Gut Microbes

Gastric acid suppressing medications have been associated with an increased risk of Clostridioides difficile (C. difficile) infection, hypothesized to be due to underlying intestinal microbiome changes. Our goal is to characterize these changes in children as their microbiome is undergoing critical development. Our study included 5 children (< 3 years old) who were started on clinically indicated gastric acid suppression and 15 healthy age-matched controls. Stool samples were collected before and after 2 months of treatment. We analyzed the microbiome using 16S rRNA sequencing. Quantitative-PCR was used to detect C. difficile toxins. Subjects and controls had similar alpha and beta diversity. We found no significant change in alpha or beta diversity of subjects after treatment. C. difficile toxins were not found and there was no increase in C. difficile carriage after treatment. A significant increase in Lactobacillus was found after treatment, which has been associated with C. difficile in adults.

The influence of genotype on defining the human gut microbiome has been extensively studied, but definite conclusions have not yet been found. To fill this knowledge gap, we leverage data from children enrolled in the Vitamin D Antenatal Asthma Reduction Trial (VDAART) from 6 months to 8 years old. We focus on a pool of 12 genes previously found to be associated with the gut microbiome in independent studies, establishing a Bonferroni corrected significance level of p-value < 2.29 x 10-6. We identified significant associations between SNPs in the FHIT gene (known to be associated with obesity and type 2 diabetes) and obesity-related microbiome features, and the childrens BMI through their childhood. Based on these associations, we defined a set of SNPs of interest and a set of taxa of interest. Taking a multi-omics approach, we integrated plasma metabolome data into our analysis and found simultaneous associations among childrens BMI, the SNPs of interest, and the taxa of interest, involving amino acids, lipids, nucleotides, and xenobiotics. Using our association results, we constructed a quadripartite graph where each disjoint node set represents SNPs in the FHIT gene, microbial taxa, plasma metabolites, or BMI measurements. Network analysis led to the discovery of patterns that identify several genetic variants, microbial taxa and metabolites as new potential markers for obesity, type 2 diabetes, or insulin resistance risk.

Preterm birth yields survivors with heightened susceptibility to long-term neurological deficits, with severity being linked to gestational age. The mechanisms behind neurodevelopmental impairment remain elusive, although research underscores the role of the gut microbiome in regulating the gut-brain axis. In this prospective observational cohort study (PROPEL-study follow-up) we evaluated whether modulation of the gut microbiota by Limosilactobacillus reuteri supplementation to 105 extremely preterm infants with extremely low birth weight (EPT-ELBW), as well as their microbiota development during the first month of life, are associated with neurodevelopment at two years of age. The gut microbiota was characterized with 16S amplicon sequencing, and neurodevelopment was assessed with clinical examination and the Bayley-III score. Our results indicate that specific microbiota composition constellations are associated with impaired neurodevelopment, as indicated by alpha- and beta-diversity being discriminative for neurodevelopment, but not a single bacterium, with increased bacterial diversity being associated to normal neurodevelopment. Microbial maturation over the first month of life was also discriminative for neurodevelopment, where higher abundance of E. coli and Enterococcus in relation to Enterobacter was associated to impaired neurodevelopment. In conclusion, the dynamics of gut microbiota maturation during early of life may have an impact on neurodevelopment in EPT-ELBW infants.

Intestinal dysbiosis is implicated in the origins of necrotising enterocolitis and late-onset sepsis in preterm babies. However, the effect of modulators of bacterial growth (e.g. antibiotics) upon the developing microbiome is not well-characterised. Using high-throughput 16S rRNA gene sequencing combined with contemporaneous clinical data collection, the within-subject relationship between antibiotic administration and microbiome development was assessed, in comparison to preterm infants with minimal antibiotic exposure. During courses of antibiotics, diversity progression fell in comparison to that seen outside periods of antibiotic use (-0.71units/week vs. +0.63units/week, p<0.01); Enterobacteriaceae relative abundance progression conversely rose (+10.6%/week vs. -8.9%/week, p<0.01). After antibiotic cessation, diversity progression remained suppressed (+0.2units/week, p=0.02). Antibiotic use has an acute and longer-lasting impact on the developing preterm intestinal microbiome. This has clinical implications with regard to the contribution of antibiotic use to evolving dysbiosis, and affects the interpretation of existing microbiome studies where this effect modulator is rarely accounted for.

Early-life gut microbiome-metabolome crosstalk has a pivotal role in the maintenance of host physiology. However, our understanding on early-life gut microbiome-metabolome maturation trajectories in humans remains limited. This study aims to explore the longitudinal patterns of gut metabolites during early life, and how they are related to gut microbiota composition in birth cohort samples of n = 670 children collected at 2.5 (n=272), 6 (n=232), 14 (n=289), and 30 months (n=157) of age. Factor analysis showed that breastfeeding has an effect on several metabolites including secondary bile acids. We found that the prevalent gut microbial abundances were associated with metabolite levels, especially in the 2.5 months-olds. We also demonstrated that the prevalent early colonizers Bacteroides, Escherichia and Bifidobacterium abundances associated with microbial metabolites bile acids especially in the breastfed infants. Taken together, our results suggests that as the microbiome matures during the early-life there is an association with the metabolome composition in an analogous fashion to how the genome information mature during early life.

Early exposure to antibiotics and prolonged hospitalization in preterm infants may perturb microbiome development and contribute to adverse health outcomes. Although nasopharyngeal microbiomes are linked to respiratory infections, their early development is underexplored and often assessed with 16S rRNA sequencing, which lacks species resolution. Here, we investigated nasopharyngeal microbiota dynamics in 66 preterm infants by performing deep shotgun metagenomics on 369 nasopharyngeal aspirates collected from birth until 6 months corrected age ([~]7-10 months chronological age). The nasopharyngeal microbiota evolved dynamically, exhibiting age-structured and individualized patterns shaped by postnatal antibiotic exposure and hospitalization. Early-life antibiotic exposure (ampicillin + gentamicin) had transient but significant effects on microbial diversity, composition, stability, and community dynamics. Conversely, an unexpected NICU outbreak left a lasting signature, with persistent S. marcescens carriage observed at 6 months corrected age, which our machine learning algorithm accurately predicted based on microbiome composition in the first days of life. A S. marcescens-dominated community type was present in 45% of total samples and exhibited remarkable stability over time, with minimal transitions to other types regardless of antibiotic exposure. These findings underscore the need for deeper insight into how antibiotics and potential outbreaks can lead to alterations in microbiome trajectories, with possible long-term health implications.

Preterm infants with very low birthweight are at serious risk for necrotizing enterocolitis. To functionally analyse the principles of three successful preventive NEC regimens, we characterized faecal samples of 54 infants (< 1,500 g, n = 383) longitudinally (two weeks) with respect to gut microbiome profiles (bacteria, archaea, fungi, viruses), microbial function, virulence factors, antibiotic resistances and metabolic profiles, including human milk oligosaccharides (HMOs) and short-chain fatty acids. Probiotic Bifidobacterium longum ssp. infantis supplementation affected microbiome development globally, pointing toward the genomic potential to convert HMOs. Engraftment of Bifidobacterium substantially reduced microbiome-associated antibiotic resistance as compared to regimens using probiotic Lactobacillus rhamnosus or no supplementation. Crucially, the beneficial effects of Bifidobacterium supplementation depended on simultaneous feeding with HMOs. We demonstrate that preventive regimens have the highest impact on early maturation of the gastrointestinal microbiome, enabling the establishment of a resilient microbial ecosystem that reduces pathogenic threats in at-risk preterm infants.

The early life gut microbiome has been suggested to be a potential driver of neurocognitive development. Evidence for this relationship in preterm children, who are at increased risk of both gut microbiome disruptions and neurodevelopmental impairment, is scarce. In a sample of 73 very preterm infants drawn from a prospective birth cohort, we assessed associations between the neonatal gut microbiome and neurodevelopment at 9 months and 2 years. The gut microbiome was profiled from stool samples collected prior to NICU discharge using shotgun metagenomics. By taking a consensus-based analytic approach, we found strong evidence for associations between the abundances of several gut bacterial species and measures related to autistic traits (e.g. Klebsiella spp.), socio-emotional development, including temperament (e.g. Enterobacter cloacae complex, Veillonella parvula), and executive functioning (Clostridium perfringens). The abundances of functional modules involved in gut-brain signalling, particularly those involved in histamine, tryptophan and quinolinic acid metabolism, were associated with measures related to executive functioning and cognitive-behavioural flexibility. This study provides evidence that the neonatal gut microbiome composition may affect longer-term neurodevelopmental profiles following preterm birth, particularly those related to socio-emotional development, autistic traits and executive functioning.

BackgroundThe prospective Kiel Inflammatory Bowel Disease (IBD) Family Cohort Study (KINDRED cohort) was initiated in 2013 to systematically and extensively collect data and biosamples from index IBD patients and their relatives (e.g., blood, stool), a population at high risk for IBD development. Regular follow-ups were conducted to collect updated health and lifestyle information, to obtain new biosamples, and to capture the incidence of IBD during development. By combining taxonomic and imputed functional microbial data collected at successive time points with extensive anthropometric, medical, nutritional, and social information, this study aimed to characterize the factors influencing the microbiota in health and disease via detailed ecological analyses. ResultsUsing two dysbiosis metrics (MD-index, GMHI) trained on the German KINDRED cohort, we identified strong and generalizable gradients within and across different IBD cohorts, which correspond strongly with IBD pathologies, physiological manifestations of inflammation (e.g., Bristol stool score, ASCA IgA/IgG), genetic risk for IBD, and general risk of disease onset. Anthropometric and medical factors influencing transit time strongly modify bacterial communities. Various Enterobacteriaceae (e.g., Klebsiella sp.) and opportunistic Clostridia pathogens (e.g., C. XIVa clostridioforme), characterize in combination with ectopic oral taxa (e.g. Veillonella sp., Cand. Saccharibacteria sp., Fusobacterium nucleatum) the distinct and chaotic IBD-specific communities. Functionally, amino acid metabolism and flagellar assembly are beneficial, while mucolytic functions are associated with IBD. ConclusionsOur findings demonstrate broad-scale ecological patterns which indicate drastic state transitions of communities into characteristically chaotic communities in IBD patients. These patterns appear to be universal across cohorts and influence physiological signs of inflammation, display high resilience, but show only little heritability/intrafamily transmission.

Stunting remains a major public health challenge in Indonesia and is increasingly associated with gut microbiota dysbiosis. This study examined 36 children aged 2-5 years through anthropometry, dietary assessment, and pooled fecal sampling. Full-length 16S rRNA sequencing using Oxford Nanopore Technologies revealed notable microbial alterations in the stunted group. Stunted children exhibited reduced alpha diversity and lower microbial richness, indicating a simplified gut ecosystem. Although both groups were dominated by Bacillota (>96%), stunted children showed higher proportions of Clostridia affiliated orders, including Eubacteriales, Peptostreptococcales, and Erysipelotrichaceae, along with enrichment of fermentative and dysbiosis associated genera such as Blautia, Romboutsia, and Terrisporobacter. Beneficial fiber degrading taxa, including Lachnospiraceae, were proportionally higher in normal children. Functional predictions using PICRUSt2 revealed greater microbial metabolic activity in the stunting group, particularly in carbohydrate, amino acid, and nucleotide metabolism, with elevated pathways such as starch and sucrose metabolism, glycolysis, and porphyrin and pyrimidine metabolism. Dietary assessment showed significantly lower intake of energy, protein, fat, and multiple micronutrients among stunted children, consistent with observed microbial and functional alterations. These findings indicate a distinct fermentative dysbiosis in Banyumas stunted children and highlight the need for integrated nutritional and microbiota targeted interventions.

The early infant gut microbiota is dominated by bifidobacteria, but there is substantial variation at the (sub)species level. Patterns of postnatal Bifidobacterium subspecies colonization in low or middle-income countries have not been widely studied. We used (sub)species-specific qPCR to quantify B. infantis, B. longum, and B. breve in stool samples from 1132 infants (0-6 months) in urban Dhaka, Bangladesh. B. infantis absolute abundance started low but increased in the first two months, whereas B. longum and B. breve abundances remained comparatively low. B. infantis emerged earlier in infants delivered by C-section, but by [~]2 months of age, infants delivered by C-section or vaginally had similar B. infantis abundances. Infant antibiotic exposure, feeding patterns, and maternal stool B. infantis were not associated with infant B. infantis. In settings where B. infantis is widespread, its patterns of postnatal colonization can be used to inform the design of targeted microbiota-modifying interventions in infancy.

Social disadvantage (SD) and psychological stressors (PS) trap some populations in poverty, resulting in health inequities. How these two factors become biologically embedded and the pathways leading to adverse health outcomes is unclear, especially in infants exposed to psychosocial adversity in utero and during early life. Variation in gut microbiome structure and functions, and systemic elevations in circulating cytokines levels as indices of inflammation, offer two possible causative pathways. Here, we interrogate the gut microbiome of mother-child dyads and maternal inflammatory markers, and compare high-SD/high-PS dyads to pairs with low-SD/Low-PS, and demonstrate that the GM of high-SD and high-PS mothers may already be compromised, resulting in the lowest observed inter-individual similarity in that group. The strong predictors of maternal high-SD and high-PS based on mothers and children microbiomes were phylogenetically very distinct bacteria indicating different GM pathways associated with SD versus PS. We identified sets of SD- and PS-discriminatory metabolic pathways in the mothers and in the children, however their predictive power was lower compared to the discriminatory bacterial species. Prediction accuracy was consistently greater for IL-6 than for the other inflammatory markers, supporting an association between systemic inflammation and psychosocial adversity. The gut microbiome of the infants can be used to predict the psychosocial adversity of mothers, and are embedded in the gut microbiota of 4-month-old infants.

The gut microbiota constitutes a complex ecosystem essential for host defense against infection and maturation of the immune system. Inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohns disease, are characterized by a severe inflammation of the intestine, arising from dysregulated control of host-microbiota crosstalk. However, neither the genetic bases of IBD nor the immune responses involved are fully understood. The pathobionts are currently under investigation for their active role in the development and the severity of IBD. These bacteria are present in the microbiota of healthy individuals without causing disease but have pathogenic potential when the intestinal environment is disturbed. Here, we highlighted Sutterella sp. as a new commensal pathobiont for its capacity to modulate the host immune functions. This anaerobic Gram-negative bacterium inhibits IL-22 production and AhR activity in mice and humans. The effectors responsible for the biological activity are large (>30 kDa) protein-based compounds secreted by Sutterella sp. These data enhance our understanding of the mechanisms that regulate host immune functions and pave the way for new therapeutic strategies to control gut inflammation.

The gut microbiota plays a pivotal role in health and disease. The use of probiotics as microbiota-targeted therapies is a promising strategy to improve host health. However, dynamic molecular mechanisms are often not elucidated, especially when targeting the small intestinal microbiota. Here, we show that supplementation of a probiotic formula (Ecologic(R)825) to the adult human small intestinal ileostoma microbiota counteracts the growth of Enterococcaceae and Enterobacteriaceae and reduces ethanol production, leading to major changes in nutrient utilization and resistance to perturbations. The observed alterations coincided with an initial increase in lactate production and decrease in pH by the probiotics, followed by a sharp increase in the levels of butyrate and propionate. Additionally, increased production of multiple N-acyl amino acids was detected in the stoma samples supplemented with the probiotic formula. Overall, this study shows how network theory can be used to improve the current or identify novel microbiota-targeted therapies. The outcome may help further understand the reported effects of these probiotic formula on the host.

The gut microbiota in early life, when critical immune maturation takes place, may influence the immunogenicity of childhood vaccinations. We assessed the association between mode of delivery, gut microbiota development in the first year of life, and mucosal antigen-specific immunoglobulin G (IgG) responses against pneumococcal and meningococcal conjugate vaccination at ages 12 and 18 months, respectively, in a prospective birth cohort of 120 infants. Birth by natural delivery was associated with higher IgG responses against both vaccines, which for the anti-pneumococcal IgG response could be explained by a gut microbial community composition with high abundances of Bifidobacterium and Escherichia coli in the first weeks of life. High E. coli abundance in the same period was also associated with higher anti-meningococcal IgG responses. Our results suggest that associations between mode of delivery and antibody responses to routine childhood vaccines are mediated by gut microbiota development.

Background and aimsFaecalibacterium prausnitzii, a highly abundant bacterium in the human gut microbiota, has been linked to overall health and is decreased in several pathological conditions, such as Inflammatory Bowel Disease (IBD). F. prausnitzii has shown anti-inflammatory properties in human and mouse models, notably through the induction of IL-10 signaling. Here, we investigated which cell types from human blood and intestinal tissue are responsible for producing IL-10 induced by F. prausnitzii, and providing the first mechanistic insights. MethodsImmune cells isolated from human blood and intestinal lamina propria of patients with IBD and non-inflamed controls, were stimulated with either F. prausnitzii EXL01 strain or Escherichia coli lipopolysaccharide (LPS) and analysed by Legendplex, ELISA, flow cytometry, RNA-sequencing (RNAseq), and Seahorse technology. ResultsF. prausnitzii EXL01 strain induced the direct and dose-dependent production of IL-10 in CD14+ monocytes from the systemic circulation and intestinal tissue of IBD patients and non-inflamed controls, without inducing a pro-inflammatory response as compared to LPS stimulation. RNAseq analysis corroborated these results and revealed that F. prausnitzii EXL01 strain differentially affects cell energy metabolism compared to LPS. The anti-inflammatory response induced by F. prausnitzii in monocytes was dependent on mitochondrial respiration. ConclusionF. prausnitzii induces an anti-inflammatory response and rewires energy metabolism in human monocytes, which might explain its beneficial impact on intestinal inflammation and human health in general. These results provide new insight into the mechanisms underlying the anti-inflammatory effects of F. prausnitzii and are crucial for a better understanding of its potential use in the treatment of IBD.

Changes in the composition of the human microbiota can negatively impact human health. Probiotic bacteria like many lactobacilli help prevent or repair dysbiosis but it is largely unclear which molecules of these bacteria mediate the probiotic effects. Given the extensive crosstalk between the immune system and microbiome members, we investigated whether lactobacilli activate the formyl-peptide receptor 2 (FPR2), a pattern recognition receptor that is expressed on the surface of intestinal epithelial cells and known to promote wound healing and immune homeostasis. Probiotic strains of Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, and Lacticaseibacillus rhamnosus were isolated from probiotic compounds and sequenced. Calcium influx experiments in FPR1 or FPR2 overexpressing HL60 cells, and primary human neutrophils, along with pharmacological inhibition of FPR2, revealed that culture filtrates of the isolated lactobacilli strongly activate FPR2, promote killing of the methicillin resistant S. aureus USA300 and induce neutrophil chemotaxis. Pretreatment of culture filtrates with proteinase K reduced FPR2 activity, indicating that the FPR2 ligands are peptides. In silico analysis of the amphipathic properties of the signal peptides of lactic acid bacteria identified selected signal peptides of L. plantarum with the ability to predominantly activate FPR2 in vitro. Thereby, via targeted activation of FPR2, peptides released by some lactobacilli are likely to positively influence the outcome of inflammatory gut diseases and could be used to treat inflammatory diseases.

Gut host physiology and the microbiome intricately interact in the complex ecosystem of the human digestive tract, playing a crucial role in maintaining overall health. In recent decades, the role of the gut microbiota in the defence against pathogens and modulating local and distal immunity has been well-established. The interactions between commensal and potential pathogenic bacteria with the intestinal epithelium can initiate immune responses in the epithelial cells, which, in turn, activate downstream immune responses in other immune cells. These intricate processes involved, especially when multiple microorganisms are present as seen in the intestinal microbiome, remain only partially understood. Previously, it was observed that in adults aged 60 years or older, the commensal Ruminococcus torques (Rt) and Escherichia coli were associated with influenza-like illness and a heightened pro-inflammatory immune profile. In this study, we used a CaCo-2 cell-based model and a human intestinal enteroid (HIE) model to explore epithelial responses to Rt and an adherent invasive E. coli (AIEC) both individually and in co-cultures under anaerobic conditions. Additionally, CaCo-2 cells were co-cultured with peripheral blood mononuclear cells, revealing downstream activation of immune cells. While both systems showed comparable cytokine profiles, they differed in their responses to the different bacteria, with the organoid system being more representative for intestinal epithelial cells in humans. We provide mechanistic evidence of the pro-inflammatory responses associated with these bacteria in the intestinal ecosystem. These models, particularly in the context of combined infections, represent a valuable and promising avenue for future research. They contribute to a deeper understanding of the complex interactions between the gut microbiota, epithelial intestinal cells and immune cells in the gut ecosystem, thereby promoting advances in the field of gut health and host response.

BackgroundDysbiosis of the microbiome has been related to the Celiac disease (CeD) progress, an autoimmune disease characterised by gluten intolerance developed in genetically susceptible individuals under certain environmental factors. The microbiome contributes to CeD pathophysiology modulating the immune response by the action of short-chain fatty acids (SCFA), affecting gut barrier integrity allowing the entrance of gluten derived proteins, and degrading immunogenic peptides of gluten through endoprolyl peptidase enzymes. We reviewed state of the art in taxonomic composition for CeD and compiled the larger dataset of 16S prokaryotic ribosomal RNA (rRNA) gene high-throughput sequencing for consensus profiling. We present for the first time an integrative analysis of metataxonomic data from CeD patients, including samples from different body sites (saliva, pharynx, duodenum, and stool). We found the presence of coordinated changes through the gastrointestinal tract characterised by an increase in Actinobacteria species in the upper tract (pharynx and duodenum), and an increase in Proteobacteria in the lower tract (duodenum and stool), as well as site- specific changes evidencing a dysbiosis in CeD patients microbiota. Moreover, we described the effect of adherence to a gluten-free diet (GFD) evidenced by an increase in beneficial bacteria and a decrease in some Betaproteobacteriales but not fully restoring CeD-related dysbiosis. We illustrate that the gut microbiota acts as an enhancer of immune response in CeD through the production of lipopolysaccharides and other bacterial components that activate the immune response and by decrease SCFA producers bacteria. Furthermore, microbial changes observed through the gastrointestinal tract of CeD patients may help manage the disease and follow-up GFD treatment.

Time-restricted feeding (TRF) during the active phase protects against high-fat diet (HFD)-induced obesity, and its impact on gut microbiota has been previously investigated using bacterial taxa as functional units. However, in the gut ecosystem, bacteria from different taxonomic backgrounds form coherent functional groups called guilds, whose members exhibit co-abundant behavior. Thirty-five co-abundance groups (CAGs), clustered from 297 prevalent amplicon sequence variants (ASVs), showed greater concordance with beta-diversity plots based on all 1131 ASVs than the 130 classifiable genera, leading to a significantly improved preservation of community-level information. TRF-enriched CAGs positively correlated with metabolic improvement, while TRF-reduced CAGs negatively correlated. TRF restored the diurnal rhythm of most of these key CAGs. Novel ASVs, unclassifiable at the genus level, were identified in these key CAGs. Overall, this suggests that the key bacterial guilds may mediate the beneficial metabolic effects of TRF through the restoration of diurnal oscillation.