Epilepsy is a common, chronic neurological disorder characterized by sudden abnormal excessive ultra-synchronized neuron discharges that result in temporary involuntary brain dysfunction (Fisher et al., 2005). Globally, there are 70 million people with epilepsy, with the highest incidence in infants and the elderly, posing a tremendous social burden throughout the world (Collaborators, 2019; Thijs et al., 2019). Despite advances and innovations in antiepileptic medications, approximately one-third of the patients suffer from drug-resistant epilepsy (de Biase et al., 2019). Therefore, further insights into the pathogenesis and the exploration of novel therapeutic targets for epilepsy are required.

There is growing evidence that the gut microbiome (GM) can regulate host homeostasis, including cardiovascular function, metabolism, and immune/inflammatory response (Le Chatelier et al., 2013). Recent research has shown that GMs play a role in neuropsychiatric disorders (Iannone et al., 2019), as they regulate brain function and behavior via the microbiota-gut-brain axis (Johnson and Foster, 2018). Differences in GM taxa have been identified in epilepsy patients compared to controls (Dong et al., 2022). The ketogenic diet (KD) is a treatment approach for intractable epilepsy (D’Andrea Meira et al., 2019). During the KD treatment of drug-resistant epilepsy, the GM pattern was altered simultaneously (Lindefeldt et al., 2019). Consequently, GMs may be involved in the crosstalk between KD and epilepsy (Fan et al., 2019). Furthermore, researchers are investigating the possibility of using the change in GM composition as a surrogate marker for the efficacy of the KD treatment in patients with drug-resistant epilepsy (Thambi et al., 2020). However, the effect of various GM taxa on epilepsy has not yet been determined. The 16S rRNA and metagenomic sequencing are the most widely used methods for identifying GM taxonomic characteristics (Durazzi et al., 2021), providing the basis for identifying the potential role of GM taxa. Recent research has increasingly focused on the causal effects of GMs on epilepsy, particularly refractory epilepsy (Lum et al., 2020). In addition, perturbations for certain GM taxa levels have been reported to affect the activity of epileptic neurons (Darch and McCafferty, 2022).

Nevertheless, the specific contribution of various GM taxa to epilepsy warrants further exploration. Similar to randomized controlled trials (RCT), the Mendelian randomization (MR) study is a novel research method for exploring the causal association between exposure and outcome (Swanson et al., 2017). In MR studies, single nucleotide polymorphisms (SNPs) are considered instrumental variables (IVs) to estimate the causal association between exposures and the outcomes of interest (Burgess et al., 2017). SNPs conform to the principle of random assignment of genetic variants at meiosis, which avoids the effect of confounding factors and the potential impact of reverse causation since genetic variants precede the onset of disease (Lawlor et al., 2008). Therefore, the causal associations of exposure factors of interest to outcomes can be identified more rapidly by MR analysis compared to RCT. For example, a recent MR study by Cai et al. has identified several blood metabolites with potential causal associations with epilepsy (Cai et al., 2022). Here, we conducted an MR study using large-scale GWAS summary statistics of GMs and epilepsy to identify potentially influential GM taxa, which could provide confidence to some existing evidence and may yield new insights into the prevention and treatment of epilepsy.

Our study comprehensively assessed the causal effect of 211 GM taxa (from phylum to genus level) on epilepsy and its sub-types. Finally, we identified a total of 23 causal relationships, of which 21 were nominal causal relationships, and two were strong causal relationships, thus highlighting the importance of GMs in epilepsy.

Accumulating evidence has suggested crosstalk between GMs and the central nervous system (CNS) (Cryan and Dinan, 2012). Investigations have shown that GMs play a vital role in the development of the enteric nervous system, blood–brain barrier, and glial cells, which are all important for cognitive development and behavior regulation (Braniste et al., 2014; Collins et al., 2014). Various neurological disorders, including multiple sclerosis (Jangi et al., 2016), autism (Mulle et al., 2013), Alzheimer’s disease (Jiang et al., 2017), and Parkinson’s disease (Parashar and Udayabanu, 2017), have been linked to intestinal dysbiosis. Recent findings also suggest that GMs may also play a role in epilepsy (Russo, 2022). Several studies have examined the effect of the KD, a treatment for refractory epilepsy, on GMs to explore the potential mechanisms of GMs in KD treatment (Lum et al., 2020). However, it is inconclusive which GM taxa have the most significant impact on epilepsy. As one-third of patients with epilepsy are diagnosed with refractory epilepsy (Dahlin and Prast-Nielsen, 2019), exploring biomarkers of epilepsy on the GMs level could offer promising alternative treatment options and potentially prevent the need for invasive treatments such as vagus nerve stimulation (VNS) or epilepsy surgery (Braakman and van Ingen, 2018).

Our study identified two strong causal relationships. Class Betaproteobacteria (OR = 1.357, 95% CI: 1.126–1.635, p = 0.001) and Order Burkholderiales (OR = 1.336, 95% CI: 1.112–1.606, p = 0.002) significantly elevated the epilepsy risk after Bonferroni correction. Burkholderiales, an order of Betaproteobacteria, was found to have a potential impact on epilepsy from our MR study, which was consistent with the findings of some previous investigations. For instance, Safak et al. identified the genus Delftia and genus Lautropia, which are members of Burkholderiales, to be significantly higher in the intestine of epilepsy patients versus healthy individuals (Safak et al., 2020). In addition, another genus of Burkholderiales, Sutterella, which was reported with increased intestinal abundance in adult patients with epilepsy (Dong et al., 2022), was also identified in our study to be nominally associated with an increased risk of focal epilepsy. The present MR study could provide evidence and confidence for the increased level of genera belonging to Order Burkholderiales in the intestines of epilepsy patients.

It’s important to note that the Bonferroni correction can result in false negatives. Our findings showed 21 GM taxa with nominal causal connections, but these correlations vanished after applying the Bonferroni correction. This may be due to the crosstalk between the gut-brain axis being usually coordinated by multiple factors and that the role of a single microbiota in the genus level in causing disease may not be as important as previously thought. In fact, several GM taxa with nominal causal relationships identified in this study corroborate the findings of previous research. For instance, Huang et al. revealed that patients with cerebral palsy and epilepsy contained a higher proportion, in comparison to healthy controls, of Bifidobacterium and Akkermansia (Huang et al., 2019). In addition, Gong and colleagues identified Bifidobacterium, Ruminococcaceae UCG 014, and Akkermansia at the genus level were increased in patients with epilepsy compared to healthy controls (Gong et al., 2020). Further, Lee et al. identified Enterococcus faecium (species of class Bacilli), Bifidobacterium longum (species of genus Bifidobacterium), and Eggerthella lenta (species of phylum Actinobacteria) as biomarkers for drug-resistant epilepsy (Lee et al., 2021). Although only nominal causal associations were identified at the genus level for these GM taxa, the coordination and crosstalk between various GM taxa remain worthy of in-depth study in the future.

The mechanisms involved in the relationship between GMs and epilepsy have not been fully determined. However, some evidence suggests potential mechanisms. (i) Studies have reported that GMs can alter neurotransmitter levels such as glutamate, gamma-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT) (Mittal et al., 2017), as well as increase levels of cytokines, chemokines, such as TNF⍺ and MCP-1, lipopolysaccharides (LPS) which led to generalized immune activation or inflammation (Blander et al., 2017), contributing to the risk of seizures. (ii) GMs have been demonstrated to interact with gut-derived metabolites, resulting in both beneficial and detrimental mechanisms for the central nervous system (Tran and Mohajeri, 2021). (iii) In addition, GMs can affect the hypothalamic–pituitary–adrenal (HPA) axis (Sudo et al., 2004) and the levels of brain-derived neurotrophic factor (BDNF) (Maqsood and Stone, 2016), which promote seizure propensity. (iv) GMs also regulate peripheral metabolites and central neurotransmitter metabolism, which affect seizure susceptibility (Lum et al., 2020). Nevertheless, the specific mechanism and crosstalk between different GM taxa remain to be verified by future studies.

The limitations of the present study should be noted: (i) Since the number of IVs fulfilling the strict threshold (p < 5 × 10⁻⁸) was extremely small, a relatively lenient threshold (p < 1 × 10⁻⁵) was adopted for screening IVs. (ii) This study included individuals of essentially European ancestry, so extrapolating the findings to other populations is limiting. (iii) The number of cases of the two subtypes of epilepsy under strict definition (generalized epilepsy and focal epilepsy) is relatively small, so future analysis based on a larger sample size of GWAS summary data is necessary to increase the confidence of the results. (iv) The GM-related GWAS summary-level dataset included in this study was based on 16S rRNA sequencing, and thus further analysis based on large-scale studies with more advanced methods, such as metagenomics sequencing, is required in the future in order to evaluate the species-level. (v) Current studies of GMs have focused only on bacteria; however, other types of GMs may also have potential functions.

Overall, by performing MR analysis of the causal effects of 211 GM taxa on epilepsy and its sub-types, we finally identified 21 nominal causal relationships and two strong causal relationships. Among them, Class Betaproteobacteria and Order Burkholderiales are significantly associated with increased epilepsy risk. However, it is essential to recognize that since the present study was conducted based on the GWAS summary-level dataset generated from 16S rRNA sequencing, further in-depth analyses based on more advanced large-scale studies generated from metagenomics sequencing are necessary. Nevertheless, our findings may provide helpful biomarkers for disease progression and potential candidate therapeutic targets for epilepsy.

Publicly available datasets were analyzed in this study. This data can be found at: https://mibiogen.gcc.rug.nl/, https://r7.finngen.fi/.

Publicly available de-identified data from participant studies approved by an ethical standards committee were used in this study. Therefore, no additional separate ethical approval was required for this study.

YZ designed the study, analyzed the data, and wrote the manuscript. SC assisted in analyzing the data and revising the manuscript. HY critically read and edited the manuscript. All authors contributed to the article and approved the submitted version.

This research was funded by the Natural Science Foundation of Hunan Province (2022JJ70069).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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