Grain versus AIN: Common rodent diets differentially affect health outcomes in adult C57BL/6j mice.

Semi-synthetic and grain-based diets are common rodent diets for biomedical research. Both diet types are considered nutritionally adequate to support breeding, growth, and long life, yet there are fundamental differences between them that may affect metabolic processes. We have characterized the effects of diet type on breeding outcomes, metabolic phenotype, and microbiota profile in adult mice. Healthy 8-week-old female and male C57BL/6J mice were fed a semi-synthetic or a grain-based diet for 12 weeks and changes in body weight and body composition were monitored. Breeding outcomes were determined. Body fat accumulation of female mice was lower on the semi-synthetic diet than on the grain-based diet. Pregnancy rate and newborn pup survival appeared to be lower in mice exposed to semi-synthetic diet compared to grain-based diet. Both female and male mice showed a profound change in fecal microbiota alpha and beta diversity depending on diet type. Our study shows that type of rodent diet may affect breeding outcomes whilst influencing metabolism and health of female laboratory mice. These factors have the potential to influence other experimental outcomes and the results suggest that semi-synthetic and grain-based diets are not interchangeable in research using rodent models. Careful consideration and increased understanding of the consequences of diet choice would lead to improvements in experimental design and reproducibility of study results.

Multi-part strategy for testing differential taxa abundance in sequencing data: A simulation study with an application to a microbiome study.

Comparing the microbiome across study arms is a recurrent goal in many studies. Standard statistical methods are often used for this purpose, however, they do not always represent the best choice in this context given the characteristics of microbiota sequencing data, e.g., non-negative, highly skewed counts with a large number of zeros. A multi-part strategy, that combines a two-part test (as described by Wagner et al., 2011), a Wilcoxon sum-rank test, a Chi-square and a Barnard's test was explored to compare the taxa abundance between study arms. The choice of the test is based on the data structure. The type I error of the multi-part strategy was evaluated by using a simulation study and the method was applied to real data. The script to perform the analysis with the multi-part approach is provided in the statistical software SAS. Several scenarios were simulated and in all of them the type I error was not inflated. Based on the statistical differences resulting from the two-part test (as described by Wagner et al., 2011) and the multi-part strategy (as proposed in this article), different biological implications can be extracted from the same comparison in the same data set. In the comparison of taxa abundance between study arms, we showed that careful attention needs to be paid on the data structure, in order to be able to choose an appropriate analysis method. Our approach selects the most suitable test according to the type of data observed, maintains a good type I error and is easily applicable by using the SAS macro provided.

Altered gut microbiome diversity and function in patients with propionic acidemia.

Propionic acidemia (PA) is an inherited metabolic disorder of propionate metabolism, where the gut microbiota may play a role in pathophysiology and therefore, represent a relevant therapeutic target. Little is known about the gut microbiota composition and activity in patients with PA. Although clinical practice varies between metabolic treatment centers, management of PA requires combined dietary and pharmaceutical treatments, both known to affect the gut microbiota. This study aimed to characterize the gut microbiota and its metabolites in fecal samples of patients with PA compared with healthy controls from the same household. Eight patients (aged 3-14y) and 8 controls (4-31y) were recruited from Center 1 (UK) and 7 patients (11-33y) and 6 controls (15-54y) from Center 2 (Austria). Stool samples were collected 4 times over 3 months, alongside data on dietary intakes and medication usage. Several microbial taxa differed between patients with PA and controls, particularly for Center 1, e.g., Proteobacteria levels were increased, whereas butyrate-producing genera, such as Roseburia and Faecalibacterium, were decreased. Most measured microbial metabolites were lower in patients with PA, and butyrate was particularly depleted in patients from Center 1. Furthermore, microbiota profile of these patients showed the lowest compositional and functional diversity, and lowest stability over 3 months. As the first study to map the gut microbiota of patients with PA, this work represents an important step forward for developing new therapeutic strategies to further improve PA clinical status. New dietary strategies should consider microbial propionate production as well as butyrate production and microbiota stability.

The Role of the Gut Microbiota in the Effects of Early-Life Stress and Dietary Fatty Acids on Later-Life Central and Metabolic Outcomes in Mice.

Early-life stress (ELS) leads to increased vulnerability for mental and metabolic disorders. We have previously shown that a low dietary ω-6/ω-3 polyunsaturated fatty acid (PUFA) ratio protects against ELS-induced cognitive impairments. Due to the importance of the gut microbiota as a determinant of long-term health, we here study the impact of ELS and dietary PUFAs on the gut microbiota and how this relates to the previously described cognitive, metabolic, and fatty acid profiles. Male mice were exposed to ELS via the limited bedding and nesting paradigm (postnatal day (P)2 to P9 and to an early diet (P2 to P42) with an either high (15) or low (1) ω-6 linoleic acid to ω-3 alpha-linolenic acid ratio. 16S rRNA was sequenced and analyzed from fecal samples at P21, P42, and P180. Age impacted α- and ß-diversity. ELS and diet together predicted variance in microbiota composition and affected the relative abundance of bacterial groups at several taxonomic levels in the short and long term. For example, age increased the abundance of the phyla Bacteroidetes, while it decreased Actinobacteria and Verrucomicrobia; ELS reduced the genera RC9 gut group and Rikenella, and the low ω-6/ω-3 diet reduced the abundance of the Firmicutes Erysipelotrichia. At P42, species abundance correlated with body fat mass and circulating leptin (e.g., Bacteroidetes and Proteobacteria taxa) and fatty acid profiles (e.g., Firmicutes taxa). This study gives novel insights into the impact of age, ELS, and dietary PUFAs on microbiota composition, providing potential targets for noninvasive (nutritional) modulation of ELS-induced deficits. IMPORTANCE Early-life stress (ELS) leads to increased vulnerability to develop mental and metabolic disorders; however, the biological mechanisms leading to such programming are not fully clear. Increased attention has been given to the importance of the gut microbiota as a determinant of long-term health and as a potential target for noninvasive nutritional strategies to protect against the negative impact of ELS. Here, we give novel insights into the complex interaction between ELS, early dietary ω-3 availability, and the gut microbiota across ages and provide new potential targets for (nutritional) modulation of the long-term effects of the early-life environment via the microbiota.

Preventive Effect of a Postbiotic and Prebiotic Mixture in a Rat Model of Early Life Rotavirus Induced-Diarrhea.

Rotavirus (RV) is the main cause of gastroenteritis in children. Prebiotics and, more recently, postbiotics are used for preventing and treating gastrointestinal infections. The aim of this study was to analyze the effects of a LactofidusTM, short-chain galacto-oligosaccharides (scGOS) and long-chain fructo-oligosaccharides (lcFOS) mixture, and their combination on RV infection, in a rat model, for early life diarrhea. Fifteen litters of suckling rats were intragastrically administered daily with the vehicle, the prebiotic mixture, the postbiotic or the combination. The RV was inoculated on day 5 and then fecal samples were clinically evaluated daily. Viral shedding, intestinal permeability assay, in vitro blocking assay, immunoglobulin profiles, and anti-RV response were assessed at day 8 and 16 of life. Cecal microbiota composition, intestinal gene expression, and short chain fatty acids (SCFAs) were analyzed at day 16. The incidence and severity of diarrhea were significantly reduced by all the supplementations. Moreover, they showed blocking activity, changes in the immunoglobulin profiles, in gut microbiota, and in the intestinal gene expression. The prebiotic mixture reduced gut permeability and changed the SCFA profile, whereas the postbiotic enhanced the expression of Toll-like receptors (TLRs). The combination preserved most of the individual observed effects, and furthermore, complementary effects, such as an increase in white blood cells and lymphocytes recruitment, as well as upregulation of TLR7 and TLR9 gene expression.

Early-life fecal microbiome and metabolome dynamics in response to an intervention with infant formula containing specific prebiotics and postbiotics.

This study examined fecal metabolome dynamics to gain greater functional insights into the interactions between nutrition and the activity of the developing gut microbiota in healthy term-born infants. The fecal samples used here originate from a randomized, controlled, double-blind clinical study that assessed the efficacy of infant formula with prebiotics and postbiotics (experimental arm) compared with a standard infant formula (control arm). A group of exclusively breast-fed term infants was used as a reference arm. First, conventional targeted physiological and microbial measurements were performed, which showed differences in fecal Bifidobacterium levels and corresponding activity (e.g., lactate levels). Next, the overall fecal microbiota composition was determined by 16S rRNA gene amplicon sequencing. The microbiota composition profiles showed several bacterial groups in the experimental arm to be significantly different from the control arm and mostly closer to the levels observed in the reference arm. Finally, we applied an untargeted UPLC-MS/MS approach to examine changes in the fecal metabolome. Fecal metabolome profiles showed the most distinct separation, up to 404 significantly different metabolites, between the study arms. Our data reveal that infant formula with specific prebiotics and postbiotics may trigger responses in the intestinal microbiota composition that brings the ensuing fecal metabolite profile of formula-fed infants closer toward those observed in breast-fed infants. Furthermore, our results demonstrate a clear need for establishing an infant gut metabolome reference database to translate these metabolite profile dynamics into functional and physiologically relevant responses.NEW & NOTEWORTHY Untargeted metabolomics techniques can provide a "snapshot" of an ecosystem in response to environmental stimuli, such as nutritional interventions. Our analyses of fecal samples from infants demonstrate the potential of phenotyping by metabolomics while deciphering the complex interactions of early-life nutrition and gut microbiome development.

Influence of timing of maternal antibiotic administration during caesarean section on infant microbial colonisation: a randomised controlled trial.

OBJECTIVE: Revised guidelines for caesarean section (CS) advise maternal antibiotic administration prior to skin incision instead of after umbilical cord clamping, unintentionally exposing the infant to antibiotics antenatally. We aimed to investigate if timing of intrapartum antibiotics contributes to the impairment of microbiota colonisation in CS born infants. DESIGN: In this randomised controlled trial, women delivering via CS received antibiotics prior to skin incision (n=20) or after umbilical cord clamping (n=20). A third control group of vaginally delivering women (n=23) was included. Faecal microbiota was determined from all infants at 1, 7 and 28 days after birth and at 3 years by 16S rRNA gene sequencing and whole-metagenome shotgun sequencing. RESULTS: Compared with vaginally born infants, profound differences were found in microbial diversity and composition in both CS groups in the first month of life. A decreased abundance in species belonging to the genera Bacteroides and Bifidobacterium was found with a concurrent increase in members belonging to the phylum Proteobacteria. These differences could not be observed at 3 years of age. No statistically significant differences were observed in taxonomic and functional composition of the microbiome between both CS groups at any of the time points. CONCLUSION: We confirmed that microbiome colonisation is strongly affected by CS delivery. Our findings suggest that maternal antibiotic administration prior to CS does not result in a second hit on the compromised microbiome. Future, larger studies should confirm that antenatal antibiotic exposure in CS born infants does not aggravate colonisation impairment and impact long-term health.

Human Milk Oligosaccharide 3'-GL Improves Influenza-Specific Vaccination Responsiveness and Immunity after Deoxynivalenol Exposure in Preclinical Models.

Deoxynivalenol (DON), a highly prevalent mycotoxin food contaminant, is known to have immunotoxic effects. In the current study, the potential of dietary interventions with specific mixtures of trans-galactosyl-oligosaccharides (TOS) to alleviate these effects were assessed in a murine influenza vaccination model. Vaccine-specific immune responses were measured in C57Bl/6JOlaHsd mice fed diets containing DON, TOS or a combination, starting 2 weeks before the first vaccination. The direct effects of TOS and its main oligosaccharide, 3'-galactosyl-lactose (3'-GL), on DON-induced damage were studied in Caco-2 cells, as an in vitro model of the intestinal epithelial barrier. Exposure to DON significantly reduced vaccine-specific immune responses and the percentages of Tbet+ Th1 cells and B cells in the spleen. DON significantly altered epithelial structure and integrity in the ileum and reduced the SCFA levels in the cecum. Adding TOS into DON-containing diets significantly improved vaccine-specific immune responses, restored the immune cell balance in the spleen and increased SCFA concentrations in the cecum. Incubating Caco-2 cells with TOS and 3'-GL in vitro further confirmed their protective effects against DON-induced barrier disruption, supporting immune modulation. Overall, dietary intervention with TOS can attenuate the adverse effects of DON on Th1-mediated immune responses and gut homeostasis. These beneficial properties might be linked to the high levels of 3'-GL in TOS.

Effects of a Postbiotic and Prebiotic Mixture on Suckling Rats' Microbiota and Immunity.

Human milk serves as a model for infant formula providing nutritional solutions for infants not able to receive enough mother's milk. Infant formulas aim to mimic the composition and functionality of human milk by providing ingredients reflecting those of the latest human milk insights, such as prebiotics, probiotics and postbiotics. The aim of this study was to examine the effects of the supplementation with a postbiotic (LactofidusTM) and its combination with the prebiotics short-chain galactooligosaccharides (scGOS) and long-chain fructooligosaccharides (lcFOS) in a preclinical model of healthy suckling rats. Pups were supplemented daily with LactofidusTM (POST group) and/or scGOS/lcFOS (P+P and PRE groups, respectively). Body weight and fecal consistency were analyzed. At the end of the study, immunoglobulin (Ig) profile, intestinal gene expression, microbiota composition and short chain fatty acid (SCFA) proportion were quantified. The supplementation with all nutritional interventions modulated the Ig profile, but the prebiotic mixture and the postbiotic induced differential effects: whereas scGOS/lcFOS induced softer feces and modulated microbiota composition and SCFA profile, Lactofidus™ upregulated Toll-like receptors gene expression. The use of the combination of scGOS/lcFOS and Lactofidus™ showed the effects observed for the oligosaccharides separately, as well as showing a synergistic impact on animal growth. Thus, the combined use of both products seems to be a good strategy to modulate immune and microbial features in early life.

Fermented infant formula (with Bifidobacterium breve C50 and Streptococcus thermophilus O65) with prebiotic oligosaccharides is safe and modulates the gut microbiota towards a microbiota closer to that of breastfed infants.

BACKGROUND & AIMS: Microbiome-modulators can help positively steer early-life microbiota development but their effects on microbiome functionality and associated safety and tolerance need to be demonstrated. We investigated the microbiome impact of a new combination of bioactive compounds, produced by the food-grade microorganisms Bifidobacterium breve C50 and Streptococcus thermophilus ST065 during a fermentation process, and prebiotics in an infant formula. Tolerance and safety were also assessed. METHODS: An exploratory prospective, randomized, double-blind, controlled, multi-centre study was designed to investigate the effect of bioactive compounds and prebiotics (short-chain galacto-oligosaccharides (scGOS)/long-chain fructo-oligosaccharides (lcFOS) 9:1). Experimental formulas containing these bioactive compounds and prebiotics (FERM/scGOS/lcFOS), prebiotics (scGOS/lcFOS), or bioactive compounds (FERM), were compared to a standard cow's milk-based control formula (Control). Exclusively breastfed infants were included as a reference arm since exclusive breastfeeding is considered as the optimal feeding for infants. The study lasted six months and included visits to health care professionals at baseline, two, four and six months of age. Stool SIgA concentration was the primary study outcome parameter. RESULTS: There were 280 infants randomized over the experimental arms and 70 infants entered the breastfed-reference arm. Demographics were balanced, growth and tolerance parameters were according to expectation and adverse events were limited. At four months of age the median SIgA concentration in the FERM/scGOS/lcFOS group was significantly higher compared to the Control group (p = 0.03) and was more similar to the concentrations found in the breastfed-reference group. Bifidobacterium increased over time in all groups. The FERM/scGOS/lcFOS combination resulted in a microbiota composition and metabolic activity closer to the breastfed infants' microbiome. CONCLUSION: The FERM/scGOS/lcFOS combination showed a significant positive effect on SIgA levels. All formulas tested were associated with normal growth and were well-tolerated. Additionally, at four months of age the FERM/scGOS/lcFOS formula brought the microbiome composition and metabolic activity closer towards that of breastfed infants. CLINICAL TRIAL REGISTRY: Registration number NTR2726 (Netherlands Trial Register; www.trialregister.nl/).

Early life nutrition influences susceptibility to chronic inflammatory colitis in later life.

The first thousand days of life are a critical time of development in humans during which the risk profile for diseases in later life can be modified. Nevertheless, long-term consequences of early environment on susceptibility to intestinal diseases have not yet been assessed. Using a mouse model of postnatal growth restriction (PNGR), we showed that early life nutrition influences intestinal maturation and gut health in later life. PNGR induced an alteration of the intestinal barrier in pups at weaning, resulting in increased intestinal permeability, and affected gut bacterial colonization. Specifically, pups with PNGR harbored a decreased bacterial diversity, higher Enterococcus spp., Staphylococcus spp., and Escherichia-Shigella spp., and lower Odoribacter spp. and several members of the Lachnospiraceae family. The lack of an efficient intestinal barrier in early life and the dysbiosis induced by PNGR were associated with a higher susceptibility to chronic colitis in adulthood.

Oligosaccharides Modulate Rotavirus-Associated Dysbiosis and TLR Gene Expression in Neonatal Rats.

Colonization of the gut in early life can be altered through multiple environmental factors. The present study aimed to investigate the effects of 2'-fucosyllactose (2'-FL), a mixture of short-chain galactooligosaccharides/long-chain fructooligosaccharides (scGOS/lcFOS) 9:1 and their combination (scGOS/lcFOS/2'-FL) on dysbiosis induced during rotavirus (RV) diarrhea in neonatal rats, elucidating crosstalk between bacteria and the immune system. The dietary interventions were administered daily by oral gavage at days 2-8 of life in neonatal Lewis rats. On day 5, RV SA11 was intragastrically delivered to induce infection and diarrhea assessment, microbiota composition, and gene expression of Toll-like receptors (TLRs) in the small intestine were studied. All dietary interventions showed reduction in clinical variables of RV-induced diarrhea. RV infection increased TLR2 expression, whereas 2'-FL boosted TLR5 and TLR7 expressions and scGOS/lcFOS increased that of TLR9. RV-infected rats displayed an intestinal dysbiosis that was effectively prevented by the dietary interventions, and consequently, their microbiota was more similar to microbiota of the noninfected groups. The preventive effect of 2'-FL, scGOS/lcFOS, and their combination on dysbiosis associated to RV diarrhea in rats could be due to changes in the crosstalk between gut microbiota and the innate immune system.

Immunomodulatory and Prebiotic Effects of 2'-Fucosyllactose in Suckling Rats.

Human milk oligosaccharides are unconjugated complex glycans present in high concentration in human milk that serve as pre-biotics and immunomodulators. They are not primarily absorbed or metabolized by the infant and reach the lower part of the intestinal tract unaltered. One of the main oligosaccharides found in human milk is 2'-fucosyllactose (2'-FL). This study aimed to investigate the effects of daily oral administration of 2'-FL in healthy suckling rats. From days 2 to 16 of life, rats were daily given the oligosaccharide (2'-FL) or vehicle (REF), weighed and their stool characteristics were assessed. On days 8 and 16 of life the morphometry, intestinal architecture, and cytokine release, mesenteric lymph nodes cell composition, plasma immunoglobulin concentrations, fecal microbiota composition, cecal short-chain fatty acids content, and the urinary metabolic profile were assessed. Animals given 2'-FL showed higher plasma IgG and IgA and more T cell subsets in the mesenteric lymph nodes on day 16. Moreover, at intestinal level, villus heights, and areas were increased on day 8. Cecal samples displayed a higher Lactobacillus proportion and a different urinary metabolic profile was observed on day 8, and a higher proportion of butyrate on day 16. In conclusion, supplementation of 2'-FL in early life has a pre-biotic and intestinal trophic effect and promotes maturation of the immune system.

Metaproteomic and 16S rRNA Gene Sequencing Analysis of the Infant Fecal Microbiome.

A metaproteomic analysis was conducted on the fecal microbiome of eight infants to characterize global protein and pathway expression. Although mass spectrometry-based proteomics is now a routine tool, analysis of the microbiome presents specific technical challenges, including the complexity and dynamic range of member taxa, the need for well-annotated metagenomic databases, and high inter-protein sequence redundancy and similarity. In this study, an approach was developed for assessment of biological phenotype and metabolic status, as a functional complement to DNA sequence analysis. Fecal samples were prepared and analysed by tandem mass spectrometry and a homology-based meta-clustering strategy was used to combine peptides from multiple species into representative proteins. In total, 15,250 unique peptides were sequenced and assigned to 2154 metaclusters, which were then assigned to pathways and functional groups. Differences were noted in several pathways, consistent with the dominant genera observed in different subjects. Although this study was not powered to draw conclusions from the comparisons, the results obtained demonstrate the applicability of this approach and provide the methods needed for performing semi-quantitative comparisons of human fecal microbiome composition, physiology and metabolism, as well as a more detailed assessment of microbial composition in comparison to 16S rRNA gene sequencing.

Synbiotics Combined with Glutamine Stimulate Brain Development and the Immune System in Preterm Pigs.

Background: Preterm infants are born with an immature gut, brain, and immune system, predisposing them to short- and long-term complications. Objective: We hypothesized that a milk diet supplemented with pre- and probiotics (i.e. synbiotics) and glutamine would improve gut, brain, and immune maturation in preterm neonates, using preterm pigs as a model. Methods: Preterm pigs (Landrace x Yorkshire x Duroc, n = 40, delivered by c-section at 90% of gestation) were reared individually until day 23 after birth under highly standardized conditions. Piglets in the intervention group (PPG, n = 20) were fed increasing volumes of bovine milk supplemented with prebiotics (short-chain galacto- and long chain fructo-oligosaccharides 9:1, 4-12 g/L), probiotics (Bifidobacterium breve M16-V, 3 × 109 CFU/d) and l-glutamine [0.15-0.30 g/(kg · d)], and compared with pigs fed bovine milk with added placebo compounds as control (CON, n = 20). Clinical, gastrointestinal, immunological, cognitive, and neurological endpoints were measured. Results: The PPG pigs showed more diarrhea but weight gain, body composition, and gut parameters were similar between the groups. Cognitive performance, assessed in a T-maze, was significantly higher in PPG pigs (P < 0.01), whereas motor function and exploratory interest were similar between the groups. Using ex vivo diffusion imaging, the orientation dispersion index in brain cortical gray matter was 50% higher (P = 0.04), and fractional anisotropy value was 7% lower (P = 0.05) in PPG pigs compared with CON pigs, consistent with increased dendritic branching in PPG. In associative fibers, radial diffusivity was lower and fractional anisotropy was higher in PPG pigs compared with CON pigs (all P < 0.05), while measures in the internal capsule showed a tendency towards reduced radial diffusivity and mean diffusivity (both P = 0.09). On day 23 pigs in the PPG group showed higher blood leukocyte numbers (+43%), neutrophil counts (+100%), and phagocytic rates (+24%), relative to CON, all P < 0.05. Conclusion: Preterm pigs supplemented with Bifidobacterium breve, galacto- and fructo-oligosaccharides, and l-glutamine showed enhanced neuronal and immunological development. The findings indicate the potential for targeted nutritional interventions after preterm birth, to support development of important systems such as immunity and brain.

Specific synbiotics in early life protect against diet-induced obesity in adult mice.

AIMS: The metabolic state of human adults is associated with their gut microbiome. The symbiosis between host and microbiome is initiated at birth, and early life microbiome perturbation can disturb health throughout life. Here, we determined how beneficial microbiome interventions in early life affect metabolic health in adulthood. METHODS: Postnatal diets were supplemented with either prebiotics (scGOS/lcFOS) or synbiotics (scGOS/lcFOS with Bifidobacterium breve M-16 V) until post-natal (PN) day 42 in a well-established rodent model for nutritional programming. Mice were subsequently challenged with a high-fat Western-style diet (WSD) for 8 weeks. Body weight and composition were monitored, as was gut microbiota composition at PN21, 42 and 98. Markers of glucose homeostasis, lipid metabolism and host transcriptomics of 6 target tissues were determined in adulthood (PN98). RESULTS: Early life synbiotics protected mice against WSD-induced excessive fat accumulation throughout life, replicable in 2 independent European animal facilities. Adult insulin sensitivity and dyslipidaemia were improved and most pronounced changes in gene expression were observed in the ileum. We observed subtle changes in faecal microbiota composition, both in early life and in adulthood, including increased abundance of Bifidobacterium. Microbiota transplantation using samples collected from synbiotics-supplemented adolescent mice at PN42 to age-matched germ-free recipients did not transfer the beneficial phenotype, indicating that synbiotics-modified microbiota at PN42 is not sufficient to transfer long-lasting protection of metabolic health status. CONCLUSION: Together, these findings show the potential and importance of timing of synbiotic interventions in early life during crucial microbiota development as a preventive measure to lower the risk of obesity and improve metabolic health throughout life.

Partly Fermented Infant Formulae With Specific Oligosaccharides Support Adequate Infant Growth and Are Well-Tolerated.

OBJECTIVE: Fermented formulae (FERM) and a specific mixture of 90% short-chain galacto-oligosaccharides and 10% long-chain fructo-oligosaccharides (scGOS/lcFOS; 9:1) have a potential beneficial effect on gastrointestinal function and microbiota development in infants. The present study assessed the safety and tolerance of the combination of partly fermented infant milk formulae and scGOS/lcFOS compared with either 1 feature, in healthy term infants. METHODS: Four hundred thirty-two infants were enrolled before 28 days of age and followed up to 17 weeks of age and assigned to 1 of the 4 groups: (i) formula with scGOS/lcFOS, (ii) scGOS/lcFOS + 15% FERM, (iii) scGOS/lcFOS + 50% FERM, or (iv) 50% fermented formula (50% FERM). Primary outcome was daily weight gain during intervention (equivalence criterion: difference in daily weight gain ≤3 g/day). Infants' anthropometrics, formula intake, number, and type of (serious) AEs were monitored monthly. Stool samples were collected at baseline and after 17 weeks for analysis of physiological and microbiological parameters. RESULTS: Equivalence of weight gain per day was demonstrated in both the intention-to-treat and per-protocol population, with a mean weight gain (SD) of 29.7 (6.1), 28.2 (4.8), 28.5 (5.0), and 28.7 (5.9) g/day for the groups i to iv respectively. No differences were observed in other growth parameters, formula intake, and the number or severity of AEs. In all scGOS/lcFOS-containing formulae, a beneficial effect of scGOS/lcFOS was observed, indicated by the lower pH, lower Clostridium difficile levels, and higher secretory immunoglobulin A levels. CONCLUSIONS: The partly fermented infant milk formulae containing the specific mixture scGOS/lcFOS were well-tolerated and resulted in normal growth in healthy infants.

Fat, fibre and cancer risk in African Americans and rural Africans.

Rates of colon cancer are much higher in African Americans (65:100,000) than in rural South Africans (<5:100,000). The higher rates are associated with higher animal protein and fat, and lower fibre consumption, higher colonic secondary bile acids, lower colonic short-chain fatty acid quantities and higher mucosal proliferative biomarkers of cancer risk in otherwise healthy middle-aged volunteers. Here we investigate further the role of fat and fibre in this association. We performed 2-week food exchanges in subjects from the same populations, where African Americans were fed a high-fibre, low-fat African-style diet and rural Africans a high-fat, low-fibre western-style diet, under close supervision. In comparison with their usual diets, the food changes resulted in remarkable reciprocal changes in mucosal biomarkers of cancer risk and in aspects of the microbiota and metabolome known to affect cancer risk, best illustrated by increased saccharolytic fermentation and butyrogenesis, and suppressed secondary bile acid synthesis in the African Americans.

Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes.

Most current approaches for analyzing metagenomic data rely on comparisons to reference genomes, but the microbial diversity of many environments extends far beyond what is covered by reference databases. De novo segregation of complex metagenomic data into specific biological entities, such as particular bacterial strains or viruses, remains a largely unsolved problem. Here we present a method, based on binning co-abundant genes across a series of metagenomic samples, that enables comprehensive discovery of new microbial organisms, viruses and co-inherited genetic entities and aids assembly of microbial genomes without the need for reference sequences. We demonstrate the method on data from 396 human gut microbiome samples and identify 7,381 co-abundance gene groups (CAGs), including 741 metagenomic species (MGS). We use these to assemble 238 high-quality microbial genomes and identify affiliations between MGS and hundreds of viruses or genetic entities. Our method provides the means for comprehensive profiling of the diversity within complex metagenomic samples.

Reset of a critically disturbed microbial ecosystem: faecal transplant in recurrent Clostridium difficile infection.

Recurrent Clostridium difficile infection (CDI) can be effectively treated by infusion of a healthy donor faeces suspension. However, it is unclear what factors determine treatment efficacy. By using a phylogenetic microarray platform, we assessed composition, diversity and dynamics of faecal microbiota before, after and during follow-up of the transplantation from a healthy donor to different patients, to elucidate the mechanism of action of faecal infusion. Global composition and network analysis of the microbiota was performed in faecal samples from nine patients with recurrent CDI. Analyses were performed before and after duodenal donor faeces infusion, and during a follow-up of 10 weeks. The microbiota data were compared with that of the healthy donors. All patients successfully recovered. Their intestinal microbiota changed from a low-diversity diseased state, dominated by Proteobacteria and Bacilli, to a more diverse ecosystem resembling that of healthy donors, dominated by Bacteroidetes and Clostridium groups, including butyrate-producing bacteria. We identified specific multi-species networks and signature microbial groups that were either depleted or restored as a result of the treatment. The changes persisted over time. Comprehensive and deep analyses of the microbiota of patients before and after treatment exposed a therapeutic reset from a diseased state towards a healthy profile. The identification of microbial groups that constitute a niche for C. difficile overgrowth, as well as those driving the reinstallation of a healthy intestinal microbiota, could contribute to the development of biomarkers predicting recurrence and treatment outcome, identifying an optimal microbiota composition that could lead to targeted treatment strategies.