Identification of a gut microbiota signature in patients with drug-resistant epilepsy upon ketogenic diet treatment

Principle Investigator

Scientific interest within the context of the graduate college:

Our research project entitled “Identification of a gut microbiota signature in patients with drug-resistant epilepsy upon ketogenic diet treatment” aims to characterize the composition of the gut microbiota of patients affected by drug-resistant epilepsy undergoing ketogenic diet treatment, with the ultimate goal of developing probiotics as therapeutic tool. This research is characterized by combining both the clinical analysis of patients with epilepsy (encephalogram, NMRI, epigenetic changes and questionnaires on lifestyle and quality of life) and the molecular and cellular analysis of patients’ blood and stool. This project stands out for its strong clinical and experimental approach in close collaboration with the Clinic for Paediatrics and Neurology, Charité. The successful candidate will benefit from a training in which he/she will be able to put into practice his/her knowledge of the medical field and learn concepts, techniques and strategies of experimental research in the laboratory, paving the way to better understand how we could modulate the course of epilepsy via microbiota-diet modulation.

In this research project, the successful candidate will be able to:

• manage human sampling collection for research purposes.
• learn about the ketogenic diet as a therapy in the clinics
• learn about patient’s group stratification according to the different profiles in response to the treatment
• prepare and analyze questionnaires to track dietary habits, lifestyle habits, and quality of life
• extract and quantify genomic DNA from human stools
• get knowledge in microbial composition bioinformatic analysis

Project description:

Epidemiology and social impact of epilepsy and drug-resistant epilepsy (DRE): Epilepsy is one of the most common neurological disorders, affecting more than 50 million people worldwide and 6 million people in Europe (Singh & Sander, 2020; Sirven, 2015). It affects people of all ages and is characterized by epileptic seizures (Berg et al., 1999; Fiest et al., 2017; Guerrini, 2006; Sirven, 2015). Uncontrolled seizures can lead to developmental delay, cognitive deficits, memory and learning difficulties. They can also cause sudden unexpected death in people with epilepsy (SUDEP) (Sperling, 2004). Current treatments include anti-seizure medicines (ASM), diet, epilepsy surgery and stimulation devices. ASM can control seizures in about two-thirds of people with epilepsy. However, one-third of patients with epilepsy cannot be controlled by two or more correctly selected and dosed ASM, what is called drug-resistant epilepsy (DRE) (Kwan et al., 2009, 2011; Picot et al., 2008; Sultana et al., 2021).

The microbiota-gut-brain axis: a modulator for epileptogenesis: The causes of epilepsy include structural, genetic, infectious, metabolic and immune factors (Guerri et al., 2020; Matin et al., 2015; Vezzani et al., 2016; Oliver et al., 2023; Rho & Boison, 2022). The gut-brain axis (GBA) has also been implicated in this respect (Iannone et al., 2019). Gut microbiota produces a variety of compounds that can affect brain function, such as neurotransmitters and metabolites, like short-chain fatty acids (SCFAs) (Chen et al., 2021; Dalile et al., 2019; Silva et al., 2020), some of which have shown anticonvulsant effects in rodents (De Caro et al., 2019; Mu et al., 2022; Olson et al., 2018). In patients, studies have confirmed that bacterial-based approaches, such as probiotics or microbiota-modifying treatments, can reduce seizure frequency. Indeed, the pilot usage of a mixture of probiotics (eight bacterial subspecies of Lactobacillus, Bacteroides and Streptococcus) reduced seizure frequency and improved the quality of life in patients with DRE (Gómez-Eguílaz et al., 2018). Similarly, fecal microbiota transplantation (FMT) for the treatment of Crohn’s disease (CD) symptoms ameliorated seizures frequency in a 22-year-old woman with a 17-year history of epilepsy, who stopped taking ASM after the FMT (He et al., 2017). Despite these promising results, the mechanisms by which the microbiota may affect the development of seizures in patients are completely unclear.

Ketogenic diet in the management of DRE: Alternative treatments for DRE include dietary treatments such as the ketogenic diet (KD) (Hemingway et al., 2001; Lyons et al., 2020). KD is a diet based on a high fat intake accompanied by moderate protein, and very low or no carbohydrate content, usually in a 3:1 or 4:1 ratio of caloric intake from lipids to both protein and carbohydrate together, and with a normal total energy intake. Restricting carbohydrate intake forces the metabolism to replace the preferred energy source, glucose, with fatty acids, which are oxidized in the liver to form ketone bodies. Although the classical KD has been used in epilepsy for many years and its efficacy in reducing seizures has been confirmed in several studies, especially when introduced in young age (Martin-McGill et al., 2020), the mechanism behind its action is not well understood in humans. In particular, it is not known why this dietary intervention has an anti-seizure effect in a proportion of the patients, while in others there is poor or no effect (Martin-McGill et al., 2020) or even worsening of seizure frequency or appearance of adverse effects (Cai et al., 2017; Newmaster et al., 2022; Yan et al., 2018). Although the exact mechanism is not yet known, several research studies in both mice and human have shown a link between the gut microbiota and the effects of the KD, topic that we recently discussed (Díaz-Marugan et al., 2024).

Hypothesis: We hypothesize that the success of KD treatment in patients with drug-resistant epilepsy is related to the composition and function of the patient’s gut microbiota, so it would be possible to use the bacterial characteristics as biomarker to assess KD efficacy and as a probiotic tool to improve the response in those patients with a low KD-responsive profile. To test our hypothesis, our objectives are:

Aim 1: Identification of a KD-specific bacterial signature.

• To identify differences in bacterial composition and function in a large cohort of patients who respond or do not respond to KD.
• To track bacterial changes throughout the KD intervention and correlate them with seizure improvement or worsening.
• To analyze the duration of the effect of the ketogenic diet on both seizure improvement and gut microbiota changes even after the end of the intervention.

Aim 2: Isolation of bacteria associated with amelioration of seizure upon KD introduction.

• To isolate the most representative bacteria from KD responder patients.

Aim 3: Preclinical Model.

• To dissect the mechanisms of action of specific bacteria isolated from KD responders in gnotobiotic preclinical mouse models of epilepsy.

The successful candidate for this fellowship will focus on addressing aim 1. However, depending on the progress of the project (Table 1) it will be possible to start the experiments related to aim 2 (isolation of potential beneficial bacteria associated with a positive outcome of KD). This will be performed in the anaerobic chamber of our lab, through the cultivation of the bacteria from the human feces’ samples and in collaboration with Dr. Thomas Clavel (Uniklinik RWTH Aachen, Germany) using methods for single-cell dispensing of bacteria from human samples, as reported (Afrizal et al., 2022).

Table 1. Timeline. Isolation of bacteria will be possible depending on the development of the aim 1 of the research project.

Abbreviations: ASM: anti-seizure medicines; DRE: drug-resistant epilepsy; FatsQ: fats-type questionnaire; FFQ: food frequency questionnaire; KD: ketogenic diet; LifeSQ: lifestyle questionnaire; QOLIE 31: quality of life in epilepsy questionnaire

References