Mitochondrial epilepsy, a challenging condition often refractory to single anti-epileptic drug (AED) therapy, often necessitates combination drug therapies. However, the potential for mitochondrial toxicity from AEDs necessitates careful consideration. The ketogenic diet (KD), a low-carbohydrate, high-fat diet that induces ketosis, has emerged as a promising adjunctive therapy. KD's efficacy has been attributed to its role in improving mitochondrial biogenesis and function, reducing oxidative stress, and potentially modulating brain excitability via mechanisms such as affecting glutamate synaptic transmission and activating ATP-sensitive potassium channels. While effective in various neurological disorders including Alzheimer's disease and autism spectrum disorders, the precise mechanisms by which KD exerts its beneficial effects on brain activity remain unclear. Recent research highlights the importance of the gut microbiota in health and disease, and its potential interaction with neurological disorders via the gut-brain axis. Dietary changes profoundly influence gut microbiota composition and function. Prior studies on KD's effects on gut microbiota in epilepsy patients have yielded varied results, possibly due to differences in dietary fat source, intervention duration, patient demographics, and epilepsy etiology. Therefore, this study aimed to investigate the impact of KD on the composition and function of gut microbiota in children with mitochondrial epilepsy, focusing on the potential links between ketosis, gut microbiota, and disease alleviation.

Several studies have explored the impact of ketogenic diets on gut microbiota in epilepsy patients. Xie et al. (2017) observed changes in the gut microbiota of epileptic infants after KD treatment, with reductions in pathogenic bacteria and increases in beneficial bacteria. Zhang et al. (2018) reported decreased alpha-diversity and altered proportions of Firmicutes and Bacteroidetes in refractory epilepsy patients following KD. In contrast, Lindefeldt et al. (2019) found no significant change in alpha-diversity but observed alterations in specific bacterial groups after KD treatment. These discrepancies highlight the need for further investigation into the relationship between KD, gut microbiota, and epilepsy.

Fifteen children with mitochondrial epilepsy, confirmed via genetic diagnosis and abnormal biomarkers, were recruited between January 2019 and December 2020. Patients were randomly assigned to a KD group (KD + AED) or a control group (regular diet + AED). Fecal samples were collected before and after 3 months of KD treatment (12 weeks) in the KD group, and in the control group, the samples were collected initially followed by 12 weeks of KD treatment. 16S rRNA gene V3-V4 region sequencing was performed to analyze the gut microbiota composition. Bioinformatics analysis using Trimmomatic, cutadapt, and dada2 within QIIME2 was performed for quality control, primer removal, and chimera removal. Alpha (Shannon and Chao1) and beta (weighted Bray-Curtis distance using PCoA) diversity were assessed. LEfSe analysis identified differentially abundant taxa between groups. Functional prediction was performed using Tax4Fun based on the KEGG database. Statistical analysis (Student's t-test, chi-square test, Spearman correlation) was performed using SPSS.

No significant differences were observed in age, sex, epilepsy type, or AED use between the KD and control groups. While Shannon diversity showed no significant difference between groups, Chao1 diversity was significantly higher in the control group (p<0.05). Beta-diversity analysis revealed distinct clustering between KD and control groups. At the phylum level, Firmicutes abundance was lower in the KD group (42.76%) compared to the control group (48.13%), while Bacteroidota abundance was significantly higher in the KD group (36.93% vs. 25.41%). LEfSe analysis showed Actinobacteriota and Phascolarctobacterium were significantly enriched in the control group, while Bacteroides (particularly Bacteroides fragilis) was significantly enriched in the KD group. Functional analysis using Tax4Fun identified significant differences in 12 KEGG pathways at level 3, impacting various metabolic processes such as the Citrate cycle, Pertussis, and Purine metabolism pathways.

The findings demonstrate that KD significantly alters the gut microbiota composition and function in children with mitochondrial epilepsy. The increased abundance of Bacteroides fragilis in the KD group is particularly noteworthy, as this bacterium has been implicated in neuroprotection and has shown promise in treating other neurological disorders. The observed changes in specific bacterial groups and metabolic pathways may serve as potential biomarkers for KD's therapeutic effect. The decrease in pathways such as Purine metabolism is also interesting, as purine metabolism is significantly linked to energy metabolism, and KD interventions increase energy levels. The alteration in other pathways should be studied further. While this study provides valuable insights, further research is needed to elucidate the precise mechanisms through which KD modulates the gut microbiota and its relationship to epilepsy alleviation.

This study demonstrates that a 3-month KD intervention significantly alters the gut microbiota composition and function in children with mitochondrial epilepsy. The increase in Bacteroides fragilis and the changes in various metabolic pathways suggest potential biomarkers for KD efficacy. However, the relatively small sample size limits the generalizability of findings, and future studies with larger sample sizes and extended KD intervention durations are necessary to confirm and further elucidate these effects. Further investigation into the specific metabolite changes within the gut and the effects on the nervous system will strengthen this area of research.

The main limitation of this study is the relatively small sample size (n=15). This limits the statistical power and the generalizability of the findings. Additionally, the study design was not blinded, which could introduce bias. The relatively short duration of the KD intervention (3 months) may not fully capture the long-term effects of the diet on the gut microbiota. Finally, the lack of detailed information on patients' dietary habits before the study began makes it challenging to fully understand the impact of the diet change on the microbiome.