Assessment of the Accuracy of High-Throughput Sequencing of the ITS1 Region of Neocallimastigomycota for Community Composition Analysis

Anaerobic fungi (Neocallimastigomycota) are common inhabitants of the digestive tract of large mammalian herbivores, where they make an important contribution to plant biomass degradation. The internal transcribed spacer 1 (ITS1) region is currently the molecular marker of choice for anaerobic fungal community analysis, despite its known size polymorphism and heterogeneity. The aim of this study was to assess the accuracy of high-throughput sequencing of the ITS1 region of anaerobic fungi for community composition analysis. To this end, full-length ITS1 clone libraries from five pure cultures, representing the ITS1 region size range, were Sanger sequenced to generate a reference dataset. Barcoded amplicons of the same five pure cultures, and four different mock communities derived from them, were then sequenced using Illumina HiSeq. The resulting sequences were then assessed in relation to either the reference dataset (for the pure cultures) or the corresponding theoretical mock communities. Annotation of sequences obtained from individual pure cultures was not always consistent at the clade or genus level, irrespective of whether data from clone libraries or high-throughput sequencing were analyzed. The detection limit of the high-throughput sequencing method appeared to be influenced by factors other than the parameters used during data processing, as some taxa with theoretical values >0.6% were not detected in the mock communities. The high number of PCR cycles used was considered to be a potential explanation for this observation. Accuracy of two of the four mock communities was limited, and this was speculated to be due to preferential amplification of smaller sized ITS1 regions. If this is true, then this is predicted to be an issue with only six of the 32 named anaerobic fungal clades. Whilst high-throughput sequencing of the ITS1 region from anaerobic fungi can be used for environmental sample analysis, we conclude that the accuracy of the method is influenced by sample community composition. Furthermore, ambiguity in the annotation of sequences within pure cultures due to ITS1 heterogeneity reinforces the limitations of the ITS1 region for the taxonomic assignment of anaerobic fungi. In order to overcome these issues, there is a need to develop an alternative taxonomic marker for anaerobic fungi.</p

Does entry to center-based childcare affect gut microbial colonization in young infants?

Entry to center-based childcare (CC) at three months of life can be an important challenge for infants as it includes major stressors such as long maternal separations and frequently changing caregivers. Stress and the new environment may in turn alter the composition of the gut microbiota with possible implications for future health outcomes. As part of an ongoing longitudinal study, we investigated whether CC, as compared to being cared for by the parents at home, alters the composition of the gut microbiota, while accounting for known covariates of the infant gut microbiota. Stool samples of infants who entered CC (n=49) and control infants (n=49) were obtained before and four weeks after CC entrance. Using Redundancy analysis, Random Forests and Bayesian linear models we found that infant gut microbiota was not affected in a uniform way by entry to CC. In line with the literature, breastfeeding, birth mode, age, and the presence of siblings were shown to significantly impact the microbial composition.Peer reviewe

Effect of antibiotics in the first week of life on faecal microbiota development

Study of the effect of antibiotics in the first week of life on fecal microbiota up to 2.5 years of age. Differences identified up to one year of age in babies who received antibiotics when compared with controls.Background Infants are frequently exposed to antibiotics (AB) in the first week of life for suspected bacterial infections. Little is known about the effect of AB on the developing intestinal microbiota. Therefore, we studied intestinal microbiota development with and without AB exposure in the first week of life in term born infants. Methods We analysed the faecal microbiota from birth until 2.5 years of age by 16S rRNA gene amplicon sequencing in a cohort with 56 term born infants, exposed to AB in the first week of life (AB+) (AB for 2-3 days (AB2, n=20), AB for 7 days (AB7, n=36)), compared with 126 healthy controls (AB-). The effects of AB and duration were examined in relation to delivery and feeding mode. Results AB+ was associated with significantly increased relative abundance of Enterobacteriaceae at 3 weeks and 1 year and a decrease of Bifidobacteriaceae, from 1 week until 3 months of age only in vaginally delivered, but not in C-section born infants. Similar deviations were noted in AB7, but not in AB2. After AB, breastfed infants had lower relative abundance of potentially pathogenic Enterobacteriaceae compared with formula fed infants and recovered 2 weeks faster towards controls. Conclusions AB exposure in the first week of life alters faecal microbiota development with deviations in the relative abundance of individual taxa until 1 year of age. These alterations can have long-term health consequences, which emphasises the need for future studies aiming at restoring intestinal microbiota after AB administration

Conserved developmental trajectories of the cecal microbiota of broiler chickens in a field study

There is great interest in identifying gut microbiota development patterns and underlying assembly rules that can inform strategies to improve broiler health and performance. Microbiota stratification using community types helps to simplify complex and dynamic ecosystem principles of the intestinal microbiota. This study aimed to identify community types to increase insight in intestinal microbiota variation between broilers and to identify factors that explain this variation. A total of 10 well-performing poultry flocks on four farms were followed. From each flock, the cecal content of nine broilers was collected at 7, 14, and 35 days posthatch. A total of two robust community types were observed using different clustering methods, one of which was dominated by 7-day-old broilers, and one by 35-day-old broilers. Broilers, 14-day-old, were divided across both community types. This is the first study that showed conserved cecal microbiota development trajectories in commercial broiler flocks. In addition to the temporal development with age, the cecal microbiota variation between broilers was explained by the flock, body weight, and the different feed components. Our data support a conserved development of cecal microbiota, despite strong influence of environmental factors. Further investigation of mechanisms underlying microbiota development and function is required to facilitate intestinal health promoting management, diagnostics, and nutritional interventions

Effect of wheat bran derived prebiotic supplementation on gastrointestinal transit, gut microbiota, and metabolic health: a randomized controlled trial in healthy adults with a slow gut transit

Acute intake of the wheat bran extract Arabinoxylan-Oligosaccharide (AXOS) modulates the gut microbiota, improves stool characteristics and postprandial glycemia in healthy humans. Yet, little is known on how long-term AXOS intake influences gastrointestinal (GI) functioning, gut microbiota, and metabolic health. In this randomized, placebo-controlled, double-blind study, we evaluated the effects of AXOS intake on GI function and metabolic health in adults with slow GI transit without constipation. Forty-eight normoglycemic adults were included with whole-gut transit time (WGTT) of >35 h receiving either 15 g/day AXOS or placebo (maltodextrin) for 12-wks. The primary outcome was WGTT, and secondary outcomes included stool parameters, gut permeability, short-chain fatty acids (SCFA), microbiota composition, energy expenditure, substrate oxidation, glucose, insulin, lipids, gut hormones, and adipose tissue (AT) function. WGTT was unchanged, but stool consistency softened after AXOS. 12-wks of AXOS intake significantly changed the microbiota by increasing Bifidobacterium and decreasing microbial alpha-diversity. With a good classification accuracy, overall microbiota composition classified responders with decreased WGTT after AXOS. The incretin hormone Glucagon-like protein 1 was reduced after AXOS compared to placebo. Energy expenditure, plasma metabolites, AT parameters, SCFA, and gut permeability were unchanged. In conclusion, intake of wheat bran extract increases fecal Bifidobacterium and softens stool consistency without major effects on energy metabolism in healthy humans with a slow GI transit. We show that overall gut microbiota classified responders with decreased WGTT after AXOS highlighting that GI transit and change thereof were associated with gut microbiota independent of Bifidobacterium. NCT02491125.</p

Associations between Pro- and Anti-Inflammatory Gastro-Intestinal Microbiota, Diet, and Cognitive Functioning in Dutch Healthy Older Adults : The NU-AGE Study

Dietary modulation of the gastro-intestinal microbiota is a potential target in improving healthy ageing and age-related functional outcomes, including cognitive decline. We explored the association between diet, gastro-intestinal microbiota and cognition in Dutch healthy older adults of the 'New dietary strategies addressing the specific needs of the elderly population for healthy aging in Europe' (NU-AGE) study. The microbiota profile of 452 fecal samples from 226 subjects was determined using a 16S ribosomal RNA gene-targeted microarray. Dietary intake was assessed by 7-day food records. Cognitive functioning was measured with an extensive cognitive test battery. We observed a dietary and microbial pro- to anti-inflammatory gradient associated with diets richer in animal- or plant-based foods. Fresh fruits, nuts, seeds and peanuts, red and processed meat and grain products were most strongly associated to microbiota composition. Plant-rich diets containing fresh fruits, nuts, seeds and peanuts were positively correlated with alpha-diversity, various taxa from the Bacteroidetes phylum and anti-inflammatory species, including those related to Faecalibacterium prausnitzii and Eubacterium rectale and E. biforme. Animal product-rich diets associated with pro-inflammatory species, including those related to Ruminococcus gnavus and Collinsella spp.. Cognition was neither associated with microbiota composition nor alpha-diversity. In conclusion, diets richer in animal- and plant-based foods were related to a pro- and anti-inflammatory microbial profile, while cognition was associated with neither.Peer reviewe

Structure-Specific Fermentation of Galacto-Oligosaccharides, Isomalto-Oligosaccharides and Isomalto/Malto-Polysaccharides by Infant Fecal Microbiota and Impact on Dendritic Cell Cytokine Responses

SCOPE: Next to galacto-oligosaccharides (GOS), starch-derived isomalto-oligosaccharide preparation (IMO) and isomalto/malto-polysaccharides (IMMP) could potentially be used as prebiotics in infant formulas. However, it remains largely unknown how the specific molecular structures of these non-digestible carbohydrates (NDCs) impact fermentability and immune responses in infants. METHODS AND RESULTS: In vitro fermentation of GOS, IMO and IMMP using infant fecal inoculum of 2- and 8-week-old infants showed that only GOS and IMO were fermented by infant fecal microbiota. The degradation of GOS and IMO coincided with an increase in Bifidobacterium and production of acetate and lactate, which was more pronounced with GOS. Individual isomers with an (1↔1)-linkage or di-substituted reducing terminal glucose residue were more resistant to fermentation. GOS, IMO and IMMP fermentation digesta attenuated cytokine profiles in immature dendritic cells (DCs), but the extent was dependent on the infants age and NDC structure. CONCLUSION: The IMO preparation, containing reducing and non-reducing isomers, showed similar fermentation patterns as GOS in fecal microbiota of 2-week-old infants. Knowledge obtained on the substrate specificities of infant fecal microbiota and the subsequent regulatory effects of GOS, IMO and IMMP on DC responses might contribute to the design of tailored NDC mixtures for infants of different age groups. This article is protected by copyright. All rights reserved

Cohort profile: LifeLines DEEP, a prospective, general population cohort study in the northern Netherlands:Study design and baseline characteristics

Purpose There is a critical need for population-based prospective cohort studies because they follow individuals before the onset of disease, allowing for studies that can identify biomarkers and disease-modifying effects, and thereby contributing to systems epidemiology. Participants This paper describes the design and baseline characteristics of an intensively examined subpopulation of the LifeLines cohort in the Netherlands. In this unique subcohort, LifeLines DEEP, we included 1539 participants aged 18 years and older. Findings to date We collected additional blood (n=1387), exhaled air (n=1425) and faecal samples (n=1248), and elicited responses to gastrointestinal health questionnaires (n=1176) for analysis of the genome, epigenome, transcriptome, microbiome, metabolome and other biological levels. Here, we provide an overview of the different data layers in LifeLines DEEP and present baseline characteristics of the study population including food intake and quality of life. We also describe how the LifeLines DEEP cohort allows for the detailed investigation of genetic, genomic and metabolic variation for a wide range of phenotypic outcomes. Finally, we examine the determinants of gastrointestinal health, an area of particular interest to us that can be addressed by LifeLines DEEP. Future plans We have established a cohort of which multiple data levels allow for the integrative analysis of populations for translation of this information into biomarkers for disease, and which will offer new insights into disease mechanisms and prevention

Mediterranean diet intervention alters the gut microbiome in older people reducing frailty and improving health status : the NU-AGE 1-year dietary intervention across five European countries

Objective Ageing is accompanied by deterioration of multiple bodily functions and inflammation, which collectively contribute to frailty. We and others have shown that frailty co-varies with alterations in the gut microbiota in a manner accelerated by consumption of a restricted diversity diet. The Mediterranean diet (MedDiet) is associated with health. In the NU-AGE project, we investigated if a 1-year MedDiet intervention could alter the gut microbiota and reduce frailty. Design We profiled the gut microbiota in 612 non-frail or pre-frail subjects across five European countries (UK, France, Netherlands, Italy and Poland) before and after the administration of a 12-month long MedDiet intervention tailored to elderly subjects (NU-AGE diet). Results Adherence to the diet was associated with specific microbiome alterations. Taxa enriched by adherence to the diet were positively associated with several markers of lower frailty and improved cognitive function, and negatively associated with inflammatory markers including C-reactive protein and interleukin-17. Analysis of the inferred microbial metabolite profiles indicated that the diet-modulated microbiome change was associated with an increase in short/branch chained fatty acid production and lower production of secondary bile acids, p-cresols, ethanol and carbon dioxide. Microbiome ecosystem network analysis showed that the bacterial taxa that responded positively to the MedDiet intervention occupy keystone interaction positions, whereas frailty-associated taxa are peripheral in the networks. Conclusion Collectively, our findings support the feasibility of improving the habitual diet to modulate the gut microbiota which in turn has the potential to promote healthier ageing.Peer reviewe

Improvement of Insulin Sensitivity after Lean Donor Feces in Metabolic Syndrome Is Driven by Baseline Intestinal Microbiota Composition

The intestinal microbiota has been implicated in insulin resistance, although evidence regarding causality in humans is scarce. We therefore studied the effect of lean donor (allogenic) versus own (autologous) fecal microbiota transplantation (FMT) to male recipients with the metabolic syndrome. Whereas we did not observe metabolic changes at 18 weeks after FMT, insulin sensitivity at 6 weeks after allogenic FMT was significantly improved, accompanied by altered microbiota composition. We also observed changes in plasma metabolites such as gamma-aminobutyric acid and show that metabolic response upon allogenic FMT (defined as improved insulin sensitivity 6 weeks after FMT) is dependent on decreased fecal microbial diversity at baseline. In conclusion, the beneficial effects of lean donor FMT on glucose metabolism are associated with changes in intestinal microbiota and plasma metabolites and can be predicted based on baseline fecal microbiota composition.Peer reviewe