Human milk oligosaccharides promote intestinal epithelium regeneration independent of the microbiota during necrotizing enterocolitis.

PURPOSE: Necrotizing enterocolitis (NEC) is a severe intestinal disease primarily affecting premature infants, marked by impaired epithelial regeneration. Breastfed infants are less susceptible to NEC than formula-fed ones, and human milk oligosaccharides (HMO) found in breast milk have prebiotic properties that can protect against NEC. However, it is unclear how HMOs influence intestinal epithelium regeneration in relation to the gut microbiota. METHODS: Broad-spectrum antibiotics were administered to pregnant dams to reduce the microbiota in offspring. NEC was induced through administration of hyperosmolar formula, lipopolysaccharide, and hypoxia from postnatal days (p) 5-9. Intestinal epithelial organoids were derived from p9 mice. HMOs were isolated from human donor breast milk and then solubilized in the formula for each feed or culture media for organoids. RESULTS: HMOs did not alter the microbiota profile in the presence of a normal or reduced microbiota. In the reduced microbiota, HMO treatment decreased NEC intestinal injury, and increased proliferation and stem cell activity. Additionally, in the complete absence of the microbiota, HMOs stimulated intestinal organoid growth. CONCLUSION: This study demonstrates that HMOs promoted intestinal epithelial regeneration independent of the gut microbiota. These findings provide further insight into the various benefits HMOs may have in the protection against NEC.

Detection and absolute quantification of Lactiplantibacillus plantarum ATCC 202195 by quantitative real-time PCR.

IMPORTANCE: When administered for seven consecutive days shortly after birth, the probiotic bacterium Lactiplantibacillus plantarum ATCC 202195 plus fructooligosaccharide (FOS) was reported to reduce sepsis and lower respiratory tract infection events during early infancy in a randomized trial in India. Since probiotic effects are often strain specific, strain-level detection and quantification by routine molecular methods enables the monitoring of safety outcomes, such as probiotic-associated bacteremia, and allows for the quality of probiotic interventions to be assessed and monitored (i.e., verify strain identity and enumerate). Despite the potential clinical applications of L. plantarum ATCC 202195, an assay to detect and quantify this strain has not previously been described. Herein, we report the design of primer and probe sequences to detect L. plantarum ATCC 202195 and the development and optimization of a real-time PCR assay to detect and quantify the strain with high specificity and high sensitivity.

Probiotics, Prebiotics, Lactoferrin, and Combination Products for Prevention of Mortality and Morbidity in Preterm Infants: A Systematic Review and Network Meta-Analysis.

Importance: Modulation of intestinal microbiome by administering probiotics, prebiotics, or both may prevent morbidity and mortality in premature infants. Objective: To assess the comparative effectiveness of alternative prophylactic strategies through a network meta-analysis (NMA) of randomized clinical trials. Data Sources: MEDLINE, EMBASE, Science Citation Index Expanded, CINAHL, Scopus, Cochrane CENTRAL, and Google Scholar from inception until May 10, 2023. Study Selection: Eligible trials tested probiotics, prebiotics, lactoferrin, and combination products for prevention of morbidity or mortality in preterm infants. Data Extraction and Synthesis: A frequentist random-effects model was used for the NMA, and the certainty of evidence and inferences regarding relative effectiveness were assessed using the GRADE approach. Main Outcomes and Measures: All-cause mortality, severe necrotizing enterocolitis, culture-proven sepsis, feeding intolerance, time to reach full enteral feeding, and duration of hospitalization. Results: A total of 106 trials involving 25 840 preterm infants were included. Only multiple-strain probiotics were associated with reduced all-cause mortality compared with placebo (risk ratio [RR], 0.69; 95% CI, 0.56 to 0.86; risk difference [RD], -1.7%; 95% CI, -2.4% to -0.8%). Multiple-strain probiotics alone (vs placebo: RR, 0.38; 95% CI, 0.30 to 0.50; RD, -3.7%; 95% CI, -4.1% to -2.9%) or in combination with oligosaccharides (vs placebo: RR, 0.13; 95% CI, 0.05 to 0.37; RD, -5.1%; 95% CI, -5.6% to -3.7%) were among the most effective interventions reducing severe necrotizing enterocolitis. Single-strain probiotics in combination with lactoferrin (vs placebo RR, 0.33; 95% CI, 0.14 to 0.78; RD, -10.7%; 95% CI, -13.7% to -3.5%) were the most effective intervention for reducing sepsis. Multiple-strain probiotics alone (RR, 0.61; 95% CI, 0.46 to 0.80; RD, -10.0%; 95% CI, -13.9% to -5.1%) or in combination with oligosaccharides (RR, 0.45; 95% CI, 0.29 to 0.67; RD, -14.1%; 95% CI, -18.3% to -8.5%) and single-strain probiotics (RR, 0.61; 95% CI, 0.51 to 0.72; RD, -10.0%; 95% CI, -12.6% to -7.2%) proved of best effectiveness in reduction of feeding intolerance vs placebo. Single-strain probiotics (MD, -1.94 days; 95% CI, -2.96 to -0.92) and multistrain probiotics (MD, -2.03 days; 95% CI, -3.04 to -1.02) proved the most effective in reducing the time to reach full enteral feeding compared with placebo. Only single-strain and multistrain probiotics were associated with greater effectiveness compared with placebo in reducing duration of hospitalization (MD, -3.31 days; 95% CI, -5.05 to -1.58; and MD, -2.20 days; 95% CI, -4.08 to -0.31, respectively). Conclusions and Relevance: In this systematic review and NMA, moderate- to high-certainty evidence demonstrated an association between multistrain probiotics and reduction in all-cause mortality; these interventions were also associated with the best effectiveness for other key outcomes. Combination products, including single- and multiple-strain probiotics combined with prebiotics or lactoferrin, were associated with the largest reduction in morbidity and mortality.

Gut microbiota promotes stem cell differentiation through macrophage and mesenchymal niches in early postnatal development.

Intestinal stem cell maturation and development coincide with gut microbiota exposure after birth. Here, we investigated how early life microbial exposure, and disruption of this process, impacts the intestinal stem cell niche and development. Single-cell transcriptional analysis revealed impaired stem cell differentiation into Paneth cells and macrophage specification upon antibiotic treatment in early life. Mouse genetic and organoid co-culture experiments demonstrated that a CD206+ subset of intestinal macrophages secreted Wnt ligands, which maintained the mesenchymal niche cells important for Paneth cell differentiation. Antibiotics and reduced numbers of Paneth cells are associated with the deadly infant disease, necrotizing enterocolitis (NEC). We showed that colonization with Lactobacillus or transfer of CD206+ macrophages promoted Paneth cell differentiation and reduced NEC severity. Together, our work defines the gut microbiota-mediated regulation of stem cell niches during early postnatal development.

Human milk nutrient fortifiers alter the developing gastrointestinal microbiota of very-low-birth-weight infants.

Nutrient fortifiers are added to human milk to support the development of very-low-birth-weight infants. Currently, bovine-milk-based fortifiers (BMBFs) are predominantly administered, with increasing interest in adopting human-milk-based fortifiers (HMBFs). Although beneficial for growth, their effects on the gastrointestinal microbiota are unclear. This triple-blind, randomized clinical trial (NCT02137473) tested how nutrient-enriching human milk with HMBF versus BMBF affects the gastrointestinal microbiota of infants born < 1,250 g during hospitalization. HMBF-fed infants (n = 63, n = 269 stools) showed lower microbial diversity, altered microbial community structure, and changes in predicted microbial functions compared with BMBF-fed infants (n = 56, n = 239 stools). HMBF-fed infants had higher relative and normalized abundances of unclassified Enterobacteriaceae and lower abundances of Clostridium sensu stricto. Post hoc analyses identified dose-dependent relationships between individual feed components (volumes of mother's milk, donor milk, and fortifiers) and the microbiota. These results highlight how nutrient fortifiers impact the microbiota of very-low-birth-weight infants during a critical developmental window.

Intestinal Microbial Composition of Children in a Randomized Controlled Trial of Probiotics to Treat Acute Gastroenteritis.

Compositional analysis of the intestinal microbiome in pre-schoolers is understudied. Effects of probiotics on the gut microbiota were evaluated in children under 4-years-old presenting to an emergency department with acute gastroenteritis. Included were 70 study participants (n=32 placebo, n=38 probiotics) with stool specimens at baseline (day 0), day 5, and after a washout period (day 28). Microbiota composition and deduced functions were profiled using 16S ribosomal RNA sequencing and predictive metagenomics, respectively. Probiotics were detected at day 5 of administration but otherwise had no discernable effects, whereas detection of bacterial infection (P<0.001) and participant age (P<0.001) had the largest effects on microbiota composition, microbial diversity, and deduced bacterial functions. Participants under 1 year had lower bacterial diversity than older aged pre-schoolers; compositional changes of individual bacterial taxa were associated with maturation of the gut microbiota. Advances in age were associated with differences in gut microbiota composition and deduced microbial functions, which have the potential to impact health later in life. Clinical Trial Registration: www.ClinicalTrials.gov, identifier: NCT01853124.

Variations in the Composition of Human Milk Oligosaccharides Correlates with Effects on Both the Intestinal Epithelial Barrier and Host Inflammation: A Pilot Study.

BACKGROUND: Human milk oligosaccharides are complex, non-digestible carbohydrates that directly interact with intestinal epithelial cells to alter barrier function and host inflammation. Oligosaccharide composition varies widely between individual mothers, but it is unclear if this inter-individual variation has any impact on intestinal epithelial barrier function and gut inflammation. METHODS: Human milk oligosaccharides were extracted from the mature human milk of four individual donors. Using an in vitro model of intestinal injury, the effects of the oligosaccharides on the intestinal epithelial barrier and select innate and adaptive immune functions were assessed. RESULTS: Individual oligosaccharide compositions shared comparable effects on increasing transepithelial electrical resistance and reducing the macromolecular permeability of polarized (Caco-2Bbe1) monolayers but exerted distinct effects on the localization of the intercellular tight junction protein zona occludins-1 in response to injury induced by a human enteric bacterial pathogen Escherichia coli, serotype O157:H7. Immunoblots showed the differential effects of oligosaccharide compositions in reducing host chemokine interleukin 8 expression and inhibiting of p38 MAP kinase activation. CONCLUSIONS: These results provide evidence of both shared and distinct effects on the host intestinal epithelial function that are attributable to inter-individual differences in the composition of human milk oligosaccharides.

Amniotic fluid stem cell administration can prevent epithelial injury from necrotizing enterocolitis.

BACKGROUND: Stem cell therapy has been proven to rescue intestinal injury and stimulate intestinal regeneration in necrotizing enterocolitis (NEC). Specifically, stem cells derived from amniotic fluid (AFSCs) and mesenchymal stem cells (MSCs) derived from bone marrow have shown promising results in the treatment of experimental NEC. This study aims to examine the effects of AFSCs and MSCs on the prevention of intestinal injury during experimental NEC. METHODS: Supernatants from AFSC and MSC cultures were collected to perform proteomic analysis. Prior to NEC induction, mice received intraperitoneal injections of phosphate-buffered saline (PBS), 2 × 106 AFSCs, or 2 × 106 MSCs. RESULTS: We found that AFSCs grew faster than MSCs. Proteomic analysis indicated that AFSCs are primarily involved in cell development and growth, while MSCs are involved in immune regulation. Administering AFSCs before NEC induction decreased NEC severity and mucosal inflammation. Intestinal proliferation and endogenous stem cell activation were increased after AFSC administration. However, administering MSCs before NEC induction had no beneficial effects. CONCLUSIONS: This study demonstrated that AFSCs and MSCs have different protein release profiles. AFSCs can potentially be used as a preventative strategy for neonates at risk of NEC, while MSCs cannot be used. IMPACT: AFSCs and MSCs have distinct protein secretory profiles, and AFSCs are primarily involved in cell development and growth, while MSCs are involved in immune regulation. AFSCs are unique in transiently enhancing healthy intestinal epithelial cell growth, which offers protection against the development of experimental NEC. The prevention of NEC via the administration of AFSCs should be evaluated in infants at great risk of developing NEC or in infants with early signs of NEC.

Structure-Function Relationships of Human Milk Oligosaccharides on the Intestinal Epithelial Transcriptome in Caco-2 Cells and a Murine Model of Necrotizing Enterocolitis.

SCOPE: Necrotizing enterocolitis (NEC) is a devastating gastrointestinal emergency affecting preterm infants. Breastmilk protects against NEC, partly due to human milk oligosaccharides (HMOs). HMO compositions are highly diverse, and it is unclear if anti-NEC properties are specific to carbohydrate motifs. Here, this study compares intestinal epithelial transcriptomes of five synthetic HMOs (sHMOs) and examines structure-function relationships of HMOs on intestinal signaling. METHODS AND RESULTS: This study interrogates the transcriptome of Caco-2Bbe1 cells in response to five synthetic HMOs (sHMOs) using RNA sequencing: 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3FL), 6'-siallyllactose (6'-SL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT). Protection against intestinal barrier dysfunction and inflammation occurred in an HMO-dependent manner. Each sHMO exerts a unique set of host transcriptome changes and modulated unique signaling pathways. There is clustering between HMOs bearing similar side chains, with little overlap in gene regulation which is shared by all sHMOs. Interestingly, most sHMOs protect pups against NEC, exerting divergent mechanisms on intestinal cell morphology and inflammation. CONCLUSIONS: These results demonstrate that while structurally distinct HMOs impact intestinal physiology, their mechanisms of action differ. This finding establishes the first structure-function relationship of HMOs in the context of intestinal cell signaling responses and offers a functional framework by which to screen and design HMO-like compounds.

Antimicrobial susceptibilities and comparative whole genome analysis of two isolates of the probiotic bacterium Lactiplantibacillus plantarum, strain ATCC 202195.

A synbiotic containing Lactiplantibacillus plantarum [American Type Culture Collection (ATCC) strain identifier 202195] and fructooligosaccharide was reported to reduce the risk of sepsis in young infants in rural India. Here, the whole genome of two isolates of L. plantarum ATCC 202195, which were deposited to the ATCC approximately 20 years apart, were sequenced and analyzed to verify their taxonomic and strain-level identities, identify potential antimicrobial resistant genes and virulence factors, and identify genetic characteristics that may explain the observed clinical effects of L. plantarum ATCC 202195. Minimum inhibitory concentrations for selected antimicrobial agents were determined using broth dilution and gradient strip diffusion techniques. The two L. plantarum ATCC 202195 isolates were genetically identical with only three high-quality single nucleotides polymorphisms identified, and with an average nucleotide identity of 99.99%. In contrast to previously published reports, this study determined that each isolate contained two putative plasmids. No concerning acquired or transferable antimicrobial resistance genes or virulence factors were identified. Both isolates were sensitive to several clinically important antibiotics including penicillin, ampicillin and gentamicin, but resistant to vancomycin. Genes involved in stress response, cellular adhesion, carbohydrate metabolism and vitamin biosynthesis are consistent with features of probiotic organisms.

Probiotic stool secretory immunoglobulin A modulation in children with gastroenteritis: a randomized clinical trial.

BACKGROUND: We previously conducted the Probiotic Regimen for Outpatient Gastroenteritis Utility of Treatment (PROGUT) study, which identified no improvements in children with acute gastroenteritis (AGE) administered a probiotic. However, the aforementioned study did not evaluate immunomodulatory benefits. OBJECTIVES: The object of this study was to determine if stool secretory immunoglobulin A (sIgA) concentrations in children with AGE increase more among participants administered a Lactobacillus rhamnosus/helveticus probiotic compared with those administered placebo. METHODS: This a priori planned multicenter, randomized, double-blinded, placebo-controlled ancillary study enrolled children presenting for emergency care who received a 5-d probiotic or placebo course. Participants submitted stool specimens on days 0, 5, and 28. The primary endpoint was the change in stool sIgA concentrations on day 5 compared with baseline. RESULTS: A total of 133 (n = 66 probiotic, 67 placebo) of 886 PROGUT participants (15.0%) provided all 3 specimens. Median stool sIgA concentrations did not differ between the probiotic and placebo groups at any of the study time points: day 0 median (IQR): 1999 (768, 4071) compared with 2198 (702, 5278) (P = 0.27, Cohen's d = 0.17); day 5: 2505 (1111, 5310) compared with 3207 (982, 7080) (P = 0.19, Cohen's d = 0.16); and day 28: 1377 (697, 2248) compared with 1779 (660, 3977) (P = 0.27, Cohen's d = 0.19), respectively. When comparing measured sIgA concentrations between days 0 and 5, we found no treatment allocation effects [ß: -0.24 (-0.65, 0.18); P = 0.26] or interaction between treatment and specimen collection day [ß: -0.003 (-0.09, 0.09); P = 0.95]. Although stool sIgA decreased between day 5 and day 28 within both groups (P < 0.001), there were no differences between the probiotic and placebo groups in the median changes in sIgA concentrations when comparing day 0 to day 5 median (IQR) [500 (-1135, 2362) compared with 362 (-1122, 4256); P = 0.77, Cohen's d = 0.075] and day 5 to day 28 [-1035 (-3130, 499) compared with -1260 (-4437, 843); P = 0.70, Cohen's d = 0.067], respectively. CONCLUSIONS: We found no effect of an L. rhamnosus/helveticus probiotic, relative to placebo, on stool IgA concentrations. This trial was registered at clinicaltrials.gov as NCT01853124.

High Fat-High Fructose Diet-Induced Changes in the Gut Microbiota Associated with Dyslipidemia in Syrian Hamsters.

Aim: The objective of this study was to characterize the early effects of high fructose diets (with and without high fat) on both the composition of the gut microbiota and lipid metabolism in Syrian hamsters, a reproducible preclinical model of diet-induced dyslipidemia. Methods: Eight-week-old male hamsters were fed diets consisting of high-fat/high-fructose, low-fat/high-fructose or a standard chow diet for 14 days. Stool was collected at baseline (day 0), day 7 and day 14. Fasting levels of plasma triglycerides and cholesterol were monitored on day 0, day 7 and day 14, and nonfasting levels were also assayed on day 15. Then, 16S rRNA sequencing of stool samples was used to determine gut microbial composition, and predictive metagenomics was performed to evaluate dietary-induced shifts in deduced microbial functions. Results: Both high-fructose diets resulted in divergent gut microbiota composition. A high-fat/high-fructose diet induced the largest shift in overall gut microbial composition, with dramatic shifts in the Firmicute/Bacteroidetes ratio, and changes in beta diversity after just seven days of dietary intervention. Significant associations between genus level taxa and dietary intervention were identified, including an association with Ruminococceace NK4A214 group in high-fat/high-fructose fed animals and an association with Butryimonas with the low-fat/high-fructose diet. High-fat/high-fructose feeding induced dyslipidemia with increases in plasma triglycerides and cholesterol, and hepatomegaly. Dietary-induced changes in several genus level taxa significantly correlated with lipid levels over the two-week period. Differences in microbial metabolic pathways between high-fat/high-fructose and low-fat/high-fructose diet fed hamsters were identified, and several of these pathways also correlated with lipid profiles in hamsters. Conclusions: The high-fat/high-fructose diet caused shifts in the host gut microbiota. These dietary-induced alterations in gut microbial composition were linked to changes in the production of secondary metabolites, which contributed to the development of metabolic syndrome in the host.

Remote ischemic conditioning counteracts the intestinal damage of necrotizing enterocolitis by improving intestinal microcirculation.

Necrotizing enterocolitis (NEC) is a devastating disease of premature infants with high mortality rate, indicating the need for precision treatment. NEC is characterized by intestinal inflammation and ischemia, as well derangements in intestinal microcirculation. Remote ischemic conditioning (RIC) has emerged as a promising tool in protecting distant organs against ischemia-induced damage. However, the effectiveness of RIC against NEC is unknown. To address this gap, we aimed to determine the efficacy and mechanism of action of RIC in experimental NEC. NEC was induced in mouse pups between postnatal day (P) 5 and 9. RIC was applied through intermittent occlusion of hind limb blood flow. RIC, when administered in the early stages of disease progression, decreases intestinal injury and prolongs survival. The mechanism of action of RIC involves increasing intestinal perfusion through vasodilation mediated by nitric oxide and hydrogen sulfide. RIC is a viable and non-invasive treatment strategy for NEC.

Human Milk Oligosaccharides Protect against Necrotizing Enterocolitis by Activating Intestinal Cell Differentiation.

SCOPE: Necrotizing enterocolitis (NEC) is a devastating gastrointestinal emergency and currently the leading cause of mortality in preterm infants. Recent studies show that human milk oligosaccharides (HMOs) reduce the frequency and incidence of NEC; however, the molecular mechanisms for their protection are largely unexplored. METHODS AND RESULTS: To address this gap, a genome-wide profiling of the intestinal epithelial transcriptome in response to HMOs using RNA-sequencing is performed. It is found that HMOs alter the host transcriptome in 225 unique target genes pertaining to cell proliferation and differentiation, including upregulation of stem cell differentiation marker HMGCS2. To validate these results, differentiation in Caco-2Bbe1 (Caco-2) intestinal cells is verified by Alcian Blue staining and transepithelial electrical resistance (TER) recordings. Furthermore, an in vivo model of NEC is also employed whereby neonatal pups are gavage fed HMOs. Interestingly, HMOs-fed pups show enhanced cell MUC2 differentiation and HMGCS2 expression. CONCLUSIONS: These findings demonstrate HMOs protect against NEC in part by altering the differentiation of the crypt-villus axis. In addition, this study suggests that pooled HMOs directly induce a series of biological processes, which provide mechanistic insights to how HMOs protect the host intestine.

Activation of Wnt signaling by amniotic fluid stem cell-derived extracellular vesicles attenuates intestinal injury in experimental necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating intestinal disease primarily affecting preterm neonates and causing high morbidity, high mortality, and huge costs for the family and society. The treatment and the outcome of the disease have not changed in recent decades. Emerging evidence has shown that stimulating the Wnt/ß-catenin pathway and enhancing intestinal regeneration are beneficial in experimental NEC, and that they could potentially be used as a novel treatment. Amniotic fluid stem cells (AFSC) and AFSC-derived extracellular vesicles (EV) can be used to improve intestinal injury in experimental NEC. However, the mechanisms by which they affect the Wnt/ß-catenin pathway and intestinal regeneration are unknown. In our current study, we demonstrated that AFSC and EV attenuate NEC intestinal injury by activating the Wnt signaling pathway. AFSC and EV stimulate intestinal recovery from NEC by increasing cellular proliferation, reducing inflammation and ultimately regenerating a normal intestinal epithelium. EV administration has a rescuing effect on intestinal injury when given during NEC induction; however, it failed to prevent injury when given prior to NEC induction. AFSC-derived EV administration is thus a potential emergent novel treatment strategy for NEC.

Mothers of Preterm Infants Have Individualized Breast Milk Microbiota that Changes Temporally Based on Maternal Characteristics.

Mother's milk contains complex microbial communities thought to be important for colonizing a preterm infant's gastrointestinal tract. However, little is known about the microbiota in the preterm mother's milk and factors influencing its composition. We characterized the temporal dynamics of microbial communities in 490 breast milk samples from 86 mothers of preterm infants (born <1,250g) over the first 8 weeks postpartum. Highly individualized microbial communities were identified in each mother's milk that changed temporally with notable alterations in predicted microbial functions. However, pre-pregnancy BMI, delivery mode, and antibiotics were associated with changes in these microbial dynamics. Individual classes of antibiotics and their duration of exposure during prenatal and postpartum periods showed unique relationships with microbial taxa abundance and diversity in mother's milk. These results highlight the temporal complexity of the preterm mother's milk microbiota and its relationship with maternal characteristics as well as the importance of discussing antibiotic stewardship for mothers.

Vitamin B12 Deficiency Alters the Gut Microbiota in a Murine Model of Colitis.

Purpose: Inflammatory bowel disease (IBD) refers to a spectrum of autoimmune diseases, which result in chronic intestinal inflammation. Previous findings suggest a role for diet, nutrition and dysbiosis of the gut microbiota in both the development and progression of the condition. Vitamin B12 is a key cofactor of methionine synthase and is produced solely by microbes. Previous work links increased levels of homocysteine, a substrate of methionine synthase, MetH, to IBD indicating a potential role for vitamin B12 deficiency in intestinal injury and inflammation. This study assessed the role of vitamin B12 in shaping the gut microbiota and determining responses to intestinal injury using a reproducible murine model of colitis. Methods: The effects of vitamin B12 supplementation and deficiency were assessed in vivo; 3-week-old post-weanling C57Bl/6 mice were divided into three dietary treatment groups: (1) sufficient vitamin B12 (50 mg/Kg), (2) deficient vitamin B12 (0 mg/Kg) and (3) supplemented vitamin B12 (200 mg/Kg) for a period of 4 weeks. Intestinal injury was induced with 2% dextran sodium sulphate (DSS) via drinking water for 5 days. The impact of varying levels of dietary vitamin B12 on gut microbiota composition was assessed using 16S rRNA gene sequencing from fecal samples collected at day 0 and day 28 of the dietary intervention, and 7 days following induction of colitis on day 38, when blood and colonic tissues were also collected. Results: No significant alterations were found in the gut microbiota composition of disease-free animals in response to dietary interventions. By contrast, after DSS-induced colitis, >30 genera were significantly altered in vitamin B12 deficient mice. Altered B12 levels produced no significant effect on composite disease-activity scores; however, administration of a B12 deficient diet resulted in reduced DSS-induced epithelial tissue damage. Conclusions: Vitamin B12 supplementation does not alter the gut microbiota composition under healthy conditions, but does contribute to differential microbial responses and intestinal dysbiosis following the induction of experimental colitis.

A randomized trial evaluating virus-specific effects of a combination probiotic in children with acute gastroenteritis.

Gastroenteritis accounts for nearly 500,000 deaths in children younger than 5 years annually. Although probiotics have been touted as having the potential to expedite diarrhea resolution, recent clinical trials question their effectiveness. A potential explanation is a shift in pathogens following the introduction of a rotavirus vaccine. Here, we report the results of a multi-center, double-blind trial of 816 children with acute gastroenteritis who completed follow-up and provided multiple stool specimens. Participants were randomized to receive a probiotic containing Lactobacillus rhamnosus and Lactobacillus helveticus or placebo. We report no virus-specific beneficial effects attributable to the probiotic, either in reducing clinical symptoms or viral nucleic acid clearance from stool specimens collected up to 28 days following enrollment. We provide pathophysiological and microbiologic evidence to support the clinical findings and conclude that our data do not support routine probiotic administration to children with acute gastroenteritis, regardless of the infecting virus.

Gut microbiota in chronic inflammatory disorders: A focus on pediatric inflammatory bowel diseases and juvenile idiopathic arthritis.

The gut microbiota is integral to human health, including maintaining the delicate balance between tolerance and protection against potentially harmful pathogens. A growing body of evidence implicates the intestinal microbiome in immune-mediated inflammatory disorders; these data span the spectrum from genetic and environmental disease risk factors, to animal studies (particularly germ-free and gnotobiotic models) and human studies, including evidence of dysbiosis in diseased individuals compared to healthy populations. In this review, we summarize both animal and human data supporting a link between the gut microbiota and inflammatory bowel diseases (IBD) and systemic inflammatory arthritis, as models for chronic inflammatory disorders, while offering a pediatric focus (pediatric IBD and juvenile idiopathic arthritis). We discuss relevant mechanisms related to the crosstalk between the gut microbiota and the innate and adaptive immune system. We close with a brief discussion of emerging microbe-altering interventions, including fecal microbial transplantation and its immunologic effects.