Medication-resistant epilepsy is associated with a unique gut microbiota signature

Abstract

Objective: Dysfunction of the microbiota-gut-brain axis is emerging as a new pathogenic mechanism in epilepsy, potentially impacting on medication response and disease outcome. We investigated the composition of the gut microbiota in a cohort of medication-resistant (MR) and medication-sensitive (MS) pediatric patients with epilepsy.

Methods: Children with epilepsy of genetic and presumed genetic etiologies were evaluated clinically and subgrouped into MR and MS. Age-matched healthy controls (HCs) were also recruited. A food diary was used to evaluate nutritional habits, and the Rome IV questionnaire was used to record gastrointestinal symptoms. The microbiota composition was assessed in stool samples through 16S rRNA. α-Diversity (AD) and β-diversity (BD) were calculated, and differential abundance analysis was performed using linear multivariable models (significance: p.adj < .05).

Results: Forty-one patients (MR:MS = 20:21) with a mean age of 7.2 years (±4.6 SD) and 27 age-matched HCs were recruited. No significant differences in AD were found when comparing patients and HCs. Significant positive correlation was found between AD and age (Chao1 p.adj = .0004, Shannon p.adj = .0004, Simpson p.adj = .0028). BD depicted a different bacterial profile in the epilepsy groups compared to HCs (MS vs. HC: Bray-Curtis F = 1.783, p = .001; Jaccard F = 1.24, p = .001; MR vs. HC: Bray-Curtis F = 2.24, p = .001; Jaccard F = 1.364, p = .001). At the genus level, the epilepsy groups were characterized by a significant increase in Hungatella (MS vs. HC: +4.95 log₂ change; MR vs. HC: +6.72 log₂ change); the [Eubacterium] siraeum group changed between the MR and MS subgroups.

Significance: Epileptic patients display unique gut metagenomic signatures compared to HCs. Moreover, a different ratio of the butyrate-producing [Eubacterium] siraeum group suggests dissimilarities between patients based on the response to antiseizure medications.

Keywords: epilepsy; gut microbiota signature; microbiota–gut–brain axis; pediatrics.

FIGURE 1

α‐Diversity (AD) correlates with aging, whereas β‐diversity depicts a different bacterial profile in the epilepsy groups compared to healthy controls (HCs). (A) Correlation plots illustrating the associations between three AD indices (Chao1, Shannon Diversity, Simpon's Index) and age. Dots represent individual subjects and are colored based on the group. Black line is the regression line, with a gray area corresponding to the 95% confidence interval. Coefficient and adjusted p‐values are obtained from respective generalized linear models. (B) Principle coordinate analysis (PCoA) plots of Bray–Curtis and Jaccard dissimilarity indices were obtained using the first two axes. The percentage of total variation explained by each axis is shown next to the axis label. Points represent each individual. Ellipses correspond to 95% confidence intervals for each group. MR, medication‐resistant; MS, medication‐sensitive. Pr(>F) is the p value of the F statistic.

FIGURE 2

Heatmap illustrating the relative abundance distribution of the two significant genera obtained from the differential abundance analysis of the three pairwise comparisons. The relative abundance (rel. abund.) percentiles were transformed into centered log‐ratio (clr) and visualized by color gradient. Samples were clustered based on group information. Right annotation columns show the significant changes obtained from each of the three pairwise comparisons, and the log₂ fold change values with the significance stars. *.01 ≤ p.adj < .05, **.001 ≤ p.adj <.01. HC, healthy control; MR, medication‐resistant; MS, medication‐sensitive.

FIGURE 3

Phylogenetic tree showing the amplicon sequence variants (ASVs) annotated to the genera found to significantly change in our population. Rooted tree constructed by alignment of the obtained ASVs was trimmed to include 24 ASVs annotated as Hungatella or [Eubacterium] siraeum group. Heatmap encircling the phylogenetic tree shows the median of centered log‐ratio (clr)‐transformed genus relative abundances in each study group. HC, healthy control; MR, medication‐resistant; MS, medication‐sensitive.

FIGURE 4

Ratio of relevant phyla and statistically significant genera. (A) Scatterplot of the ratio between abundance of Bacteroidota and Firmicutes phyla in the three groups. (B) Scatterplot of Hungatella (Hung) dominance over [Eubacterium] siraeum group (Eub). (C) Hung/Eub ratio at each sample level in the three groups studied. The relative abundance of Hung and Eub were summed and used as an isolated community. A binary score was assigned based on their proportional abundance: “1” indicated Hung dominance (≥50% abundance) and “0” indicated Eub dominance. *p ≤ .05, ***p ≤ .0001. HC, healthy control; MR, medication‐resistant; MS, medication‐sensitive.

FIGURE 5

Significant changes in Kyoto Encyclopedia of Genes and Genomes pathway relative abundances between groups. Individual samples are represented in dots on left‐hand side, whereas the boxplots on the right side depict the median line in black and the interquartile interval with minimum and maximum values. The triangle shapes show outlier data points. Raw and adjusted p values are shown for significant changes. HC, healthy control; MR, medication‐resistant; MS, medication‐sensitive.