BACKGROUND:Honey has well-described antimicrobial properties in wound healing, but little is known about its effects within the gastrointestinal tract. OBJECTIVE:We used in vitro gastric digestion and fermentation of clover honey to investigate its potential interactions with small intestine bacteria (SIB), the foodborne pathogen enterotoxigenic Escherichia coli (ETEC), and small intestine epithelial cells. METHODS:In vitro gastric digestions of sterile-filtered honey, and a simple sugar control were performed with 1 × 108 colony-forming units (CFU)/mL ETEC H10407. ETEC CFU and pH were measured, and the resulting digesta were added to batch fermentations with SIB, in the form of a mock community or ileal aspirate from 6 healthy donors. SIB fermentations were characterized by 16S rRNA amplicon sequencing and application of fermentation supernatants to mature Caco-2 cell monolayers for analysis of changes in transepithelial electrical resistance (TEER) and interleukin (IL)-8 production. Differential abundance of bacterial taxa detected by 16S rRNA sequencing between the honey and sugar conditions was tested using an Analysis of Compositions of Microbiomes with Bias Correction 2. Differences in ETEC CFU, media pH, and changes in TEER and IL-8 production between the honey and sugar conditions were tested by linear mixed effects modeling with post hoc least-squares means analysis. RESULTS:Under pH 2.5 in vitro gastric digestion conditions, ETEC CFU decreased significantly (P < 0.0001) more in the presence of clover honey (0.0035% input) than in a sugar control (0.036% input). The pH of SIB fermentation media was significantly reduced (P < 0.0001) with digested honey relative to sugar. Honey fermentation supernatant from human ileal communities challenged with ETEC elicited a greater improvement in TEER (3.68 compared with -0.80, P = 0.0479) relative to the same fermentations performed with control. CONCLUSIONS:Honey limits the survival of ETEC H10407 within an in vitro gastric environment at low pH. Clover honey may also impart decreased pH and potential improvement in in vitro epithelial cell barrier integrity in the presence of a pathogen.

ABSTRACT Gastroenteritis is an important cause of ailment among infants in high-income countries, including Denmark. There are many cases of diarrhea among Danish children for which no etiological agent is detected. This study investigated the associations between gastroenteritis-related morbidities (diarrhea, fever, and vomiting) and gut microbial community in well-nourished, breastfeeding Danish infants. Infant stool samples, morbidity questionnaires, and diet/breastfeeding questionnaires were collected at three time points during the first 8.5 months of life. The V4-V5 region of the 16S rRNA gene was amplified from stool DNA extracts, sequenced with Illumina MiSeq, and analyzed using QIIME2. PERMANOVA, linear mixed-effects modeling, and ANCOM-BC2 were used to identify associations between infant gut microbiome and gastroenteritis-related morbidities across all visits. Logistic regression and random forest models were employed to determine whether early gut microbial alpha diversity or abundance, respectively, could predict morbidity later in infancy. Alpha diversity was negatively associated with diarrhea. Granulicatella abundance was positively associated with diarrhea. From 3.5 to 6 months of age, Bacteroidales abundance was negatively associated with fever. Faith’s phylogenetic diversity, Staphylococcales abundance, and Haemophilus abundance during 3.5 months of life were positively associated with future diarrhea during ages 3.5–8.5 months. Pielou’s evenness and Actinobacteriota abundance within 3.5 months of life had negative and positive associations, respectively, with fever during ages 3.5–8.5 months. This study reveals diarrhea is associated with stool Granulicatella in well-nourished, breastfeeding Danish infants, as it is in children from low-income countries, and that the early gut microbiome may contribute to risks of diarrhea and fever morbidities later in infancy.CLINICAL TRIALSThis trial was registered at ClinicalTrials.gov as NCT03254329.IMPORTANCEGastroenteritis continues to cause much morbidity among infants in high-income countries, and the relationship with the gut microbiome is not fully understood, especially for well-nourished and breastfeeding infants. In the study presented here, infant stool Staphylococcales abundance (comprised of Staphylococcus and Gemella) and Haemophilus abundance during the first few months of life were positively associated with later diarrhea in well-nourished and breastfeeding Danish infants. Meanwhile, the abundance of Granulicatella (a facultative anaerobe with pathogenic potential) was greater in stool from infants who had recent diarrhea, suggesting further research is needed to determine its possible role in diarrhea and recovery from diarrhea. Fever usually did not co-occur with diarrhea or vomiting. Early life Actinobacteriota abundance was positively associated with later fever. This phylum was represented here by both pathobionts (Actinomyces) and mutualists (bifidobacteria), which may have contributed to fever differently—pathobionts through infection and mutualists through promotion of effective immune response to infection.

Dietary polyphenols are bioactive compounds with a bidirectional impact on the gut microbiome; they shape the microbial community and are transformed through bacterial metabolism. However, there are limited studies pairing metagenomic and dietary data to investigate the relationship between polyphenol intake and the taxonomic and functional profiles of the human gut microbiome. We examined if dietary polyphenol intake associates with microbial composition and polyphenol utilization capacity. Healthy adults participated in a cross-sectional study balanced for age, sex, and BMI. Polyphenol intake was previously estimated by mapping multiple 24 h dietary recalls to the Food Database (FooDB). We coupled intake with microbial taxonomic and functional profiles from shotgun-sequenced fecal metagenomes (n = 313). Microbial reads were mapped to dbPUP, a database with 60 experimentally characterized, gut-associated polyphenol utilization proteins (PUPs). We assessed the relationship of polyphenol intake on microbial diversity, abundance of microbes with PUP genes, PUP gene counts, and select lipopolysaccharide (LPS) producers, accounting for age, sex, BMI, fiber intake, and diet quality. Specific polyphenols associated with an increased abundance of nine PUP-containing genera. We found 117 associations between polyphenol intake and microbial PUP genes, with 85 associations involving hydrolysis PUPs. Diversity in polyphenol intake was positively associated with diversity in PUP genes but not with microbial diversity. Lastly, we detected a positive relationship between intake of olive-related polyphenol classes and abundance of order Bacteroidales, a producer of immunoinhibitory LPS. Dietary polyphenol intake may influence the gut microbiome's capacity for polyphenol utilization, particularly its hydrolytic activity, without impacting taxonomic diversity or composition.

Moringa oleifera is associated with several nutritional and therapeutic benefits. However, there is limited research on how much these health benefits are mediated directly by the plant or through fermentation with intestinal microbes. We examined the interaction between M. oleifera aqueous leaf extract and three common gut microbes whose abundance was significantly altered in previous intervention studies. Growth curves of Escherichia coli, Bifidobacterium longum, and Bacteroides thetaiotaomicron were examined in the presence of increasing concentrations of M. oleifera leaf extract in YCFA media with and without carbohydrates and short chain fatty acids (SCFAs). Anthrone and Fast Blue BB assays were conducted on spent media to measure carbohydrate and phenolic content, respectively. Sterile fermentation supernatants were applied to an in vitro gut barrier model consisting of differentiated Caco-2 monolayers on permeable cell culture inserts and the transepithelial resistance (TEER) was measured. Growth curve analysis demonstrated that the three bacterial isolates tested could grow in the presence of M. oleifera. However, B. longum had a greater increase in total growth, consumed more soluble carbohydrates, and produced more soluble polyphenols using M. oleifera leaf extract as a sole carbohydrate source than the other two microbes. Additionally, B. longum fermentation of both glucose and M. oleifera increased TEER in Caco-2 monolayers significantly more than E. coli fermentation of both carbohydrate sources (p = 0.0007). These results suggest a potential mechanism through which consumption of M. oleifera may promote the growth of probiotic organisms within the human gut to improve gut barrier integrity.

Diet modulates gut microbiome composition and function. However, determining causal links between diet-gut microbiome interactions and human health is complicated by inconsistencies in the evidence, arising partially from variability in research methods and reporting. Widespread adoption of standardized best practices would advance the field but require those practices to be identified, consolidated, and discussed. This umbrella review aimed to identify recommended best practices, define existing gaps, and collate considerations for conducting research on diet-gut microbiome interactions and their impact on human health outcomes. Reviews meeting inclusion criteria and published after 2013 were identified using a systematic search. Recommendations, considerations, and gaps relating to the best practices associated with study design, participant selection, dietary intervention/assessment, biological sample collection, and data analysis and reporting were extracted and consolidated. Eight narrative reviews were included. Several general points of agreement were identified, and a recurring theme was that best practices are dependent upon the research aims, outcomes, and feasibility. Multiple gaps were also identified. Some, such as suboptimal diet assessment methods and lack of validated dietary intake biomarkers, are particularly relevant to nutrition science. Others, including defining a "healthy" gut microbiome and the absence of standardized sample and data collection/analysis protocols, were relevant specifically to gut microbiome research. Gaps specific to diet-gut microbiome research include the underrepresentation of microbiome-modulating dietary components in food databases, lack of knowledge regarding interventions eliciting changes in the gut microbiome to confer health benefits, lack of in situ measurement methods, and the need to further develop and refine statistical approaches for integrating diet and gut microbiome data. Future research and cross-disciplinary exchange will address these gaps and evolve the best practices. In the interim, the best practices and considerations discussed herein, and the publications from which that information was extracted provide a roadmap for conducting diet-gut microbiome research. This trial was registered at PROSPERO as CRD42023437645.

Lactase persistence is a genetically inherited trait that enables continued lactose digestion into adulthood. Lactase non-persistence (LNP) individuals often experience incomplete lactose digestion, allowing undigested lactose to reach the colon, where it may shape microbial composition and function. We investigated the relationship between the lactase persistence (LP) genotype, lactose consumption, and the taxonomic and functional profiles of the fecal microbiome. Participants from the USDA Nutritional Phenotyping Study, a cross-sectional observational study designed to assess how dietary factors impact human health, whose fecal microbiome profile was measured using shotgun metagenomic sequencing (n = 330) were included in this analysis. Fecal SCFA levels were measured using GC-MS. Fecal microbiome taxonomy and gene abundance were quantified using shotgun metagenomic sequencing. Lactose consumption and yogurt intake were estimated based on Automated Self-Administered 24h Dietary Assessment Tool (ASA24®) dietary recalls or Food Frequency Questionnaire. The LP/LNP genotype was determined by a single nucleotide polymorphism (SNP ID: rs4988235). Several genera of lactic acid bacteria (Veillonella, Lactobacillus, Lacticaseibacillus, and Lactococcus) were differentially abundant between recent high-lactose consuming (>10.0 g lactose per day) and low-lactose consuming (<3.3 g lactose per day) individuals. Among the LNP participants who self-identified as Caucasian or Hispanic, high-lactose consumers (>10.0 g per day via 24-h recall) had significantly higher relative abundances of lactic acid bacteria and lactate-utilizing bacteria (Lacticaseibacillus, Lactobacillus, Megamonas, and Veillonella) than low-lactose consumers (<3.3 g per day). Independent of lactose intake, LNP participants had a higher abundance of fecal microbial β-galactosidase genes than LP participants. Among the LNP participants, those with high recent lactose consumption also showed a significant shift towards more fecal propionate. The abundance of the yogurt-associated microbe, Streptococcus thermophilus, was positively associated with yogurt intake independent of the genotype. Alternative milk consumption was significantly negatively associated with fecal SCFAs both in the full cohort and the Caucasian/Hispanic subset, regardless of the genotype. Our results suggest that functional and persistent host lactase enzymes may work to competitively exclude lactic acid bacteria, contributing to a smaller realized niche for lactic acid bacteria in LP individuals compared to LNP individuals. However, regardless of the host genotype, consumption of alternative milk may be associated with reduced production of health-promoting intestinal metabolites, such as SCFAs.

BACKGROUND:Gut microbes produce short-chain fatty acids (SCFAs), which are associated with broad health benefits. However, it is not fully known how diet and/or the gut microbiome could be modulated to improve SCFA production. OBJECTIVES:The objective of this study was to identify dietary, inflammatory, and/or microbiome predictors of SCFAs in a cohort of healthy adults. METHODS:SCFAs were measured in fecal and plasma samples from 359 healthy adults in the United States Department of Agriculture Nutritional Phenotyping Study. Habitual and recent diet was assessed using a Food Frequency Questionnaire and Automated Self-Administered 24-h Dietary Assesment Tool dietary recalls. Markers of systemic and gut inflammation were measured in fecal and plasma samples. The gut microbiome was assessed using shotgun metagenomics. Using statistics and machine learning, we determined how the abundance and composition of SCFAs varied with measures of diet, inflammation, and the gut microbiome. RESULTS:We show that fecal pH may be a good proxy for fecal SCFA abundance. A higher Healthy Eating Index for a habitual diet was associated with a compositional increase in fecal butyrate relative to acetate and propionate. SCFAs were associated with markers of subclinical gastrointestinal (GI) inflammation. Fecal SCFA abundance was inversely related to plasma lipopolysaccharide-binding protein. When we analyzed hierarchically organized diet and microbiome data with taxonomy-aware algorithms, we observed that diet and microbiome features were far more predictive of fecal SCFA abundances compared to plasma SCFA abundances. The top diet and microbiome predictors of fecal butyrate included potatoes and the thiamine biosynthesis pathway, respectively. CONCLUSIONS:These results suggest that resistant starch in the form of potatoes and microbially produced thiamine provide a substrate and essential cofactor, respectively, for butyrate synthesis. Thiamine may be a rate-limiting nutrient for butyrate production in adults. Overall, these findings illustrate the complex biology underpinning SCFA production in the gut. This trial was registered at clinicaltrials.gov as NCT02367287.

Background: Lactase persistence (LP) is a heritable trait in which lactose can be digested throughout adulthood. Lactase nonpersistent (LNP) individuals who consume lactose may experience microbial adaptations in response to undigested lactose.Objectives: The objective of the study was to estimate lactose from foods reported in the Automated Self-Administered 24-Hour Dietary Assessment Tool (ASA24) and determine the interaction between lactose consumption, LP genotype, and gut microbiome in an observational cross-sectional study of healthy adults in the United States (US).Methods: Average daily lactose consumption was estimated for 279 healthy US adults, genotyped for the lactase gene-13910G>A polymorphism (rs4988235) by matching ASA24-reported foods to foods in the Nutrition Coordinating Center Food and Nutrient Database. Analysis of covariance was used to identify whether the A genotype (LP) influenced lactose and total dairy consumption, with total energy intake and weight as covariates. The 16S rRNA V4/V5 region, amplified from bacterial DNA extracted from each frozen stool sample, was sequenced using Illumina MiSeq (300 bp paired-end) and analyzed using Quantitative Insights Into Microbial Ecology (QIIME)2 (version 2019.10). Differential abundances of bacterial taxa were analyzed using DESeq2 likelihood ratio tests.Results: Across a diverse set of ethnicities, LP subjects consumed more lactose than LNP subjects. Lactobacillaceae abundance was highest in LNP subjects who consumed more than 12.46 g/d (upper tercile). Within Caucasians and Hispanics, family Lachnospiraceae was significantly enriched in the gut microbiota of LNP individuals consuming the upper tercile of lactose across both sexes.Conclusions: Elevated lactose consumption in individuals with the LNP genotype is associated with increased abundance of family Lactobacillaceae and Lachnospriaceae, taxa that contain multiple genera capable of utilizing lactose. This trial was registered on clinicaltrials.gov as NCT02367287.

Bovine milk can harbor microbes that cause mastitis, milk spoilage, and foodborne illness. Fatty acids found in milk can be antimicrobial and milk oligosaccharides can have antiadhesive, prebiotic, and immune-modulatory effects.

ABSTRACT Interactions among intestinal bacteria and the immune system contribute to the maintenance of a functional intestinal barrier in healthy individuals, and possibly to systemic immune activity. We hypothesized that intestinal bacteria would be associated with systemic biomarkers of innate and adaptive immune responses in healthy adults. 79 immune function markers were subjected to factor analysis resulting in 17 Immune Factors (IFs), each composed of 2–10 immune variables. Bacterial taxa from stool samples were identified at the family and genus levels by 16S rRNA amplicon sequence analysis and their read counts and relative abundances were utilized in a multiple linear regression model to identify microbial taxa associated with the IFs. A total of 10 significant associations were identified between bacterial taxa and IFs. The family Rikenellaceae showed a positive association with innate IF5 (including 5 chemokines, 2 cytokines, 2 adhesion molecules, and the macrophage metabolite neopterin) and a negative association with adaptive IF4 (including T-cells with activation marker HLA-DR). Additionally, Pseudomonadaceae and its genus Pseudomonas showed a negative relationship with innate IF5, and adaptive IF13 (including T-cell cytokines IL-10, IL-17, and IFN-γ) was negatively associated with Butyrivibrio and positively associated with Slackia. These associations suggest ongoing interactions between gut bacteria and the systemic immune system in healthy adults. The association of these taxa with the IFs may result from specific microbial-immune system interactions that play a role in maintenance of a healthy barrier integrity in our cohort of healthy adults. IMPORTANCE Chronic inflammation may develop over time in healthy adults as a result of a variety of factors, such as poor diet directly affecting the composition of the intestinal microbiome, or by causing obesity, which may also affect the intestinal microbiome. These effects may trigger the activation of an immune response that could eventually lead to an inflammation-related disease, such as colon cancer. Before disease develops it may be possible to identify subclinical inflammation or immune activation attributable to specific intestinal bacteria normally found in the gut that could result in future adverse health impacts. In the present study, we examined a group of healthy men and women across a wide age range with and without obesity to determine which bacteria were associated with particular types of immune activation to identify potential preclinical markers of inflammatory disease risk. Several associations were found that may help develop dietary interventions to lower disease risk.