Development ofa Quantitative Serial LC-MS/MS Methodfor Gut Microbiota Metabolomics

Human Microbiota in Health and Disease

Sleep disorders are closely linked to stroke, while the role of gut microbiota in human health and disease is increasingly recognized. However, the causal relationships between sleep disorders, gut microbiota, and stroke remain unclear. This study aimed to investigate the causal relationships between sleep disorders, gut microbiota, and stroke (including its subtypes), as well as the potential mediating role of gut microbiota in the association between sleep disorders and stroke. Using a two-sample Mendelian randomization approach, we analyzed causal relationships among various sleep disorders, gut microbiota, and stroke. The inverse variance weighted method served as the primary analysis, supplemented by multiple sensitivity analyses to ensure robustness. Mediation effects of gut microbiota in the sleep disorder-stroke relationship were assessed using the coefficient product method. Insomnia was causally associated with transient ischemic attacks (OR = 1.592, 95% CI: 1.116-2.270), excessive daytime sleepiness with ischemic stroke (IS) (OR = 1.887, 95% CI: 1.041-3.420), and sleep apnea with overall stroke risk (OR = 1.247, 95% CI: 1.015-1.532). Several gut microbiota taxa showed causal associations with stroke and its subtypes, with Ruminococcus E sp900314705 increasing the risk of overall stroke, IS, and transient ischemic attacks. Ezakiellaceae partially mediated the causal relationship between daytime sleepiness and IS, accounting for 15.9% of the effect. The current study established and elucidated causal relationships between sleep disorders and stroke, identified specific gut microbiota associated with stroke risk, and highlighted the mediating role of Ezakiellaceae in the causal relationship between daytime sleepiness and IS. These findings underscore the potential importance of improving sleep quality and modulating gut microbiota for stroke prevention and management.

Background: Convergent evidence implicates gut microbiota in human health and disease. Hitherto, relatively few studies have evaluated the gut microbiota profile in individuals with bipolar disorder (BD) relative to healthy controls (HC). Methods: Fecal samples were collected from subjects (aged 18–65) meeting DSM-5-defined criteria for BD and age- and sex-matched HC without current or past history of mental or major medical disorders. Samples were sequenced using Illumina sequencing and association of specific taxa and co-occurrence of taxa with sample groups including the effect of diet was assessed using cluster analysis and analysis of communities of microorganisms (ANCOM). Nutritional composition was evaluated using the Dietary Questionnaire for Epidemiological Studies (DQES v2) Food Frequency Questionnaire. Results: Forty-six subjects were enrolled (n=23 BD, n=23 HC). Cluster analyses did not identify any significant differences between BD and HC (p=0.38). Lower microbiota diversity was observed among BD subjects relative to HC (p=0.04). A greater abundance of a Clostridiaceae OTU was observed among BD subjects when compared to HC and of Collinsella among BD-II subjects relative to BD-I. Cluster analysis revealed that neither diagnosis (p=0.38) nor diet (p=0.43) had a significant effect on overall gut microbiota composition. Limitations: This study has a small sample size and insufficient control for some potential moderating factors (e.g. psychotropic medication and smoking). Conclusion: This study suggests that individuals with BD may have a distinct gut microbiota profile compared to healthy controls, with a greater abundance of Clostridiaceae and Collinsella. These findings need to be replicated in future studies with larger sample sizes.

The Development of Early Life Microbiota in Human Health and Disease

Metagenome-scale community metabolic modelling for understanding the role of gut microbiota in human health

Obesity is currently recognized as an epidemic and one of the most important health problems worldwide. World Health Organization data indicates that in developing countries, obesity in adults is more frequent than malnutrition. More than 1.9 billion adults are overweight, with 650 million of these being obese. More than 200 million school-aged children are overweight, making this generation more likely to have a shorter productive life and life expectancy than their parents. This portion of the population has a greatly increased risk of developing cardiovascular disease, diabetes, dyslipidemia, hypertension, hepatic steatosis, certain types of tumor and infertility, negatively impacting their quality of life. Obesity can be prevented and is treatable with the adoption of a healthy and appropriate diet, and with regular physical exercise. However,lifestyle modification therapy for the obese population remains unsatisfactory. In addition, it is important to emphasize that obesity treatment presents better results when accompanied by a multidisciplinary team, using diet therapy, prescribed physical exercise, psychotherapy and drug therapy, according to the needs of each patient. Obesity is a multifactorial, complex, chronic and relapsing disease involving gene-environment interaction and should therefore be treated with a systemic and ecological approach. This is because many roles of intestinal microbiota in human health have recently been discovered, mainly in relation to weight gain or loss. Additionally, recent studies suggest that human gut microbiota may contribute to the regulation of multiple neurochemical and neurometabolic pathways through complex systems that interact and interconnect the gastrointestinal tract, skin, liver, and other organs, such as the central nervous system. The brain and intestine form a complex nervous, endocrine and immune bidirectional communication axis, involving neurotransmitters and neuromodulators. Changes in one organ will affect other organs, and disturbances in the composition and number of intestinal microorganisms can affect the enteric and central nervous systems. Alterations in intestinal microbiota may increase intestinal barrier permeability, raising the risk of developing chronic diseases, like obesity. Furthermore, the imbalance between gut microbiota and its host leads to dysbiosis, which, in turn, contributes to the establishment of an inflammatory and oxidative process, impaired glucose metabolism, insulin resistance, obesity, increased risk of developing of metabolic syndrome, type 2 diabetes, inflammatory bowel, autoimmune diseases, and cancers. Studies have shown that less-industrialized populations, such as those in Africa, present a more diversified gut microbiota that is richer in terms of bacterial genera, which encode enzymes that hydrolyze cellulose and xylan. This finding suggests these individuals have a fiber-rich diet, a different situation from industrialized populations who obtain energy from an ultra-processed diet, rich in fats, salt, sugars and preservatives, and who present a high prevalence of obesity and other chronic diseases. In this sense, it is fundamental to reflect on what type of anti-obesity drug should be developed to treat this pathology, as most of these drugs act on the central nervous system and, therefore, interfere in communication between the brain and intestine. It is important to develop an anti-obesity drug that acts not only on appetite control and on increased satiety, but also engages with and is friendly to intestinal microbiota, via increased diversity of this ecosystem, decreased fat storage and chronic oxidation inflammation, and correct modulation of the immune system. Nonetheless, we must not forget that obesity is a complex and multifactorial disease, and that its drug treatment must be combined with a healthy and adequate diet, physical exercise and cognitive-behavioral therapy. Within this context, this chapter aims to promote reflection around this theme and to provide the theoretical foundation for development of the next generation of anti-obesogenic drugs.

<p>Research into the effect of the gut microbiota on host immune response is continuing to shed new light on the underappreciated role of the microbiota in human health. Recent research using mice has shown that the microbiota is critical to the host immune response to influenza infection. Whilst there is great variation in the human gut microbiota, classifications called stool community types can be used to classify individuals based on the abundance of major bacterial taxa. The primary objective of this study was to investigate the feasibility of using the study protocol for a large randomised controlled trial. Healthy adult participants (n=125) aged 18 to 64 were recruited from the general population and vaccinated with the seasonal trivalent influenza vaccine. Participants were followed up over a period of six months, during which time, both stool and blood samples were collected. Blood samples were collected at Day Zero, Three, Seven, 28 and 180 to measure immune response. The immune response to vaccination was measured by HAI antibody titres at Day Zero and Day 28. Stool samples were collected at Day Zero and Day 28 to assign participants to one of the four stool community types and assess stability over time. Stool samples were assigned to stool community types using the proportions of major taxa present. The association between stool community type and either post vaccination HAI titre, seroconversion rates or seroprotection rates was also assessed. The results obtained in this study supported the feasibility of a large randomised controlled trial using the study protocol. The study demonstrated a high participant retention rate (97.6%; 95% CI = 93.1% to 99.5%), as well as high participant adherence to the study protocol and good success obtaining the required blood and stool samples.</p>

The gut microbiota in patients with food allergy, and the skin microbiota in atopic dermatitis patients differ from those of healthy people. We hypothesize that relationships may exist between gut and skin microbiota in patients with allergies. The aim of this study was to determine the possible relationship between gut and skin microbiota in patients with allergies, hence simultaneous analysis of the two compartments of microbiota was performed in infants with and without allergic symptoms. Fifty-nine infants with food allergy and/or atopic dermatitis and 28 healthy children were enrolled in the study. The skin and gut microbiota were evaluated using 16S rRNA gene amplicon sequencing. No significant differences in the α-diversity of dermal or fecal microbiota were observed between allergic and non-allergic infants; however, a significant relationship was found between bacterial community structure and allergy phenotypes, especially in the fecal samples. Certain clinical conditions were associated with characteristic bacterial taxa in the skin and gut microbiota. Positive correlations were found between skin and fecal samples in the abundance of Gemella among allergic infants, and Lactobacillus and Bacteroides among healthy infants. Although infants with allergies and healthy infants demonstrate microbiota with similar α-diversity, some differences in β-diversity and bacterial species abundance can be seen, which may depend on the phenotype of the allergy. For some organisms, their abundance in skin and feces samples may be correlated, and these correlations might serve as indicators of the host’s allergic state.

Background: Peanut oral immunotherapy has emerged as a novel, active management approach for peanut-allergic sufferers, but limited data exist currently on the role of the microbiome in successful desensitization. Objective: We examined the oral and gut microbiome in a cohort of 17 children undergoing peanut oral immunotherapy with the aim to identify the microbiome signatures associated with successful desensitization. We also set out to characterize their fecal metabolic profiles after successful therapy. Methods: Participants gradually built up their daily dose from 2 mg (starting dose) to 300 mg (maintenance dose) within approximately 40 weeks. We collected a buccal and stool specimen from each subject at two different time points: at baseline and post-therapy (1 month after reaching maintenance). The oral (buccal) and gut (fecal) microbiome was characterized based on sequencing of 16S rRNA gene amplicons with Illumina MiSeq. Fecal short chain fatty acid levels were measured using liquid chromatography-tandem mass spectrometry. Results: We report increased alpha diversity of the oral microbiome post-therapy and have also identified a significant increase in the relative abundance of oral Actinobacteria, associated with the desensitized state. However, the baseline gut microbiome did not differ from the post-therapy. Additionally, fecal short chain fatty acids increased after therapy, but not significantly. Conclusion: Our research adds to the limited current knowledge on microbiome and metabolic signatures in pediatric patients completing oral immunotherapy. Post-therapy increased trends of fecal fatty acid levels support a role in modulating the allergic response and potentially exerting protective and anti-inflammatory effects alongside successful desensitization. A better understanding of the microbiome-related mechanisms underlying desensitization may allow development of smarter therapeutic approaches in the near future. Clinical implication: The oral microbiome composition is altered following successful peanut oral immunotherapy, with a significant increase in alpha diversity and the relative abundance of phylum Actinobacteria. Capsule summary: Significant microbiome changes in children completing peanut immunotherapy include increase in alpha-diversity and overrepresentation of Actinobacteria in the oral microbiome, and increased trends for fecal short chain fatty acids, suggesting a protective effect against the allergic response.

The beneficial effect of consuming fruits and vegetables in the prevention of chronic non-communicable diseases and healthy aging is well known. This is attributed to food and vegetable antioxidant and fiber content. The aim of this publication is to communicate the results of recent research on pectin in humans, to propose an increased consumption of fruits and vegetables, or their possible use as a food supplement. A comprehensive narrative review was conducted considering recent publications on pectin. The description of starch, pectin, the physicochemical changes caused by pectin, and the effect of pectin on the activity of amylase are reported. Dietary fiber and gut microbiota in human health are also described, with the production of saturated fatty acids with fewer than six carbon atoms. Finally, health effects such as anti-hyperglycemic and anti-hyperlipidemic activities, preventing and controlling obesity and heart disease, are analyzed, as well as other health effects in tumors, the gastrointestinal tract, and immunity. Considering the beneficial effects of pectin in health and the low consumption throughout the world, it is recommended to promote the consumption of fruits and vegetables to increase pectin intake in the human diet.

With the deepening understanding of the role of gut microbiota in human health and disease, fecal microbiota transplantation has gained widespread attention as an emerging therapeutic approach in recent years. This technique involves the transplantation of microbial communities from the feces of healthy donors into patients to reconstruct or improve the gut microbiota structure, thereby achieving therapeutic goals. Fecal microbiota transplantation has become an effective method for treating recurrent or refractory Clostridium difficile infections and has shown good therapeutic effects and safety in clinical trials for various gastrointestinal diseases, including inflammatory bowel disease, irritable bowel syndrome, slow transit constipation, and chronic diarrhea. Moreover, its application has been extended to research in metabolic diseases and neurological disorders, which are not directly related to the gut. However, the clinical efficacy of fecal microbiota transplantation still needs improvement, and there are many challenges regarding specific application strategies that remain to be addressed. This article discusses the current progress and challenges of fecal microbiota transplantation strategies and reviews cutting-edge interventional methods such as small intestine microbiota intervention and bacteriophage therapy, aiming to provide reference for further research in fecal microbiota transplantation.

Although the presence of an intestinal flora has been known for a long time, the discovery of the role of gut microbiota in human health and disease has been widely recognized as one of the most important advances in the recent years. Chronic diseases may result from dysbiosis, i.e. a disruption of the balance within the bacterial population hosted by the human body. These developments open new prospects in terms of prevention and treatment, including the design of adapted diets, the development of functional foods and fecal transplantation. These discoveries have profoundly altered our view of microbes, of health and disease, of self and non-self, as well as our representations of the body and its relationship with its ecosystem. Gut microbiota is now generally considered as an organ in its own right. A model of the "microbiotic person" thus arises, in which the human organism is defined as an ecosystem, a chimeric superorganism with a double genome, both human and microbial. Thought should be given to the way in which these new paradigms modify lay perceptions of the human body.

Antibiotics have benefitted human health since their introduction nearly a century ago. However, the rise of antibiotic resistance may portend the dawn of the "post-antibiotic age." With the narrow pipeline for novel antimicrobials, we need new approaches to deal with the rise of multidrug resistant organisms. In the last 2 decades, the role of the intestinal microbiota in human health has been acknowledged and studied widely. Of the various activities carried out by the gut microbiota, colonization resistance is a key function that helps maintain homeostasis. Therefore, re-establishing a healthy microbiota is a novel strategy for treating drug resistance organisms. Preliminary studies suggest that this is a viable approach. However, the extent of their success still needs to be examined. Herein, we will review work in this area and suggest where future studies can further investigate this method for dealing with the threat of antibiotic resistance.

Novel microbes are either newly isolated genera and species from natural sources or bacterial strains derived from existing bacteria. Novel microbes are gaining increasing attention for the general aims to preserve and modify foods and to modulate gut microbiota. The use of novel microbes to improve health outcomes is of particular interest because growing evidence points to the importance of gut microbiota in human health. As well, some recently isolated microorganisms have promise for use as probiotics, although in-depth assessment of their safety is necessary. Recent examples of microorganisms calling for more detailed evaluation include Bacteroides xylanisolvens, Akkermansia muciniphila, fructophilic lactic acid bacteria (FLAB), and Faecalibacterium prausnitzii. This paper discusses each candidate's safety evaluation for novel food or novel food ingredient approval according to European Union (EU) regulations. The factors evaluated include their beneficial properties, antibiotic resistance profiling, history of safe use (if available), publication of the genomic sequence, toxicological studies in agreement with novel food regulations, and the qualified presumptions of safety. Sufficient evidences have made possible to support and authorize the use of heat-inactivated B. xylanisolvens in the European Union. In the case of A. muciniphila, the discussion focuses on earlier safety studies and the strain's suitability. FLAB are also subjected to standard safety assessments, which, along with their proximity to lactic acid bacteria generally considered to be safe, may lead to novel food authorization in the future. Further research with F. prausnitzii will increase knowledge about its safety and probiotic properties and may lead to its future use as novel food. Upcoming changes in EUU Regulation 2015/2283 on novel food will facilitate the authorization of future novel products and might increase the presence of novel microbes in the food market.

The aim of current study was to determine variations in sow’s gut microbiota, serum immunity, and milk metabolite profile mediated by lysozyme supplementation. Twenty-four pregnant sows were assigned to a control group without supplementation and two treatments with 0.5 kg/t and 1.0 kg/t lysozyme provided in formula feed for 21 days (n = 8 per treatment). Microbiota analysis and metagenomic predictions were based on 16s RNA high-throughput sequencing. Milk metabolome was assessed by untargeted liquid chromatography tandem mass spectrometry. Serum biochemical indicators and immunoglobulins were also determined. Gut microbial diversity of sows receiving 1.0 kg/t lysozyme treatment was significantly reduced after the trial. Spirochaetes, Euryarchaeota, and Actinobacteria significantly increased while Firmicutes showed a remarkable reduction in 1.0 kg/t group compared with control. Lysozyme addition rebuilt sow’s gut microbiota to beneficial composition identified by reduced richness of Escherichia coli and increased abundance of Lactobacillus amylovorus. Accordingly, microbial metabolic functions including pyrimidine metabolism, purine metabolism, and amino acid related enzymes were significantly up-regulated in 1.0 kg/t group. Microbial metabolic phenotypes like the richness of Gram-positive bacteria and oxidative stress tolerance were also significantly reduced by lysozyme treatment. Serum alanine transaminase (ALT) activity and IgA levels were significantly down-regulated in the 1.0 kg/t group compared with control, but IgM levels showed a significantly increase in 1.0 kg/t group. Milk metabolites such as L-glutamine, creatine, and L-arginine showed significantly dose-dependent changes after treatment. Overall, lysozyme supplementation could effectively improve the composition, metabolic functions, and phenotypes of sow’s gut microbiota and it also benefit sows with better serum immunity and milk composition. This research could provide theoretical support for further application of lysozyme in promoting animal gut health and prevent pathogenic infections in livestock production.