Differences in the Concentration of the Fecal Neurotransmitters GABA and Glutamate Are Associated with Microbial Composition among Healthy Human Subjects

Abstract

Recent studies have shown that the gut microbiota modulates the physical and psychological functions of the host through several modes of action. One of them is mediating the production of active neurotransmitters, such as serotonin and gamma-aminobutyric acid (GABA). GABA is the major inhibitory neurotransmitter in the central nervous system. Here, we analyzed the relationship between fecal GABA concentration and microbial composition in more than 70 human participants. The gut microbiome composition was analyzed using next-generation sequencing based on 16S ribosomal RNA. High-performance liquid chromatography was used to evaluate the neurotransmitters GABA and glutamate. The GABA level was detected in a broad range (0-330 µg/g feces). The participants' samples were classified into high (>100 µg/g), medium (10-100 µg/g), and low (<10 µg/g) groups, based on fecal GABA concentration. The results reveal that the microbiome of the high-GABA samples had lower alpha diversity than the other samples. Beta diversity analysis showed significant (p < 0.05) separation between the high-GABA samples and others. Furthermore, we surveyed the abundance of specific GABA producer biomarkers among the microbiomes of tested samples. The family Bifidobacteriaceae exhibited high abundance in the microbiome of the high-GABA group. This study demonstrated that Bifidobacterium abundance was associated with high fecal GABA content in healthy human subjects. These results may aid the development of potential probiotics to improve microbial GABA production, which can support the maintenance of the physical and psychiatric health of the host.

Keywords: Bifidobacterium; GABA; gut microbiota; gut–brain axis; neurotransmitters.

Figure 1

Fecal GABA and glutamate concentrations. Fecal GABA and glutamate contents were analyzed among healthy human participants. Participants were divided based on GABA productivity into high, medium, and low groups. Circle, diamond, and triangle symbols represent participants categorized as low-, medium-, and high-GABA productivity groups, respectively. Different colors represent the geographical origin of each participant: B, South Asia; E, Northeast Africa; I, Southeast Asia; J, East Asia. Regression curve is displayed on the figure with a deep gray line, showing a negative correlation between fecal GABA and glutamate concentrations. Confidence interval (95%) is expressed in a light gray color. The correlation coefficient (R) and p-value of the regression curve are also shown on the plot.

Figure 2

Alpha diversity among fecal GABA groups. Alpha diversity, measured by observed species (A) and Chao1 (B), Shannon diversity (C), and inverse Simpson indices (D), is plotted for examined samples, i.e., in high-, medium-, and low-GABA groups. Box plots depict microbiome diversity and abundance differences according to each test. The horizontal line inside the box represents the median. Outliers and individual sample values are represented by dots. Different colors represent the geographical origin of each participant: B, South Asia; E, Northeast Africa; I, Southeast Asia; J, East Asia. All alpha diversity measurements shown here significantly decreased in the high-GABA group compared to those in the low group.

Figure 3

Beta diversity and community similarity analysis among fecal gamma-aminobutyric acid (GABA) groups. (A) Non-metric multidimensional scaling (NMDS) plot based on the distance matrix of operational taxonomic units (OTUs) designated at the family level of taxonomic classification calculated using the Bray–Curtis model. (B) Distance-based redundancy analysis (db-RDA) using the Bray–Curtis dissimilarity matrix calculated with OTUs designated at the family level of taxonomy. The environmental variables were statistically significant (p < 0.01), and the top 10 most abundant taxa are displayed. Circle, diamond, and triangle symbols represent participants categorized as low-, medium-, and high-GABA productivity groups, respectively. Different colors represent the geographical origin of each participant: B, South Asia; E, Northeast Africa; I, Southeast Asia; J, East Asia.

Figure 4

Heatmap of the top 20 abundant taxa in the examined human fecal samples. Operational taxonomic units (OTUs) were collapsed at the family-level taxonomy. A dendrogram was constructed with the beta flexible method based on the distance matrix of OTUs, calculated using the Bray–Curtis model. The geographical origin of each participant is represented by different colors: B, South Asia; E, Northeast Africa; I, Southeast Asia; J, East Asia.

Figure 5

Linear discriminate analysis (LDA) effect size (LEfSe) showing the characteristics of the microbial community composition between the low- and medium-gamma-aminobutyric acid (GABA) groups in panels (A,B) as well as between the medium- and high-GABA groups in panels (C,D). (A,C) LEfSe (LDA scores 10⁴ and more) displaying statistical and differentially abundant taxa in each group. (B,D) Cladogram showing the microbiome differences at different phylogenetic levels. The central point represents the root of the tree (bacteria) and each ring displays the next (lower) taxonomic rank (p__, phylum; c__, class; o__ order; f__, family; g__, genus). The diameter of each circle represents the relative abundance of each taxon.

Figure 6

Comparison of the relative taxon abundance of the phyla Firmicutes (panel A) and Actinobacteria (panel B) among the three gamma-aminobutyric acid (GABA) groups (high, medium, and low). Significance codes: <0.001 ***, <0.01 **, <0.1 “NS”.

Figure 7

Proportion of the taxa that occurred abundantly in the high-gamma-aminobutyric acid (GABA) group. Two taxa at the order level, comprising Bifidobacteriales (Panel A) and Lactobacillales (Panel B), are displayed. Three taxa at the family level are demonstrated incorporating Bifidobacteraceae (Panel C), Streptococcaceae (Panel D), and Enterococcaceae (Panel E). Significance codes: <0.001 ***, <0.01 **, <0.05 *, <0.1 “NS”.

Figure 8

Proportion of taxa that occurred abundantly in the low-gamma-aminobutyric acid (GABA) group including Clostridiales (Panel A), Lachnospiraceae (Panel B), and Ruminococcaceae (Panel C). Significance codes: <0.001 ***, <0.01 **, <0.05 *, <0.1 “NS”.