Supplementation with inulin-type fructans affects gut microbiota and attenuates some of the cardiometabolic benefits of a plant-based diet in individuals with overweight or obesity.

Background: The gut microbiota has emerged as a potential therapeutic target to improve the management of obesity and its comorbidities. Objective: We investigated the impact of a high fiber (∼38 g/d) plant-based diet, consumed ad libitum, with or without added inulin-type fructans (ITF), on the gut microbiota composition and cardiometabolic outcomes in subjects with obesity. We also tested if baseline Prevotella/Bacteroides (P/B) ratio predicts weight loss outcomes. Methods: This is a secondary exploratory analysis from the PREVENTOMICS study, in which 100 subjects (82 completers) aged 18-65 years with body mass index 27-40 kg/m2 were randomized to 10 weeks of double-blinded treatment with a personalized or a generic plant-based diet. Changes from baseline to end-of-trial in gut microbiota composition (16S rRNA gene amplicon sequencing), body composition, cardiometabolic health and inflammatory markers were evaluated in the whole cohort (n = 82), and also compared in the subgroup of subjects who were supplemented with an additional 20 g/d ITF-prebiotics (n = 21) or their controls (n = 22). Results: In response to the plant-based diet, all subjects lost weight (-3.2 [95% CI -3.9, -2.5] kg) and experienced significant improvements in body composition and cardiometabolic health indices. Addition of ITF to the plant-based diet reduced microbial diversity (Shannon index) and selectively increased Bifidobacterium and Faecalibacterium (q < 0.05). The change in the latter was significantly associated with higher values of insulin and HOMA-IR and lower HDL cholesterol. In addition, the LDL:HDL ratio and the concentrations of IL-10, MCP-1 and TNFα were significantly elevated in the ITF-subgroup. There was no relationship between baseline P/B ratio and changes in body weight (r = -0.07, p = 0.53). Conclusion: A plant-based diet consumed ad libitum modestly decreases body weight and has multiple health benefits in individuals with obesity. Addition of ITF-prebiotics on top this naturally fiber-rich background selectively changes gut microbiota composition and attenuates some of the realized cardiometabolic benefits. Clinical trial registration: [https://clinicaltrials.gov/ct2/show/NCT04590989], identifier [NCT04590989].

Human milk oligosaccharides induce acute yet reversible compositional changes in the gut microbiota of conventional mice linked to a reduction of butyrate levels.

Human Milk Oligosaccharides (HMOs) are glycans with prebiotic properties known to drive microbial selection in the infant gut, which in turn influences immune development and future health. Bifidobacteria are specialized in HMO degradation and frequently dominate the gut microbiota of breastfed infants. However, some species of Bacteroidaceae also degrade HMOs, which may prompt selection also of these species in the gut microbiota. To investigate to what extent specific HMOs affect the abundance of naturally occurring Bacteroidaceae species in a complex mammalian gut environment, we conducted a study in 40 female NMRI mice administered three structurally different HMOs, namely 6'sialyllactose (6'SL, n = 8), 3-fucosyllactose (3FL, n = 16), and Lacto-N-Tetraose (LNT, n = 8), through drinking water (5%). Compared to a control group receiving unsupplemented drinking water (n = 8), supplementation with each of the HMOs significantly increased both the absolute and relative abundance of Bacteroidaceae species in faecal samples and affected the overall microbial composition analyzed by 16s rRNA amplicon sequencing. The compositional differences were mainly attributed to an increase in the relative abundance of the genus Phocaeicola (formerly Bacteroides) and a concomitant decrease of the genus Lacrimispora (formerly Clostridium XIVa cluster). During a 1-week washout period performed specifically for the 3FL group, this effect was reversed. Short-chain fatty acid analysis of faecal water revealed a decrease in acetate, butyrate and isobutyrate levels in animals supplemented with 3FL, which may reflect the observed decrease in the Lacrimispora genus. This study highlights HMO-driven Bacteroidaceae selection in the gut environment, which may cause a reduction of butyrate-producing clostridia.

Escherichia coli Promoters with Consistent Expression throughout the Murine Gut.

Advanced microbial therapeutics have great potential as a novel modality to diagnose and treat a wide range of diseases. Yet, to realize this potential, robust parts for regulating gene expression and consequent therapeutic activity in situ are needed. In this study, we characterized the expression level of more than 8000 variants of the Escherichia coli sigma factor 70 (σ70) promoter in a range of different environmental conditions and growth states using fluorescence-activated cell sorting and deep sequencing. Sampled conditions include aerobic and anaerobic culture in the laboratory as well as growth in several locations of the murine gastrointestinal tract. We found that σ70 promoters in E. coli generally maintain consistent expression levels across the murine gut (R2: 0.55-0.85, p value < 1 × 10-5), suggesting a limited environmental influence but a higher variability between in vitro and in vivo expression levels, highlighting the challenges of translating in vitro promoter activity to in vivo applications. Based on these data, we design the Schantzetta library, composed of eight promoters spanning a wide expression range and displaying a high degree of robustness in both laboratory and in vivo conditions (R2 = 0.98, p = 0.000827). This study provides a systematic assessment of the σ70 promoter activity in E. coli as it transits the murine gut leading to the definition of robust expression cassettes that could be a valuable tool for reliable engineering and development of advanced microbial therapeutics.

Gut Microbiota Development: Influence of Diet from Infancy to Toddlerhood.

Early life is a critical period as our gut microbiota establishes here and may impact both current and future health. Thus, it is of importance to understand how different factors govern the complex microbial colonization patterns in this period. The gut microbiota changes substantially during infancy and toddlerhood in terms of both taxonomic composition and diversity. This developmental trajectory differs by a variety of factors, including term of birth, mode of birth, intake of antibiotics, presence of furred pets, siblings and family members, host genetics, local environment, geographical location, and maternal and infant/toddler diet. The type of milk feeding and complementary feeding is particularly important in early and late infancy/toddlerhood, respectively. Breastfeeding, due to the supply of human milk oligosaccharide into the gut, promotes the growth of specific human milk oligosaccharide (HMO)-utilizing Bifidobacterium species that dominate the ecosystem as long as the infant is primarily breastfed. These species perform saccharolytic fermentation in the gut and produce metabolites with physiological effects that may contribute to protection against infectious and immune-related diseases. Formula feeding, due to its lack of HMOs and higher protein content, give rise to a more diverse gut microbiota that contains more opportunistic pathogens and results in a more proteolytic metabolism in the gut. Complementary feeding, due to the introduction of dietary fibers and new protein sources, induces a shift in the gut microbiota and metabolism away from the milk-adapted and toward a more mature and diverse adult-like community with increased abundances of short chain fatty acid-producing bacterial taxa. While the physiological implication of these complementary diet-induced changes remains to be established, a few recent studies indicate that an inadequately matured gut microbiota may be causally related to poor growth and development. Further studies are required to expand our knowledge on interactions between diet, gut microbiota, and health in the early life setting.

Settlers of our inner surface - factors shaping the gut microbiota from birth to toddlerhood.

During the first 3 years of life, the microbial ecosystem within the human gut undergoes a process that is unlike what happens in this ecosystem at any other time of our life. This period in time is considered a highly important developmental window, where the gut microbiota is much less resilient and much more responsive to external and environmental factors than seen in the adult gut. While advanced bioinformatics and clinical correlation studies have received extensive focus within studies of the human microbiome, basic microbial growth physiology has attracted much less attention, although it plays a pivotal role to understand the developing gut microbiota during early life. In this review, we will thus take a microbial ecology perspective on the analysis of factors that influence the temporal development of the infant gut microbiota. Such factors include sources of microbes that seed the intestinal environment, physico-chemical (abiotic) conditions influencing microbial growth and the availability of nutrients needed by the intestinal microbes.

Maternal milk microbiota and oligosaccharides contribute to the infant gut microbiota assembly.

Breastfeeding protects against diseases, with potential mechanisms driving this being human milk oligosaccharides (HMOs) and the seeding of milk-associated bacteria in the infant gut. In a cohort of 34 mother-infant dyads we analyzed the microbiota and HMO profiles in breast milk samples and infant's feces. The microbiota in foremilk and hindmilk samples of breast milk was compositionally similar, however hindmilk had higher bacterial load and absolute abundance of oral-associated bacteria, but a lower absolute abundance of skin-associated Staphylococcus spp. The microbial communities within both milk and infant's feces changed significantly over the lactation period. On average 33% and 23% of the bacterial taxa detected in infant's feces were shared with the corresponding mother's milk at 5 and 9 months of age, respectively, with Streptococcus, Veillonella and Bifidobacterium spp. among the most frequently shared. The predominant HMOs in feces associated with the infant's fecal microbiota, and the dominating infant species B. longum ssp. infantis and B. bifidum correlated inversely with HMOs. Our results show that breast milk microbiota changes over time and within a feeding session, likely due to transfer of infant oral bacteria during breastfeeding and suggest that milk-associated bacteria and HMOs direct the assembly of the infant gut microbiota.

Intestinal Enterococcus abundance correlates inversely with excessive weight gain and increased plasma leptin in breastfed infants.

Epidemiological evidence indicates that breastfeeding provides protection against development of overweight/obesity. Nonetheless, a small subgroup of infants undergo excessive weight gain during exclusive breastfeeding, a phenomenon that remains unexplained. Breast milk contains both gut-seeding microbes and substrates for microbial growth in the gut of infants, and a large body of evidence suggests a role for gut microbes in host metabolism. Based on the recently established SKOT III cohort, we investigated the role of the infant gut microbiota in excessive infant weight gain during breastfeeding, including 30 exclusively breastfed infants, 13 of which exhibited excessive weight gain and 17 controls which exhibited normal weight gain during infancy. Infants undergoing excessive weight gain during breastfeeding had a reduced abundance of gut Enterococcus as compared with that observed in the controls. Within the complete cohort, Enterococcus abundance correlated inversely with age/gender-adjusted body-weight, body-mass index and waist circumference, body fat and levels of plasma leptin. The reduced abundance of Enterococcus in infants with excessive weight gain was coupled to a lower content of Enterococcus in breast milk samples of their mothers than seen for mothers in the control group. Together, this suggests that lack of breast milk-derived gut-seeding Enterococci may contribute to excessive weight gain in breastfed infants.

Faecalibacterium Gut Colonization Is Accelerated by Presence of Older Siblings.

Faecalibacterium prausnitzii is a highly abundant human gut microbe in healthy individuals, but it is present at reduced levels in individuals with gastrointestinal inflammatory diseases. It has therefore been suggested to constitute a marker of a healthy gut and is associated with anti-inflammatory properties. However, factors affecting the colonization of F. prausnitzii in the human gut during early life are very poorly understood. By analysis of 16S rRNA amplicon sequencing data from three separate infant study populations, we determined the colonization dynamics of Faecalibacterium and factors affecting its establishment in the gut. We found that in particular, the presence of older siblings was consistently associated with Faecalibacterium gut colonization during late infancy and conclude that acquisition of Faecalibacterium is very likely to be accelerated through transfer between siblings. IMPORTANCEFaecalibacterium prausnitzii has been suggested to constitute a key marker of a healthy gut, yet the factors shaping the colonization of this highly oxygen-sensitive, non-spore-forming species in the intestinal environment remain poorly understood. Here, we provide evidence from three separate infant study populations that F. prausnitzii colonization in the gut happens during late infancy and is affected by the number of older siblings in the family. We conclude that Faecalibacterium acquisition is highly likely to be accelerated by contact between siblings. Bearing in mind the immunoregulatory properties of F. prausnitzii and the well-established protective effects against allergic disorders related to the presence of older siblings, early colonization of this species may have profound consequences for child health.

Administration of two probiotic strains during early childhood does not affect the endogenous gut microbiota composition despite probiotic proliferation.

BACKGROUND: Probiotics are increasingly applied to prevent and treat a range of infectious, immune related and gastrointestinal diseases. Despite this, the mechanisms behind the putative effects of probiotics are poorly understood. One of the suggested modes of probiotic action is modulation of the endogenous gut microbiota, however probiotic intervention studies in adults have failed to show significant effects on gut microbiota composition. The gut microbiota of young children is known to be unstable and more responsive to external factors than that of adults. Therefore, potential effects of probiotic intervention on gut microbiota may be easier detectable in early life. We thus investigated the effects of a 6 month placebo-controlled probiotic intervention with Bifidobacterium animalis subsp. lactis (BB-12®) and Lactobacillus rhamnosus (LGG®) on gut microbiota composition and diversity in more than 200 Danish infants (N = 290 enrolled; N = 201 all samples analyzed), as assessed by 16S rRNA amplicon sequencing. Further, we evaluated probiotic presence and proliferation by use of specific quantitative polymerase chain reaction (qPCR). RESULTS: Probiotic administration did not significantly alter gut microbiota community structure or diversity as compared to placebo. The probiotic strains were detected in 91.3% of the fecal samples from children receiving probiotics and in 1% of the placebo treated children. Baseline gut microbiota was not found to predict the ability of probiotics to establish in the gut after the 6 month intervention. Within the probiotics group, proliferation of the strains LGG® and BB-12® in the gut was detected in 44.7% and 83.5% of the participants, respectively. A sub-analysis of the gut microbiota including only individuals with detected growth of the probiotics LGG® or BB-12® and comparing these to placebo revealed no differences in community structure or diversity. CONCLUSION: Six months of probiotic administration during early life did not change gut microbiota community structure or diversity, despite active proliferation of the administered probiotic strains. Therefore, alteration of the healthy infant gut microbiota is not likely to be a prominent mechanism by which these specific probiotics works to exert beneficial effects on host health. TRIAL REGISTRATION: NCT02180581 . Registered 30 June 2014.

Infant Gut Microbiota Development Is Driven by Transition to Family Foods Independent of Maternal Obesity.

The first years of life are paramount in establishing our endogenous gut microbiota, which is strongly affected by diet and has repeatedly been linked with obesity. However, very few studies have addressed the influence of maternal obesity on infant gut microbiota, which may occur either through vertically transmitted microbes or through the dietary habits of the family. Additionally, very little is known about the effect of diet during the complementary feeding period, which is potentially important for gut microbiota development. Here, the gut microbiotas of two different cohorts of infants, born either of a random sample of healthy mothers (n = 114), or of obese mothers (n = 113), were profiled by 16S rRNA amplicon sequencing. Gut microbiota data were compared to breastfeeding patterns and detailed individual dietary recordings to assess effects of the complementary diet. We found that maternal obesity did not influence microbial diversity or specific taxon abundances during the complementary feeding period. Across cohorts, breastfeeding duration and composition of the complementary diet were found to be the major determinants of gut microbiota development. In both cohorts, gut microbial composition and alpha diversity were thus strongly affected by introduction of family foods with high protein and fiber contents. Specifically, intake of meats, cheeses, and Danish rye bread, rich in protein and fiber, were associated with increased alpha diversity. Our results reveal that the transition from early infant feeding to family foods is a major determinant for gut microbiota development. IMPORTANCE The potential influence of maternal obesity on infant gut microbiota may occur either through vertically transmitted microbes or through the dietary habits of the family. Recent studies have suggested that the heritability of obesity may partly be caused by the transmission of "obesogenic" gut microbes. However, the findings presented here suggest that maternal obesity per se does not affect the overall composition of the gut microbiota and its development after introduction of complementary foods. Rather, progression in complementary feeding is found to be the major determinant for gut microbiota establishment. Expanding our understanding of the influence of complementary diet on the development and establishment of the gut microbiota will provide us with the knowledge to tailor a beneficial progression of our intestinal microbial community.

Having older siblings is associated with gut microbiota development during early childhood.

BACKGROUND: Evidence suggests that early life infections, presence of older siblings and furred pets in the household affect the risk of developing allergic diseases through altered microbial exposure. Recently, low gut microbial diversity during infancy has also been linked with later development of allergies. We investigated whether presence of older siblings, furred pets and early life infections affected gut microbial communities at 9 and 18 months of age and whether these differences were associated with the cumulative prevalence of atopic symptoms of eczema and asthmatic bronchitis at 3 years of age. Bacterial compositions and diversity indices were determined in fecal samples collected from 114 infants in the SKOT I cohort at age 9 and 18 months by 16S rRNA gene sequencing. These were compared to the presence of older siblings, furred pets and early life infections and the cumulative prevalence of diagnosed asthmatic bronchitis and self-reported eczema at 3 years of age. RESULTS: The number of older siblings correlated positively with bacterial diversity (p = 0.030), diversity of the phyla Firmicutes (p = 0.013) and Bacteroidetes (p = 0.004) and bacterial richness (p = 0.006) at 18 months. Further, having older siblings was associated with increased relative abundance of several bacterial taxa at both 9 and 18 months of age. Compared to the effect of having siblings, presence of household furred pets and early life infections had less pronounced effects on the gut microbiota. Gut microbiota characteristics were not significantly associated with cumulative occurrence of eczema and asthmatic bronchitis during the first 3 years of life. CONCLUSIONS: Presence of older siblings is associated with increased gut microbial diversity and richness during early childhood, which could contribute to the substantiation of the hygiene hypothesis. However, no associations were found between gut microbiota and atopic symptoms of eczema and asthmatic bronchitis during early childhood and thus further studies are required to elucidate whether sibling-associated gut microbial changes influence development of allergies later in childhood.