligation and puncture; FMT, fecal microbiota transplantation.

Figure 2

Composition of fecal microbiota plotted by group and experimental day. (A) Composition of fecal microbiota at the phylum level. (B) Composition of fecal microbiota at the class level. (C) Composition of fecal microbiota at the genus level. (D) Composition of fecal microbiota at the species level. ABX, antibiotic treatment; FMT, fecal microbiota transplantation.

Figure 3

Relative abundance of each dominant phylum in the fecal microbiota of each group. The bar graph displays the mean values for each group, with error bars representing the mean plus one standard deviation. The y-axis represents the relative abundance of each phylum. (A) Changes in the abundance of each dominant phylum of fecal microbiota over time in each group. (B) Comparison of the relative abundance of each dominant phylum constituting the fecal microbiota on day 7 between each group. (C) Comparison of the relative abundance of each dominant phylum constituting the fecal microbiota on day 14 between each group. ABX: antibiotic treatment, FMT: fecal microbiota transplantation. Asterisks indicate statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 with two-tailed Welch’s t-test.

Figure 4

The Shannon and Simpson’s indices of fecal microbiota. Data are presented as individual values with long and short horizontal bars representing the mean plus one standard deviation. The y-axis represents either the Shannon or Simpson’s indices, depending on the specific panel. (A) Changes in the Shannon and Simpson’s indices for the control group over time. (B) Changes in the Shannon and Simpson’s indices for the ABX-FMT group over time. (C) Changes in the Shannon and Simpson’s indices for the ABX group over time. (D) Comparison of the Shannon or Simpson’s indices of fecal microbiota between each group on day 7 and day 14. ABX, antibiotic treatment; FMT, fecal microbiota transplantation. Asterisks indicate statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 with two-tailed Welch’s t-test.

Figure 5

Beta diversity of the overall fecal microbiota. (A) Non-metric multidimensional scaling plots based on the Bray-Curtis distances for dissimilarity. (B) Non-metric multidimensional scaling plots based on the Euclidean distances for dissimilarity. ABX, antibiotic treatment, D0: day 0, D7: day 7, D14: day 14, D16: day 16, FMT, fecal microbiota transplantation.

Figure 6

Changes in blood cytokine concentrations (pg/ml) in each group from immediately before CLP to 12 hours after. Grey horizontal bars indicate the mean values, and vertical lines represent the mean plus one standard deviation. Sample sizes: n = 7 at all time points in all groups, except for the ABX-FMT group at pre-CLP (n = 5, excluding two subjects due to sample errors). ABX, antibiotic treatment; CLP, cecal ligation and puncture; CXCL1, C-X-C motif chemokine ligand 1; FMT, fecal microbiota transplantation; GM-CSF, granulocyte macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor. Asterisks indicate statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 with two-tailed Welch’s t-test. Colored dotted lines represent comparisons across time within the same group, and solid black lines represent comparisons between groups at the same time.

Figure 7

Kaplan-Meier survival curve. Sample sizes: n = 5 for the ABX group (two early deaths dropped out), and n = 7 for other groups. ABX, antibiotic treatment; FMT, fecal microbiota transplantation, *p < 0.05.

Figure 8

Representative histological features from each group showing the extent of infiltration of inflammatory cells. (A) Inflammatory cell infiltration in the lamina propria of the mucosa. (B) Inflammatory cell infiltration in the mesentery, indicating peritonitis. Annotations: neutrophils (thick short arrows), plasma cells (long arrows), macrophages (thick open arrows), and lymphocytes (arrowheads). Staining: H&E. Magnification: ×400; scale bars = 25 µm. ABX, antibiotic treatment; FMT, fecal microbiota transplantation.