Comparative characterization of the infant gut microbiome and their maternal lineage by a multi-omics approach.

The human gut microbiome establishes and matures during infancy, and dysregulation at this stage may lead to pathologies later in life. We conducted a multi-omics study comprising three generations of family members to investigate the early development of the gut microbiota. Fecal samples from 200 individuals, including infants (0-12 months old; 55% females, 45% males) and their respective mothers and grandmothers, were analyzed using two independent metabolomics platforms and metagenomics. For metabolomics, gas chromatography and capillary electrophoresis coupled to mass spectrometry were applied. For metagenomics, both 16S rRNA gene and shotgun sequencing were performed. Here we show that infants greatly vary from their elders in fecal microbiota populations, function, and metabolome. Infants have a less diverse microbiota than adults and present differences in several metabolite classes, such as short- and branched-chain fatty acids, which are associated with shifts in bacterial populations. These findings provide innovative biochemical insights into the shaping of the gut microbiome within the same generational line that could be beneficial in improving childhood health outcomes.

Evaluation of Tannin-Delivery Approaches for Gut Microbiota Modulation: Comparison of Pectin-Based Microcapsules and Unencapsulated Extracts.

The aim of this study was to investigate the impact of tannins on gut microbiota composition and activity, and to evaluate the use of pectin-microencapsulation of tannins as a potential mode of tannin delivery. Thus, pectin-tannin microcapsules and unencapsulated tannin extracts were in vitro digested and fermented, and polyphenol content, antioxidant capacity, microbiota modulation, and short-chain fatty acid (SCFA) production were analyzed. Pectin microcapsules were not able to release their tannin content, keeping it trapped after the digestive process, and are therefore not recommended for tannin delivery. Unencapsulated tannin extracts were found to exert a positive effect on the human gut microbiota. The digestion step resulted to be a fundamental requirement in order to maximize tannin bioactive effects, especially with regard to condensed tannins, as the antioxidant capacity exerted and the SCFAs produced were greater when tannins were submitted to digestion prior to fermentation. Moreover, tannins interacted differently with the intestinal microbiota depending on whether they underwent prior digestion or not. Polyphenol content and antioxidant capacity correlated with SCFA production and with the abundance of several bacterial taxa.

Effects of different foods and cooking methods on the gut microbiota: an in vitro approach.

To support personalized diets targeting the gut microbiota, we employed an in vitro digestion-fermentation model and 16S rRNA gene sequencing to analyze the microbiota growing on representative foods of the Mediterranean and Western diets, as well as the influence of cooking methods. Plant- and animal-derived foods had significantly different impacts on the abundances of bacterial taxa. Animal and vegetable fats, fish and dairy products led to increases in many taxa, mainly within the Lachnospiraceae. In particular, fats favored increases in the beneficial bacteria Faecalibacterium, Blautia, and Roseburia. However, butter, as well as gouda cheese and fish, also resulted in the increase of Lachnoclostridium, associated to several diseases. Frying and boiling produced the most distinct effects on the microbiota, with members of the Lachnospiraceae and Ruminococcaceae responding the most to the cooking method employed. Nevertheless, cooking effects were highly individualized and food-dependent, challenging the investigation of their role in personalized diets.

Influence of perinatal and childhood exposure to tobacco and mercury in children's gut microbiota.

Background: Early life determinants of the development of gut microbiome composition in infants have been widely investigated; however, if early life pollutant exposures, such as tobacco or mercury, have a persistent influence on the gut microbial community, its stabilization at later childhood remains largely unknown. Objective: In this exposome-wide study, we aimed at identifying the contribution of exposure to tobacco and mercury from the prenatal period to childhood, to individual differences in the fecal microbiome composition of 7-year-old children, considering co-exposure to a width of established lifestyle and clinical determinants. Methods: Gut microbiome was studied by 16S rRNA amplicon sequencing in 151 children at the genus level. Exposure to tobacco was quantified during pregnancy through questionnaire (active tobacco consumption, second-hand smoking -SHS) and biomonitoring (urinary cotinine) at 4 years (urinary cotinine, SHS) and 7 years (SHS). Exposure to mercury was quantified during pregnancy (cord blood) and at 4 years (hair). Forty nine other potential environmental determinants (12 at pregnancy/birth/infancy, 15 at 4 years and 22 at 7 years, such as diet, demographics, quality of living/social environment, and clinical records) were registered. We used multiple models to determine microbiome associations with pollutants including multi-determinant multivariate analysis of variance and linear correlations (wUnifrac, Bray-Curtis and Aitchison ß-diversity distances), single-pollutant permutational multivariate analysis of variance adjusting for co-variates (Aitchison), and multivariable association model with single taxa (MaAsLin2; genus). Sensitivity analysis was performed including genetic data in a subset of 107 children. Results: Active smoking in pregnancy was systematically associated with microbiome composition and ß-diversity (R2 2-4%, p < 0.05, Aitchison), independently of other co-determinants. However, in the adjusted single pollutant models (PERMANOVA), we did not find any significant association. An increased relative abundance of Dorea and decreased relative abundance of Akkermansia were associated with smoking during pregnancy (q < 0.05). Discussion: Our findings suggest a long-term sustainable effect of prenatal tobacco exposure on the children's gut microbiota. This effect was not found for mercury exposure or tobacco exposure during childhood. Assessing the role of these exposures on the children's microbiota, considering multiple environmental factors, should be further investigated.

Nutrition, Gut Microbiota, and Allergy Development in Infants.

The process of gut microbiota development in infants is currently being challenged by numerous factors associated with the contemporary lifestyle, including diet. A thorough understanding of all aspects of microbiota development will be necessary for engineering strategies that can modulate it in a beneficial direction. The long-term consequences for human development and health of alterations in the succession pattern that forms the gut microbiota are just beginning to be explored and require much further investigation. Nevertheless, it is clear that gut microbiota development in infancy bears strong associations with the risk for allergic disease. A useful understanding of microbial succession in the gut of infants needs to reveal not only changes in taxonomic composition but also the development of functional capacities through time and how these are related to diet and various environmental factors. Metagenomic and metatranscriptomic studies have started to produce insights into the trends of functional repertoire and gene expression change within the first year after birth. This understanding is critical as during this period the most substantial development of the gut microbiota takes place and the relations between gut microbes and host immunity are established. However, further research needs to focus on the impact of diet on these changes and on how diet can be used to counteract the challenges posed by modern lifestyles to microbiota development and reduce the risk of allergic disease.

Prediction of degradation pathways of phenolic compounds in the human gut microbiota through enzyme promiscuity methods.

The relevance of phenolic compounds in the human diet has increased in recent years, particularly due to their role as natural antioxidants and chemopreventive agents in different diseases. In the human body, phenolic compounds are mainly metabolized by the gut microbiota; however, their metabolism is not well represented in public databases and existing reconstructions. In a previous work, using different sources of knowledge, bioinformatic and modelling tools, we developed AGREDA, an extended metabolic network more amenable to analyze the interaction of the human gut microbiota with diet. Despite the substantial improvement achieved by AGREDA, it was not sufficient to represent the diverse metabolic space of phenolic compounds. In this article, we make use of an enzyme promiscuity approach to complete further the metabolism of phenolic compounds in the human gut microbiota. In particular, we apply RetroPath RL, a previously developed approach based on Monte Carlo Tree Search strategy reinforcement learning, in order to predict the degradation pathways of compounds present in Phenol-Explorer, the largest database of phenolic compounds in the literature. Reactions predicted by RetroPath RL were integrated with AGREDA, leading to a more complete version of the human gut microbiota metabolic network. We assess the impact of our improvements in the metabolic processing of various foods, finding previously undetected connections with output microbial metabolites. By means of untargeted metabolomics data, we present in vitro experimental validation for output microbial metabolites released in the fermentation of lentils with feces of children representing different clinical conditions.

Roles of Secretory Immunoglobulin A in Host-Microbiota Interactions in the Gut Ecosystem.

In the gastrointestinal tract (GIT), the immune system interacts with a variety of microorganisms, including pathogens as well as beneficial symbionts that perform important physiological functions for the host and are crucial to sustain intestinal homeostasis. In normal conditions, secretory immunoglobulin A (SIgA) is the principal antibody produced by B cells in the GIT mucosa. Polyreactivity provides certain SIgA molecules with the ability of binding different antigens in the bacterial surface, such as O-antigens and teichoic acids, while cross-species reactivity allows them to recognize and interact with different types of bacteria. These functions may be crucial in allowing SIgA to modulate the complex gut microbiota in an efficient manner. Several studies suggest that SIgA can help with the retention and proliferation of helpful members of the gut microbiota. Gut microbiota alterations in people with IgA deficiency include the lack of some species that are known to be normally coated by SIgA. Here, we discuss the different ways in which SIgA behaves in relation to pathogens and beneficial bacteria of the gut microbiota and how the immune system might protect and facilitate the establishment and maintenance of certain gut symbionts.

Evaluation of the Effects of a Short Supplementation With Tannins on the Gut Microbiota of Healthy Subjects.

Western diet, high in fats and sugars and low in greens, contributes to dysbiosis of the gut microbiota, which can lead to a variety of chronic diseases related with inflammation. Supplementation with bioactive compounds can help to maintain a healthy eubiotic state. Thus, we performed a 4-weeks nutritional intervention on healthy volunteers to investigate whether a blend of natural tannin extracts could induce healthy changes in the microbial intestinal ecosystem. Changes in the composition and functionality of the microbiota could be observed from the first two weeks onward. 16S rRNA amplicon next-generation sequencing (NGS) revealed a significant increase in microbial diversity at the end of the intervention, as well as trends toward increases in the relative abundances of several beneficial taxa, such as Ruminococcus bicirculans, Faecalibacterium prausnitzii, Lachnospiraceae UCG 010, Lachnospiraceae NK4A136, Bacteroides thetaiotaomicron and B. uniformis. Remarkably, some of the identified taxa were also identified as responsible for an increase in the production of short-chain fatty acids (SCFAs), microbial metabolites that contribute to the modulation of the immune system and have various other anti-inflammatory functions in the gut. Taken together, these results suggest that the tannin supplementation could exert a prebiotic effect by selectively stimulating the growth and the activity of bacteria that are advantageous for the host.

The Stance4Health Project: Evaluating a Smart Personalised Nutrition Service for Gut Microbiota Modulation in Normal- and Overweight Adults and Children with Obesity, Gluten-Related Disorders or Allergy/Intolerance to Cow's Milk.

Unhealthy diets represent a major risk for the pathogenesis of metabolic and chronic inflammatory diseases. Improving the quality of diet is important to prevent chronic diseases, and diet-induced modifications of the gut microbiota (GM) community likely play an important role. The EU-funded Stance4Health project aims at performing a randomized clinical trial based on a nutritional intervention program in the context of normal weight and overweight adults as well as children with obesity and gluten-related disorders or allergy/intolerance to cow's milk. The trial will evaluate the efficacy of a Smart Personalised Nutrition (SPN) service in modifying GM composition and metabolic function and improving consumer empowerment through technology adoption.

Association of Dietary Patterns with MRI Markers of Hepatic Inflammation and Fibrosis in the MAST4HEALTH Study.

Whereas the etiology of non-alcoholic fatty liver disease (NAFLD) is complex, the role of nutrition as a causing and preventive factor is not fully explored. The aim of this study is to associate dietary patterns with magnetic resonance imaging (MRI) parameters in a European population (Greece, Italy, and Serbia) affected by NAFLD. For the first time, iron-corrected T1 (cT1), proton density fat fraction (PDFF), and the liver inflammation fibrosis score (LIF) were examined in relation to diet. A total of 97 obese patients with NAFLD from the MAST4HEALTH study were included in the analysis. A validated semi-quantitative food frequency questionnaire (FFQ) was used to assess the quality of diet and food combinations. Other variables investigated include anthropometric measurements, total type 2 diabetes risk, physical activity level (PAL), and smoking status. Principal component analysis (PCA) was performed to identify dietary patterns. Six dietary patterns were identified, namely "High-Sugar", "Prudent", "Western", "High-Fat and Salt", "Plant-Based", and "Low-Fat Dairy and Poultry". The "Western" pattern was positively associated with cT1 in the unadjusted model (beta: 0.020, p-value: 0.025) and even after adjusting for age, sex, body mass index (BMI), PAL, smoking, the center of the study, and the other five dietary patterns (beta: 0.024, p-value: 0.020). On the contrary, compared with low-intake patients, those with medium intake of the "Low-Fat Dairy and Poultry" pattern were associated with lower values of cT1, PDFF, and LIF. However, patients with a "Low-Fat Dairy and Poultry" dietary pattern were negatively associated with MRI parameters (cT1: beta: -0.052, p-value: 0.046, PDFF: beta: -0.448, p-value: 0.030, LIF: beta: -0.408, p-value: 0.025). Our findings indicate several associations between MRI parameters and dietary patterns in NAFLD patients, highlighting the importance of diet in NAFLD.

Effect of roasting conditions on cocoa bioactivity and gut microbiota modulation.

Cocoa is a highly consumed food with beneficial effects on human health. Cocoa roasting has an important influence on its sensory and nutritional characteristics; therefore, roasting could also play a role in cocoa bioactivity. Thus, the aim of this paper is to unravel the effect of cocoa roasting conditions on its antioxidant capacity and modifications of gut microbiota after in vitro digestion-fermentation. HMF and furfural, chemical markers of non-enzymatic browning, were analyzed in unroasted and roasted cocoa powder at different temperatures, as well as different chocolates. The antioxidant capacity decreased with roasting, most probably due to the loss of phenolic compounds during heating. In the case of the evaluated chocolates, the antioxidant capacity was 2-3 times higher in the fermented fraction. On the other hand, HMF and furfural content increased during roasting due to increasing temperatures. Moreover, unroasted and roasted cocoa powder have different effects on gut microbial communities. Roasted cocoa favored butyrate production, whereas unroasted cocoa favored acetate and propionate production in a significant manner. In addition, unroasted and roasted cocoa produced significantly different gut microbial communities in terms of composition. Although many bacteria were affected, Veillonella and Faecalibacterium were some of the most discriminant ones; whereas the former is a propionate producer, the latter is a butyrate producer that has also been linked to positive effects on the inflammatory health of the gut and the immune system. Therefore, unroasted and roasted cocoa (regardless of the roasting temperature) promote different bacteria and a different SCFA production.

Nutrigenetic Interactions Might Modulate the Antioxidant and Anti-Inflammatory Status in Mastiha-Supplemented Patients With NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease with no therapeutic consensus. Oxidation and inflammation are hallmarks in the progression of this complex disease, which also involves interactions between the genetic background and the environment. Mastiha is a natural nutritional supplement known to possess antioxidant and anti-inflammatory properties. This study investigated how a 6-month Mastiha supplementation (2.1 g/day) could impact the antioxidant and inflammatory status of patients with NAFLD, and whether genetic variants significantly mediate these effects. We recruited 98 patients with obesity (BMI ≥ 30 kg/m2) and NAFLD and randomly allocated them to either the Mastiha or the placebo group for 6 months. The anti-oxidative and inflammatory status was assessed at baseline and post-treatment. Genome-wide genetic data was also obtained from all participants, to investigate gene-by-Mastiha interactions. NAFLD patients with severe obesity (BMI > 35kg/m2) taking the Mastiha had significantly higher total antioxidant status (TAS) compared to the corresponding placebo group (P value=0.008). We did not observe any other significant change in the investigated biomarkers as a result of Mastiha supplementation alone. We identified several novel gene-by-Mastiha interaction associations with levels of cytokines and antioxidant biomarkers. Some of the identified genetic loci are implicated in the pathological pathways of NAFLD, including the lanosterol synthase gene (LSS) associated with glutathione peroxidase activity (Gpx) levels, the mitochondrial pyruvate carrier-1 gene (MPC1) and the sphingolipid transporter-1 gene (SPNS1) associated with hemoglobin levels, the transforming growth factor-beta-induced gene (TGFBI) and the micro-RNA 129-1 (MIR129-1) associated with IL-6 and the granzyme B gene (GZMB) associated with IL-10 levels. Within the MAST4HEALTH randomized clinical trial (NCT03135873, www.clinicaltrials.gov) Mastiha supplementation improved the TAS levels among NAFLD patients with severe obesity. We identified several novel genome-wide significant nutrigenetic interactions, influencing the antioxidant and inflammatory status in NAFLD. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT03135873.

Enrichment of Food With Tannin Extracts Promotes Healthy Changes in the Human Gut Microbiota.

Food and food bioactive components are major drivers of modulation of the human gut microbiota. Tannin extracts consist of a mix of bioactive compounds, which are already exploited in the food industry for their chemical and sensorial properties. The aim of our study was to explore the viability of associations between tannin wood extracts of different origin and food as gut microbiota modulators. 16S rRNA amplicon next-generation sequencing (NGS) was used to test the effects on the gut microbiota of tannin extracts from quebracho, chestnut, and tara associated with commercial food products with different composition in macronutrients. The different tannin-enriched and non-enriched foods were submitted to in vitro digestion and fermentation by the gut microbiota of healthy subjects. The profile of the short chain fatty acids (SCFAs) produced by the microbiota was also investigated. The presence of tannin extracts in food promoted an increase of the relative abundance of the genus Akkermansia, recognized as a marker of a healthy gut, and of various members of the Lachnospiraceae and Ruminococcaceae families, involved in SCFA production. The enrichment of foods with tannin extracts had a booster effect on the production of SCFAs, without altering the profile given by the foods alone. These preliminary results suggest a positive modulation of the gut microbiota with potential benefits for human health through the enrichment of foods with tannin extracts.

Effect of Mastiha supplementation on NAFLD: The MAST4HEALTH Randomised, Controlled Trial.

SCOPE: Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease with poor therapeutic strategies. Mastiha possesses antioxidant/anti-inflammatory and lipid-lowering properties. The authors investigate the effectiveness of Mastiha as a nonpharmacological intervention in NAFLD. METHODS AND RESULTS: Ninety-eight patients with NAFLD in three countries (Greece, Italy, Serbia) are randomly allocated to either Mastiha or Placebo for 6 months, as part of a multicenter, randomized, double-blind, placebo-controlled, parallel-group clinical trial. The authors assess NAFLD severity via magnetic resonance imaging (MRI) scanning and LiverMultiScan technique and evaluate the effectiveness of Mastiha through medical, anthropometric, biochemical, metabolomic, and microbiota assessment. Mastiha is not superior to Placebo on changes in iron-corrected T1 (cT1) and Liver Inflammation Fibrosis score (LIF) in entire patient population; however, after BMI stratification (BMI ≤ 35 kg m-2 and BMI > 35 kg m-2 ), severely obese patients show an improvement in cT1 and LIF in Mastiha versus Placebo. Mastiha increases dissimilarity of gut microbiota, as shown by the Bray-Curtis index, downregulates Flavonifractor, a known inflammatory taxon and decreases Lysophosphatidylcholines-(LysoPC) 18:1, Lysophosphatidylethanolamines-(LysoPE) 18:1, and cholic acid compared to Placebo. CONCLUSION: Mastiha supplementation improves microbiota dysbiosis and lipid metabolite levels in patients with NAFLD, although it reduces parameters of liver inflammation/fibrosis only in severely obese patients.

Mastiha (Pistacia lentiscus) Improves Gut Microbiota Diversity, Hepatic Steatosis, and Disease Activity in a Biopsy-Confirmed Mouse Model of Advanced Non-Alcoholic Steatohepatitis and Fibrosis.

SCOPE: As a result of the obesity epidemic, the prevalence of non-alcoholic steatohepatitis (NASH) is increasing. No drug is approved for the treatment of NASH. In this study, the effect of a nutritional supplement, Mastiha or Chios mastic gum, on metabolic and histological parameters and on the gut microbiome in mice with NASH and fibrosis was investigated. METHODS AND RESULTS: Advanced NASH was induced by feeding C57BL/6J mice a diet rich in fat, sucrose, and cholesterol for 41 weeks. After randomization, animals received the NASH-inducing diet with or without 0.2% (w/w) Mastiha for a further 8 weeks. Disease activity was assessed by liver histology and determination of plasma transaminase activities. Fecal microbiota DNA extraction and 16S rRNA amplicon sequencing were used to determine the composition of the gut microbiome. Mastiha supplementation led to a significant reduction in circulating alanine aminotransferase (ALT) activity, improvement in hepatic steatosis and collagen content, and a reduction in NAFLD activity score. Furthermore, it resulted in a partial but significant recovery of gut microbiota diversity and changes in identity and abundance of specific taxa. CONCLUSION: This is the first study demonstrating an improvement in disease activity in mice with advanced NASH with fibrosis by a diet containing Mastiha.

Adaptation of the Human Gut Microbiota Metabolic Network During the First Year After Birth.

Predicting the metabolic behavior of the human gut microbiota in different contexts is one of the most promising areas of constraint-based modeling. Recently, we presented a supra-organismal approach to build context-specific metabolic networks of bacterial communities using functional and taxonomic assignments of meta-omics data. In this work, this algorithm is applied to elucidate the metabolic changes induced over the first year after birth in the gut microbiota of a cohort of Spanish infants. We used metagenomics data of fecal samples and nutritional data of 13 infants at five time points. The resulting networks for each time point were analyzed, finding significant alterations once solid food is introduced in the diet. Our work shows that solid food leads to a different pattern of output metabolites that can be potentially released from the gut microbiota to the host. Experimental validation is presented for ferulate, a neuroprotective metabolite involved in the gut-brain axis.

Spent Coffee Grounds Extract, Rich in Mannooligosaccharides, Promotes a Healthier Gut Microbial Community in a Dose-Dependent Manner.

Coffee is one of the most consumed beverages around the world, and as a consequence, spent coffee grounds are a massively produced residue that is causing environmental problems. Reusing them is a major focus of interest presently. We extracted mannooligosaccharides (MOS) from spent coffee grounds and submitted them to an in vitro fermentation with human feces. Results obtained suggest that MOS are able to exert a prebiotic effect on gut microbiota by stimulating the growth of some beneficial genera, such as Barnesiella, Odoribacter, Coprococcus, Butyricicoccus, Intestinimonas, Pseudoflavonifractor, and Veillonella. Moreover, short-chain fatty acids (SCFA) production also increased in a dose-dependent manner. However, we observed that 5-(hydroxymethyl)furfural, furfural, and polyphenols (which are either produced or released from the spent coffee grounds matrix during hydrolysis) could have an inhibitory effect on other beneficial genera, such as Faecalibacterium, Ruminococcus, Blautia, Butyricimonas, Dialister, Collinsella, and Anaerostipes, which could negatively affect the prebiotic activity of MOS.

Metabolic adaptation in the human gut microbiota during pregnancy and the first year of life.

BACKGROUND: The relationship between the gut microbiome and the human host is dynamic and we may expect adjustments in microbiome function if host physiology changes. Metatranscriptomic approaches should be key in unraveling how such adjustments occur. METHODS: We employ metatranscriptomic sequencing analyses to study gene expression in the gut microbiota of infants through their first year of life, and of their mothers days before delivery and one year afterwards. FINDINGS: In infants, hallmarks of aerobic metabolism disappear from the microbial metatranscriptome as development proceeds, while the expression of functions related to carbohydrate transport and metabolism increases and diversifies, approaching that observed in non-pregnant women. Butyrate synthesis enzymes are overexpressed at three months of age, even though most butyrate-producing organisms are still rare. In late pregnancy, the microbiota readjusts the expression of carbohydrate-related functions in a manner consistent with a high availability of glucose. INTERPRETATION: Our findings suggest that butyrate production may be ensured in the gut of young infants before the typical butyrate synthesizers of the adult gut become abundant. The late pregnancy gut microbiota may be able to access the high levels of blood glucose characteristic of this period. Moreover, late pregnancy gut bacteria may reach stationary phase, which may affect their likelihood of translocating across the intestinal epithelium. FUNDS: This work was supported by grants CSD2009-00006 (CONSOLIDER Program) and SAF2009-13032-C02-02 from MICINN (Ministry of Science and Innovation, Spain), and by grant SAF2012-31187 from MINECO (Ministry of Economics and Competitiveness, Spain).