Compositions and methods for the treatment of seizures and related disorders

Abstract

Provided herein are methods and compositions for treating or preventing seizures.

Description

[0001] COMPOSITIONS AND METHODS FOR THE TREATMENT OF SEIZURES AND

[0002] RELATED DISORDERS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This patent application claims priority to U.S. Provisional Patent Application No. 63 / 677,089, filed July 30, 2024, which is hereby incorporated by reference in its entirety.

[0005] GOVERNMENT SUPPORT

[0006] This invention was made with government support under NS 115537 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] BACKGROUND

[0008] Epilepsy is characterized by recurrent seizures that can lead to loss of awareness, loss of consciousness, and / or disturbances of movement, autonomic function, sensation (including vision, hearing and taste), mood, and / or mental function. Epilepsy afflicts 1-2% of the population in the developed world.

[0009] The low-carbohydrate, high-fat ketogenic diet (KD) is a treatment for refractory epilepsy, wherein more than one-third of epileptic individuals do not respond to existing anticonvulsant medications. The efficacy of the KD is supported by multiple retrospective and prospective studies, which estimate that -30% of patients become seizure-free, and -60% experience significant benefit. However, despite its value for treating epilepsy and its increasing application to other disorders, including autism, Alzheimer’s disease, Parkinson’s disease, metabolic syndrome and cancer, use of the KD remains low due to difficulties with implementation, dietary compliance and adverse side effects. In fact, even with successful seizure reduction, epileptic patient retention on the KD is only an estimated 12% by the third year of dietary therapy. Moreover, mechanisms underlying the beneficial effects of the KD are poorly understood, and molecular and / or cellular targets for intervention are lacking. That the diet succeeds in controlling various types of symptoms in cases when drugs fail suggests that it enhances endogenous neuroprotective pathways that are not targeted by existing medications. Compositions and methods for replicating these effects would be highly useful. SUMMARY OF THE INVENTION

[0010] The gut microbiome is emerging as an important modulator of the anti-seizure effects of the classic ketogenic diet. However, many variations of the ketogenic diet are used clinically to treat refractory epilepsy, and how different dietary formulations differentially modify the gut microbiome in ways that impact seizure outcome is poorly understood. Clinically prescribed ketogenic infant formulas vary in macronutrient ratio, fat source, and fiber content and also in their ability to promote resistance to 6-Hz psychomotor seizures in mice. By screening specific dietary variables for their effects on a model human infant microbial community, the instant disclosure demonstrates that particular bacteria in the gut microbiome drives substantial metagenomic shifts. Distinct subsets of metagenomic responses in the model human infant microbial community have been identified that correspond with increased seizure resistance in mice. Supplementation with seizure- protective fibers and bacteria enriches microbial representation of genes related to L-alanine biosynthesis, queuosine biosynthesis, and preQo biosynthesis, which is also seen in seizure- protected mice that are fed fiber-containing ketogenic infant formulas. The present disclosure shows that different formulations of clinical ketogenic diets, and dietary fiber content in particular, differentially impact seizure outcome in mice through modification of the gut microbiome. Leveraging interactions between dietary components of the ketogenic diet, the gut microbiome, and host susceptibility to seizures informs novel microbiome-guided approaches to treat refractory epilepsy.

[0011] In some aspects, provided herein are methods and compositions for preventing or treating a condition responsive to a ketogenic diet in a subject, comprising administering to the subject a composition comprising bacteria of the Bacteroidetes phylum, Proteobacteria phylum, Deferribacteres phylum, Streptococcaceae family, Coriobacteriia class, Bifidobacterium genera, Bacteroides genera, and / or Clostridium genera. In some embodiments, the composition comprises Bifidobacterium infantis (e.g., Bifidobacterium longum sups, infantis DSM 20088). In some embodiments, the composition comprises Bifidobacterium longum (e.g., Bifidobacterium longum sups, longum ATCC BAA-999). In some embodiments, the composition comprises Bifidobacterium breve (e.g., Bifidobacterium breve DSM 20213). In some embodiments, the composition comprises Bacteroides fragilis (e.g., Bacteroides fragilis ATCC 25285). In some embodiments, the composition comprises Clostridium perfringens (e.g., Clostridium perfringens ATCC 13124). Also provided herein are combinations of the agents / bacteria disclosed herein for use in the methods described herein. Such combinations may include a bacteria disclosed herein, such as Bacteroidetes phylum, Proteobacteria phylum, Deferribacteres phylum, Streptococcaceae family, Coriobacteriia class, Bifidobacterium genera, Bacteroides genera, and / or Clostridium, in combination with an enriching agent, such as dietary fiber or another agent disclosed herein. In some embodiments, the method comprises a combination method comprising administering a bacteria of the Streptococcaceae family and a bacteria of the Coriobacteriia class. In some embodiments, the method comprises a combination method comprising administering a bacteria of Streptococcaceae family and a bacteria of the Deferribacteres phylum. In some embodiments, the method comprises a combination method comprising administering a bacteria of the Deferribacteres phylum and a bacteria of the Coriobacteriia class. In some embodiments, the method further comprising administering one or more types of dietary fiber, such as FOS, inulin, gum arabic and / or cellulose. In some embodiments, the method further comprising administering a mixture of dietary fiber, such as a mixture comprising FOS, inulin, gum arabic and cellulose.

[0012] In another aspect, provided herein are methods and compositions for preventing or treating a condition responsive to a ketogenic diet in a subject, comprising administering to the subject an agent that decreases levels of one or more bacteria in the subject’s gut, wherein the one or more bacteria comprise bacteria of the Actinobacteria phylum, Erysipelotrichia class, Escherichia genera, Enterococcus genera, Mammaliicoccus genera, Bifidobacterium species, Bacteroides species, and / or Klebsiella species.

[0013] In some embodiments, the agent decreases levels of Escherichia coli (e.g., Escherichia coli K-12 ATCC 10798). In some embodiments, the agent decreases levels of Enterococcus faecalis (e.g., Enterococcus faecalis ATCC 19433). In some embodiments, the agent decreases levels of Mammaliicoccus sciuri. In some embodiments, the agent decreases levels of Bifidobacterium breve (e.g., Bifidobacterium breve DSM 20213). In some embodiments, the agent decreases levels of Bifidobacterium infantis (e.g., Bifidobacterium longum sups, infantis DSM 20088). In some embodiments, the agent decreases levels of Bacteroides vulgatus (e.g., Bacteroides vulgatus ATCC8482). In some embodiments, the agent decreases levels of Klebsiella pneumoniae (e.g., Klebsiella pneumoniae subsp. pneumoniae ATCC 13883).

[0014] Also provided herein are combinations of the agents disclosed herein for use in the methods described herein. In some embodiments, the method comprises a combination method comprising administering lactose or a suppressing agent. In some embodiments, the method further comprising administering one or more types of lactose. In some embodiments, the method further comprising administering a mixture of lactose.

[0015] In another aspect, provided herein are methods and compositions for preventing or treating a condition responsive to a ketogenic diet in a subject, comprising administering to the subject a composition comprising L-alanine, queuosine, preQo, guanosine, guanosine triphosphate, alpha-ketoglutarate (2-oxoglutarate), citrate, succinate and sucrose and / or lactose.

[0016] In some embodiments, the condition is seizures, optionally wherein subject has a neurodevelopmental condition, e.g., selected from epilepsy, autism spectrum disorder, Rett syndrome, attention deficit disorder, and fragile X syndrome. In some embodiments, the subject has a condition selected from Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), cancer, stroke, a metabolic disease, a mitochondrial disorder, depression, migraines, and traumatic brain injury (TBI).

[0017] In some embodiments, the method further comprises administering to the subject a fiber supplement (e.g., inulin, fructooligosaccharides, acacia fiber, gum arabic, cellulose, and / or orange fiber). In some embodiments, the method further comprises administering to a subject a composition comprising L-alanine, queuosine, preQo, guanosine, guanosine triphosphate, alpha-ketoglutarate (2-oxoglutarate), citrate, succinate and sucrose and / or lactose.

[0018] Also provided herein are compositions. The composition may be self-administered. The composition may be a food product, such as yogurt or fiber supplement. In some embodiments, the composition is a synbiotic product, such as one that combines bacteria (e.g., probiotic, such as bacteria of the Bacteroidetes phylum, Proteobacteria phylum, Deferribacteres phylum, Streptococcaceae family, Coriobacteriia class, Bifidobacterium genera, Bacteroides genera, and / or Clostridium genera) and a fiber mixture that supports the growth of bacteria or bacterial mixture. In some embodiments, the composition is infant formula. In some embodiments, the food product is a fiber supplement or mixture containing one or more of the dietary fiber types disclosed herein.

[0019] In some embodiments, the subject is on a diet (e.g., a ketogenic diet) that includes at least one dietary fiber (e.g., a dietary fiber disclosed herein) BRIEF DESCRIPTION OF THE FIGURES

[0020] FIG. 1A-1C shows the different formulations of medical ketogenic diets (KD) and the resulting differential responses to 6-Hz seizures in mice who receive the diets. FIG. 1A shows macronutrient composition without fiber (for determining KD fat ratio), macronutrient composition with fiber, absence / presence of fat sources, and percent carbohydrate composition for the commercial KD infant formulas KD4: 1, KD3: 1, and MCT2.5: 1, relative to standard infant formula as control diet (CD). FIG. IB shows the experimental design: 4 week old conventional (specific pathogen free, SPF) Swiss Webster (SW) mice (n=14 mice / group) were fed each medical KD or CD as liquid diets for 7 days. FIG. 1C shows 6-Hz seizure threshold (left) and latency to exploration (right) for mice fed KDs or CD as liquid diet (left, one-way ANOVA with Bonferroni, n=14 mice / group, ***p<0.001). The line at y = 10 s represents threshold for scoring seizures.

[0021] FIG. 2A-2E shows that medical KDs induce differential alterations in the gut microbiome that associate with resistance vs. susceptibility to 6-Hz seizures. FIG. 2A shows alpha diversity from fecal metagenomic sequencing data after treatment with KDs or CD (Kruskal-Wallis with Dunn’s test: *p < 0.05,**p <0.01 n.s., not statistically significant; n=4 cages / group. Data are presented as mean ± SEM). FIG. 2B shows principal coordinates analysis (PCoA) of Bray-Curtis dissimilarity (left) and weighted UniFrac distance (right) based on fecal metagenomic sequencing data after dietary treatment. (PERMANOVA, n = 4 cages / group). FIG. 2C shows the top 10 most abundant metagenomic superclass pathways (left). Differentially abundant pathways that are significantly altered in seizure susceptible group KD3 : 1 and / or shared between seizure protected groups KD4: 1 and MCT2.5: 1. (Kruskal-Wallis with Dunn’s test: *p < 0.05, **p< 0.01, ***p<0.001; n=4 cages / group. Data are presented as mean ± SEM.) FIG. 2D shows a venn diagram of differential metagenomic pathways (q<0.05) for each KD relative to CD. (MaAsLin2, General Linear Model (GLM); n=4 cages / group). FIG. 2E shows a heatmap of differential metagenomic pathways (q<0.05) that are shared between seizure-protected groups KD4: 1 and MCT2.5: 1 and not significant in seizure-susceptible group KD3: 1. (GLM statistical test, n=4 / condition)

[0022] FIG. 3A-3E shows the addition of dietary fiber to KDs enriches metagenomic features associated with seizure protection in a model human infant gut community and restores resistance to 6-Hz seizures in mice. FIG. 3A shows experimental design: Fiber mix containing inulin, gum arabic, cellulose, and fructooligosaccharide (FOS), or lactose as a non-fiber carbohydrate control, was added to KD-based synthetic culture media for anaerobic culture of a model human infant gut microbial community. FIG. 3B shows principal coordinates plots of metagenomic pathway abundance data for human infant microbes grown in KD-based media containing fiber mix versus lactose. (PERMANOVA, n=7 / condition). FIG. 3C shows a venn diagram of differential metagenomic pathways (q<0.05) shared across all fiber-containing KD media groups relative to corresponding lactose-containing media groups as controls (left). 15 fiber-induced differential metagenomic pathways (q<0.05) that are similarly seen in seizure protective mice fed KD4: 1 or MCT2.5: 1 (right). (General Linear Model, n=7 / condition). FIG. 3D shows the experimental design: 4 week old conventional (specific pathogen free, SPF) Swiss Webster (SW) mice (n=14-16 mice / group) were fed KD3: 1 supplemented with fiber mix, KD3: 1 alone, or CD as liquid diets for 7 days. FIG. 3E shows 6-Hz seizure threshold (left) and latency to exploration (right) for mice fed KD3 : 1+fiber mix, KD3 : 1, or CD as liquid diet (left, one-way ANOVA with Bonferroni, n=14-16 mice / group, ***p<0.001). The line at y = 10 s represents threshold for scoring seizures.

[0023] FIG. 4-4E shows the addition of excess dietary fiber to fiber-containing KD4: 1 further potentiates seizure resistance. FIG. 4A shows the experimental design: 4 week old conventional (specific pathogen free, SPF) Swiss Webster (SW) mice (n=16 mice / group) were fed KD4: 1 supplemented with fiber mix or KD4: 1 alone as liquid diets for 7 days. FIG. 4B shows the 6-Hz seizure threshold (left) and latency to exploration (right) for mice fed KD4: 1 and KD4: 1+fiber mix as liquid diet (left student t-test, n=16 mice / group, ***p<0.001). The line at y = 10 s represents threshold for scoring seizures. FIG. 4C shows experimental design: 13 dietary fiber sources and types were supplemented to KD4: 1 infant formula for anaerobic culture of a model human infant gut microbial community. FIG. 4D shows a heatmap of 15 fiber-induced differential metagenomic pathways (q<0.05) that were similarly seen in seizure-protected mice fed KD4: 1 or MCT2.5: 1 (right). Groupings were denoted on top of the dendrogram. (General Linear Model statistical test, n=10 / condition, * q<0.05 for fiber source / type relative to KD4: 1 as a control). FIG. 4E shows 6-Hz seizure threshold (left) and latency to exploration (right) for mice fed KD4: 1 supplemented with dietary fiber mix (Group 1), gum arabic (Group 2), or oat fiber (Group 3), or KD4: 1 alone as paste diet (left, one-way ANOVA with Bonferroni, n=14 mice / group, **p<0.01, ***p<0.001). The line at y = 10 s represents threshold for scoring seizures.

[0024] FIG. 5A-5D shows medical KDs administered as solid diets phenocopy differential seizure responses seen with liquid diets. FIG. 5A shows average caloric intake per cage for KDs and CD administered as liquid diet (n=3-4 cages). FIG. 5B shows serum betahydroxybutyrate from mice fed liquid KDs or CD. (One way ANOVA with Bonferroni: *p < 0.05, **p<0.01, ***p<0.00; n=14 mice / group. Data are presented as mean ± SEM). FIG. 5C shows 6-Hz seizure threshold (left) and latency to exploration (right) for mice fed KDs or CD as solid diet (left, one-way ANOVA with Bonferroni, n=16 mice / group, ***p<0.001). The line at y = 10 s represents threshold for scoring seizures. FIG. 5D shows average consumption of solid diets (n=4 cages; Kruskal-Wallis with Dunn’s test: *p < 0.05, **p<0.01. Data are presented as mean ± SEM).

[0025] FIG. 6A-6E shows the effects of KDs on taxonomic and metagenomic signatures of the fecal microbiome in mice. FIG. 6A shows taxonomic distributions of bacterial phyla from fecal metagenomics data of mice fed liquid KDs or CD (left, n = 4 cages / group). Relative abundances of Actinobacteria, Bacteroidetes, Bacteria unclassified, Proteobacteria, and Deferribacteres (right, n = 4 cages / group. Kruskal-Wallis with Dunn’s test: *p < 0.05, **p< 0.01 n.s., not statistically significant). FIG. 6B shows relative abundances bacterial taxa differentially altered by KD4: 1 and MCT2.5: l, but not KD3: l relative to CD. (n=4 cages / group. Kruskal -Wallis with Dunn’s test. *p < 0.05, **p< 0.01, n.s., not statistically significant. Data are presented as mean ± SEM). FIG. 6C shows relative abundances of bacterial taxa differentially altered by KD3: 1, but not KD4: 1 and MCT2.5: 1, relative to CD. (n=4 cages / group. Kruskal-Wallis with Dunn’s test. *p < 0.05, **p< 0.01. Data are presented as mean ± SEM). FIG. 6D shows a heatmap of differential metagenomic pathways (q<0.05) seen in seizure susceptible group KD3: 1, but not seizure protective groups KD4: 1 and MCT2.5: 1. (General Linear Model, q<0.05, n=4 / condition). FIG. 6E shows a heatmap of metagenomic pathways that are significantly associated with macronutrient composition (General Linear Model, *q<0.05, n=4 / condition).

[0026] FIG. 7A-7I shows the effects of fat ratio, fat source / type, and carbohydrate source for KD-based synthetic culture media on metagenomic profiles of a model human infant microbial community. FIG. 7A shows bacterial species comprising the model human infant microbial community, as compared to published data from human infants. FIG. 7B shows change in bacterial species abundance after 24 hour culture in rich complex medium as a control (average of n=10). FIG. 7C shows the experimental design: KD-based synthetic culture media was formulated with differing fat ratios for anaerobic culture of a model human infant gut microbial community. FIG. 7D shows the change in bacterial species abundance (left) and PCoA analysis of microbial taxonomic data (right) after 24 hour culture of model human infant gut microbial community in KD-based media with differing fat ratios (PERMANOVA, n=8 / condition). FIG. 7E shows the experimental design: KD-based synthetic culture media was formulated with differing fat sources that vary in level of saturation for anaerobic culture of a model human infant gut microbial community. FIG. 7F shows the change in bacterial species abundance (left) and PCoA analysis of microbial taxonomic data (right) after 24 hour culture of model human infant gut microbial community in KD-based media with differing fat sources (PERMANOVA, n=5-7 / condition). FIG. 7G shows the experimental design: KD-based synthetic culture media was formulated with differing fat types for anaerobic culture of a model human infant gut microbial community. FIG. 7H shows the change in bacterial species abundance (left) and PCoA analysis of microbial taxonomic data (right) after 24 hour culture of model human infant gut microbial community in KD-based media with differing fat types (PERMANOVA, n=5 / condition). FIG. 71 shows the change in bacterial species abundance (left) and PCoA analysis of microbial taxonomic data (right) after 24 hour culture of model human infant gut microbial community in KD-based media with differing carbohydrate sources (PERMANOVA, n=7 / condition).

[0027] FIG. 8 shows the addition of dietary fiber to KD-based synthetic culture media alters metagenomic signatures in a model human infant gut microbial community through a heatmap of differential metagenomic pathways (q<0.05) seen in model human infant gut microbial community after 24 anaerobic culture in fiber-containing KD-based media compared to lactose-containing KD-based media (General Linear Model, n=7 / condition).

[0028] FIG. 9A-9B shows the addition of fiber to KD3 : 1 as a solid diet phenocopies increases in seizure resistance seen with liquid diet. FIG. 9A shows 6-Hz seizure threshold (left) and latency to exploration (right) for mice fed KD3:l+fiber mix, KD3: 1, or CD as solid diet (left, one-way ANOVA with Bonferroni, n=14 mice / group, ***p<0.001). The line at y = 10 s represents threshold for scoring seizures. FIG. 9B shows the average consumption of solid diets (n=4 cages; Kruskal -Wallis with Dunn’s test. Data are presented as mean ± SEM).

[0029] FIG. 10A-10C shows the addition of excess fiber to KD4: 1 as a solid diet phenocopies increases in seizure resistance seen with liquid diet. FIG. 10A shows the average caloric intake per cage for KD4: 1 and KD4: 1+fiber administered as liquid diet (n=4 cages). FIG. 10B shows the 6-Hz seizure threshold (left) and latency to exploration (right) for mice fed KD4: 1+fiber mix or KD4: 1 as solid diet (left, one-way ANOVA with Bonferroni, n=14 mice / group, ***p<0.001). The line at y = 10 s represents threshold for scoring seizures. FIG. IOC shows the average consumption of solid diets (n=4 cages; Kruskal -Wallis with Dunn’s test. Data are presented as mean ± SEM).

[0030] FIG. 11A-11B shows that SCFA supplementation does not phenocopy effects of fiber supplementation on KD-induced response to 6-Hz seizures. FIG. 11A shows 6-Hz seizure threshold (left) and latency to exploration (middle) for mice fed KD4: 1 paste diet and supplemented with SCFAs or vehicle (NaCl) control in the drinking water (left, one-way ANOVA with Bonferroni, n=14 mice / group, ****p<0.0001). The line at y = 10 s represents threshold for scoring seizures. Average consumption of paste diets (right, n=4 cages; Kruskal -Wallis Wilcoxon signed-rank test and Benjamini -Hochberg adjustment. Data are presented as mean ± SEM) FIG. 11B shows 6-Hz seizure threshold (left) and latency to exploration (middle) for mice fed KD4: 1 + SCFAs or vehicle (NaCl) control as a paste diet (left, one-way ANOVA with Bonferroni, n=14 mice / group, ****p<0.0001). The line at y = 10 s represents threshold for scoring seizures. Average consumption of paste diets (right, n=4 cages; Kruskal-Wallis Wilcoxon signed-rank test and Benjamini -Hochberg adjustment. Data are presented as mean ± SEM).

[0031] FIG. 12 shows the supplementation of 13 dietary fiber sources and types to KD4: 1 infant formula differentially alters the taxonomic composition of a model human infant gut microbial community. Bacterial species abundance changes after 24-hour culture of model human infant gut microbial community in KD4: 1 infant formula with differing fiber sources and types, relative to KD4: 1 alone (n=10. Kruskal-Wallis with Dunn’s test, *p < 0.05. Data are presented as mean ± SEM).

[0032] DETAILED DESCRIPTION

[0033] In some aspects, provided herein are methods and compositions for preventing or treating a condition responsive to a ketogenic diet in a subject, comprising administering to the subject a composition comprising bacteria of the Bacteroidetes phylum, Proteobacteria phylum, Deferribacteres phylum, Streptococcaceae family, Coriobacteriia class, Bifidobacterium genera, Bacteroides genera, and / or Clostridium genera.

[0034] For example, the methods described herein may include administering a combination of bacteria, such a bacteria of the Bacteroidetes phylum and a bacteria of the Proteobacteria phylum. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Bacteroidetes phylum and a bacteria of the Deferribacteres phylum. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Bacteroidetes phylum and a bacteria of the Streptococcaceae family. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Bacteroidetes phylum and a bacteria of the Coriobacteriia class. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Bacteroidetes phylum and a bacteria of the Bifidobacterium genera. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Bacteroidetes phylum and a bacteria of the Bifidobacterium genera. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Bacteroidetes phylum and a bacteria of the Clostridium genera.

[0035] For example, the methods described herein may include administering a combination of bacteria, such a bacteria of the Proteobacteria phylum and a bacteria of the Deferribacteres phylum. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Proteobacteria phylum and a bacteria of the Streptococcaceae family. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Proteobacteria phylum and a bacteria of the Coriobacteriia class. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Proteobacteria phylum and a bacteria of the Bifidobacterium genera. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Proteobacteria phylum and a bacteria of the Bifidobacterium genera. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Proteobacteria phylum and a bacteria of the Clostridium genera.

[0036] For example, the methods described herein may include administering a combination of bacteria, such a bacteria of the Deferribacteres phylum and a bacteria of the Streptococcaceae family. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Deferribacteres phylum and a bacteria of the Coriobacteriia class. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Deferribacteres phylum and a bacteria of the Bifidobacterium genera. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Deferribacteres phylum and a bacteria of the Bifidobacterium genera. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Deferribacteres phylum and a bacteria of the Clostridium genera.

[0037] For example, the methods described herein may include administering a combination of bacteria, such a bacteria of the Streptococcaceae family and a bacteria of the Coriobacteriia class. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Streptococcaceae phylum and a bacteria of the Bifidobacterium genera. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Streptococcaceae phylum and a bacteria of the Bifidobacterium genera. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Streptococcaceae phylum and a bacteria of the Clostridium genera.

[0038] For example, the methods described herein may include administering a combination of bacteria, such a bacteria of the Coriobacteriia class and a bacteria of the Bifidobacterium genera. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Coriobacteriia class and a bacteria of the Bifidobacterium genera. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Coriobacteriia class and a bacteria of the Clostridium genera.

[0039] In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Bifidobacterium genera and a bacteria of the Clostridium genera.

[0040] In some embodiments, the composition comprises Bifidobacterium infantis (e.g., Bifidobacterium longum sups, infantis DSM 20088). In some embodiments, the composition comprises Bifidobacterium longum (e.g., Bifidobacterium longum sups, longum ATCC BAA- 999). In some embodiments, the composition comprises Bifidobacterium breve e.g., Bifidobacterium breve DSM 20213). In some embodiments, the composition comprises Bacteroides fragilis (e.g., Bacteroides fragilis ATCC 25285). In some embodiments, the composition comprises Clostridium perfringens (e.g., Clostridium perfringens ATCC 13124).

[0041] In another aspect, provided herein are methods and compositions for preventing or treating a condition responsive to a ketogenic diet in a subject, comprising administering to the subject an agent that decreases levels of one or more bacteria in the subject’s gut, wherein the one or more bacteria comprise bacteria of the Actinobacteria phylum, Erysipelotrichia class, Escherichia genera, Enterococcus genera, Mammaliicoccus genera, Bifidobacterium species, Bacteroides species, and / or Klebsiella species.

[0042] In some embodiments, the agent is a small molecule that specifically decreases the level of bacteria of the Actinobacteria phylum. In some embodiments, the agent is a small molecule that specifically decreases the level of bacteria of the Erysipelotrichia class. In some embodiments, the agent is a small molecule that specifically decreases the level of bacteria of the Escherichia genera. In some embodiments, the agent is a small molecule that specifically decreases the level of bacteria of the Enterococcus genera. In some embodiments, the agent is a small molecule that specifically decreases the level of bacteria of the Mammaliicoccus genera. In some embodiments, the agent is a small molecule that specifically decreases the level of bacteria of the Bifidobacterium breve species. In some embodiments, the agent is a small molecule that specifically decreases the level of bacteria of the Bifidobacterium infantis. In some embodiments, the agent is a small molecule that specifically decreases the level of bacteria of the Bacteroides vulgatus species. In some embodiments, the agent is a small molecule that specifically decreases the level of bacteria of the and / or Klebsiella pneumoniae.

[0043] In some embodiments, the agent is an antibiotic specific for a particular bacterial phylum. In some embodiments, the agent is an antibiotic that specifically reduces levels of bacteria of the Actinobacteria phylum, Erysipelotrichia class, Escherichia genera, Enterococcus genera, Mammaliicoccus genera, Bifidobacterium species, Bacteroides vulgatus species, or Klebsiella species.

[0044] In some embodiments, the agent decreases levels of Escherichia coli (e.g., Escherichia coli K-12 ATCC 10798). In some embodiments, the agent decreases levels of Enterococcus faecalis (e.g., Enterococcus faecalis ATCC 19433). In some embodiments, the agent decreases levels of Mammaliicoccus sciuri. In some embodiments, the agent decreases levels of Bifidobacterium breve e.g., Bifidobacterium breve DSM 20213). In some embodiments, the agent decreases levels of Bifidobacterium infantis (e.g., Bifidobacterium longum sups, infantis DSM 20088). In some embodiments, the agent decreases levels of Bacteroides vulgatus (e.g., Bacteroides vulgatus ATCC8482). In some embodiments, the agent decreases levels of Klebsiella pneumoniae (e.g., Klebsiella pneumoniae subsp. pneumoniae ATCC 13883).

[0045] In some embodiments, the methods further comprise administering a bacteria or a molecule that causes alterations in the pathways related to L-alanine biosysnthesis, preQO biosynthesis, sucrose degradation and partial TCA cycle in the gut microbiome, such as L- alanine, queuosine, preQo, guanosine, guanosine triphosphate, alpha-ketoglutarate (2- oxoglutarate), citrate, succinate and sucrose and / or lactose.

[0046] In another aspect, provided herein are methods and compositions for preventing or treating a condition responsive to a ketogenic diet in a subject, comprising administering to the subject a composition comprising L-alanine, queuosine, preQo, guanosine, guanosine triphosphate, alpha-ketoglutarate (2-oxoglutarate), citrate, succinate and sucrose and / or lactose.

[0047] In some embodiments, the condition is seizures, optionally wherein subject has a neurodevelopmental condition, e.g., selected from epilepsy, autism spectrum disorder, Rett syndrome, attention deficit disorder, and fragile X syndrome. In some embodiments, the subject has a condition selected from Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), cancer, stroke, a metabolic disease, a mitochondrial disorder, depression, migraines, and traumatic brain injury (TBI).

[0048] In some embodiments, the method further comprises administering to the subject a fiber supplement (e.g., inulin, fructooligosaccharides, acacia fiber, gum arabic, cellulose, and / or orange fiber). In some embodiments, the method further comprises administering to a subject a composition comprising L-alanine, queuosine, preQo, guanosine, guanosine triphosphate, alpha-ketoglutarate (2-oxoglutarate), citrate, succinate and / or sucrose and / or lactose.

[0049] Also provided herein are compositions. The composition may be self-administered. The composition may be a food product. In some embodiments, the composition is infant formula.

[0050] The subject may be a pediatric subject (e.g., the subject is under 18 years of age). The subject may be an adult (e.g., 18 years old or older). In some embodiments, the subject is no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 years of age. In some embodiments, the condition disclosed herein may be a condition in children. Thus, the condition may be a pediatric condition. The child may be less than about 1 week old. The child may be less than about 1 month old. The child may be less than about 6 months old. The child may be less than about 12 months old. The child may be less than about 2 years old. The child may be less than about 3 years old. The child may be less than about 4 years old. The child may be less than about 5 years old. The child may be less than about 6 years old. The child may be less than about 7 years old. The child may be less than about 8 years old. The child may be less than about 9 years old. The child may be less than about 10 years old. The child may be less than about 12 years old.

[0051] Definitions

[0052] As used herein in the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one. As used herein “another” may mean at least a second or more.

[0053] The term “agent” is used herein to include, but is not limited to, a chemical compound, a small molecule, and / or a mixture of chemical compounds. “Agent” includes, but is not limited to, microbially produced metabolites that are product of microbial activity from carbohydrates, proteins and fats. The activity of such agents may render them suitable as a “therapeutic agent” which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.

[0054] The phrase “ketogenic diet” is used herein to include, but is not limited to, a high-fat diet, and / or a low-carbohydrate diet. The ketogenic diet may comprise additional fiber supplements (e.g., inulin, fructooligosaccharides, acacia fiber, gum arabic, cellulose, and / or orange fiber). The ketogenic diet may comprise the components of Table 1. The diet may consist of more than 60-75% fats. The diet may consist of less than 5-10% carbohydrates.

[0055] The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0056] The term "preventing' is art-recognized, and when used in relation to a condition, such as a local recurrence, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of seizures includes, for example, reducing the number of seizures in a population of patients receiving a prophylactic treatment relative to an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0057] The term “prophylactic” or “therapeutic” treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic (i.e., it protects the host against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

[0058] The term “small molecule” is a term of the art and includes molecules that are less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not exclusively comprise peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds which can be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. (1998) Science 282:63), and natural product extract libraries. In another embodiment, the compounds are small, organic non-peptidic compounds. In a further embodiment, a small molecule is not biosynthetic.

[0059] The term “subject” refers to a mammal, including, but not limited to, a human or nonhuman mammal, such as a bovine, equine, canine, ovine, or feline.

[0060] A “therapeutically effective amount” of a compound with respect to the subject method of treatment refers to an amount of the compound(s) in a preparation which, when administered as part of a desired dosage regimen (to a mammal, preferably a human) alleviates a symptom, ameliorates a condition, or slows the onset of disease conditions according to clinically acceptable standards for the disorder or condition to be treated or the cosmetic purpose, e.g., at a reasonable benefit / risk ratio applicable to any medical treatment.

[0061] As used herein, the term “ treating" or “treatment includes reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in a manner to improve or stabilize a subject's condition.

[0062] Therapeutic Methods

[0063] A ketogenic diet (KD) with fiber induces substantial changes in the gut microbiome, and enriching KD-associated bacteria via probiotic administration, fecal transplant, or selective microbial reconstitution of the native microbiome mimics the beneficial effects of such a diet. Provided herein are methods and compositions that can replace or supplement the KD diet in the treatment or prevention of a condition as described. The methods and compositions described herein can be used separately or in conjunction with the KD diet or another diet disclosed herein in the treatment or prevention of a condition described herein. Therefore, in some embodiments, the subject is on a ketogenic diet that includes at least one dietary fiber (e.g., a dietary fiber disclosed herein).

[0064] In some embodiments, the subject is given antibiotics or antimicrobial agents to deplete the subject’s gut microbiota.

[0065] In certain embodiments, the methods treat or prevent seizures in a subject. In some aspects, provided herein are methods for preventing or treating a condition responsive to a ketogenic diet in a subject, comprising administering to the subject a composition comprising particular bacterial species. In some aspects, provided herein are methods for increasing the expression of genes or products related to L-alanine biosynthesis, queuosine biosynthesis, and / or preQo biosynthesis.

[0066] In some embodiments, the subject has epilepsy (e.g., pediatric epilepsy; refractory or non-refractory epilepsy). The disclosed epilepsy disorder may be benign Rolandic epilepsy, frontal lobe epilepsy, infantile spasms, juvenile myoclonic epilepsy (JME), juvenile absence epilepsy, childhood absence epilepsy (e.g. pyknolepsy), febrile seizures, progressive myoclonus epilepsy of Lafora, Lennox-Gastaut syndrome, Landau -KI effner syndrome, Dravet syndrome, Generalized Epilepsy with Febrile Seizures (GEFS+), Severe Myoclonic Epilepsy of Infancy (SMEI), Benign Neonatal Familial Convulsions (BFNC), West Syndrome, Ohtahara Syndrome, early myoclonic encephalopathies, migrating partial epilepsy, infantile epileptic encephalopathies, Tuberous Sclerosis Complex (TSC), focal cortical dysplasia, Type I Lissencephaly, Miller-Dieker Syndrome, Angelman's syndrome, Fragile X syndrome, epilepsy in autism spectrum disorders, subcortical band heterotopia, Walker-Warburg syndrome, Alzheimer's disease, posttraumatic epilepsy, progressive myoclonus epilepsies, reflex epilepsy, Rasmussen's syndrome, temporal lobe epilepsy, limbic epilepsy, status epilepticus, abdominal epilepsy, massive bilateral myoclonus, catamenial epilepsy, Jacksonian seizure disorder, Unverricht-Lundborg disease, or photosensitive epilepsy. The epilepsy may include generalized seizures or partial (i.e. focal) seizures.

[0067] Epilepsy disorder, as disclosed herein, may be Dravet Syndrome, Lennox-Gastaut Syndrome, infantile spasm, or Ohtahara Syndrome. The epilepsy disorder may be Dravet Syndrome, Lennox-Gastaut Syndrome, infantile spasm, or Ohtahara Syndrome, or a pediatric epilepsy disorder. The pediatric epilepsy disorder may be benign childhood epilepsy, Benign Neonatal Familial Convulsions (BFNC), febrile seizures, Dravet Syndrome, Lennox-Gastaut Syndrome, infantile spasm, Ohtahara Syndrome, juvenile myoclonic epilepsy, juvenile absence epilepsy, childhood absence epilepsy (e.g. pyknolepsy), infantile spasms. In embodiments the epilepsy disorder is Dravet Syndrome. According to the invention, the epilepsy disorder is Dravet syndrome or Lennox-Gastaut syndrome.

[0068] The disclosed pediatric epilepsy disorder may be benign childhood epilepsy. The disclosed pediatric epilepsy disorder may be Benign Neonatal Familial Convulsions (BFNC). The disclosed pediatric epilepsy disorder may be febrile seizures. In embodiments according to the invention the pediatric epilepsy disorder is Dravet Syndrome. In embodiments according to the invention the pediatric epilepsy disorder is Lennox-Gastaut Syndrome. The disclosed pediatric epilepsy disorder may be infantile spasm. The disclosed pediatric epilepsy disorder may be Ohtahara Syndrome. The disclosed pediatric epilepsy disorder may be juvenile myoclonic epilepsy. The disclosed pediatric epilepsy disorder may be juvenile absence epilepsy. The disclosed pediatric epilepsy disorder may be childhood absence epilepsy (e.g. pyknolepsy). The disclosed pediatric epilepsy disorder may be infantile spasms.

[0069] In some embodiments, the epilepsy disorder may be a result of a neurological disease or injury such as, for example, encephalitis, cerebritis, abscess, stroke, tumor, trauma, genetic, tuberous sclerosis, cerebral dysgenesis, or hypoxic-ischemic encephalophathy. The epilepsy disorder may be associated with a neurodegenerative disease such as, for example, Alzheimer's disease or Parkinson's Disease. The epilepsy disorder may be associated with autism. The epilepsy disorder may be associated with a single gene mutation. The epilepsy disease may be associated with compulsive behaviors or electrographic seizures. The epilepsy disorder may be an epilepsy disorder which is non-responsive to treatment with an antiepileptic drug (AED). The AED may be acetazolamide. The AED may be benzodiazepine. The AED may be cannabadiols. The AED may be carbamazepine. The AED may be clobazam. The AED may be clonazepam. The AED may be eslicarbazepine acetate. The AED may be ethosuximide. The AED may be ethotoin. The AED may be felbamate. The AED may be fenfluramine. The AED may be fosphenytoin. The AED may be gabapentin. The AED may be ganaxolone. The AED may be huperzine A. The AED may be lacosamide. The AED may be lamotrigine. The AED may be levetiracetam. The AED may be nitrazepam. The AED may be oxcarbazepine. The AED may be perampanel. The AED may be piracetam. The AED may be phenobarbital. The AED may be phenytoin. The AED may be potassium bromide. The AED may be pregabalin. The AED may be primidone. The AED may be retigabine. The AED may be rufinamide. The AED may be valproic acid. The AED may be sodium valproate. The AED may be stiripentol. The AED may be tiagabine. The AED may be topiramate. The AED may be vigabatrin. The AED may be zonisamide.

[0070] In some embodiments, the subject has a neurodevelopmental disorder. Representative neurodevelopmental disorders include autism spectrum disorder, Rett syndrome, fragile X, attention deficit disorder, and attention-deficit / hyperactivity disorder. In some embodiments, the neurodevelopmental disorder is a disorder known to be comorbid with seizures. As used herein, a condition “responsive to a ketogenic diet” includes, but is not limited to, epilepsy, autism spectrum disorder, Rett syndrome, fragile X, attention deficit disorder, attention- deficit / hyperactivity disorder, seizures, autism spectrum disorder, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), cancer, stroke, a metabolic disease (e.g., diabetes or obesity), a mitochondrial disorder, depression, migraines (e.g., chronic migraines), or traumatic brain injury (TBI).

[0071] In some embodiments, the subject is refractory to anti-conversant drug or anti-seizure drug. An "anti-seizure drug", "anti-epilepsy drug", "AED" or "anticonvulsant" are used interchangeably herein and according to their common and ordinary meaning and include compositions for reducing or eliminating seizures. Anticonvulsants include, but are not limited to acetazolamide, benzodiazepine, cannabidiols, carbamazepine, clobazam, clonazepam, eslicarbazepine acetate, ethosuximide, ethotoin, felbamate, fenfluramine, fosphenytoin, gabapentin, ganaxolone, huperzine A, lacosamide, lamotrigine, levetiracetam, nitrazepam, oxcarbazepine, perampanel, piracetam, phenobarbital, phenytoin, potassium bromide, pregabalin, primidone, retigabine, rufinamide, valproic acid, sodium valproate, stiripentol, tiagabine, topiramate, vigabatrin, or zonisamide.

[0072] In some embodiments, the agents and / or compositions described herein may be administered conjointly. In some embodiments, a composition described herein may be conjointly administered with an anticonvulsant.

[0073] In other embodiments, the subject has a condition responsive to a ketogenic diet. The condition may be Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), cancer, stroke, a metabolic disease, a mitochondrial disorder, depression, migraines (e.g., chronic migraines), or traumatic brain injury (TBI). In some embodiments, the methods and compositions comprise administering to the subject a composition provided herein. In some embodiments, the condition is epilepsy, seizures, autism spectrum disorder, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), cancer, stroke, a metabolic disease (e.g., obesity or diabetes), a mitochondrial disorder, depression, migraines (e.g., chronic migraines), Rett syndrome, attention deficit disorder, fragile X syndrome, or traumatic brain injury (TBI). In some embodiments, the compositions and methods provided herein are useful in treating or preventing aging or aging-associating conditions. In some embodiments, the compositions and methods provided herein can replace the ketogenic diet in the treatment or prevention of a condition described herein; in other embodiments, the compositions and methods provided herein can be combined with the ketogenic diet. More information on conditions may be found in Stafstrom et al. (2012) Front. Pharmacol. 3:59, hereby incorporated in its entirety.

[0074] The composition may be formulated for oral delivery. In some embodiments, the composition may comprise probiotics. In some embodiments, the compositions disclosed herein are food products. The composition may be in the form of a pill, tablet, or capsule. In some embodiments, the subject may be a mammal (e.g., a human). In some embodiments, the composition is self-administered.

[0075] In some embodiments, the above methods include reducing the amount of pathogenic bacteria in a subject (i.e., in the gastrointestinal tract of the subject) prior to administration of an agent disclosed herein. In some embodiments, this includes any such therapy that achieves the same goal of reducing the number of pathogenic organisms, when used in combination with the compositions described herein, would lead to replacement of the pathogenic microflora involved in the diseased state with microflora associated with a non- diseased state, or less pathogenic species occupying the same ecological niche as the type causing a disease state. For example, a subject may undergo treatment with antibiotics (e.g., antimicrobial compounds) or a composition comprising antibiotics to target and decrease the prevalence of pathogenic organisms, and subsequently be treated with a composition described herein. The treatment may also comprise an antifungal or anti-viral compound.

[0076] Suitable antimicrobial compounds include capreomycins, including capreomycin IA, capreomycin IB, capreomycin IIA and capreomycin IIB; carbomycins, including carbomycin A; carumonam; cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, cefbuperazone, cefcapene pivoxil, cefclidin, cefdinir, cefditoren, cefime, ceftamet, cefmenoxime, cefmetzole, cefminox, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotetan, cefotiam, cefoxitin, cefpimizole, cefpiramide, cefpirome, cefprozil, cefroxadine, cefsulodin, ceftazidime, cefteram, ceftezole, ceftibuten, ceftiofur, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam, cephalexin, cephalogycin, cephaloridine, cephalosporin C, cephalothin, cephapirin, cephamycins, such as cephamycin C, cephradine, chlortetracycline, chlarithromycin, clindamycin, clometocillin, clomocycline, cioxacillin, cyclacillin, danofloxacin, demeclocyclin, destomycin A, dicloxacillin, dirithromycin, doxycyclin, epicillin, erythromycin A, ethanbutol, fenbenicillin, flomoxef, florfenicol, floxacillin, flumequine, fortimicin A, fortimicin B, forfomycin, foraltadone, fusidic acid, gentamycin, glyconiazide, guamecycline, hetacillin, idarubicin, imipenem, isepamicin, josamycin, kanamycin, leumycins such as leumycin Al, lincomycin, lomefloxacin, loracarbef, lymecycline, meropenam, metampicillin, methacycline, methicillin, mezlocillin, micronomicin, midecamycins such as midecamycin Al, mikamycin, minocycline, mitomycins such as mitomycin C, moxalactam, mupirocin, nafcillin, netilicin, norcardians such as norcardian A, oleandomycin, oxytetracycline, panipenam, pazufloxacin, penamecillin, penicillins such as penicillin G, penicillin N and penicillin O, penillic acid, pentylpenicillin, peplomycin, phenethicillin, pipacyclin, piperacilin, pirlimycin, pivampicillin, pivcefalexin, porfiromycin, propiallin, quinacillin, ribostamycin, rifabutin, rifamide, rifampin, rifamycin SV, rifapentine, rifaximin, ritipenem, rekitamycin, rolitetracycline, rosaramicin, roxithromycin, sancycline, sisomicin, sparfloxacin, spectinomycin, streptozocin, sulbenicillin, sultamicillin, talampicillin, teicoplanin, temocillin, tetracyclin, thostrepton, tiamulin, ticarcillin, tigemonam, tilmicosin, tobramycin, tropospectromycin, trovafloxacin, tylosin, and vancomycin, and analogs, derivatives, pharmaceutically acceptable salts, esters, prodrugs, and protected forms thereof. Suitable anti-fungal compounds include ketoconazole, miconazole, fluconazole, clotrimazole, undecylenic acid, sertaconazole, terbinafine, butenafine, clioquinol, haloprogin, nystatin, naftifine, tolnaftate, ciclopirox, amphotericin B, or tea tree oil and analogs, derivatives, pharmaceutically acceptable salts, esters, prodrugs, and protected forms thereof.

[0077] Compositions

[0078] Provided herein are compositions (e.g., compositions comprising an agent disclosed herein and a pharmaceutically acceptable carrier). The composition may comprise bacteria of the Bacteroidetes phylum, Proteobacteria phylum, Deferribacteres phylum, Streptococcaceae family, Coriobacteriia class, Bifidobacterium genera, Bacteroides genera, and / or Clostridium genera.

[0079] The composition may comprise a pharmaceutically acceptable carrier. The composition may comprise probiotics. The compostion may comprise one or more types of bacteria disclosed herein, and optionally further comprising and agent disclosed herein and / or a fiber disclosed herein. The pharmaceutical compositions disclosed herein may be delivered by any suitable route of administration, including orally, buccally, sublingually, parenterally, and rectally, as by powders, ointments, drops, liquids, gels, tablets, capsules, pills, or creams. In certain embodiments, the pharmaceutical compositions are delivered generally (e.g., via oral administration). In certain other embodiments, the compositions disclosed herein are delivered rectally.

[0080] The pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier may be one or more substance that may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0081] A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

[0082] Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in- water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.

[0083] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0084] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0085] The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and / or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro- encapsulated form, if appropriate, with one or more of the above-described excipients.

[0086] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0087] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0088] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0089] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0090] The selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0091] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could prescribe and / or administer doses of the compounds employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0092] In general, a suitable daily dose of the compositions and methods of the invention will be that amount of the composition that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. If desired, the effective daily dose of the composition may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present invention, the composition may be administered two or three times daily. In preferred embodiments, the composition will be administered once daily.

[0093] The patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines, cattle, swine, sheep, cats, and dogs; poultry; and pets in general.

[0094] In certain embodiments, compositions of the invention may be used alone or conjointly administered with another type of therapeutic agent.

[0095] Bacterial Compositions

[0096] In certain aspects, provided herein are bacterial compositions comprising bacteria of the Bacteroidetes phylum, Proteobacteria phylum, Deferribacteres phylum, Streptococcaceae family, Coriobacteriia class, Bifidobacterium genera, Bacteroides genera, and / or Clostridium genera, or a combination thereof. In certain embodiments, substantially all of the bacteria in the bacterial composition are selected from the species of Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium breve, Bacteroides fragilis, and / or Clostridium perfringens.

[0097] In some embodiments, the bacterial formulation comprises a bacterium and / or a combination of bacteria described herein and a pharmaceutically acceptable carrier.

[0098] In certain embodiments, at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the bacteria in the bacterial composition are selected from bacteria of the Bacteroidetes phylum, Proteobacteria phylum, Deferribacteres phylum, Streptococcaceae family, Coriobacteriia class, Bifidobacterium genera, Bacteroides genera, and / or Clostridium genera, or a combination thereof. In certain embodiments, substantially all of the bacteria in the bacterial composition are selected from the species of Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium breve, Bacteroides fragilis, and / or Clostridium perfringens.

[0099] In some embodiments, the composition comprises Bifidobacterium infantis (e.g., Bifidobacterium longum sups, infantis DSM 20088). In some embodiments, the composition comprises Bifidobacterium longum (e.g., Bifidobacterium longum sups, longum ATCC BAA- 999). In some embodiments, the composition comprises Bifidobacterium breve e.g., Bifidobacterium breve DSM 20213). In some embodiments, the composition comprises Bacteroides fragilis (e.g., Bacteroides fragilis ATCC 25285). In some embodiments, the composition comprises Clostridium perfringens (e.g., Clostridium perfringens ATCC 13124).

[0100] In certain embodiments, the bacterial composition comprises at least 10 colony forming units (CFUs), at least 100 colony forming units (CFUs), at least 1 x 103colony forming units (CFUs), at least 1 x 104colony forming units (CFUs), at least 1 x 105colony forming units (CFUs), at least 5 x 105colony forming units (CFUs), at least 1 x 106colony forming units (CFUs), at least 2 x 106colony forming units (CFUs), at least 3 x 106colony forming units (CFUs), at least 4 x 106colony forming units (CFUs), at least 5 x 106colony forming units (CFUs), at least 6 x 106colony forming units (CFUs), at least 7 x 106colony forming units (CFUs), at least 8 x 106colony forming units (CFUs), at least 9 x 106colony forming units (CFUs), at least 1 x 107colony forming units (CFUs), at least 2 x 107colony forming units (CFUs), at least 3 x 107colony forming units (CFUs), at least 4 x 107colony forming units (CFUs), at least 5 x 107colony forming units (CFUs), at least 6 x 107colony forming units (CFUs), at least 7 x 107colony forming units (CFUs), at least 8 x 107colony forming units (CFUs), at least 9 x 107colony forming units (CFUs), at least 1 x 108colony forming units (CFUs), at least 2 x 108colony forming units (CFUs), at least 3 x 108colony forming units (CFUs), at least 4 x 108colony forming units (CFUs), at least 5 x 108colony forming units (CFUs), at least 6 x 108colony forming units (CFUs), at least 7 x 108colony forming units (CFUs), at least 8 x 108colony forming units (CFUs), at least 9 x 108colony forming units (CFUs), at least 1 x 109colony forming units (CFUs), at least 5 x 109colony forming units (CFUs), at least 1 x 1010colony forming units (CFUs), at least 5 x 1010colony forming units (CFUs), at least 1 x 1011colony forming units (CFUs), at least 5 x 1011colony forming units (CFUs), at least 1 x 1012colony forming units (CFUs), at least 5 x 1012colony forming units (CFUs), or at least 1 x 1013colony forming units (CFUs) of bacteria. In some preferred embodiments, the bacterial composition comprises 1 x 109to 1 x 1011colony forming units of bacteria. The selected dosage level will depend upon a variety of factors including the subject’s diet, the route of administration, the time of administration, the residence time of the particular microorganism being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0101] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the bacterial composition required. For example, the physician or veterinarian could prescribe and / or administer doses of the bacteria employed in the composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0102] In some embodiments, the bacterial formulation comprises an enteric coating (e.g., for duodenal release at pH 5.5) or micro encapsulation. In certain embodiments, the enteric coating or micro encapsulation improves targeting to a desired region of the gastrointestinal tract. For example, in certain embodiments, the bacterial composition comprises an enteric coating and / or microcapsules that dissolve at a pH associated with a particular region of the gastrointestinal tract. In some embodiments, the enteric coating and / or microcapsules dissolve at a pH of about 5.5 - 6.2 to release in the duodenum, at a pH value of about 7.2 - 7.5 to release in the ileum, and / or at a pH value of about 5.6 - 6.2 to release in the colon. Exemplary enteric coatings and microcapsules are described, for example, in U.S. Pat. Pub. No. 2016 / 0022592, which is hereby incorporated by reference in its entirety.

[0103] In some embodiments, the composition is a food product (e.g., a food or beverage) such as an infant formula, a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed. Specific examples of the foods and beverages include various beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauces, and Chinese soups; soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, yogurts, fermented beverages, and pickles; bean products; various confectionery products, including biscuits, cookies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups; microwavable foods; and the like. Further, the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, capsules, liquids, pastes, and jellies. The composition may be a fermented food product, such as, but not limited to, a fermented milk product. Non-limiting examples of fermented food products include kombucha, sauerkraut, pickles, miso, tempeh, natto, kimchi, raw cheese, and yogurt. The composition may also be a food additive, such as, but not limited to, an acidulent (e.g., vinegar). Food additives can be divided into several groups based on their effects. Non-limiting examples of food additives include acidulents (e.g., vinegar, citric acid, tartaric acid, malic acid, fumaric acid, and lactic acid), acidity regulators, anticaking agents, antifoaming agents, foaming agents, antioxidants (e.g., vitamin C), bulking agents (e.g., starch), food coloring, fortifying agents, color retention agents, emulsifiers, flavors and flavor enhancers (e.g., monosodium glutamate), flour treatment agents, glazing agents, humectants, tracer gas, preservatives, stabilizers, sweeteners, and thickeners.

[0104] EXEMPLIFICATION

[0105] Example 1: Dietary fiber content in clinical ketogenic diets modifies the gut microbiome and seizure resistance in mice

[0106] The low-carbohydrate, high-fat ketogenic diet (KD) is used to treat epilepsy in children who do not respond positively to existing anti-seizure medications. While it is well integrated into the healthcare system, KD therapies have variable effectiveness in reducing seizures, ranging from 45 to 85% in infants and children that exhibit high compliance and with substantially lower rates in adults. Recent reports highlight a key role for the gut microbiome in mediating effects of the KD on various host physiologies, including glucose and lipid metabolism, immune function, brain activity, and behavior. The KD alters the gut microbiome across several human and animal epilepsy studies, and relationships are seen between the gut microbiome and seizure resistance in various rodent epilepsy models. Findings from the field are converging upon the notion that variation in the gut microbiome may contribute to variability in patient responsiveness to the KD, and that microbiome- targeted interventions could be used to promote the efficacy of the KD in treating refractory epilepsy.

[0107] While existing studies of the microbiome and KD have focused predominantly on the classic KD, many variations of the KD with different macronutrient ratios and types are used clinically to treat epilepsy, depending on factors such as the age of the patient, seizure type, and tolerability of the dietary regimen. For example, the KD is commonly administered as a 4: 1 or 3 : 1 fat to carbohydrate and protein ratio, depending on patient tolerance. The medium chain triglyceride (MCT) diet, often derived from MCT-rich coconut oil, is thought to promote enhanced ketone production while being less restrictive than the classic KD. The Modified Atkins Diet (MAD), which does not require strict weighing of food or fluids, and Low Glycemic Index Treatment (LGIT), which focuses on carbohydrates with low glycemic index rather than removal, are additional less restrictive variations of the KD that are frequently used in older children and adults.

[0108] In a retrospective open label trial of patients with drug resistant epilepsy, transitioning to a polyunsaturated fatty acid (PUFA)-based KD enhanced seizure control in individuals who responded poorly to the classic KD. Moreover, differences in dietary formulation can have substantial impacts on microbiome-dependent host phenotypes - KDs with different fat ratio and / or source resulted in differential influences of the microbiome on host glucose and lipid metabolism, as well as immune function. In addition, supplementation with dietary fiber, a key energy source for gut bacteria that modulates myriad host metabolic, immune, and neural functions, is incorporated into some clinical KD regimens to ease gastrointestinal symptoms, but whether it alters seizure response is unclear. Overall, increasing research indicating that the gut microbiome modifies seizure susceptibility and the anti-seizure effects of the KD raises the important question of how variations in the formulation of medical KDs differentially shape the microbiome in ways that impact seizure outcome.

[0109] In this study, the effects of three clinically prescribed KD infant formulas on the mouse gut microbiome and resistance to 6-Hz psychomotor seizures were tested as a benchmark model of refractory epilepsy. To determine which dietary variables serve as key drivers of microbiome response, a model human infant microbial consortium was established, and the effects of fat ratio, fat source, and carbohydrate source on shaping its functional potential was assessed. Thirteen fiber sources and types were screened for their differential impacts on the model infant microbial community and tested top candidates for their ability to restore and / or potentiate seizure protective effects of clinical KD infant formulas. Results from this study reveal key diet-microbiome interactions that promote the seizure protective effects of medical KDs.

[0110] Different clinical KD infant formulas elicit differential seizure responses in mice

[0111] Mechanistic studies of the KD on seizure resistance often rely on commercial KD chows that are formulated for lab animals and not directly relevant to medical KD therapies used for human epilepsy. At the same time, clinical KD regimens vary widely in nutritional content and are often tailored to the particular individual’s needs and tolerability, making it difficult to identify standard regimens. To examine how clinically relevant formulations of the KD elicit differential effects on seizure outcome, three commonly prescribed commercial KD infant formulas - KD4: 1, KD3: 1, and MCT2.5: 1 - were used due to their reproducible composition, direct clinical relevance, frequent prescription, and importance for infants as an especially vulnerable subset of refractory epilepsy patients for which improved interventions are needed. Compared to a standard infant formula as a control diet (CD), the three KD infant formulas all exhibit high fat content relative to carbohydrate and protein, but they display nuanced differences in formulation (FIG. 1A, Table 1). In addition to differences in fat ratio, fat source varies between the formulations, where KD4: 1 contains soy lecithin but lacks coconut oil and linoleic acid, KD3 : 1 contains linoleic acid but lacks soy lecithin and coconut oil, and MCT2.5: 1 contains coconut oil but lacks soy lecithin and linoleic acid. There are also differences in carbohydrate content, where both KD4: 1 and MCT2.5: 1 contain corn syrup solids, high amylose com starch, chicory root inulin, gum arabic (acacia fiber), cellulose, fructooligosaccharides (FOS), soy fiber, and maltodextrin, whereas KD3: 1 contains only lactose and corn syrup solids, with none of the dietary fibers. The CD contains lactose and less than 2% dietary fiber comprised of galactooligosaccharides, which differs from the types of fibers included in KD4 : 1 and MCT2.5: 1.

[0112] Table 1. Dietary Information

[0113] Control Medium

[0114] Ketogenic Ketogenic infant Chain

[0115] Ingredients (g / kcal) Diet (KD) Diet (KD) formula Triglyceride 4:1 3:1 (CD (MCT) 2.5:1

[0116] Fat 97.0 93.5

[0117] Saturated 37.0 31.4

[0118] Medium Chain Triglycerides 23.5

[0119] Monounsaturated 34.0 52.3

[0120] Polyunsaturated 25.0 9.8

[0121] DHA <2 % 0.3 0.38

[0122] ARA <2 % 0.3

[0123] Linoleic Acid 10.0 22.0

[0124] Linolenic Acid 1.8

[0125] Carbohydrates 105.0 10.0 14.4

[0126] Dietary Fiber 7.2

[0127] Soluble 3.7

[0128] Insoluble 3.7

[0129] Protein 22.0 29.4

[0130] Vitamins (ug / kcal)

[0131] Vitamin A (lU / kcal) 3000.0 1750.0 1640.0

[0132] Vitamin D (lU / kcal) 600.0 569.0 863.0

[0133] Choline 240000.0 455000.0 285000.0

[0134] Vitamin C 90000.0 57000.0 83000.0

[0135] Vitamin E (lU / kcal) 15.0 23.0 17.0

[0136] Niacin 10500.0 7000.0 6300.0

[0137] Pantothenate 4500.0 4100.0 7600.0

[0138] Vitamin K 80.0 44.0 70.0

[0139] Riboflavin 1500.0 1000.0 900.0

[0140] Thiamin 1000.0 1300.0 900.0

[0141] Vitamin B6 600.0 1000.0 900.0 Folic Acid 150.0 165.0 135.0

[0142] Biotin 44.0 27.0 18.0

[0143] Vitamin B12 2.5 1.8 1.8

[0144] Inositol 47000 27000 197000

[0145] Ingredients

[0146] Fat

[0147] High Oleic Safflower Oil

[0148] Soy Oil

[0149] Coconut Oil (or MCT)

[0150] High Oleic Sunflower Oil

[0151] Palm Oil

[0152] C. Cohnii Oil

[0153] M. Alpina Oil

[0154] Soy Lecithin

[0155] Carbohydrates

[0156] Lactose

[0157] Short-Chain Fructooligosaccharide

[0158] Com Syrup Solids

[0159] Chicory Root Inulin

[0160] High Amylose Com Starch

[0161] Gum Arabic

[0162] Microcrystaline Cellulose

[0163] Soy Fiber

[0164] Maltodextrin

[0165] Protein

[0166] Whey Protein Concentrate

[0167] Nonfat Milk (Casein)

[0168] Minerals

[0169] Manganese (ug / kcal) 50.0 1200.0 200.0 1176.3 Iron (mg / kcal) 18.0 9.9 15.0 9.1 Zinc (mg / kcal) 7.5 5.1 11.0 6.1 Copper (ug / kcal) 900.0 710.0 647.0 782.4 Calcium (mg / kcal) 780.0 993.0 1030.0 752.1 Potassium (mg / kcal) 1050.0 1090.0 1170.0 1633.6 Phosphorus (mg / kcal) 420.0 596.0 692.0 586.8 Chloride (mg / kcal) 650.0 1030.0 691.0 1352.6 Sodium (mg / kcal) 240.0 682.0 450.0 763.1 Magnesium (mg / kcal) 60.0 137.0 99.0 1697.0 Selenium (ug / kcal) 20.0 38.0 34.0 35.3 Molybdenum (ug / kcal) 41.0 44.0 35.8 Chromium (ug / kcal) 21.0 37.0 16.3

[0170] L-Camitine (mg / kcal) 64.0 62.0 55.1

[0171] Taurine (mg / kcal) 47.0 62.0 41.0

[0172] Iodine (ug / kcal) 150.0 122.0 132.0 124.0

[0173] To determine how different KD formulations impact seizure susceptibility, cohorts of conventional 4 week-old mice were fed the KD4: 1, KD3: 1, MCT2.5: 1, or CD formula as liquid diet for 1 week, and then tested for susceptibility to 6-Hz psychomotor seizures (FIG. IB). Juvenile mice were selected to mimic the typical use of the KD to treat pediatric epilepsy, to align the timing of mouse brain development to early brain development in humans, and to preclude effects of pre-weaning treatment, where effects of the diets on maternal behavior and physiology would confound their direct effects on offspring. One week of feeding was selected based on prior longitudinal characterization, which indicated that KD chow shifts the gut microbiome and confers seizure protection by day 4 of treatment in mice. Finally, the 6-Hz seizure assay was selected as a benchmark model of refractory epilepsy that is used to screen for new anti-seizure medications and involves low-frequency corneal stimulation to induce complex partial seizures related to human temporal lobe epilepsy. KD chow protects against 6-Hz seizures, as indicated by increases in current intensity required to elicit a seizure in 50% of the subjects tested (CC50, seizure threshold).

[0174] As seen previously for KD chows, it was observed that feeding mice clinical KD4: 1 infant formula increased seizure thresholds compared to controls fed a CD infant formula (FIG. 1C). MCT2.5: 1 also increased seizure thresholds albeit to a lesser degree than KD4: 1, which may be due to its comparatively lower fat ratio or different fat source. In contrast, however, KD3 : 1 infant formula yielded decreased seizure thresholds compared to all other groups, including CD-fed controls, suggesting that the KD3: 1 formulation increases susceptibility to 6-Hz seizures in mice. There was no correlation of seizure threshold with average calories consumed for the different KDs or with degree of ketosis as assessed by serum levels of beta-hydroxybutyrate (FIG. 5A, B). To further assess whether the differences in seizure outcome may be confounded by nuances of providing the diet in liquid form, such as differences in density or leakage from the bottle, the experiment was repeated by providing the infant formula diets in solid form following dehydration. Consistent with previous observation, solid KD4: 1 and MCT2.5: 1 increased seizure threshold relative to controls fed solid CD, whereas solid KD3:1 decreased resistance to 6-Hz seizures, with no correlation with total diet consumed (FIG. 5C, D) These data indicate that variations in clinical KD formulations differentially modify host resistance versus susceptibility to 6-Hz seizures in mice.

[0175] Clinical KD infant formulas differentially alter the mouse gut microbiome

[0176] Classic KD-induced changes in the mouse and human microbiome are necessary and / or sufficient to confer resistance to 6-Hz seizures in mice. To determine how the different clinical KD infant formulas impact the gut microbiome, metagenomic sequencing of fecal microbiota from mice fed KD4: 1, KD3: 1, MCT2.5: 1, or CD for 1 week was performed. In contrast to results from KD vs. standard chow, KD4: 1 and MCT2.5: 1 significantly increased a-diversity of the microbiome, as indicated by elevated Shannon’s diversity index, when compared to CD controls (FIG. 2A). However, there was no significant effect of KD3: 1 on Shannon diversity levels, despite comparable increases across all KD formula groups in species richness of the fecal microbiota. This suggests that the main driver of a-diversity differences between the KD groups is differential alteration in species evenness — indeed, KD3: 1 yielded fecal microbiota with significantly reduced Pielou’s evenness compared to KD4: 1 and MCT2.5: 1 groups. 0-diversity analysis of the gut microbiota based on Bray-Curtis dissimilarity and weighted Unifrac distances showed that KD samples clustered distinctly from CD controls along PCoAl, with KD4: 1 and MCT2.5: 1 samples showing further separation from CD than KD3: 1 samples (PERMANOVA, p=0.001, R2=0.6, FIG. 2B). In particular, all KD groups exhibited significantly decreased relative abundances of Actinobacteria and increased Bacteroidetes and unclassified Bacteria compared to CD controls (FIG. 6A). However, only KD4: 1 and MCT2.5: l shared statistically significant decreases in Erysipelotrichia and increases in Streptococcaceae, Coriobacleriia. and Deferribacteres, whereas KD3 : 1 exhibited no significant changes in these taxa compared to CD (FIG. 6A-C). Rather, KD3 : 1 showed significantly increased relative abundance of Proteobacteria, Escherichia coli, Enterococcus faecalis, and Mammaliicoccus sciuri compared to CD, KD4: 1, and / or MCT2.5:1 controls (FIG. 6A-C).

[0177] The seizure susceptible KD3: 1 group also exhibited decreased representation of the top 10 most abundant metagenomic superclass pathways (FIG. 2C), suggesting that the KD3: 1 limits the presence of microbial taxa associated with prevalent functions and / or enriches the representation of previously rare metagenomic pathways. Among the top 10, the relative abundance of superclass pathways related to amino acid, carbohydrate, and nucleoside and nucleotide biosyntheses were significantly lower in KD3: 1 relative to MCT2.5: 1, CD, and / or KD4:1 groups. In contrast, superclass pathways related to carboxylic acid, fatty acid and lipid, and secondary metabolite degradation were significantly elevated in KD3 : 1 compared to other groups. When considering specific alterations at the more resolved pathway level, all three KDs shared subsets of metagenomic changes compared to CD controls, where KD4: 1 and MCT2.5: 1 shared greater overlap than with KD3.1 (FIG. 2D). Namely, KD4: 1 and MCT2.5:l (but not KD3:1) similarly induced significant metagenomic increases in select pathways related to carbohydrate biosynthesis (UDP-N-acetyl-D- galactosamine II and UDP-N-acetyl-D-glucosamine biosynthesis II), carboxylic acid degradation (biotin-dependent malonate degradation), and cofactor, carrier, and vitamin biosynthesis (biotin biosynthesis), and decreases in select pathways related to carbohydrate degradation (hexitol and galactitol degradation, sucrose, lactose, galactose degradation, and Entner-Doudoroff pathway), amino acid biosynthesis (L-lysine and L-alanine biosynthesis), carbohydrate biosynthesis (UDP-N-acetyl-D-glucosamine biosynthesis I and UDP-glucose- derived-O-antigen building blocks biosynthesis), and pentose phosphate pathway compared to CD controls (FIG. 2E). KD3: 1 displayed the most differentially abundant metagenomic pathways compared to CD, which were distinct from those seen in the other KD groups (FIG. 6D). The majority of differentially abundant pathways were elevated by KD3: 1 and related to amide, amidine, amine, and polyamine degradation, fatty acid and lipid biosynthesis, carboxylic acid degradation, and fermentation (FIG. 6D). In particular, pathways for phospholipid remodeling, lactate fermentation, and biosynthesis of octanoyl and myristate, and degradation of erythronate, threonate, galactitol, and allantoin were all significantly increased by KD3: 1, decreased by KD4: 1 and MCT2.5: 1 (FIG. 6D), and associated with low dietary fiber content (FIG. 6E). The only pathway decreased by KD3: 1, but elevated by KD4: 1 and MCT2.5: 1, was L-glutamate and L-glutamine biosynthesis (FIG. 6D), which was further positively associated with dietary fiber (FIG. 6E). Taken together, these results indicate that resistance vs. susceptibility to 6-Hz seizures in response to different KD infant formulas is associated with differential alterations in the composition and functional potential of the gut microbiome.

[0178] Fiber content in the KD drives microbial alterations and promotes seizure resistance The gut microbiome is shaped by changes in host diet and can be responsive to the presence, abundance, and sources of dietary macronutrients. To gain insight into how different clinical KD formulas differentially alter the gut microbiome, various dietary parameters were screened for their effects on a model human infant microbial community. Nine bacterial strains were selected based on their prevalence and relative abundances across multiple large studies of the infant gut microbiome (FIG. 7A, Table 2). All community members were confirmed to grow stably together in a rich complex medium as a positive control (FIG. 7B). To test the effects of KD fat ratio, the model infant gut microbial community was cultured in synthetic KD media prepared in ratios from KD4: 1 to KD1.5: 1 (FIG. 7C, Table 3). There were no statistically significant differences in taxonomic response to the KDs with different fat ratio (PERMANOVA, p=0.13, R2=0.14, FIG. 7D). To examine effects of KD fat source, the model infant gut microbial community was cultured in synthetic media representing KD4: 1, KD3 : 1, or MCT2.5: 1, each using sunflower oil (6% saturated fat), soy lecithin (23% saturated fat and dominant in KD4: 1 infant formula), or palm oil (50% saturated fat), as fat sources with different levels of saturation (FIG. 7E). The media prepared with soy lecithin increased the absolute abundance of B. infantis, B.fragilis, and C. perfringens, resulting in distinct separation along PCoAl from the sunflower and palm oil groups (PERMANOVA, p<0.05; FIG. 7F). This may be due to the presence of free sugars (8%) in the commercial soy lecithin and / or the emulsifying properties of soy lecithin, compared to the other fat sources. There were no statistically significant differences between the sunflower and palm oil groups across all media conditions (FIG. 7F), suggesting that the differential effects of soy lecithin are driven by its fat source rather than saturation level.

[0179] Table 2. Simplified model of the infant microbiome

[0180] Species Composition Volume (mL)

[0181] Actinobacteria 21% 3

[0182] Bifidobacterium longum sups, infantis DSM 20088 1

[0183] Bifidobacterium longum sups, longum ATCC BAA-999 1

[0184] Bifidobacterium breve DSM 20213 1

[0185] Bacteroidetes 14% 2

[0186] Bacteroides fragilis ATCC 25285 1

[0187] Bacteroides vulgatus ATCC8482 1

[0188] Firmicutes 28% 4

[0189] Enterococcus faecalis ATCC 19433 2

[0190] Clostridium perfringens ATCC 13124 2

[0191] Proteobacteria 37% 5.3

[0192] Escherichia coli K- 12 ATCC 10798 3

[0193] Klebsiella pneumoniae subsp. pneumoniae ATCC 13883 2.3 Table 3. Composition of Synthetic Media

[0194] To test effects of additional fat sources, KD-based media were also prepared with addition of MCT, dominant in MCT2:5: 1 infant formula, or linoleic acid, dominant in KD3: 1 infant formula (FIG. 7G). Addition of MCT increased the absolute abundance of B. breve, B. infantis, and B. longum compared to corresponding controls, resulting in notable shifts in diversity when added to KD4: 1 and KD3:1 media (PERMANOVA p=0.05, R2=0.33; p=0.017, R2=0.32), but KD2.5: l media (PERMANOVA p=0.55, R2=0.04) (FIG. 7H). In contrast, addition of linoleic acid decreased the absolute abundance of B. infantis and B. vulgatus, which resulted in statistically significant shifts across PCoAl relative to all media groups (FIG. 7H). This raises the question of whether differential effects of linoleic acid on the microbiome could contribute to the failure of KD3 : 1 infant formula to protect against 6- Hz seizures (FIG. 1C, FIG. 5C).

[0195] Finally, to evaluate effects of carbohydrate type, the model infant gut microbial community was cultured in synthetic media representing KD4: 1, KD3: 1, and MCT2.5: 1 and containing either lactose or a fiber mix, comprised of equal amounts of FOS, inulin, cellulose, and gum arabic, as the fiber sources that distinguish KD4: 1 and MCT2.5: 1 infant formula from KD3: 1 and CD formulas (FIG. 3A). The presence of dietary fiber led to substantial shifts in the model infant gut microbial community across all media conditions, with particular enrichment of B. fragilis and decreases in B. breve and B. infantis (FIG. 71). PCoA analysis of synthetic metagenomic data assembled from quantitative taxonomic profiles showed significant clustering of fiber mix groups away from lactose controls (PERMANOVA, p=0.013 (KD4:1), p=0.012 (KD3: 1), p=0.001 (MCT2.5:1), FIG. 3B), with greater discrimination than seen with alterations in fat ratio or source (FIG. 7D-H). In particular, fiber mix yielded statistically significant decreases in several pathways related to amino acid biosynthesis, nucleotide and nucleoside biosynthesis, and carbohydrate degradation, among many others (FIG. 3C and FIG. 7G). Among the 110 metagenomic pathways that were significantly altered by in vitro culture of the simplified infant microbial community with fiber mix compared to lactose, 15 pathways (13.6%) were similarly significantly altered in the fecal microbiome of mice fed the fiber-containing KD4: 1 and MCT2.5: 1, as compared to lactose-containing CD controls (FIG. 3C). Specifically, queuosine biosynthesis and its intermediate preQo biosynthesis were significantly enriched by fiber in the in vitro system and by fiber-containing KDs in the mouse. Similarly, fiber- induced decreases in pentose phosphate pathways, pathways related carbohydrate degradation (sucrose, glucose, xylose, and glycogen degradation), carbohydrate biosynthesis (UDP-N- acetyl-D-glucosamine biosynthesis and UDP -glucose derived O-antigen building blocks biosynthesis), amino acid biosynthesis (L-alanine, L-lysine and L-aspartate and L-asparagine biosynthesis), partial TCA cycle, and methylerythritol phosphate pathway were also shared with mouse metagenomes of KD4: 1 and MCT2.5: 1 groups (FIG. 3C and 2E). The results suggest that dietary fiber, more so than fat ratio or source, exerts a strong influence on community structure and functional potential of a model infant gut microbial community. Select alterations are consistent with those seen in the mouse microbiome in response to host consumption of fiber-containing clinical KD infant formulas (KD4: 1 and MCT2.5: 1), which confer resistance to 6-Hz seizures (FIG. 8). The results suggest that these particular metagenomic signatures may serve as biomarkers for seizure resistance.

[0196] To test whether dietary fiber content has a causal impact on resistance to 6-Hz seizures, the fiber mix was supplemented into the KD3 : 1 infant formula to match reported fiber levels in KD4: 1 infant formula, and mice were tested for seizure susceptibility at 7 days after dietary treatment (FIG. 3D). As previously demonstrated, mice fed liquid KD3.1 exhibited decreased seizure threshold compared to CD controls (FIG. 3E). Notably, addition of fiber to the KD3 : 1 elevated seizure thresholds to levels that exceeded those seen in CD controls. The fiber supplementation was further repeated using the solid diet paradigm, where the same infant formulas were dehydrated and administered as chow instead of liquid diet. As seen in liquid form, supplementation with fiber mix significantly increased seizure threshold of mice fed KD3: 1, with no significant differences in diet consumption (FIG. 9). These data demonstrate that addition of fiber to the low fiber KD3 : 1 infant formula restores its antiseizure effects toward levels seen with fiber-containing KD4: 1 and MCT2.5: 1.

[0197] To determine whether dietary fiber supplementation can potentiate KD-induced seizure protection, the fiber-containing KD4: 1 infant formula, which yielded the highest seizure thresholds of all KD variants (FIG. 1), was supplemented with the dietary fiber mix that is already existing in the formula and tested mice for resistance to 6-Hz seizures after 7 days of feeding with the liquid diet (FIG. 4A). The additional fiber added to KD4: 1 formula increased fiber content from 5.3% to -10.3%. Dietary fiber supplementation significantly increased seizure thresholds to levels that exceeded those seen with KD4: 1 alone (FIG. 4B). There were no significant differences between groups in dietary consumption (FIG. 10A). The ability of fiber supplementation to further promote the anti-seizure effects of KD4: 1 was similarly seen when administered as solid diet, instead of liquid diet, also with no significant differences in food consumption (FIG. 10B, C). To further ask whether fiber supplementation promotes seizure resistance via bacterial production of short-chain fatty acids (SCFAs), KD4: 1 infant formula was supplemented with the SCFAs acetate, butyrate, and propionate, at concentrations predicted to match those achieved produced by fermentation of the dietary fiber mix. In both liquid and solid form, SCFA supplementation failed to phenocopy effects of dietary fiber supplementation and instead yielded mice with modest reductions in resistance to 6-Hz seizures, as compared to controls supplemented with vehicle solution (FIG. 11 A, B). Taken together, these data indicate that dietary fiber supplementation both restores the anti-seizure effects of the low fiber KD3 : 1 and further potentiates the anti-seizure effects of the fiber-containing KD4: 1, through mechanisms that are not sufficiently recapitulated by oral SCFA supplementation.

[0198] Different fiber types and sources elicit differential microbial alterations and seizure outcomes Dietary fibers are fermented by select gut bacteria and shape the composition and activity of the gut microbiome. To gain insight into whether particular fiber types or sources interact with KD4: 1 to differentially alter the infant gut microbiome, 13 different fiber conditions, comprised of commercially available fiber products or purified fiber types, were screened for their additional effects on the model infant microbial community when grown directly in KD4: 1 infant formula (rather than in a diet-based synthetic culture medium, as in prior experiments) (FIG. 4C). Taxonomic profiles showed that 8 out of the 13 fiber conditions significantly increased the absolute abundance of B.fragilis, and 11 fiber conditions significantly decreased B. breve (FIG. 12), both of which align with previous in vitro results from fiber supplementation into synthetic media (FIG. 71). 7 of the 13 fiber conditions yielded reductions in E. coif which parallel the increases in E. coli observed with mouse consumption of fiber-deficient KD3 : 1 (FIG. 6C). Next, synthetic metagenomic profiles were generated for the 13 fiber supplementation conditions and filtered results to prioritize the 15 protective features that were shared between mouse consumption of the KD4: 1 and MCT2.5: 1 (FIG. 2E) and model human infant microbial community responses to fiber in synthetic diet-based media (FIG. 3C, FIG. 8). The results revealed 4 subgroupings of model infant microbial responses to the 13 different fibers in KD4: 1 infant formula (FIG. 4D). Group la consisted of fiber mix, FOS, and orange fiber and was characterized by increases in genes related to preQO biosynthesis and L-alanine biosynthesis, with reductions in sucrose degradation and partial TCA cycle (FIG. 4D). Group lb consisting of pea, acacia, and psyllium husk fibers, clustered together with Group la and exhibited a similar general pattern of metagenomic features but with reductions in L-alanine biosynthesis and less substantial shifts in preQO biosynthesis and sucrose degradation (FIG. 4D). Group 2 consisted of inulin, cellulose, and gum arabic, which was characterized by significant decreases in genes related to 5-7 pathways (glycogen and sucrose degradation, L-alanine, L- lysine, L-aspartate, L-asparagine, and UDF-N-acetyl-D-glucosamine biosynthesis, partial TCA cycle, and methylerythritol phosphate pathway) and significant increases in preQO biosynthesis genes (FIG. 4D). Group 3, consisting of oat, potato, wheat, and apple fibers, was characterized by notable increases in representation of L-alanine biosynthesis and UDP- glucose-derived O-antigen building blocks biosynthesis, with decreases in queuosine biosynthesis (FIG. 4D).

[0199] Based on these patterns of microbial representation for key metagenomic features conserved in mice fed fiber-containing KDs and infant microbial communities cultured with fiber-supplemented media, one representative fiber condition per primary grouping (Group 1 : fiber mix, Group 2: gum arabic, Group 3: oat fiber) was selected to test for causal effects on seizure resistance. Representative fibers from each group were supplemented into KD4: 1 infant formula to raise fiber content from 5.3% to -10.3%, and tested mice for resistance to 6- Hz seizures after 7 days of feeding in paste form. As previously observed in liquid and solid diet form (FIG. 4B, FIG. 9B), supplementation of KD4: 1 paste with fiber mix significantly increased resistance to 6-Hz seizures (FIG. 4E). In contrast, supplementation with gum arabic (Group 2) had no overt effects on seizure threshold compared KD4: 1 controls (FIG. 4E). In addition, supplementation with oat fiber (Group 3) had a detrimental effect, significantly decreasing seizure thresholds compared to KD4: 1 controls and all other fiber conditions (FIG. 4E). Overall, these data reveal that the ability of fiber supplementation to potentiate the seizure protective effects of KD4: 1 infant formula is specific to particular sources and types of fibers that alter key metagenomic features of the gut microbiome.

[0200] Discussion

[0201] Findings from this study demonstrate that different clinical KD infant formulas have varying effects on seizure resistance in mice, likely due to differences in how specific dietary components affect the function of the gut microbiome. This disclosure demonstrates that fiber-containing commercial infant formulas KD4: 1 and MCT2.5: 1 promote resistance to 6- Hz seizures in mice, whereas the fiber-deficient commercial infant formula KD3 : 1 increases susceptibility to 6-Hz seizures. Correspondingly, the protective KD4: 1 and MCT2.5: 1 induce several shared metagenomic alterations in the gut microbiome, which are not seen with KD3: 1. In particular, KD4: 1 and MCT2.5: 1, but not KD3: l, reduce representation of select genes related to carbohydrate degradation, which were significantly associated with the presence of dietary fiber and similarly induced by fiber supplementation to a cultured infant gut microbial community. Adding a fiber mixture to the KD3 : 1 to match levels present in KD4: 1 and MCT2.5: 1 restores seizure protection in mice. Moreover, supplementing the fiber mixture to the already protective KD4: 1 infant formula further enhances seizure resistance in mice.

[0202] Only a few small human studies have tested the effects of different medical KD regimens on seizure reduction, reporting no significant differences between the MAD, MCT, and LGIT diets relative to the classic KD in controlling seizures in children with refractory epilepsy. However, none of these examined the role of fiber or any specific dietary constituents on patient responses to KD therapy. A cross-sectional study of 150 epileptic individuals reported insufficient intake of fiber, among several other vitamins and minerals, and that patients with low intake of vegetables exhibited greater likelihood of uncontrolled seizures. When considering specific macronutrients and micronutrients that distinguish patients with controlled and uncontrolled seizures, percent intake of fiber was the closest to statistical significance (reported p=0.05). In addition, a human study of KD therapy in children with refractory epilepsy reported changes in 29 metagenomic pathways, including the reduction of seven pathways involved in carbohydrate metabolism and fermentation such as fructooligosaccharides (FOS) and raffinose utilization, sucrose utilization, glycogen metabolism, lacto-N-biose I and galacto-N-biose metabolic pathway; lactate, pentose phosphate pathway; and formaldehyde assimilation: ribulose monophosphate pathway.

[0203] Dietary fibers are resistant to digestion by the host and specifically fermented by gut bacteria that together encode hundreds of glycoside hydrolases with varying specificity for different fiber types. As such, not only does the gut microbiome degrade fiber, it also responds to and is shaped in composition and function by dietary fiber. The disclosure demonstrates that supplementing mice with SCFAs, as the microbial byproducts of fiber fermentation, fails to phenocopy the beneficial effects of fiber supplementation on potentiating seizure protection in mice fed the KD4: 1. This may align with prior human studies reporting that epilepsy is associated with deficient levels of SCFA-producing bacteria, which are further reduced by KD therapy to promote seizure control. This suggests that the fiber effects on seizure resistance are mediated not by direct products of fiber degradation, but rather by indirect effects of fiber fermentation on the microbiome and host. Indeed, alterations in the gut microbiome are increasingly implicated in risk for epilepsy and seizure responsiveness to the KD across several human studies.

[0204] Findings from animal models demonstrate that KD-induced alterations in the gut microbiome contribute to seizure resistance, suggesting that differential effects of dietary formulations on the gut microbiome may lead to variation in seizure protection. By screening various dietary factors that distinguish the KD infant formulas, including fat ratio, fat source, fat saturation, and carbohydrate type, on a model human infant microbial community, it was found that addition of fiber to a diet-based synthetic culture media elicits substantial shifts in microbial metagenomic profiles. Many key metagenomic features seen in response to fiber supplementation in the in vitro system are consistent with those seen in the gut microbiome of seizure-protected mice fed fiber-containing KD4: 1 and MCT2.5: 1, suggesting direct interactions between dietary fiber and the microbiome that are effectively modeled in simplified microbial culture systems.

[0205] Different fiber types and sources can vary greatly in their chemical structure, fermentability, and effects on the gut microbiome. The in vitro screening approach was expanded to include 13 different soluble or insoluble fiber types and sources, as supplemented directly into the commercial KD4: 1 infant formula (rather than a diet-based synthetic culture medium). By using key fiber-associated metagenomic features to stratify microbial responses to the 13 fiber conditions, a specific subset of fibers that potentiates the seizure protective effects of the KD4: 1 in mice was identified. This subgroup, including fiber mix (inulin, FOS, gum arabic, cellulose), FOS alone, and orange fiber, is characterized by metagenomic enrichment of pathways related to preQo biosynthesis and L-alanine biosynthesis, and decreases in metagenomic representation of sucrose degradation and partial TCA cycle. PreQo is a deazapurine nucleoside. In mice that exhibited seizure resistance in response to transplantation of the clinical KD-induced human microbiota, microbial preQo biosynthesis was associated with alterations in hippocampal expression of genes related to neuron generation and migration protection. L-alanine is an essential amino acid that is modulated by ketosis and regulates the function of glutamatergic neurons and astrocytes. L- alanine levels were diminished significantly in the cerebrospinal fluid of children after four months of KD therapy, and genes related to L-alanine metabolism were elevated in imputed microbial metagenomic pathways from epileptic individuals relative to healthy controls. Microbial sucrose utilization is a carbohydrate pathway reduced after KD therapy in humans, likely due to the low availability of carbohydrates in the diet. In bacteria, the TCA pathway fuels aerobic respiration, wherein acetyl-CoA is converted to intermediate organic acids such as citrate, 2-oxoglutarate, and succinate. Overall, these results suggest that increases in microbial biosynthetic pathways for preQo and L-alanine and reductions in microbial carbohydrate metabolism may serve as biomarkers for diet-induced seizure resistance. Results from this study reveal that nuanced differences in the formulation of KDs that are used to treat refractory epilepsy can lead to major differences in treatment efficacy and in the functional potential of the gut microbiome. Dietary fiber has a role in restoring and potentiating the seizure protective effects of commercial KD formulas when fed to mice. Dietary fiber shifts key metagenomic features in both the mouse gut microbiome and a model human infant microbial community, which can be used to identify specific fiber types that potentiate the seizure protective effects of a classical KD formula in mice. These findings align with increasing evidence that the gut microbiome modifies the anti-seizure effects of the KD and that microbiome-targeted diets can be used to shape the structure and function of the gut microbiome. It further supports the growing notion that careful consideration of dietary effects on host-microbial interactions is needed to inform the design of more effective and personalized dietary interventions for disease.

[0206] Methods

[0207] Mice

[0208] All mouse experiment protocols were approved by the UCLA Institutional Animal Care and Use Committee. Juvenile (4-week old) specific pathogen free (SPF), male Swiss Webster (Taconic Farms) mice were used for all animal experiments, fed standard chow (Labdiet 5010, 28.7%: 13.1%: 58.2% protein: fat: carbohydrate by calories), and housed in sterile caging under a 12 h: 12 h light:dark cycle with standard temperature and humidity control.

[0209] Dietary treatment

[0210] Experimental animals were fed commercially available KD infant formulas (KetoCai, Nutricia North America, FIG. la,) or a popular commercially available, standard infant formula as control diet (Abbott Nutrition, FIG. la) for 7 days. For liquid diet paradigm, 90 g of powder formula or 90 mL of liquid formula was mixed with 600 mL water at 60°C. Before adding to cages, the diet solution was brought to IL and each cage containing 3-4 mice was supplemented with liquid diets in water bottles. The water bottles were filled with liquid diets and the cages were changed every 1-2 days. For the solid diet paradigm, 90 g of powder formula was mixed with 600 mL water and dehydrated using a food dehydrator (CASORI). The diets were administered in sterile petri dishes and cages were provided with standard sterile water. For the pasted diet paradigm, 30 g of powder was mixed with water and administered as a paste in sterile petri dishes.

[0211] For fiber supplementation experiments, 5 g of individual fiber or fiber mixture (fructooligosaccharides (FOS), inulin, cellulose and gum arabic from Sigma-Aldrich, mixed at 1 : 1 (w / w)) was added to 90 g of KD formula prior to administering as a paste as described above. For SCFA supplementation, the following concentrations were reference values for SCFAs reported in SPF mice fed standard chow containing 15% fiber: acetate (67.5 mM), propionate (25 mM) and butyrate (40 mM). To model 5% fiber content present in KD4: 1, sodium acetate (22.55 mM), sodium propionate (8.33 mM) and sodium butyrate (13.35 mM) were administered in sterile drinking water. For paste diets, 1 : 10 of the SCFA mixture were mixed with water and added to the powder diets at the following concentrations: sodium acetate (2.255 mM), sodium propionate (0.833 mM) and sodium butyrate (1.335 mM). As a negative control, sodium chloride (NaCl) was supplemented to match amounts in SCFA salts in water (132.5 mM) and in diet (13.25mM).

[0212] 6-Hz psychomotor seizure assay

[0213] The 6-Hz psychomotor seizure test was conducted as previously described in Olson, C. A. et al. The Gut Microbiota Mediates the Anti-Seizure Effects of the Ketogenic Diet. Cell 173, 1728-1741. el3 (2018). One drop (~50 ul) of 0.5% tetracaine hydrochloride ophthalmic solution was applied to the corneas of each mouse 10-15 min before stimulation. Corneal electrodes were coated with a thin layer of electrode gel (Parker Signagel). A current device (ECT Unit 57800, Ugo Basile) was used to deliver current at 3 s duration, 0.2 ms pulse-width and 6 pulses / s frequency. CC50 (the intensity of current required to elicit seizures in 50% of the experimental group) was measured as a metric for seizure susceptibility. Pilot experiments were conducted to identify 28 mA as the CC50 for SPF wild-type Swiss Webster mice when they are on liquid and solid diet and 44 mA when they are on paste diet. Each mouse was seizure-tested only once, and thus n=14-16 mice were used to adequately power each experimental group. 28 or 44 mA currents were administered to the first mouse per cohort, followed by fixed increases or decreases by 2 mA intervals. Mice were restrained manually during stimulation and then released into a new cage for behavioral observation. Locomotor behavior was recorded using a camera and quantitative measures for stunned fixture, falling, tail dorsiflexion (Straub tail), forelimb clonus, eye / vibrissae twitching, and behavioral remission were scored manually. Latency to exploration (time elapsed from when an experimental mouse is released into the observation cage (after corneal stimulation) to its normal exploratory behavior) was scored manually with an electronic timer. Mice were blindly scored as protected from seizures if they did not show seizure behavior and resumed normal exploratory behavior within 10 s. Seizure threshold (CC50) was determined, using the average log interval of current steps per experimental group, where sample n is defined as the subset of animals displaying the less frequent seizure behavior. Data used to calculate CC50 are also displayed as latency to explore for each current intensity, where n represents the total number of biological replicates per group regardless of seizure outcome.

[0214] Fecal shotgun metagenomics

[0215] Frozen stool samples from mice pre- and post-dietary treatment were subjected to DNA extraction using the ZymoBIOMICS DNA Miniprep kit (Zymo), with bead beating used to lyse cells. Briefly, the samples were transferred into PowerBead tubes containing lysis solution and bead beaded at maximum speed for 1 min five times with 1 min of ice incubation in between cycles. The rest of the protocol followed the manufacturer’s instructions. The DNA was eluted in 60 pL elution buffer provided by the kit. Purified DNAs were sent to Novogene Corporation Inc for paired end (PE) metagenomic sequencing. Sequencing was performed on the Illumina NovaSeq platform with PE reads of 150 bp for each sample averaging around 3GB data. Raw reads were subjected to kneaddata to remove host contaminants. Metagenomic data was analyzed using HUMAnN3 and MetaCyc database to profile gene families and pathway abundance. MetaPhlAn4 was used for metagenomic taxonomic profiling, a-diversity indexes for taxonomic profiling were determined by Shannon’s index, richness, and Pielou's Evenness using vegan v2.6-4 in R. For P-diversities, calculate diversity.R script were run within the MetaPhlAn4. For Unifrac distances, mpa_vOct22_CHOCOPhlAnSGB_202212.nwk was used for SGB-level phylogenetic tree as reference. R packages tidyverse v2.0.0, vegan v2.6-4, and phyloseq vl.38.0 was used for Principal coordinate analysis (PCoA) of taxanomic distribution. Alterations in microbial diversity were assessed using PERMANOVA with adonis2 with 999 permutations from the vegan package in R. File2meco R package was used for MetaCyc pathway hierarchical classification. MaAsLin 2.0 was used to assess significant pathway associations between dietary treatments with an adjusted p value (q value) cutoff of 0.05, where indicated in the figure by asterisk. Beta-hydroxybutyrate (BHB) measurements

[0216] Blood was collected via a capillary tube from the medial canthus of the eye, allowed to clot 30 min at room temperature, and spun through SST vacutainers (Becton Dickinson) at 1500g for 90 sec for serum separation. Samples were immediately snap frozen in liquid nitrogen and stored at -80°C until further processing. BHB levels were quantified by colorimetric assay according to the manufacturer’s instructions (Cayman Chemical).

[0217] Bacterial strains and culturing

[0218] The following strains were selected to represent the taxonomically and functionally human infant gut microbiome (FIG. 6A) and obtained either from ATCC or DSMZ collection and propagated as instructed: Bifidobacterium longum subsp. infantis DSM 20088, Bifidobacterium longum subsp. longum ATCC BAA-999, Bifidobacterium breve DSM 20213, Bacteroides fragilis ATCC 25285, Bacteroides vulgatus ATCC8482, Enterococcus faecalis ATCC 19433, Clostridium perfringens ATCC 13124, Escherichia coli K-12 ATCC 10798, Klebsiella pneumoniae subsp. pneumoniae ATCC 13883. The cultures were routinely grown anaerobically in their respective media and temperature (Table 2). The growth of species were tested on a rich complex medium for 24 h to confirm stable relative abundances over the duration of anaerobic culture, as confirmed by cfu plating and qPCR (Table 4).

[0219] Table 4. Composition of Rich Bacterial Medium

[0220] Amount

[0221] Ingredient . (g / L) .

[0222] Yeast Extract (Sigma- Aldrich) 4.5

[0223] Peptone from meat, peptic digest (Sigma-Aldrich) 5 Tryptone (VWR) 5 Inulin (Sigma-Aldrich) 1 Mucin (Sigma- Aldrich) 4 Soluble Starch (Sigma-Aldrich) 5 Gum Arabic (Sigma-Aldrich) 1 Minimal media

[0224] Sodium Chloride (Sigma-Aldrich) 4.5 Potasium Chloride (EMD) 4.5 Magnesium sulfate heptahydrate (Sigma-Aldrich) 1.25 Calcium chloride dihyrate (Sigma-Aldrich) 0.1 Potasium dihydrogen phosphate. (Alfa Aesar) 0.5 Sodium bicarbonate (Sigma-Aldrich) 1.5 Bile bovine (Sigma- Aldrich) 0.05 L-cysteine hydrochloride (Acros Organics) 0.8 Iron (II) sulfate heptahydrate (Sigma- Aldrich) 0.005

[0225] Hemin (Sigma- Aldrich) 0.005

[0226] Vitamin mix solution (ATCC) (mL) 1

[0227] Vitamin Ki (HiMedia) 0.002

[0228] Tween 80 (Sigma-Aldrich) (mL) 1

[0229] Vitamin mix solution

[0230] Folic acid 2

[0231] Pyridoxine hydrochloride 10

[0232] Riboflavin 5

[0233] Biotin 2

[0234] Thiamine 5

[0235] Nicotinic acid 5

[0236] Calcium Pantothenate 5

[0237] Vitamin B 12 0.1 p-Aminobenzoic acid 5

[0238] Thioctic acid 5

[0239] Monopotassium phosphate 900

[0240] In vitro batch culture fermentations

[0241] Synthetic KDs with different ratios, fat and carbohydrate source were prepared using sunflower oil (Baja Precious), vegetable shortening (Crisco), palm oil (Okonatur), soy lecithin (Modernist Pantry), linoleic acid (Sigma-Aldrich), and Medium Chain Triglycerides (MCT, Nutriticia) as fat sources, whey protein isolate (Bulk Supplements) as protein source, and lactose (modernist pantry) and dietary fiber mixture of fructooligosaccharides (Sigma- Aldrich), inulin from chicory (Sigma-Aldrich), crystalline cellulose (Sigma-Aldrich), and gum arabic from Acacia Tree (Sigma-Aldrich) as carbohydrate sources. Additionally, for fiber supplementation fermentation experiments, wheat, pea, potato, and apple fiber from J. Rettenmaier USA LP, orange (citrus) fiber from Citri-Fi Naturals, oat (NuNaturals), acacia (Nutricost organic), and psyllium husk (It’s just) were used. The powders were ultraviolet (UV)-sterilized and confirmed to be sterile by aerobic and anaerobic culture. They were then mixed with simulated saliva solution, gastric solution, and intestinal fluid as described in INFOGEST model without enzymatic solution to simulate the gastric and intestinal bolus entering to the colon and was subjected to an in vitro batch culture fermentation. The representative bacterial strains were mixed in a minimal media at the dilution factor (1 : 100) needed to achieve a ratio of 21% Actinobacteria, 14% of Bacteroidetes, 28% of Firmicutes, and 37% of Proteobacteria, reflective of relative abundances seen in a typical infant gut (Table 2). Species that comprise Actinobacteria, Bacteroidetes and Firmicutes were mixed at 1 : 1 ratio, whereas Proteobacteria consists of 57 % of Escherichia coli and 42% of Klebsiella pneumoniae. The bacterial mixture was then mixed with each diet bolus (1 : 1 v / v) and subjected to 24-hour anaerobic culture. After 24 hours, the bacterial pellets were separated from the media and stored at -80°C until further analysis. The pellets from pre- fermentation were also collected as a control.

[0242] Bacterial quantification via qRT-PCR

[0243] Total DNA was extracted from the pellets collected after fermentation, following standard procedures for the ZymoBIOMICS DNA Miniprep kit. The microbial composition was determined using quantitative RT-PCR with species specific primers and respective qPCR conditions. DNA extracted from individual overnight cultures were used to generate a standard curve. The copy numbers for each sample were calculated based on the standard curve and normalized to DNA concentration of the original sample. Absolute quantification of growth after anaerobic culture of each sample was determined by subtracting the prefermentation quantities and presented as log values. Any species that exhibited negative values after subtraction were regarded as zero or no growth. Data are presented in bar plots as a mean of each bacteria. PCoA plots were created using cmdscale from the distance matrix created using Euclidean distances in vegan package in R.

[0244] Production of synthetic metagenome reads and synthetic metagenome analysis

[0245] The genome fastq files for each species were obtained from ATCC.org. Open source BBMap v38.94 randomreads. sh plugin was used to randomly produce paired reads at 150 bp length from each genome based on the qPCR absolute quantification multiplied by a million.

[0246] For each sample, between 50-80 million metagenomic reads were produced. Metagenomes were analyzed using Humann3 and significant pathway associations were determined with MaAsLin2 package in R as described above.

[0247] Statistical analysis

[0248] All statistical analyses were conducted using R version 4.1.2. Data for boxplots were plotted as mean ± SEM. Data for parametric data sets was analyzed using one-way ANOVA with Bonferroni adjustment between multiple groups. For differences between two sample conditions from non-parametric data sets were analyzed using Wilcoxon signed rank test with Benjamini -Hochberg adjustment. For non-parametric distributions with more than two groups, data was analyzed by Kruskal -Wallis with Dunn’s test. For PCoA plots, the distance matrix created within vegan package was initially subjected to betadisper and permutest for multivariate homogeneity of groups dispersions (variances), then PERMANOVA with adonis2 with 999 permutations was used to determine statistical differences between groups. Significant differences from the tests were donated as follows: * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Notable non-significant differences were denoted as n.s.

[0249] INCORPORATION BY REFERENCE

[0250] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0251] EQUIVALENTS

[0252] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We Claim:

1. A method of preventing or treating a condition responsive to a ketogenic diet in a subject, comprising administering to the subject a composition comprising bacteria of at least one of the Bacteroidetes phylum, Proteobacteria phylum, Deferribacteres phylum, Streptococcaceae family, Coriobacteriia class, Bifidobacterium genera, Bacteroides genera, and Clostridium genera.

2. The method of claim 1, wherein the composition comprises Bifidobacterium infantis (e.g., Bifidobacterium longum sups, infantis DSM 20088).

3. The method of claim 1 or 2, wherein the composition comprises Bifidobacterium longum (e.g., Bifidobacterium longum sups, longum ATCC BAA-999).

4. The method of any one of claims 1-3, wherein the composition comprises Bifidobacterium breve e.g., Bifidobacterium breve DSM 20213).

5. The method of any one of claims 1-4, wherein the composition comprises Bacteroides fragilis (e.g., Bacteroides fragilis ATCC 25285).

6. The method of any one of claims 1-5, wherein the composition comprises Clostridium perfringens (e.g., Clostridium perfringens ATCC 13124).

7. A method of preventing or treating a condition responsive to a ketogenic diet in a subject, comprising administering to the subject an agent that decreases levels of one or more bacteria in the subject’s gut, wherein the one or more bacteria comprise bacteria of at least one of the Actinobacteria phylum, Erysipelotrichia class, Escherichia genera, Enterococcus genera, Mammaliicoccus genera, Bifidobacterium species, Bacteroides species, and Klebsiella species.

8. The method of claim 7, wherein the agent decreases levels of Escherichia coli (e.g., Escherichia coli K- 12 ATCC 10798).

9. The method of claim 7 or 8, wherein the agent decreases levels of Enterococcus faecalis (e.g., Enterococcus faecalis ATCC 19433).

10. The method of any one of claims 7-9, wherein the agent decreases levels of Mammaliicoccus sciuri.

11. The method of any one of claims 7-10, wherein the agent decreases levels of Bifidobacterium breve (e.g., Bifidobacterium breve DSM 20213).

12. The method of any one of claims 7-11, wherein the agent decreases levels of Bifidobacterium infantis (e.g., Bifidobacterium longum sups, infantis DSM 20088).

13. The method of any one of claims 7-12, wherein the agent decreases levels of Bacteroides vulgatus (e.g., Bacteroides vulgatus ATCC8482).

14. The method of any one of claims 7-13, wherein the agent decreases levels of Klebsiella pneumoniae (e.g., Klebsiella pneumoniae subsp. pneumoniae ATCC 13883).

15. The method of any one of claims 7-14, wherein the agent is a small molecule.

16. The method of any one of claims 7-15, wherein the agent is an antibiotic specific for bacteria of the Actinobacteria phylum, Erysipelotrichia class, Escherichia genera, Enterococcus genera, Mammaliicoccus genera, Bifidobacterium species, Bacteroides species, o Klebsiella species.

17. The method of any one of claims 1-16, comprising administering to a subject a composition comprising at least one of L-alanine, queuosine, preQo, guanosine, guanosine triphosphate , alpha-ketoglutarate (2-oxoglutarate), citrate, succinate and sucrose and / or lactose.

18. A method of preventing or treating a condition responsive to a ketogenic diet in a subject, comprising administering to the subject a composition comprising at least one of L- alanine, queuosine, preQo, guanosine, guanosine triphosphate, alpha-ketoglutarate (2- oxoglutarate), citrate, succinate and sucrose and / or lactose.

19. The method of any one of claims 1-18, wherein the condition is seizures, optionally wherein subject has a neurodevel opmental condition, e.g., selected from epilepsy, autism spectrum disorder, Rett syndrome, attention deficit disorder, and fragile X syndrome.

20. The method of any one of claims 1-19 wherein the subject has a condition selected from Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), cancer, stroke, a metabolic disease, a mitochondrial disorder, depression, migraines, and traumatic brain injury (TBI).

21. The method of any one of claims 1-20, comprising administering a fiber supplement to the subject.

22. The method of claim 20, wherein the fiber supplement comprises at least one of inulin, fructooligosaccharides (FOS), acacia fiber (e.g., gum arabic), cellulose, and orange fiber.

23. The method of any one of claims 1-22, wherein the subject is on a ketogenic diet, a high fat diet, a low carbohydrate diet, or a diet with a fiber supplement.

24. The method of any one of claims 1-23, wherein the composition is formulated for oral delivery.

25. The method of any one of claims 1-24, wherein the composition is a food product.

26. The method of claim 25, wherein the food product is infant formula.

27. The method of claim 25, wherein the food product is a fiber supplement.

28. The method of any one of claims 1-23, wherein the composition is formulated for rectal delivery.

29. The method of any one of claims 1-28, wherein the composition is self-administered.

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