Background: Galacto-oligosaccharides (GOS) intake has been linked to health benefits via modulation of the gut microbiome. Milk, where the majority of lactose is enzymatically converted to GOS (called here Novel or “N milk”), retains milk’s nutritional value with reduced lactose and a high amount of prebiotic GOS. Objectives: The aim of this study was to investigate the effect of N milk on the gut microbiome and related changes in health-related biomarkers, complemented by ex vivo fermentation experiments. Methods: In a 2-arm crossover, double-blind, randomized controlled clinical trial, 26 healthy adults consumed either N milk (containing 9 g GOS and 1.7 g of lactose per serving) or lactose-free milk (control), for 2 wk with a 2-wk washout period. Stool and fasting blood samples were collected at the start and the end of the intervention periods. Gut microbiome was analyzed using shotgun metagenomics, and metabolites using both targeted and untargeted methods. In addition, we tested lactose-free milk, N milk, and GOS in ex vivo colonic fermentation to obtain insights into the bacterial processing of substrates. Results: N milk intake led to a 3-fold increase in median gut bifidobacteria (P < 0.0001) and significant increases in plasma acetate, octanoic acid, β-alanine, and nicotinamide (all P < 0.05). Untargeted plasma metabolomics revealed a shift in amino acid metabolism, with an increase in 3-indole propionate, accompanied by a decrease in 2 uremic toxins, p-cresol sulfate, and indoxyl-sulfate (P < 0.05 without false discovery rate adjustment). Ex vivo fermentation experiments supported the results of the clinical study, whereby N milk increased bifidobacteria accompanied by higher production of short-chain fatty acids and a shift in microbial tryptophan metabolism, and indicated unique effects of N milk compared with GOS. Conclusions: N milk resulted in a significant increase in gut bifidobacteria, along with changes in plasma metabolites previously associated with immune and metabolic health benefits.This study was registered at clinicaltrials.gov as NCT05207839.

Dietary fiber supplementation can reduce cardiometabolic risk, but its effective use is limited by incomplete understanding of fibre-microbiome interactions and highly individualized responses. We tested acacia gum (AG; fermentable fibre), resistant starch type 4 (RS4; fermentable fibre), and microcrystalline cellulose (MCC; non-fermentable control fibre) in a six-week randomized trial in adults with excess body weight. Multi-omics profiling revealed distinct, structure-specific microbiota and short-chain fatty acid shifts with AG and RS4, which were not directly linked to physiological outcomes. Improvements in inflammation, gut barrier function, and satiety occurred across all arms, indicating fermentation-independent effects. AG reduced plasma ghrelin, linked to microbial carbohydrate-active enzyme genes targeting its structures. Machine-learning models predicted individualized, fiber-specific effects on blood pressure (AG) and C-reactive protein (RS4) from microbial pathways and fecal bile acids. These findings delineate fermentation-dependent and independent mechanisms of fibre action and provide a mechanistic basis for personalized fibre supplementation. Trial registration:ClinicalTrials.gov NCT02322112.

AbstractKey PointsThis section will be completed furtherImportanceToddlerhood is a key window of opportunity for development of musculoskeletal system and microbiome. In this study we tested the efficacy of a synbiotic-based young child formula on bone and muscle strength and microbiome maturation in young children during motor-skill development.InterventionIn this randomized, double-blind controlled trial, children aged 2-3 years received either an experimental young child formula (EYCF) containing a combination ofLimosilactobacillus reuteriDSM 17938 and galacto-oligosaccharides (GOS) or a minimally fortified milk (CM) for 6 months. A third arm remained on their habitual diet.Main outcomes and measuresBone quality (tibia speed of sound), muscle strength (handgrip), microbiota composition (shotgun metagenomics) and functionality (fecal metabolome) were evaluated at baseline, and after 3 months and 6 months of intervention. Microbiota and metabolomic features were associated to each other and to clinical bone and muscle readouts at the same timepoints.ResultsTibial speed of sound was significantly increased after 6 months (primary end point, p<0.01) and 3 months (p<0.05) of EYCF vs CM feeding. These effects on bone strength were paralleled by significantly higher muscle strength after 6 months in EYCF vs CM. The intervention significantly remodeled microbiome composition, with enrichment ofL. reuteri, and higher bifidobacteria presence in the stools of EYCF vs CM children at both 3 and 6 months. IncreasedL. reuteriabundance after 6 months of EYCF consumption was associated with higher bone quality and muscle strength. Stool metabolomics were significantly modulated by EYCF consumption with 45 metabolites significantly modified and associated to microbiome compositional changes such asBifidobacteriumspp. andL. reuteriexpansion. Pairing of metagenomic and metabolomic signatures induced by EYCF revealed an enrichment of tryptophane and indole metabolism which significantly associated to bone and muscle strength clinical outcomes.Conclusions and relevanceConsumption of an experimental young child formula containing aL. reuteri+ GOS synbiotic improves musculoskeletal development in toddlers that was associated with a modulation of microbiota composition and functionality. These results provide novel mechanistic insights on gut-musculoskeletal crosstalk during early life and demonstrate that nutritional interventions targeting the microbiome can support healthy bone and muscle development and may contribute to functional motorskills acquisition during childhood.Trial registrationThe trial was registered atclinicaltrial.govasNCT04799028

Cow’s milk protein allergy (CMPA) is a prevalent food allergy among infants and young children. We conducted a randomized, multicenter intervention study involving 194 non-breastfed infants with CMPA until 12 months of age (clinical trial registration: NCT03085134). One exploratory objective was to assess the effects of a whey-based extensively hydrolyzed formula (EHF) supplemented with 2′-fucosyllactose (2′-FL) and lacto-N-neotetraose (LNnT) on the fecal microbiome and metabolome in this population. Thus, fecal samples were collected at baseline, 1 and 3 months from enrollment, as well as at 12 months of age. Human milk oligosaccharides (HMO) supplementation led to the enrichment of bifidobacteria in the gut microbiome and delayed the shift of the microbiome composition toward an adult-like pattern. We identified specific HMO-mediated changes in fecal amino acid degradation and bile acid conjugation, particularly in infants commencing the HMO-supplemented formula before the age of three months. Thus, HMO supplementation partially corrected the dysbiosis commonly observed in infants with CMPA. Further investigation is necessary to determine the clinical significance of these findings in terms of a reduced incidence of respiratory infections and other potential health benefits.

Faecalibacterium prausnitzii (F. prausnitzii) is a bacterial taxon in the human gut with anti-inflammatory properties, and this may contribute to the beneficial effects of healthy eating habits. However, little is known about the nutrients that enhance the growth of F. prausnitzii other than simple sugars and fibers. Here, we combined dietary and microbiome data from the American Gut Project (AGP) to identify nutrients that may be linked to the relative abundance of F. prausnitzii. Using a machine learning approach in combination with univariate analyses, we identified that sugar alcohols, carbocyclic sugar, and vitamins may contribute to F. prausnitzii growth. We next explored the effects of these nutrients on the growth of two F. prausnitzii strains in vitro and observed robust and strain-dependent growth patterns on sorbitol and inositol, respectively. In the context of a complex community using in vitro fermentation, neither inositol alone nor in combinations with vitamin B exerted a significant growth-promoting effect on F. prausnitzii, partly due to high variability among the fecal microbiota community from four healthy donors. However, the fecal communities that showed an increase in F. prausnitzii on inulin also responded with at least 60% more F. prausnitzii on any of inositol containing media than control. Future nutritional studies aiming to increase the relative abundance of F. prausnitzii should explore a personalized approach accounting for strain-level genetic variations and community-level microbiome composition.

ABSTRACT Shotgun metagenomic sequencing has the potential to provide bacterial strain-level resolution which is of key importance to tackle a host of clinical questions. While bioinformatic tools that achieve strain-level resolution are available, thorough benchmarking is needed to validate their use for less investigated and low biomass microbiomes like those from the upper respiratory tract. We analyzed a previously published data set of longitudinally collected nasopharyngeal samples from Bangladeshi infants (Microbiota and Health study) and a novel data set of oropharyngeal samples from Swiss children with cystic fibrosis. Data from bacterial cultures were used for benchmarking the parameters of StrainPhlAn 3, a bioinformatic tool designed for strain-level resolution. In addition, StrainPhlAn 3 results were compared with metagenomic assemblies derived from StrainGE and newly derived whole-genome sequencing data. After optimizing the analytical parameters, we compared StrainPhlAn 3 results to culture gold standard methods and achieved sensitivity values of 87% ( Streptococcus pneumoniae ), 80% ( Moraxella catarrhalis ), 75% ( Haemophilus influenzae ), and 57% ( Staphylococcus aureus ) for 420 nasopharyngeal and 75% ( H. influenzae ) and 46% ( S. aureus ) for 260 oropharyngeal samples. Comparing the phylogenetic tree of the core genome of 50 S . aureus isolates with a corresponding marker gene tree generated by StrainPhlAn 3 revealed a striking similarity in tree topology for all but three samples indicating adequate strain resolution. In conclusion, a comparison of StrainPhlAn 3 results to data from bacterial cultures revealed that strain-level tracking of the respiratory microbiome is feasible despite the high content of host DNA when parameters are carefully optimized to fit low biomass microbiomes. IMPORTANCE The usage of 16S rRNA gene sequencing has become the state-of-the-art method for the characterization of the microbiota in health and respiratory disease. The method is reliable for low biomass samples due to prior amplification of the 16S rRNA gene but has limitations as species and certainly strain identification is not possible. However, the usage of metagenomic tools for the analyses of microbiome data from low biomass samples is not straight forward, and careful optimization is needed. In this work, we show that by validating StrainPhlAn 3 results with the data from bacterial cultures, the strain-level tracking of the respiratory microbiome is feasible despite the high content of host DNA being present when parameters are carefully optimized to fit low biomass microbiomes. This work further proposes that strain retention analyses are feasible, at least for more abundant species. This will help to better understand the longitudinal dynamics of the upper respiratory microbiome during health and disease.

Pain-related functional gastrointestinal disorders (FGIDs) are characterized by visceral hypersensitivity (VHS) associated with alterations in the microbiota-gut-brain axis. Since human milk oligosaccharides (HMOs) modulate microbiota, gut and brain, we investigated whether HMOs impact VHS, and explored the role of gut microbiota. To induce VHS, C57BL/6JRj mice received hourly water avoidance stress (WAS) sessions for 10 d, or antibiotics (ATB) for 12 d. Challenged and unchallenged (Sham) animals were fed AIN93M diet (Cont) or AIN93M containing 1% of a 6-HMO mix (HMO6). VHS was assessed by monitoring the visceromotor response to colorectal distension. Fecal microbiome was analyzed by shotgun metagenomics. The effect of HMO6 sub-blends on VHS and nociceptive pathways was further tested using the WAS model. In mice fed Cont, WAS and ATB increased the visceromotor response to distension. HMO6 decreased WAS-mediated electromyographic rise at most distension volumes and overall Area Under Curve (AUC=6.12±0.50 in WAS/HMO6 vs. 9.46±0.50 in WAS/Cont; P<.0001). In contrast, VHS in ATB animals was not improved by HMO6. In WAS, HMO6 promoted most microbiota taxa and several functional pathways associated with low VHS and decreased those associated with high VHS. Among the sub-blends, 2’FL+DFL and LNT+6’SL reduced visceromotor response close to Sham/Cont values and modulated serotoninergic and CGRPα-related pathways. This research further substantiates the capacity of HMOs to modulate the microbiota-gut-brain communication and identifies mitigation of abdominal pain as a new HMO benefit. Ultimately, our findings suggest the value of specific HMO blends to alleviate pain associated FGIDs such as infantile colic or Irritable Bowel Syndrome.

The gut microbiome has an important role in infant health and development. We characterized the fecal mi-crobiome and metabolome of 222 young children in Dhaka, Bangladesh during the first two years of life. A distinct Bifidobacterium longum clade expanded with introduction of solid foods and harbored enzymes for utilizing both breast milk and solid food substrates. The clade was highly prevalent in Bangladesh, present globally (at lower prevalence), and correlated with many other gut taxa and metabolites, indicating an impor-tant role in gut ecology. We also found that the B. longum clades and associated metabolites were implicated in childhood diarrhea and early growth, including positive associations between growth measures and B. longum subsp. infantis, indolelactate and N-acetylglutamate. Our data demonstrate geographic, cultural, seasonal, and ecological heterogeneity that should be accounted for when identifying microbiome factors implicated in and potentially benefiting infant development.

ABSTRACT Faecalibacterium prausnitzii ( F. prausnitzii ) is a bacterial taxon of the human gut with anti-inflammatory properties and negative associations with chronic inflammatory conditions. F. prausnitzii may be one of key species contributing to the effects of healthy eating habits, and yet little is known about the nutrients that enhance the growth of F. prausnitzii other than simple sugars and fibers. Here we combined dietary and microbiome data from the American Gut Project (AGP) to identify nutrients that may be linked to the relative abundance of F. prausnitzii . Using a machine learning approach in combination with univariate analyses, we identified that sugar alcohols, carbocyclic sugar and vitamins may contribute to F. prausnitzii growth. We next explored the effects of these nutrients on the growth of two F. prausnitzii strains in vitro and observed strain dependent growth patterns on the nutrient tested. In the context of a complex community using in vitro fermentation, none of the tested nutrients and nutrient combinations exerted a significant growth-promoting effect on F. prausnitzii due to high variability in batch responses. A positive association between F. prausnitzii and butyrate concentrations was observed. Future nutritional studies aiming to increase relative abundance of F. prausnitzii should explore a personalized approach accounting for strain-level genetic variations and community-level microbiome composition.

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Unhealthy lifestyle choices, such as bad eating behaviors and cigarette smoking, have major detrimental impacts on health. However, the inter-relations between obesity and smoking are still not fully understood. We thus developed an experimental model of high-fat diet-fed obese C57BL/6 male mice chronically exposed to cigarette smoke. Our study evaluated for the first time the resulting effects of the combined exposure to unhealthy diet and cigarette smoke on several metabolic, pulmonary, intestinal, and cardiac parameters. We showed that the chronic exposure to cigarette smoke modified the pattern of body fat distribution in favor of the visceral depots in obese mice, impaired the respiratory function, triggered pulmonary inflammation and emphysema, and was associated with gut microbiota dysbiosis, cardiac hypertrophy and myocardial fibrosis.

Human milk is the sole and recommended nutrition for the newborn infant and contains one of the largest constituents of diverse oligosaccharides, dubbed human milk oligosaccharides (HMOs). Preclinical and clinical association studies indicate that HMOs have multiple physiological functions largely mediated through the establishment of the gut microbiome. Until recently, HMOs were not available to investigate their role in randomized controlled intervention trials. To our knowledge, this is the first report on the effects of 2 HMOs on establishing microbiota in newborn infants. We provide a detailed description of the microbiota changes observed upon feeding a formula with 2 HMOs in comparison to breastfed reference infants' microbiota. Then, we associate the microbiota to long-term health as assessed by prescribed antibiotic use.

A role for the gut microbiota in the aging process was postulated a century ago, but it is only with the recent advent of high-throughput genomics technologies that intestinal microbes have been put in the spotlight of healthy aging research. Numerous studies over the past few years have aimed to draw a comprehensive picture of gut microbiota changes in the elderly in order to design targeted dietary interventions towards healthy aging. However, the link between diet, aging and gut microbiota is only partially understood as high interindividual variations and lack of mechanical insights have limited findings primarily to descriptive associations. The present entry is a critical evaluation of the available data from the diet/gut microbiota/healthy aging field to provide a balanced scientific overview.

Targeted metagenomics is the solution of choice to reveal differential microbial profiles (defined by richness, diversity and composition) as part of case-control studies. It is well documented that each data processing step may have the potential to introduce bias in the results. However, selecting a bioinformatics pipeline to analyze high-throughput sequencing data from A to Z remains one of the critical considerations in a case-control microbiota study design. Consequently, the aim of this study was to assess whether the same biological conclusions regarding human gut microbiota composition and diversity could be reached using different bioinformatics pipelines. In this work, we considered four pipelines (mothur, QIIME, kraken and CLARK) with different versions and databases, and examined their impact on the outcome of metagenetic analysis of Ion Torrent 16S sequencing data. We re-analyzed a case-control study evaluating the impact of the colonization of the intestinal protozoa Blastocystis sp. on the human gut microbial profile. Although most pipelines reported the same trends in this case-control study, we demonstrated how the use of different pipelines affects the biological conclusions that can be drawn. Targeted metagenomics must therefore rather be considered as a profiling tool to obtain a broad sense of the variations of the microbiota, rather than an accurate identification tool.

Targeted metagenomics, also known as metagenetics, is a high-throughput sequencing application focusing on a nucleotide target in a microbiome to describe its taxonomic content. A wide range of bioinformatics pipelines are available to analyze sequencing outputs, and the choice of an appropriate tool is crucial and not trivial. No standard evaluation method exists for estimating the accuracy of a pipeline for targeted metagenomics analyses. This article proposes an evaluation protocol containing real and simulated targeted metagenomics datasets, and adequate metrics allowing us to study the impact of different variables on the biological interpretation of results. This protocol was used to compare six different bioinformatics pipelines in the basic user context: Three common ones (mothur, QIIME and BMP) based on a clustering-first approach and three emerging ones (Kraken, CLARK and One Codex) using an assignment-first approach. This study surprisingly reveals that the effect of sequencing errors has a bigger impact on the results that choosing different amplified regions. Moreover, increasing sequencing throughput increases richness overestimation, even more so for microbiota of high complexity. Finally, the choice of the reference database has a bigger impact on richness estimation for clustering-first pipelines, and on correct taxa identification for assignment-first pipelines. Using emerging assignment-first pipelines is a valid approach for targeted metagenomics analyses, with a quality of results comparable to popular clustering-first pipelines, even with an error-prone sequencing technology like Ion Torrent. However, those pipelines are highly sensitive to the quality of databases and their annotations, which makes clustering-first pipelines still the only reliable approach for studying microbiomes that are not well described.

In the past decade, metagenomics studies have become widespread due to the arrival of second-generation sequencing platforms characterized by low costs, high throughput and short read lengths. Today, although benchtop sequencers are considered to be accurate platforms to deliver data for targeted metagenomics studies, the limiting factor has become the analysis of these data. In a previous paper, we performed an Ion Torrent PGM 16S rDNA gene sequencing of faecal DNAs from 48 Blastocystis-colonized patients and 48 Blastocystis-negative subjects, in order to decipher the impact of this widespread protist on gut microbiota composition and diversity. We report here on the Ion Torrent targeted metagenomic sequencing and analysis of these 96 human faecal samples, and the complete datasets from raw to analysed data. We also provide the key steps of the bioinformatic analyses, from library preparation to data filtering and OTUs tables generation. This data represents a valuable resource for the scientific community, enabling re-processing of these targeted metagenomic datasets through various pipelines and a comparative evaluation of microbiota analysis methods.

This compressed file contains the OTU_count_tables tsv file that is the output of the second analytical step (clustering analysis and OTU classification) of the home-made bioinformatics pipeline. It contains, for each sample, four columns: the first column is the consensus read name associated to the OTU, the second column is the OTU raw counts, the third column is the consensus read name (same as first column) and the fourth column is the associated taxon.

Introduction : Blastocystis is a highly prevalent protozoa of the intestinal tract belonging to the Stramenopile group. Its prevalence in human often exceeds 5% in industrialized countries reaching as high as 76% in developing countries. Although the role of Blastocystis as human pathogen remains unclear, it can cause acute or chronic digestive disorders and some studies have suggested an association with irritable bowel syndrome. The life cycle of the parasite is poorly documented, the vacuolar stage being the most easily recognizable and the most frequently observed in both laboratory culture and stool samples (Fig.