Analysis of the fermentation kinetics and gut microbiota modulatory effect of dried chicory root reveals the impact of the plant-cell matrix rationalizing its conversion in the distal colon.

Aim: The cell matrix of plant foods has received little attention in prebiotic fiber research. We aimed to understand the impact of the plant cell matrix in dried chicory root on its breakdown in the human gut to explain its reported beneficial effects on gut and metabolic health. Methods: We applied in vitro digestion and fermentation models together with an ex vivo gut barrier integrity model. Plant cell matrix intactness in the upper gastrointestinal tract was investigated by scanning electron microscopy. Colonic breakdown of inulin, and chicory root cubes and powder was assessed by gut microbiota analysis using 16S rRNA gene amplicon sequencing and determining the kinetics of changes in pH, gas, and short-chain fatty acid (SCFA) production. Finally, effects on gut barrier integrity were explored by exposing colonic biopsies to fermentation supernatants in an Ussing chamber model. Results: The plant cell matrix of dried chicory root cubes remained intact throughout upper gastrointestinal transit. Dried chicory root fermentation resulted in higher final relative abundances of pectin-degrading Monoglobus and butyrate-producing Roseburia spp. compared to inulin and a seven-fold increase in Bifidobacterium spp. in donors where these species were present. Dried chicory root cubes yielded similar total SCFAs but higher final butyrate levels than chicory root powder or isolated inulin with less gas produced. No uniform but donor-specific effects of fermentation supernatants on the maintenance of gut barrier integrity were detected. Conclusion: The intact plant cell matrix of dried chicory root affected its colonic breakdown kinetics and microbiota, underpinning its beneficial effect in vivo.

Concepts and criteria defining emerging microbiome applications.

In recent years, microbiomes and their potential applications for human, animal or plant health, food production and environmental management came into the spotlight of major national and international policies and strategies. This has been accompanied by substantial R&D investments in both public and private sectors, with an increasing number of products entering the market. Despite widespread agreement on the potential of microbiomes and their uses across disciplines, stakeholders and countries, there is no consensus on what defines a microbiome application. This often results in non-comprehensive communication or insufficient documentation making commercialisation and acceptance of the novel products challenging. To showcase the complexity of this issue we discuss two selected, well-established applications and propose criteria defining a microbiome application and their conditions of use for clear communication, facilitating suitable regulatory frameworks and building trust among stakeholders.

Metatranscriptomic analysis indicates prebiotic effect of isomalto/malto-polysaccharides on human colonic microbiota in-vitro.

Isomalto/malto-polysaccharides (IMMPs) are a novel type of soluble dietary fibres with a prebiotic potential promoting growth of beneficial microbes in the gut. However, the mode of action of IMMPs remains unknown. Previous studies on IMMPs showed an increase in total bacteria, especially lactobacilli, and higher production of short chain fatty acids (SCFA) when IMMPs were fed to rats or used during in vitro fermentation. Here we used metatranscriptomics to investigate how IMMPs with different amounts of α - (1 → 6) glycosidic linkages affected microbial function during incubation with human fecal inoculum. We showed that active microbial community dynamics during fermentation varied depending on the type of IMMP used and that the observed changes were reflected in the community gene expression profiles. Based on metatranscriptome analysis, members of Bacteroides, Lactobacillus and Bifidobacterium were the predominant degraders of IMMPs, and the increased gene expression in these bacteria correlated with high amounts of α - (1 → 6) glycosidic linkages. We also noted an increase in relative abundance of these bacteria and an activation of pathways involved in SCFA synthesis. Our findings could provide a baseline for more targeted approaches in designing prebiotics for specific bacteria and to achieve more controlled modulation of microbial activity towards desired health outcomes.

Spatio-temporal dynamics of the human small intestinal microbiome and its response to a synbiotic.

Although fecal microbiota composition is considered to preserve relevant and representative information for distal colonic content, it is evident that it does not represent microbial communities inhabiting the small intestine. Nevertheless, studies investigating the human small intestinal microbiome and its response to dietary intervention are still scarce. The current study investigated the spatio-temporal dynamics of the small intestinal microbiome within a day and over 20 days, as well as its responses to a 14-day synbiotic or placebo control supplementation in 20 healthy subjects. Microbial composition and metabolome of luminal content of duodenum, jejunum, proximal ileum and feces differed significantly from each other. Additionally, differences in microbiota composition along the small intestine were most pronounced in the morning after overnight fasting, whereas differences in composition were not always measurable around noon or in the afternoon. Although overall small intestinal microbiota composition did not change significantly within 1 day and during 20 days, remarkable, individual-specific temporal dynamics were observed in individual subjects. In response to the synbiotic supplementation, only the microbial diversity in jejunum changed significantly. Increased metabolic activity of probiotic strains during intestinal passage, as assessed by metatranscriptome analysis, was not observed. Nevertheless, synbiotic supplementation led to a short-term spike in the relative abundance of genera included in the product in the small intestine approximately 2 hours post-ingestion. Collectively, small intestinal microbiota are highly dynamic. Ingested probiotic bacteria could lead to a transient spike in the relative abundance of corresponding genera and ASVs, suggesting their passage through the entire gastrointestinal tract. This study was registered to http://www.clinicaltrials.gov, NCT02018900.

Effect of prebiotic oligosaccharides on bowel habit and the gut microbiota in children with functional constipation (Inside study): study protocol for a randomised, placebo-controlled, multi-centre trial.

BACKGROUND: Functional constipation (FC) in children is a common gastrointestinal disorder with a worldwide-pooled prevalence of 9.5%. Complaints include infrequent bowel movements, painful defecation due to hard and/or large stools, faecal incontinence, and abdominal pain. Prebiotic oligosaccharides have been shown to relieve constipation symptoms in young adults and elderly. However, sufficient evidence is lacking linking additional prebiotic intake to improve symptoms in children with FC. We hypothesise that prebiotic oligosaccharides are able to relieve symptoms of constipation in young children as well. METHODS: In the present randomised, double-blind, placebo-controlled, multi-centre study, we will study the effects of two prebiotic oligosaccharides in comparison to placebo on constipation symptoms in children of 1-5 years (12 to 72 months) of age diagnosed with FC according to the Rome IV criteria for functional gastrointestinal disorders. The primary outcome measure will be change in stool consistency. Secondary outcomes include stool frequency and stool consistency in a number of cases (%). Tertiary outcomes include among others painful defecation, use of rescue medication, and quality of life. In addition, the impact on gut microbiome outcomes such as faecal microbiota composition and metabolites will be investigated. Participants start with a run-in period, after which they will receive supplements delivered in tins with scoops for 8 weeks, containing one of the two prebiotic oligosaccharides or placebo, followed by a 4-week wash-out period. DISCUSSION: This randomised double-blind, placebo-controlled multi-centre study will investigate the effectiveness of prebiotic oligosaccharides in children aged 1-5 years with FC. TRIAL REGISTRATION: ClinicalTrials.gov NCT04282551. Registered on 24 February 2020.

Biobased short chain fatty acid production - Exploring microbial community dynamics and metabolic networks through kinetic and microbial modeling approaches.

In recent years, there has been growing interest in harnessing anaerobic digestion technology for resource recovery from waste streams. This approach has evolved beyond its traditional role in energy generation to encompass the production of valuable carboxylic acids, especially volatile fatty acids (VFAs) like acetic acid, propionic acid, and butyric acid. VFAs hold great potential for various industries and biobased applications due to their versatile properties. Despite increasing global demand, over 90% of VFAs are currently produced synthetically from petrochemicals. Realizing the potential of large-scale biobased VFA production from waste streams offers significant eco-friendly opportunities but comes with several key challenges. These include low VFA production yields, unstable acid compositions, complex and expensive purification methods, and post-processing needs. Among these, production yield and acid composition stand out as the most critical obstacles impacting economic viability and competitiveness. This paper seeks to offer a comprehensive view of combining complementary modeling approaches, including kinetic and microbial modeling, to understand the workings of microbial communities and metabolic pathways in VFA production, enhance production efficiency, and regulate acid profiles through the integration of omics and bioreactor data.

Methodological recommendations for human microbiota-gut-brain axis research.

Observational studies have determined numerous correlations between sequence-based gut microbiota data and human mental traits. However, these associations are often inconsistent across studies. This inconsistency is one of the reasons that mechanistic validation studies of the observed correlations are lagging, making it difficult to establish causal associations. The absence of consistent study findings may partially be due to the lack of clear guidelines for identifying confounders of relations between complex microbial communities and mental conditions. Gut microbial complexity also impedes deciphering microbiota-host relations by using a single analytical approach. The aim of the current review is to help solve these problems by providing methodological recommendations for future human microbiota-gut-brain axis research on the selection of confounders, the use of integrative biostatistical methods, and the steps needed to translate correlative findings into causal conclusions.

Bioaugmentation has temporary effect on anaerobic pesticide biodegradation in simulated groundwater systems.

Groundwater is the most important source for drinking water in The Netherlands. Groundwater quality is threatened by the presence of pesticides, and biodegradation is a natural process that can contribute to pesticide removal. Groundwater conditions are oligotrophic and thus biodegradation can be limited by the presence and development of microbial communities capable of biodegrading pesticides. For that reason, bioremediation technologies such as bioaugmentation (BA) can help to enhance pesticide biodegradation. We studied the effect of BA using enriched mixed inocula in two column bioreactors that simulate groundwater systems at naturally occurring redox conditions (iron and sulfate-reducing conditions). Columns were operated for around 800 days, and two BA inoculations (BA1 and BA2) were conducted in each column. Inocula were enriched from different wastewater treatment plants (WWTPs) under different redox-conditions. We observed a temporary effect of BA1, reaching 100% removal efficiency of the pesticide 2,4-D after 100 days in both columns. In the iron-reducing column, 2,4-D removal was in general higher than under sulfate-reducing conditions demonstrating the influence of redox conditions on overall biodegradation. We observed a temporary shift in microbial communities after BA1 that is relatable to the increase in 2,4-D removal efficiency. After BA2 under sulfate-reducing conditions, 2,4-D removal efficiency decreased, but no change in the column microbial communities was observed. The present study demonstrates that BA with a mixed inoculum can be a valuable technique for improving biodegradation in anoxic groundwater systems at different redox-conditions.

Physiology of γ-aminobutyric acid production by Akkermansia muciniphila.

Gut bacteria hold the potential to produce a broad range of metabolites that can modulate human functions, including molecules with neuroactive potential. One such molecule is γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter of the central nervous system in animals. Metagenomic analyses suggest that the genomes of many gut bacteria encode glutamate decarboxylase (GAD), the enzyme that catalyzes GABA production. The genome of Akkermansia muciniphila, a mucin specialist and potential next-generation probiotic from the human gut, is predicted to encode GAD, suggesting a contributing role in GABA production in the human gut. In this study, A. muciniphila was grown in batch cultures with and without pH control. In both experiments, A. muciniphila was found to produce GABA as a response to acid (pH <5.5), although only when GABA precursors, either glutamate or glutamine, were present in the medium. Proteomic analysis comparing A. muciniphila grown with and without precursors at pH 4 did not show a difference in GAD expression, suggesting that it is expressed regardless of the presence of GABA precursors. To further investigate the function of A. muciniphila GAD, we heterologously expressed the gad gene (encoded by locus tag Amuc_0372) with a His tag in Escherichia coli and purified the GAD protein. Enzyme assays showed GAD activity in a pH range between 4 and 6, with the highest specific activity at pH 5 of 144 ± 16 µM GABA/min/mg. Overall, our results demonstrate the ability of A. muciniphila to produce GABA as an acid response and unravel the conditions under which GABA production in A. muciniphila occurs.IMPORTANCEAkkermansia muciniphila is considered to be a beneficial bacterium from the human gut, but the exact mechanisms by which A. muciniphila influences its host are not yet fully understood. To this end, it is important to identify which metabolites are produced and consumed by A. muciniphila that may contribute to a healthy gut. In the present study, we demonstrate the ability of A. muciniphila to produce γ-aminobutyric acid (GABA) when grown in an acidic environment, which often occurs in the gut. GABA is the major inhibitory neurotransmitter in the central nervous system and is present in the human gut. For this reason, it is considered an important bacterial metabolite. Our finding that A. muciniphila produces GABA in acidic environments adds to the growing body of understanding of its relationship with host health and provides an explanation on how it can survive acid stress in the human gut.

Development of the gut microbiota in the first 14 years of life and its relations to internalizing and externalizing difficulties and social anxiety during puberty.

Relations between the gut microbiota and host mental health have been suggested by a growing number of case-control and cross-sectional studies, while supporting evidence is limited in large community samples followed during an extended period. Therefore, the current preregistered study ( https://osf.io/8ymav , September 7, 2022) described child gut microbiota development in the first 14 years of life and explored its relations to internalizing and externalizing difficulties and social anxiety in puberty, a period of high relevance for the development of mental health problems. Fecal microbiota composition was analysed by 16S ribosomal RNA gene amplicon sequencing in a total of 1003 samples from 193 children. Through a clustering method, four distinct microbial clusters were newly identified in puberty. Most children within three of these clusters remained in the same clusters from the age of 12 to 14 years, suggesting stability in microbial development and transition during this period. These three clusters were compositionally similar to enterotypes (i.e., a robust classification of the gut microbiota based on its composition across different populations) enriched in Bacteroides, Prevotella, and Ruminococcus, respectively. Two Prevotella 9-predominated clusters, including one reported by us earlier in middle childhood and the other one in puberty, were associated with more externalizing behavior at age 14. One Faecalibacterium-depleted pubertal cluster was related to more social anxiety at age 14. This finding was confirmed by a negative cross-sectional relation between Faecalibacterium and social anxiety in the 14-year-olds. The findings of this study continue to map gut microbiota development in a relatively large community sample followed from birth onwards, importantly extending our knowledge to puberty. Results indicate that Prevotella 9 and Faecalibacterium may be relevant microbial taxa in relation to externalizing behavior and social anxiety, respectively. These correlational findings need validations from other similar cohort studies, as well as well-designed mechanistic pre-clinical investigations before inferring cause and effect.

Effects of antidepressant exposure on aquatic communities assessed by a combination of morphological identification, functional measurements, environmental DNA metabarcoding and bioassays.

The antidepressant fluoxetine is frequently detected in aquatic ecosystems, yet the effects on aquatic communities and ecosystems are still largely unknown. Therefore the aim of this study is to assess the effects of the long-term application of fluoxetine on key components of aquatic ecosystems including macroinvertebrate-, zooplankton-, phytoplankton- and microbial communities and organic matter decomposition by using traditional and non-traditional assessment methods. For this, we exposed 18 outdoor mesocosms (water volume of 1530 L and 10 cm of sediment) to five different concentrations of fluoxetine (0.2, 2, 20 and 200 µg/L) for eight weeks, followed by an eight-week recovery period. We quantified population and community effects by morphological identification, environmental DNA metabarcoding, in vitro and in vivo bioassays and measured organic matter decomposition as a measure of ecosystem functioning. We found effects of fluoxetine on bacterial, algal, zooplankton and macroinvertebrate communities and decomposition rates, mainly for the highest (200 µg/L) treatment. Treatment-related decreases in abundances were found for damselfly larvae (NOEC of 0.2 µg/L) and Sphaeriidae bivalves (NOEC of 20 µg/L), whereas Asellus aquaticus increased in abundance (NOEC <0.2 µg/L). Fluoxetine decreased photosynthetic activity and primary production of the suspended algae community. eDNA assessment provided additional insights by revealing that the algae belonging to the class Cryptophyceae and certain cyanobacteria taxa were the most negatively responding taxa to fluoxetine. Our results, together with results of others, suggest that fluoxetine can alter community structure and ecosystem functioning and that some impacts of fluoxetine on certain taxa can already be observed at environmentally realistic concentrations.

Assessing ecological responses to exposure to the antibiotic sulfamethoxazole in freshwater mesocosms.

Antibiotics are a contaminant class of worldwide concern as they are frequently detected in aquatic ecosystems. To better understand the impacts of antibiotics on aquatic ecosystems, we conducted an outdoor mesocosm experiment in which aquatic communities were exposed to different concentrations of the antibiotic sulfamethoxazole (0, 0.15, 1.5, 15 and 150 µg/L). These concentrations include mean (0.15 µg/L) and maximum detected concentrations (15 and 150 µg/L) in aquatic ecosystems worldwide. Sulfamethoxazole was applied once a week for eight consecutive weeks to 1530 L outdoor mesocosms in the Netherlands, followed by an eight-week recovery period. We evaluated phytoplankton-, bacterial- and invertebrate responses during and after sulfamethoxazole exposure and assessed impacts on organic matter decomposition. Contrary to our expectations, consistent treatment-related effects on algal and bacterial communities could not be demonstrated. In addition, sulfamethoxazole did not significantly affect zooplankton and macroinvertebrate communities. However, some effects on specific taxa were observed, with an increase in Mesostoma flatworm abundance (NOEC of <0.15 µg/L). In addition, eDNA analyses indicated negative impacts on the insects Odonata at a sulfamethoxazole concentration of 15 µg/L. Overall, environmentally relevant sulfamethoxazole concentration did not result in direct or indirect impairment of entire aquatic communities and ecological processes in our mesocosms. However, several specific macroinvertebrate taxa demonstrated significant (in)direct effects from sulfamethoxazole. Comparison of the results with the literature showed inconsistent results between studies using comparable, environmentally relevant, concentrations. Therefore, our study highlights the importance of testing the ecological impacts of pharmaceuticals (such as sulfamethoxazole) across multiple trophic levels spanning multiple aquatic communities, to fully understand its potential ecological threats.

Gut microbiota and child behavior in early puberty: does child sex play a role?

A growing number of studies have indicated relations between the gut microbiota and mental health. However, to date, there is a scarcity of microbiota studies in community samples in early puberty. The current preregistered study (https://osf.io/wu2vt) investigated gut microbiota composition in relation to sex in low-risk children and explored behavioral associations with gut microbiota composition and metabolites in the same samples, together with the potential role of sex. Fecal microbiota composition was analyzed in 12-year-old children (N = 137) by 16S rRNA gene sequencing and quantitative PCR. Modest sex differences were observed in beta diversity. Generalized linear models showed consistent behavioral relations to both relative and absolute abundances of individual taxa, including positive associations between Parasutterella and mother-reported internalizing behavior, and negative associations between Odoribacter and mother-reported externalizing behavior. Additionally, Prevotella 9 was positively related to mother-reported externalizing behavior, confirming earlier findings on the same cohort at 5 years of age. Sex-related differences were found in behavioral relations to Ruminiclostridium 5, Alistipes, Streptococcus, Ruminiclostridium 9, Ruminococcaceae UCG-5, and Dialister, for relative abundances, as well as to Family XIII AD3011 group and an unidentified bacterium within the Tenericutes, for absolute abundances. Limited behavioral relations were observed regarding alpha diversity and fecal metabolites. Our findings describe links between the gut microbiota and child behavior, together with differences between child sexes in these relations, in low-risk early pubertal children. Importantly, this study confirmed earlier findings in this cohort of positive relations between Prevotella 9 and externalizing behavior at age 10 years. Results also show the merit of including absolute abundances in microbiota studies.

A longitudinal study of the gut microbiota during the first three years of life: Links with problem behavior and executive functions at preschool age.

Early life is a sensitive period when microbiota-gut-brain interactions may have important impact on development. This study investigated the associations of the gut microbiota in the first three years of life (two, six, and 12 weeks, and one and three years) with problem behavior and executive functions in N = 64 three-year-old children. Higher relative abundance of Streptococcus at the age of two weeks, as well as its trajectory over time (including ages two, six and 12 weeks, and one and three years), was related to worse executive functions. Higher relative abundance of [Ruminococcus] torques group at the age of three years, as well as its trajectory from one to three years, was associated with less internalizing behavior. Besides, several robust age-specific associations were identified: higher Bifidobacterium relative abundance (age three years) was associated with more internalizing and externalizing issues; higher Blautia relative abundance (age three years) was linked to less internalizing behavior; and increased relative abundance of an unidentified Enterobacteriaceae genus (age two weeks) was related to more externalizing behavior. Our findings provide important longitudinal evidence that early-life gut microbiota may be linked to behavioral and cognitive development in low-risk children.

In vitro interactions between Blautia hydrogenotrophica, Desulfovibrio piger and Methanobrevibacter smithii under hydrogenotrophic conditions.

Methanogens, reductive acetogens and sulfate-reducing bacteria play an important role in disposing of hydrogen in gut ecosystems. However, how they interact with each other remains largely unknown. This in vitro study cocultured Blautia hydrogenotrophica (reductive acetogen), Desulfovibrio piger (sulfate reducer) and Methanobrevibacter smithii (methanogen). Results revealed that these three species coexisted and did not compete for hydrogen in the early phase of incubations. Sulfate reduction was not affected by B. hydrogenotrophica and M. smithii. D. piger inhibited the growth of B. hydrogenotrophica and M. smithii after 10 h incubations, and the inhibition on M. smithii was associated with increased sulfide concentration. Remarkably, M. smithii growth lag phase was shortened by coculturing with B. hydrogenotrophica and D. piger. Formate was rapidly used by M. smithii under high acetate concentration. Overall, these findings indicated that the interactions of the hydrogenotrophic microbes are condition-dependent, suggesting their interactions may vary in gut ecosystems.

Candidatus Nemesobacterales is a sponge-specific clade of the candidate phylum Desulfobacterota adapted to a symbiotic lifestyle.

Members of the candidate phylum Dadabacteria, recently reassigned to the phylum Candidatus Desulfobacterota, are cosmopolitan in the marine environment found both free-living and associated with hosts that are mainly marine sponges. Yet, these microorganisms are poorly characterized, with no cultured representatives and an ambiguous phylogenetic position in the tree of life. Here, we performed genome-centric metagenomics to elucidate their phylogenomic placement and predict the metabolism of the sponge-associated members of this lineage. Rank-based phylogenomics revealed several new species and a novel family (Candidatus Spongomicrobiaceae) within a sponge-specific order, named here Candidatus Nemesobacterales. Metabolic reconstruction suggests that Ca. Nemesobacterales are aerobic heterotrophs, capable of synthesizing most amino acids, vitamins and cofactors and degrading complex carbohydrates. We also report functional divergence between sponge- and seawater-associated metagenome-assembled genomes. Niche-specific adaptations to the sponge holobiont were evident from significantly enriched genes involved in defense mechanisms against foreign DNA and environmental stressors, host-symbiont interactions and secondary metabolite production. Fluorescence in situ hybridization gave a first glimpse of the morphology and lifestyle of a member of Ca. Desulfobacterota. Candidatus Nemesobacterales spp. were found both inside sponge cells centred around sponge nuclei and in the mesohyl of the sponge Geodia barretti. This study sheds light on the enigmatic group Ca. Nemesobacterales and their functional characteristics that reflect a symbiotic lifestyle.

What are Normal Defecation Patterns in Healthy Children up to Four Years of Age? A Systematic Review and Meta-Analysis.

OBJECTIVE: To summarize available data on defecation frequency and stool consistency of healthy children up to age 4 in order to estimate normal references values. STUDY DESIGN: Systematic review including cross-sectional, observational, and interventional studies published in English, that reported on defecation frequency and/or stool consistency in healthy children 0-4 years old. RESULTS: Seventy-five studies were included with 16 393 children and 40 033 measurements of defecation frequency and/or stool consistency. Based on visual inspection of defecation frequency data, a differentiation was made between two age categories: young infants (0-14 weeks old) and young children (15 weeks-4 years old). Young infants had a mean defecation frequency of 21.8 per week (95 % CI, 3.9-35.2) compared with 10.9 (CI, 5.7-16.7) in young children (P < .001). Among young infants, human milk-fed (HMF) infants had the highest mean defecation frequency per week (23.2 [CI, 8.8-38.1]), followed by formula-fed (FF) infants (13.7 [CI 5.4-23.9]), and mixed-fed (MF) infants (20.7 [CI, 7.0-30.2]). Hard stools were infrequently reported in young infants (1.5%) compared with young children (10.5%), and a reduction in the frequency of soft/watery stools was observed with higher age (27.0% in young infants compared with 6.2% in young children). HMF young infants had softer stools compared with FF young infants. CONCLUSIONS: Young infants (0-14 weeks old) have softer and more frequent stools compared with young children (15 weeks-4 years old).