Exogenous ketones exert antiseizure effects and modulate the gut microbiome and mycobiome in a clinically relevant murine model of epilepsy.

OBJECTIVE: Despite growing interest in the potential use of exogenous ketones for the treatment of epilepsy, their impact on seizures and the gut microbiome and mycobiome remain unclear. METHODS: Here, we examined the effects of both oral gavage and subcutaneous (SC) injection of a ketone ester (KE) in spontaneously epileptic Kcna1-null (KO) mice that model seminal aspects of human temporal lobe epilepsy. Electroencephalographic recordings and biochemical analyses were performed in KE-treated KO mice. Fecal microbial and fungal communities were profiled to determine whether the antiseizure activity of KE involves changes in the gut microbiome. RESULTS: We found that exogenous KE administration by SC injection was more effective than oral gavage in terms of rendering antiseizure effects while generating similar degrees of ketonemia. However, reductions in mean daily seizure counts were accompanied by overall alterations in the fecal bacterial microbiome. Either oral or SC injection imposed a greater impact on the microbiome in male than female mice. In males, oral KE decreased Bacteroidota phylum and genera of Ligilactobacillus and Muribaculaceae, whereas SC injection decreased Bacteroides, Lactobacillus, and Lachnospiraceae. The fecal mycobiome was affected by KE injection to a greater degree than by oral gavage, and more in females than in males, as reflected by an increase in Ascomycota and Saccharomyces. Correlation analysis between microbiome and seizure counts revealed that in mice receiving KE injection, the seizure count was positively correlated with an amplicon sequencing variant of Lactobacillus (Spearman rho = .64, p = .03) and tended toward a negative correlation with Saccharomyces (Spearman rho = -.57, p = .057). SIGNIFICANCE: Our findings demonstrate that exogenous ketone administration alone can induce antiseizure effects equally via different routes of administration, and that they induce differential shifts in both the bacterial microbiome and mycobiome.

Fructo-oligosaccharides promote butyrate production over citrus pectin during in vitro fermentation by colonic inoculum from pig.

OBJECTIVES: Fructo-oligosaccharide (FOS) and citrus pectin (CP) are soluble fibers with different chemical composition. However, their fermentation pattern in large intestine remains unclear. METHODS: An in vitro batch fermentation using colonic digesta from pigs as inoculum was employed to investigate the fermentation dynamics of FOS and CP. The monosaccharides and SCFAs contents were assayed by high-performance liquid chromatography and gas chromatography, respectively. And the microbiota community was assessed by 16S rRNA gene high-throughput sequencing. RESULTS: Both FOS and CP were degarded after 6 h, especially to a neglected level in FOS. FOS group showed higher abundances of butyrate-producing bacteria such as Eubacterium rectale, Roseburia faecis and Coprococcus comes and butyrate compared to CP. CP stimulated the growth of pectinolytic microbes Lachnospira pectinoschiza, succinate-producing bacteria Succinivibrio dextrinosolvens, succinate-utilizing bacteria Phascolarctobacterium succinatutens and the production of acetate and propionate compared to FOS. Moreover, the relative abundances of key enzymes (e.g. butyrate kinase) involving in butyrate formation via the butyrate kinase route were upregulated in the FOS group. And the key enzymes (e.g. acetyl-CoA synthetase) associated with propionate production through the succinate pathway were upregulated in the CP group. CONCLUSIONS: FOS was preferred to ferment by butyrate-producing bacteria to yield a higher level of butyrate via the butyrate kinase pathway, while CP enhanced the cross-feeding of succinate-producing and succinate-utilizing bacteria to form propionate through the succinate pathway. These findings deepen our understanding on the fermentation characteristics of the soluble fibers, and also provide guidelines for fiber choice in precisely modulating the microbial composition and metabolism in large intestine.

Breed-Driven Microbiome Heterogeneity Regulates Intestinal Stem Cell Proliferation via Lactobacillus-Lactate-GPR81 Signaling.

Genetically lean and obese individuals have distinct intestinal microbiota and function. However, the underlying mechanisms of the microbiome heterogeneity and its regulation on epithelial function such as intestinal stem cell (ISC) fate remain unclear. Employing pigs of genetically distinct breeds (obese Meishan and lean Yorkshire), this study reveals transcriptome-wide variations in microbial ecology of the jejunum, characterized by enrichment of active Lactobacillus species, notably the predominant Lactobacillus amylovorus (L. amylovorus), and lactate metabolism network in obese breeds. The L. amylovorus-dominant heterogeneity is paralleled with epithelial functionality difference as reflected by highly expressed GPR81, more proliferative ISCs and activated Wnt/ß-catenin signaling. Experiments using in-house developed porcine jejunal organoids prove that live L. amylovorus and its metabolite lactate promote intestinal organoid growth. Mechanistically, L. amylovorus and lactate activate Wnt/ß-catenin signaling in a GPR81-dependent manner to promote ISC-mediated epithelial proliferation. However, heat-killed L. amylovorus fail to cause these changes. These findings uncover a previously underrepresented role of L. amylovorus in regulating jejunal stem cells via Lactobacillus-lactate-GPR81 axis, a key mechanism bridging breed-driven intestinal microbiome heterogeneity with ISC fate. Thus, results from this study provide new insights into the role of gut microbiome and stem cell interactions in maintaining intestinal homeostasis.

Host-microbiota interaction in intestinal stem cell homeostasis.

Intestinal stem cells (ISCs) play a pivotal role in gut physiology by governing intestinal epithelium renewal through the precise regulation of proliferation and differentiation. The gut microbiota interacts closely with the epithelium through myriad of actions, including immune and metabolic interactions, which translate into tight connections between microbial activity and ISC function. Given the diverse functions of the gut microbiota in affecting the metabolism of macronutrients and micronutrients, dietary nutrients exert pronounced effects on host-microbiota interactions and, consequently, the ISC fate. Therefore, understanding the intricate host-microbiota interaction in regulating ISC homeostasis is imperative for improving gut health. Here, we review recent advances in understanding host-microbiota immune and metabolic interactions that shape ISC function, such as the role of pattern-recognition receptors and microbial metabolites, including lactate and indole metabolites. Additionally, the diverse regulatory effects of the microbiota on dietary nutrients, including proteins, carbohydrates, vitamins, and minerals (e.g. iron and zinc), are thoroughly explored in relation to their impact on ISCs. Thus, we highlight the multifaceted mechanisms governing host-microbiota interactions in ISC homeostasis. Insights gained from this review provide strategies for the development of dietary or microbiota-based interventions to foster gut health.

Microbial Features Linked to Medication Strategies in Cardiometabolic Disease Management.

Human gut microbiota are recognized as critical players in both metabolic disease and drug metabolism. However, medication-microbiota interactions in cardiometabolic diseases are not well understood. To gain a comprehensive understanding of how medication intake impacts the gut microbiota, we investigated the association of microbial structure with the use of single or multiple medications in a cohort of 134 middle-aged adults diagnosed with cardiometabolic disease, recruited from Alberta's Tomorrow Project. Predominant cardiometabolic prescription medication classes (12 total) were included in our analysis. Multivariate Association with Linear Model (MaAsLin2) was employed and results were corrected for age, BMI, sex, and diet to evaluate the relationship between microbial features and single- or multimedication use. Highly individualized microbiota profiles were observed across participants, and increasing medication use was negatively correlated with α-diversity. A total of 46 associations were identified between microbial composition and single medications, exemplified by the depletion of Akkermansia muciniphila by ß-blockers and statins, and the enrichment of Escherichia/Shigella and depletion of Bacteroides xylanisolvens by metformin. Metagenomics prediction further indicated alterations in microbial functions associated with single medications such as the depletion of enzymes involved in energy metabolism encoded by Eggerthella lenta due to ß-blocker use. Specific dual medication combinations also had profound impacts, including the depletion of Romboutsia and Butyriciocccus by statin plus metformin. Together, these results show reductions in bacterial diversity as well as species and microbial functional potential associated with both single- and multimedication use in cardiometabolic disease.

Maternal Acylcarnitine Disruption as a Potential Predictor of Preterm Birth in Primigravida: A Preliminary Investigation.

Preterm birth, defined as any birth before 37 weeks of completed gestation, poses adverse health risks to both mothers and infants. Despite preterm birth being associated with several risk factors, its relationship to maternal metabolism remains unclear, especially in first-time mothers. Aims of the present study were to identify maternal metabolic disruptions associated with preterm birth and to evaluate their predictive potentials. Blood was collected, and the serum harvested from the mothers of 24 preterm and 42 term births at 28-32 weeks gestation (onset of the 3rd trimester). Serum samples were assayed by untargeted metabolomic analyses via liquid chromatography/mass spectrometry (QTOF-LC/MS). Metabolites were annotated by inputting the observed mass-to-charge ratio into the Human Metabolome Database (HMDB). Analysis of 181 identified metabolites by PLS-DA modeling using SIMCA (v17) showed reasonable separation between the two groups (CV-ANOVA, p = 0.02). Further statistical analysis revealed lower serum levels of various acyl carnitines and amino acid metabolites in preterm mothers. Butenylcarnitine (C4:1), a short-chain acylcarnitine, was found to be the most predictive of preterm birth (AUROC = 0.73, [CI] 0.60-0.86). These observations, in conjuncture with past literature, reveal disruptions in fatty acid oxidation and energy metabolism in preterm primigravida. While these findings require validation, they reflect altered metabolic pathways that may be predictive of preterm delivery in primigravida.

Dose-Responsive Effects of Iron Supplementation on the Gut Microbiota in Middle-Aged Women.

Oral iron supplementation is the first-line treatment for addressing iron deficiency, a concern particularly relevant to women who are susceptible to sub-optimal iron levels. Nevertheless, the impact of iron supplementation on the gut microbiota of middle-aged women remains unclear. To investigate the association between iron supplementation and the gut microbiota, healthy females aged 40-65 years (n = 56, BMI = 23 ± 2.6 kg/m2) were retrospectively analyzed from the Alberta's Tomorrow Project. Fecal samples along with various lifestyle, diet, and health questionnaires were obtained. The gut microbiota was assessed by 16S rRNA sequencing. Individuals were matched by age and BMI and classified as either taking no iron supplement, a low-dose iron supplement (6-10 mg iron/day), or high-dose iron (>100 mg/day). Compositional and functional analyses of microbiome data in relation to iron supplementation were investigated using various bioinformatics tools. Results revealed that iron supplementation had a dose-dependent effect on microbial communities. Elevated iron intake (>100 mg) was associated with an augmentation of Proteobacteria and a reduction in various taxa, including Akkermansia, Butyricicoccus, Verrucomicrobia, Ruminococcus, Alistipes, and Faecalibacterium. Metagenomic prediction further suggested the upregulation of iron acquisition and siderophore biosynthesis following high iron intake. In conclusion, adequate iron levels are essential for the overall health and wellbeing of women through their various life stages. Our findings offer insights into the complex relationships between iron supplementation and the gut microbiota in middle-aged women and underscore the significance of iron dosage in maintaining optimal gut health.

Protocol for nutritional intervention in neonatal rats using the "pup-in-a-cup" artificial rearing system.

Early-life nutrition fundamentally influences newborn development and health. Here, we present a protocol for nutritional intervention in neonatal rats using the "pup-in-a-cup" artificial rearing system. We describe steps for rat milk substitute preparation, cheek cannulation and maintenance, and nutritional manipulation during the suckling period. This protocol enables investigation into the role of nutritional factors in newborns by artificially rearing rats away from the mother with experimental diets starting at postnatal day 4 for up to 18 days. For complete details on the use and execution of this protocol, please refer to Wang et al.,1 Choudhary et al.,2 and Mu et al.3,4.

Magnolol-driven microbiota modulation elicits changes in tryptophan metabolism resulting in reduced skatole formation in pigs.

Skatole of gut origin has garnered significant attention as a malodorous pollutant due to its escalating emissions, recalcitrance to biodegradation and harm to animal and human health. Magnolol is a health-promoting polyphenol with potential to considerably mitigate the skatole production in the intestines. To investigate the impact of magnolol and its underlying mechanism on the skatole formation, in vivo and in vitro experiments were conducted in pigs. Our results revealed that skatole concentrations in the cecum, colon, and faeces decreased by 58.24% (P = 0.088), 44.98% (P < 0.05) and 43.52% (P < 0.05), respectively, following magnolol supplementation. Magnolol supplementation significantly decreased the abundance of Lachnospira, Faecalibacterium, Paramuribaculum, Faecalimonas, Desulfovibrio, Bariatricus, and Mogibacterium within the colon (P < 0.05). Moreover, a strong positive correlation (P < 0.05) between skatole concentration and Desulfovibrio abundance was observed. Subsequent in silico studies showed that magnolol could dock well with indolepyruvate decarboxylase (IPDC) within Desulfovibrio. Further in vitro investigation unveiled that magnolol addition led to less indole-3-pyruvate diverted towards the oxidative skatole pathway by the potential docking of magnolol towards IPDC, thereby diminishing the conversion of substrate into skatole. Our findings offer novel targets and strategies for mitigating skatole emission from the source.

Protocol for measuring respiratory function of mitochondria in frozen colon tissue from rats.

Mitochondrial respirometry allows for the comprehensive study of oxygen consumption within the electron transport system in tissues. However, limited techniques exist for analyzing frozen or biobanked intestinal tissues. Here, we present a protocol to evaluate the respiratory function of mitochondria in colonic tissues after cryopreservation at -80°C. We describe steps for rat dissection, respirometry calibration, and tissue preparation. We then detail measurement of oxygen respiration and protein concentration. This protocol facilitates the retrospective analysis of mitochondrial respiration in frozen tissue.

Metagenomics-based inference of microbial metabolism towards neuroactive amino acids and the response to antibiotics in piglet colon.

Gut-derived neuroactive metabolites from amino acids perform a broad range of physiological roles in the body. However, the interaction between microbiota and epithelium in the metabolism of amino acids with neuroactive properties remains unclear in the colon of piglets. To investigate the microbial and epithelial metabolism, metagenomics and mucosal metabolomics were performed using colonic samples from 12 ileum-canulated piglets subjected to a 25-day infusion with saline or antibiotics. We categorized 23 metabolites derived from the metabolism of tryptophan, glutamate, and tyrosine, known as precursors of neuroactive metabolites. Microbial enzymes involved in the kynurenine synthesis via arylformamidase, 4-aminobutyric acid (GABA) synthesis via putrescine aminotransferase, and tyramine synthesis via tyrosine decarboxylase were identified in Clostridiales bacterium, uncultured Blautia sp., and Methanobrevibacter wolinii, respectively. Antibiotics significantly affected the microbiota involved in tryptophan-kynurenine and glutamate-GABA metabolism. An increase in the relative abundance of putrescine aminotransferase and Blautia sp. correlated positively with an increase in luminal GABA concentration. Overall, our findings provide new insights into the microbial ability to metabolize key amino acids that are precursors of neuroactive metabolites.

Regulation of serotonin production by specific microbes from piglet gut.

BACKGROUND: Serotonin is an important signaling molecule that regulates secretory and sensory functions in the gut. Gut microbiota has been demonstrated to affect serotonin synthesis in rodent models. However, how gut microbes regulate intestinal serotonin production in piglets remains vague. To investigate the relationship between microbiota and serotonin specifically in the colon, microbial composition and serotonin concentration were analyzed in ileum-cannulated piglets subjected to antibiotic infusion from the ileum when comparing with saline infusion. Microbes that correlated positively with serotonin production were isolated from piglet colon and were further used to investigate the regulation mechanisms on serotonin production in IPEC-J2 and a putative enterochromaffin cell line RIN-14B cells. RESULTS: Antibiotic infusion increased quantities of Lactobacillus amylovorus (LA) that positively correlated with increased serotonin concentrations in the colon, while no effects observed for Limosilactobacillus reuteri (LR). To understand how microbes regulate serotonin, representative strains of LA, LR, and Streptococcus alactolyticus (SA, enriched in feces from prior observation) were selected for cell culture studies. Compared to the control group, LA, LR and SA supernatants significantly up-regulated tryptophan hydroxylase 1 (TPH1) expression and promoted serotonin production in IPEC-J2 cells, while in RIN-14B cells only LA exerted similar action. To investigate potential mechanisms mediated by microbe-derived molecules, microbial metabolites including lactate, acetate, glutamine, and γ-aminobutyric acid were selected for cell treatment based on computational and metabolite profiling in bacterial supernatant. Among these metabolites, acetate upregulated the expression of free fatty acid receptor 3 and TPH1 while downregulated indoleamine 2,3-dioxygenase 1. Similar effects were also recapitulated when treating the cells with AR420626, an agonist targeting free fatty acid receptor 3. CONCLUSIONS: Overall, these results suggest that Lactobacillus amylovorus showed a positive correlation with serotonin production in the pig gut and exhibited a remarkable ability to regulate serotonin production in cell cultures. These findings provide evidence that microbial metabolites mediate the dialogue between microbes and host, which reveals a potential approach using microbial manipulation to regulate intestinal serotonin biosynthesis.

Lactiplantibacillus plantarum L47 and inulin alleviate enterotoxigenic Escherichia coli induced ileal inflammation in piglets by upregulating the levels of α-linolenic acid and 12,13-epoxyoctadecenoic acid.

Alternatives to antibiotics for preventing bacteria-induced inflammation in early-weaned farm animals are sorely needed. Our previous study showed that Lactiplantibacillus plantarum L47 and inulin could alleviate dextran sulfate sodium (DSS)-induced colitis in mice. To explore the protective effects of L. plantarum L47 and inulin on the ileal inflammatory response in weaned piglets challenged with enterotoxigenic Escherichia coli (ETEC), 28 weaned piglets were assigned into four groups, namely, CON group-orally given 10 mL/d phosphate buffer saline (PBS), LI47 group-orally given a mixture of 10 mL/d L. plantarum L47 and inulin, ECON group-orally given 10 mL/d PBS and challenged by ETEC, and ELI47 group-orally given 10 mL/d L. plantarum L47 and inulin mixture and challenged by ETEC. The results demonstrated that the combination of L. plantarum L47 and inulin reduced inflammatory responses and relieved the inflammatory damage caused by ETEC, including ileal morphological damage, reduced protein expression of ileal tight junction, decreased antioxidant capacity, and decreased anti-inflammatory factors. Transcriptome analysis revealed that L. plantarum L47 and inulin up-regulated the gene expression of phospholipase A2 group IIA (PLA2G2A) (P < 0.05) as well as affected alpha-linolenic acid (ALA) metabolism and linoleic acid metabolism. Moreover, L. plantarum L47 and inulin increased the levels of ALA (P < 0.05), lipoteichoic acid (LTA) (P < 0.05), and 12,13-epoxyoctadecenoic acid (12,13-EpOME) (P < 0.05) and the protein expression of Toll-like receptor 2 (TLR2) (P = 0.05) in the ileal mucosa. In conclusion, L. plantarum L47 and inulin together alleviated ETEC-induced ileal inflammation in piglets by up-regulating the levels of ALA and 12,13-EpOME via the LTA/TLR2/PLA2G2A pathway.

Intermittent vs continuous ketogenic diet: Impact on seizures, gut microbiota, and mitochondrial metabolism.

We have shown previously that the ketogenic diet (KD) is effective in reducing seizures associated with infantile spasms syndrome (ISS) and that this benefit is related to alterations in the gut microbiota. However, it remains unclear whether the efficacy of the KD persists after switching to a normal diet. Employing a neonatal rat model of ISS, we tested the hypothesis that the impact of the KD would diminish when switched to a normal diet. Following epilepsy induction, neonatal rats were divided into two groups: continuous KD for 6 days; and a group fed with KD for 3 days and then a normal diet for 3 days. Spasms frequency, mitochondrial bioenergetics in the hippocampus, and fecal microbiota were evaluated as major readouts. We found that the anti-epileptic effect of the KD was reversible, as evidenced by the increased spasms frequency in rats that were switched from the KD to a normal diet. The spasms frequency was correlated inversely with mitochondrial bioenergetic function and a set of gut microbes, including Streptococcus thermophilus and Streptococcus azizii. These findings suggest that the anti-epileptic and metabolic benefits of the KD decline rapidly in concert with gut microbial alterations in the ISS model.

Physical activity-induced alterations of the gut microbiota are BMI dependent.

Physical inactivity is one of the leading causes of chronic metabolic disease including obesity. Increasing physical activity (PA) has been shown to improve cardiometabolic and musculoskeletal health and to be associated with a distinct gut microbiota composition in trained athletes. However, the impact of PA on the gut microbiota is inconclusive for individuals performing PA in their day-to-day life. This study examined the role of PA and hand-grip strength on gut microbiome composition in middle-aged adults (40-65 years, n = 350) with normal (18.5-24.9 kg/m2 ) and overweight (25-29.9 kg/m2 ) body mass index (BMI). PA was recorded using the International Physical Activity Questionnaire, and hand-grip strength was measured using a dynamometer. Serum samples were assessed for lipidomics while DNA was extracted from fecal samples for microbiome analysis. Overweight participants showed a higher concentration of triacylglycerols, and lower concentrations of cholesteryl esters, sphingomyelin, and lyso-phosphotidylcholine lipids (p < .05) compared with those with normal BMI. Additionally, overweight participants had a lower abundance of the Oscillibacter genus (p < .05). The impact of PA duration on the gut microbiome was BMI dependent. In normal but not overweight participants, high PA duration showed greater relative abundance of commensal taxa such as Actinobacteria and Proteobacteria phyla, as well as Collinsella and Prevotella genera (p < .05). Furthermore, in males with normal BMI, a stronger grip strength was associated with a higher relative abundance of Faecalibacterium and F. prausnitzii (p < .05) compared with lower grip strength. Taken together, data suggest that BMI plays a significant role in modeling PA-induced changes in gut microbiota.

Duodenal infusion of soy protein hydrolysate activates cAMP signaling and hypothalamic neurotransmitter synthesis in pigs.

Neurotransmitters in the brain are important for cognition and memory. As bioactive substrates, whether increased soy protein levels in pigs can promote hypothalamic neurotransmitter synthesis remains unclear. The effect of increased soy protein hydrolysate (SPH) levels in the small intestine of pigs on neurotransmitter precursor supply, hypothalamic neurotransmitter synthesis and underlying molecular processes was investigated by using sixteen pigs (35.2 ± 0.3 kg) infused either with SPH (70 g day-1) or sterile saline (control) twice daily for 15 days via a duodenal fistula. It demonstrated that SPH infusion increased the expression of the neutral amino acid transporter B0AT1 in the jejunal mucosa, the serum tyrosine/large neutral amino acid ratio, the concentrations of serum tyrosine, hypothalamic tyrosine, dopamine and brain-derived neurotrophic factor (BDNF) (P < 0.05). It also increased the jejunal and serum choline, hypothalamic choline and acetylcholine levels (P < 0.05). Hypothalamic transcriptome revealed that differential genes were significantly enriched in the cholinergic synapse, dopaminergic synapse and cyclic adenosine monophosphate (cAMP) signalling pathways, and that the expression of key enzyme genes in the synthesis of acetylcholine and dopamine and dopamine receptors 1 (DRD1) was upregulated by SPH (P < 0.05). Western blotting showed that SPH infusion activated the hypothalamic cAMP signalling pathways. Overall, SPH infusion promoted the synthesis of hypothalamic dopamine and acetylcholine, and the synthesised dopamine promoted BDNF production most likely through the activation of the cAMP signalling pathways by the DRD1.

Dietary citrus pectin drives more ileal microbial protein metabolism and stronger fecal carbohydrate fermentation over fructo-oligosaccharide in growing pigs.

Fructo-oligosaccharide (FOS) and pectin are known soluble dietary fibers and can influence gut microbiota and consequently modulate gut health. To understand the differential impact patterns of pectin vs. FOS in modulating gut microbiota in the small and large intestine, an ileal-cannulated pig model was adopted to compare the temporal and spatial effects of FOS and citrus pectin (CP) on the gut microbiota. Sixteen terminal ileal-cannulated pigs were randomly divided into 2 groups and fed with a standard diet supplemented with either 3% FOS or 3% CP for 28 d. The CP group and FOS group showed different microbial composition, especially in the feces, with time and location as major factors affecting microbiota in the CP group, and with only location contribution in the FOS group. In the feces, relative to the FOS group, the CP group showed higher abundance of Christensenellaceae R-7 group and Ruminococcaceae UCG-010 and lower abundance of Mitsuokella and Olsenella (adjusted P < 0.05), a higher level of short-chain fatty acids and a lower level of lactate at both d 14 and 25 (P < 0.05), and more copy numbers of genes encoding key enzymes related to propionate (mmdA) and butyrate (BCoAT) production and lactate utilization (LcdA) (P < 0.05), indicating a greater degree of microbial carbohydrate fermentation. In the ileum, as compared with FOS, CP increased the bacteria with high capability of fermenting amino acids, including Escherichia-Shigella and Klebsiella (adjusted P < 0.05), and the expression of enzymes responsible for amino acid fermentation (i.e. lysine decarboxylase), as well as the amino acid fermentation products (cadaverine and tyramine) (P < 0.05), indicating a greater degree of amino acid fermentation. Overall, our results highlight a differential dynamic impact of dietary CP vs. FOS on microbial composition and metabolism in the gut. The dietary CP has a stronger ability to promote microbial amino acid fermentation in the ileum and carbohydrate fermentation in the feces than FOS. These findings provide a new insight into the role of different fibers in gut nutrition and guidelines for the choice of fibers in manipulating gut health.

Amino acid utilization allows intestinal dominance of Lactobacillus amylovorus.

The mammalian intestine harbors heterogeneous distribution of microbes among which specific taxa (e.g. Lactobacillus) dominate across mammals. Deterministic factors such as nutrient availability and utilization may affect microbial distributions. Due to physiological complexity, mechanisms linking nutrient utilization and the dominance of key taxa remain unclear. Lactobacillus amylovorus is a predominant species in the small intestine of pigs. Employing a pig model, we found that the small intestine was dominated by Lactobacillus and particularly L. amylovorus, and enriched with peptide-bound amino acids (PBAAs), all of which were further boosted after a peptide-rich diet. To investigate the bacterial growth dominance mechanism, a representative strain L. amylovorus S1 was isolated from the small intestine and anaerobically cultured in media with free amino acids or peptides as sole nitrogen sources. L. amylovorus S1 grew preferentially with peptide-rich rather than amino acid-rich substrates, as reflected by enhanced growth and PBAA utilization, and peptide transporter upregulations. Utilization of free amino acids (e.g. methionine, valine, lysine) and expressions of transporters and metabolic enzymes were enhanced simultaneously in peptide-rich substrate. Additionally, lactate was elevated in peptide-rich substrates while acetate in amino acid-rich substrates, indicating distinct metabolic patterns depending on substrate forms. These results suggest that an increased capability of utilizing PBAAs contributes to the dominance of L. amylovorus, indicating amino acid utilization as a deterministic factor affecting intestinal microbial distribution. These findings may provide new insights into the microbe-gut nutrition interplay and guidelines for dietary manipulations toward gut health especially small intestine health.

Selective Probiotic Treatment Positively Modulates the Microbiota-Gut-Brain Axis in the BTBR Mouse Model of Autism.

Recent studies have shown promise for the use of probiotics in modulating behaviour through the microbiota-gut-brain axis. In the present study, we assessed the impact of two probiotic strains in mitigating autism-related symptomology in the BTBR T+ Itpr3tf/J mouse model of autism spectrum disorder (ASD). Male juvenile BTBR mice were randomized into: (1) control, (2) Lr probiotic (1 × 109 CFU/mL Lacticaseibacillus rhamnosus HA-114), and (3) Ls probiotic groups (1 × 109 CFU/mL Ligilactobacillus salivarius HA-118) (n = 18-21/group), receiving treatments in drinking water for 4 weeks. Gut microbiota profiling by 16S rRNA showed Lr, but not Ls supplementation, to increase microbial richness and phylogenetic diversity, with a rise in potential anti-inflammatory and butyrate-producing taxa. Assessing serum and brain metabolites, Lr and Ls supplementation produced distinct metabolic profiles, with Lr treatment elevating concentrations of potentially beneficial neuroactive compounds, such as 5-aminovaleric acid and choline. As mitochondrial dysfunction is often observed in ASD, we assessed mitochondrial oxygen consumption rates in the prefrontal cortex and hippocampus. No differences were observed for either treatment. Both Lr and Ls treatment reduced behavioural deficits in social novelty preference. However, no changes in hyperactivity, repetitive behaviour, and sociability were observed. Results show Lr to impart positive changes along the microbiota-gut-brain axis, exhibiting beneficial effects on selected behaviour, gut microbial diversity, and metabolism in BTBR mice.

Targeted gut microbiota manipulation attenuates seizures in a model of infantile spasms syndrome.

Infantile spasms syndrome (IS) is a devastating early-onset epileptic encephalopathy associated with poor neurodevelopmental outcomes. When first-line treatment options, including adrenocorticotropic hormone and vigabatrin, are ineffective, the ketogenic diet (KD) is often employed to control seizures. Since the therapeutic impact of the KD is influenced by the gut microbiota, we examined whether targeted microbiota manipulation, mimicking changes induced by the KD, would be valuable in mitigating seizures. Employing a rodent model of symptomatic IS, we show that both the KD and antibiotic administration reduce spasm frequency and are associated with improved developmental outcomes. Spasm reductions were accompanied by specific gut microbial alterations, including increases in Streptococcus thermophilus and Lactococcus lactis. Mimicking the fecal microbial alterations in a targeted probiotic, we administered these species in a 5:1 ratio. Targeted probiotic administration reduced seizures and improved locomotor activities in control diet-fed animals, similar to KD-fed animals, while a negative control (Ligilactobacillus salivarius) had no impact. Probiotic administration also increased antioxidant status and decreased proinflammatory cytokines. Results suggest that a targeted probiotic reduces seizure frequency, improves locomotor activity in a rodent model of IS, and provides insights into microbiota manipulation as a potential therapeutic avenue for pediatric epileptic encephalopathies.