Altered gut microbiota in temporal lobe epilepsy with anxiety disorders

Abstract

Introduction: Patients with epilepsy are particularly vulnerable to the negative effects of anxiety disorders. In particular, temporal lobe epilepsy with anxiety disorders (TLEA) has attracted more attention in epilepsy research. The link between intestinal dysbiosis and TLEA has not been established yet. To gain deeper insight into the link between gut microbiota dysbiosis and factors affecting TLEA, the composition of the gut microbiome, including bacteria and fungi, has been examined.

Methods: The gut microbiota from 51 temporal lobe epilepsy patients has been subjected to sequencing targeting 16S rDNA (Illumina MiSeq) and from 45 temporal lobe epilepsy patients targeting the ITS-1 region (through pyrosequencing). A differential analysis has been conducted on the gut microbiota from the phylum to the genus level.

Results: TLEA patients' gut bacteria and fungal microbiota exhibited distinct characteristics and diversity as evidenced by high-throughput sequencing (HTS). TLEA patients showed higher abundances of Escherichia-Shigella (genus), Enterobacterales (order), Enterobacteriaceae (family), Proteobacteria (phylum), Gammaproteobacteria (class), and lower abundances of Clostridia (class), Firmicutes, Lachnospiraceae (family), Lachnospirales (order), and Ruminococcus (genus). Among fungi, Saccharomycetales fam. incertae sedis (family), Saccharomycetales (order), Saccharomycetes (class), and Ascomycota (phylum) were significantly more abundant in TLEA patients than in patients with temporal lobe epilepsy but without anxiety. Adoption and perception of seizure control significantly affected TLEA bacterial community structure, while yearly hospitalization frequency affected fungal community structures in TLEA patients.

Conclusion: Here, our study validated the gut microbiota dysbiosis of TLEA. Moreover, the pioneering study of bacterial and fungal microbiota profiles will help in understanding the course of TLEA and drive us toward preventing TLEA gut microbiota dysbiosis.

Keywords: anxiety disorder; bacteria; fungi; gut microbiota; temporal lobe epilepsy patient.

Figure 1

Geographic distribution of the study population. Only Han Chinese individuals residing in populations in tropical China were included, thus mitigating the effect of ethnic and geographic bias.

Figure 2

Anonymous questionnaire for collecting demographic characteristics, disease characteristics, and treatment characteristics. (A) QR code for logging in to the anonymous questionnaire; (B) the user interface of the anonymous questionnaire.

Figure 3

α-diversity of different groups of gut bacteria at the OTU level. (A–D) α-diversity of different groups of gut bacteria at the level of operational taxonomic units (OTUs) and index values represent species diversity. (A) Variations in Chao (A), Ace (B), Simpson (C), and Shannon (D) diversity indices between TLEA and TLEW. (E) Comparing the type and number of OTUs. Rare microbial OTUs were eliminated from the Venn diagram, and no subsampling was done. Wilcoxon rank-sum test. TLEA, temporal lobe epilepsy with anxiety disorders; TLEW, temporal lobe epilepsy but without anxiety disorder; OTU, operational taxonomic unit.

Figure 4

β-diversity of different groups of gut bacteria at the OTU level. Biodiversity of bacterial microbiota. (A) PCA of microbiota in TLEA and TLEW patients. (B) PCoA of microbiota in TLEA and TLEW patients. TLEA, temporal lobe epilepsy with anxiety disorders; TLEW, temporal lobe epilepsy but without anxiety disorder; OTU, operational taxonomic unit; PCA, principal component analysis; PCoA, principal coordinate analysis.

Figure 5

The abundance of gut bacteria in TLEA. The abundance of bacterial communities in TLEA patients at the phylum (A), class (B), order (C), family (D), and genus (E) levels. TLEA, temporal lobe epilepsy with anxiety disorders; TLEW, temporal lobe epilepsy but without anxiety disorders.

Figure 6

Different levels of bacterial biomarkers in TLEA patients. The abundance of bacterial communities at the phylum (A), class (B), order (C), family (D), and genus (E) levels for TLEA samples. (F) Macrogenomic biomarkers were analyzed utilizing LEfSe. Significant taxonomic variations in intestinal bacterial communities were identified between the three groups (LDA > 4, non-parametric factor Kruskal–Wallis rank-sum test, P < 0.05). (G) Branching map of bacterial compartments. Nodes of different colors indicate enriched microbiota and show significant differences between the groups. The size of the circles in the branching map is proportional to fungal abundance. From inside to outside, the circles represent the phylum, order, phylum, and family of fungi, respectively. *P < 0.05; **P < 0.01; and ***P < 0.001. LDA, linear discriminant analysis; LEfSe, LDA effect size; TLEA, temporal lobe epilepsy with anxiety disorders; TLEW, temporal lobe epilepsy but without anxiety disorders.

Figure 7

Diversity of gut fungi in TLEA patients. Sobs (A), Ace (B), Chao (C), and Simpson (D) differences in OTU levels of fungal flora in patients with TLEA and TLEW groups. Values represent the diversity of species. (E) Analysis of fungal β-diversity utilizing principal component analysis (PCA). There was no significant clustering among the groups of samples. (F) Analysis of similarity between fungi (ANOSIM/Adonis). The vertical coordinate indicates the distance rank calculated at the level of operational taxonomic units (OTUs) for each group. TLEA, temporal lobe epilepsy with anxiety disorders; TLEW, temporal lobe epilepsy but without anxiety disorder; OTU, operational taxonomic unit; ANOSIM, analysis of similarities; TLEA, temporal lobe epilepsy with anxiety disorders; TLEW, temporal lobe epilepsy but without anxiety disorders.

Figure 8

The abundance of fungal communities in TLEA patients at the phylum (A), class (B), order (C), family (D), and genus (E) levels. TLEA, temporal lobe epilepsy with anxiety disorders; TLEW, temporal lobe epilepsy but without anxiety disorders.

Figure 9

Different levels of fungal biomarkers in TLEA patients. The abundance of fungal communities at the phylum (A), class (B), order (C), family (D), and genus (E) levels for TLEA samples. (F, G) LEfSe bar plot of fungal communities. Linear discriminant analysis (LDA) has been performed to assess the influence of each component's abundance on differential effects. *P < 0.05; **P < 0.01; and ***P < 0.001. LDA, Linear discriminant analysis; LEfSe, LDA effect size; TLEA, temporal lobe epilepsy with anxiety disorders; TLEW, temporal lobe epilepsy but without anxiety disorders.

Figure 10

Predicted microbial functions altered in TLEA patients. (A) A heatmap of the module based on PICRUSt 1. Differences among TLEA and TLEW. (B) Variations in the composition of fungal functional groups inferred by FUNGuild. Differences in fungal functional groups among TLEA and TLEW patients. TLEA, temporal lobe epilepsy with anxiety disorders; TLEW, temporal lobe epilepsy but without anxiety disorders.

Figure 11

RDA of anxiety impact factors (arrows). Bacterial and fungal flora are shown in (A, B), respectively. The values of axis 1 and axis 2 are the percentages explained by the corresponding factor. RDA, redundancy analysis; TLEA, temporal lobe epilepsy with anxiety disorders; TLEW, temporal lobe epilepsy but without anxiety disorders.