Association of intestinal bacteria with immune activation in a 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.

Methods in Nutrition & Gut Microbiome Research: An American Society for Nutrition Satellite Session [13 October 2022].

The microbial cells colonizing the human body form an ecosystem that is integral to the regulation and maintenance of human health. Elucidation of specific associations between the human microbiome and health outcomes is facilitating the development of microbiome-targeted recommendations and treatments (e.g., fecal microbiota transplant; pre-, pro-, and post-biotics) to help prevent and treat disease. However, the potential of such recommendations and treatments to improve human health has yet to be fully realized. Technological advances have led to the development and proliferation of a wide range of tools and methods to collect, store, sequence, and analyze microbiome samples. However, differences in methodology at each step in these analytic processes can lead to variability in results due to the unique biases and limitations of each component. This technical variability hampers the detection and validation of associations with small to medium effect sizes. Therefore, the American Society for Nutrition (ASN) Nutritional Microbiology Group Engaging Members (GEM), sponsored by the Institute for the Advancement of Food and Nutrition Sciences (IAFNS), hosted a satellite session on methods in nutrition and gut microbiome research to review currently available methods for microbiome research, best practices, as well as tools and standards to aid in comparability of methods and results. This manuscript summarizes the topics and research discussed at the session. Consideration of the guidelines and principles reviewed in this session will increase the accuracy, precision, and comparability of microbiome research and ultimately the understanding of the associations between the human microbiome and health.

Association of Estimated Daily Lactose Consumption, Lactase Persistence Genotype (rs4988235), and Gut Microbiota in Healthy Adults in the United States.

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.

Associations among Milk Microbiota, Milk Fatty Acids, Milk Glycans, and Inflammation from Lactating Holstein Cows.

Milk oligosaccharides (MOs) can be prebiotic and antiadhesive, while fatty acids (MFAs) can be antimicrobial. Both have been associated with milk microbes or mammary gland inflammation in humans. Relationships between these milk components and milk microbes or inflammation have not been determined for cows and could help elucidate a novel approach for the dairy industry to promote desired milk microbial composition for improvement of milk quality and reduction of milk waste. We aimed to determine relationships among milk microbiota, MFAs, MOs, lactose, and somatic cell counts (SCC) from Holstein cows, using our previously published data. Raw milk samples were collected at three time points, ranging from early to late lactation. Data were analyzed using linear mixed-effects modeling and repeated-measures correlation. Unsaturated MFA and short-chain MFA had mostly negative relationships with potentially pathogenic genera, including Corynebacterium, Pseudomonas, and an unknown Enterobacteriaceae genus but numerous positive relationships with symbionts Bifidobacterium and Bacteroides. Conversely, many MOs were positively correlated with potentially pathogenic genera (e.g., Corynebacterium, Enterococcus, and Pseudomonas), and numerous MOs were negatively correlated with the symbiont Bifidobacterium. The neutral, nonfucosylated MO composed of eight hexoses had a positive relationship with SCC, while lactose had a negative relationship with SCC. One interpretation of these trends might be that in milk, MFAs disrupt primarily pathogenic bacterial cells, causing a relative increase in abundance of beneficial microbial taxa, while MOs respond to and act on pathogenic taxa primarily through antiadhesive methods. Further research is needed to confirm the potential mechanisms driving these correlations. IMPORTANCE 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. Relationships among milk microbes, fatty acids, oligosaccharides, and inflammation have been reported for humans. To our knowledge, associations among the milk microbial composition, fatty acids, oligosaccharides, and lactose have not been reported for healthy lactating cows. Identifying these potential relationships in bovine milk will inform future efforts to characterize direct and indirect interactions of the milk components with the milk microbiota. Since many milk components are associated with herd management practices, determining if these milk components impact milk microbes may provide valuable information for dairy cow management and breeding practices aimed at minimizing harmful and spoilage-causing microbes in raw milk.

Dietary Intake of Monosaccharides from Foods is Associated with Characteristics of the Gut Microbiota and Gastrointestinal Inflammation in Healthy US Adults.

BACKGROUND: Current assessment of dietary carbohydrates does not adequately reflect the nutritional properties and effects on gut microbial structure and function. Deeper characterization of food carbohydrate composition can serve to strengthen the link between diet and gastrointestinal health outcomes. OBJECTIVES: The present study aims to characterize the monosaccharide composition of diets in a healthy US adult cohort and use these features to assess the relationship between monosaccharide intake, diet quality, characteristics of the gut microbiota, and gastrointestinal inflammation. METHODS: This observational, cross-sectional study enrolled males and females across age (18-33 y, 34-49 y, and 50-65 y) and body mass index (normal, 18.5-24.99 kg/m2; overweight, 25-29.99 kg/m2; and obese, 30-44 kg/m2) categories. Recent dietary intake was assessed by the automated self-administered 24-h dietary recall system, and gut microbiota were assessed with shotgun metagenome sequencing. Dietary recalls were mapped to the Davis Food Glycopedia to estimate monosaccharide intake. Participants with >75% of carbohydrate intake mappable to the glycopedia were included (N = 180). RESULTS: Diversity of monosaccharide intake was positively associated with the total Healthy Eating Index score (Pearson's r = 0.520, P = 1.2 × 10-13) and negatively associated with fecal neopterin (Pearson's r = -0.247, P = 3.0 × 10-3). Comparing high with low intake of specific monosaccharides revealed differentially abundant taxa (Wald test, P < 0.05), which was associated with the functional capacity to break down these monomers (Wilcoxon rank-sum test, P < 0.05). CONCLUSIONS: Monosaccharide intake was associated with diet quality, gut microbial diversity, microbial metabolism, and gastrointestinal inflammation in healthy adults. As specific food sources were rich in particular monosaccharides, it may be possible in the future to tailor diets to fine-tune the gut microbiota and gastrointestinal function. This trial is registered at www. CLINICALTRIALS: gov as NCT02367287.

Trimethylamine N-Oxide Response to a Mixed Macronutrient Tolerance Test in a Cohort of Healthy United States Adults.

Plasma trimethylamine n-oxide (TMAO) concentration increases in responses to feeding TMAO, choline, phosphatidylcholine, L-carnitine, and betaine but it is unknown whether concentrations change following a mixed macronutrient tolerance test (MMTT) with limited amounts of TMAO precursors. In this proof-of-concept study, we provided healthy female and male adults (n = 97) ranging in age (18-65 years) and BMI (18-44 kg/m2) a MMTT (60% fat, 25% sucrose; 42% of a standard 2000 kilo calorie diet) and recorded their metabolic response at fasting and at 30 min, 3 h, and 6 h postprandially. We quantified total exposure to TMAO (AUC-TMAO) and classified individuals by the blood draw at which they experienced their maximal TMAO concentration (TMAO-response groups). We related AUC-TMAO to the 16S rRNA microbiome, to two SNPs in the exons of the FMO3 gene (rs2266782, G>A, p.Glu158Lys; and rs2266780, A>G, p.Glu308Gly), and to a priori plasma metabolites. We observed varying TMAO responses (timing and magnitude) and identified a sex by age interaction such that AUC-TMAO increased with age in females but not in males (p-value = 0.0112). Few relationships between AUC-TMAO and the fecal microbiome and FMO3 genotype were identified. We observed a strong correlation between AUC-TMAO and TNF-α that depended on TMAO-response group. These findings promote precision nutrition and have important ramifications for the eating behavior of adults who could benefit from reducing TMAO exposure, and for understanding factors that generate plasma TMAO.

Associations of microbial and indoleamine-2,3-dioxygenase-derived tryptophan metabolites with immune activation in healthy adults.

Background: Tryptophan (Trp) metabolites from intestinal bacteria (indole, indole acetic acid [IAA] and indole propionic acid [IPA]), and the Trp metabolite kynurenine (Kyn) from the indoleamine 2,3-dioxygenase (IDO) pathway, are aryl hydrocarbon receptor (AhR) agonists and thus, can regulate immune activity via the AhR pathway. We hypothesized that plasma concentrations of these metabolites would be associated with markers of immune activation in a cohort of healthy adults in a manner consistent with AhR-mediated immune-regulation. We also hypothesized that the plasma Kyn/Trp ratio, a marker of IDO activity, would be associated with immune markers reflecting IDO activation in innate immune cells. Finally, we hypothesized that some intestinal bacteria would be associated with plasma indole, IPA and IAA, and that these bacteria themselves would be associated with immune markers. Methods: A novel set of 88 immune markers, and plasma Trp metabolites, were measured in 362 healthy adults. Bacterial taxa from stool were identified by 16S rRNA gene analysis. Multiple linear regression analysis was used to identify significant associations with immune markers. Results: The sum of indole and IAA was positively associated with natural killer T-cells levels. Kyn and Kyn/Trp were positively associated with neopterin and IP-10, markers of type 1 immunity, and TNF-α and C-reactive protein (CRP), markers of the acute phase response, and the regulatory cytokine IL-10. Three bacteria negatively associated with Trp metabolites were associated with markers of immune activation: the family Lachnospiraceae with higher lymphocyte counts but lower level of activated CD4 T-cells, the genus Dorea with higher production of IFN-γ by T-cells in PBMC cultures, and the genus Ruminococcus with higher production IL-6 in PBMC cultures stimulated with bacterial lipopolysaccharide (LPS). Conclusions: In this cohort of healthy adults bacterial Trp metabolites were not strongly associated with immune markers. Conversely, the Kyn/Trp ratio was strongly associated with markers of systemic inflammation and the acute phase response, consistent with IDO activation in innate immune cells. Finally, commensal bacteria associated with lower plasma (and perhaps intestinal) levels of bacterial Trp metabolites were associated with greater immune activation, possibly reflecting decreased regulatory immune activity related to lower intestinal levels of bacterial indole metabolites.

A Low-Starch and High-Fiber Diet Intervention Impacts the Microbial Community of Raw Bovine Milk.

Background: A more sustainable dairy cow diet was designed that minimizes use of feed components digestible by monogastric animals by increasing the quantity of forages. Objectives: This study determined if feeding lactating cows the more sustainable, low-starch and high-fiber (LSHF) diet was associated with changes in raw milk microbiota composition and somatic cell count (SCC). Methods: In a crossover design, 76 lactating Holstein cows were assigned to an LSHF diet or a high-starch and low-fiber (HSLF) diet, similar to common dairy cow diets in the United States, for 10 wk then placed on the opposite diet for 10 wk. The LSHF diet contained greater quantities of forages, beet pulp, and corn distillers' grain, but contained less canola meal and no high-moisture corn compared with the HSLF diet. Raw milk samples were collected from each cow 4-5 d before intervention and 5 wk into each diet treatment. Within 4 d, additional milk samples were collected for measurement of SCC using Fossmatic 7. The microbial community was determined by sequencing the 16S rRNA gene V4-V5 region and analyzing sequences with QIIME2. After quality filtering, 53 cows remained. Results: Raw milk microbial communities differed by diet and time. Taxa associated with fiber consumption, such as Lachnospiraceae, Lactobacillus, Bacteroides, and Methanobrevibacter, were enriched with the LSHF diet. Meanwhile, taxa associated with mastitis, such as Pseudomonas, Stenotrophomonas, and Enterobacteriaceae, were enriched with the HSLF diet. Relatedly, an interaction of diet and time was found to impact SCC. Conclusions: In raw milk, consumption of an LSHF diet compared with an HSLF diet was associated with changes in abundance of microbes previously associated with fiber consumption, udder health, and milk spoilage. Further research is needed to determine if an LSHF diet indeed leads to lower rates of mastitis and milk spoilage, which could benefit the dairy industry.

Association of Diet and Antimicrobial Resistance in Healthy U.S. Adults.

Antimicrobial resistance (AMR) represents a significant source of morbidity and mortality worldwide, with expectations that AMR-associated consequences will continue to worsen throughout the coming decades. Since resistance to antibiotics is encoded in the microbiome, interventions aimed at altering the taxonomic composition of the gut might allow us to prophylactically engineer microbiomes that harbor fewer antibiotic resistant genes (ARGs). Diet is one method of intervention, and yet little is known about the association between diet and antimicrobial resistance. To address this knowledge gap, we examined diet using the food frequency questionnaire (FFQ; habitual diet) and 24-h dietary recalls (Automated Self-Administered 24-h [ASA24®] tool) coupled with an analysis of the microbiome using shotgun metagenome sequencing in 290 healthy adult participants of the United States Department of Agriculture (USDA) Nutritional Phenotyping Study. We found that aminoglycosides were the most abundant and prevalent mechanism of AMR in these healthy adults and that aminoglycoside-O-phosphotransferases (aph3-dprime) correlated negatively with total calories and soluble fiber intake. Individuals in the lowest quartile of ARGs (low-ARG) consumed significantly more fiber in their diets than medium- and high-ARG individuals, which was concomitant with increased abundances of obligate anaerobes, especially from the family Clostridiaceae, in their gut microbiota. Finally, we applied machine learning to examine 387 dietary, physiological, and lifestyle features for associations with antimicrobial resistance, finding that increased phylogenetic diversity of diet was associated with low-ARG individuals. These data suggest diet may be a potential method for reducing the burden of AMR. IMPORTANCE Antimicrobial resistance (AMR) represents a considerable burden to health care systems, with the public health community largely in consensus that AMR will be a major cause of death worldwide in the coming decades. Humans carry antibiotic resistance in the microbes that live in and on us, collectively known as the human microbiome. Diet is a powerful method for shaping the human gut microbiome and may be a tractable method for lessening antibiotic resistance, and yet little is known about the relationship between diet and AMR. We examined this relationship in healthy individuals who contained various abundances of antibiotic resistance genes and found that individuals who consumed diverse diets that were high in fiber and low in animal protein had fewer antibiotic resistance genes. Dietary interventions may be useful for lessening the burden of antimicrobial resistance and might ultimately motivate dietary guidelines which will consider how nutrition can reduce the impact of infectious disease.

Diet, Fecal Microbiome, and Trimethylamine N-Oxide in a Cohort of Metabolically Healthy United States Adults.

TMAO is elevated in individuals with cardiometabolic diseases, but it is unknown whether the metabolite is a biomarker of concern in healthy individuals. We conducted a cross-sectional study in metabolically healthy adults aged 18-66 years with BMI 18-44 kg/m2 and assessed the relationship between TMAO and diet, the fecal microbiome, and cardiometabolic risk factors. TMAO was measured in fasted plasma samples by liquid chromatography mass spectrometry. The fecal microbiome was assessed by 16S ribosomal RNA sequencing and recent food intake was captured by multiple ASA24 dietary recalls. Endothelial function was assessed via EndoPAT. Descriptive statistics were computed by fasting plasma TMAO tertiles and evaluated by ANOVA and Tukey's post-hoc test. Multiple linear regression was used to assess the relationship between plasma TMAO and dietary food intake and metabolic health parameters. TMAO concentrations were not associated with average intake of animal protein foods, fruits, vegetables, dairy, or grains. TMAO was related to the fecal microbiome and the genera Butyribrio, Roseburia, Coprobaciullus, and Catenibacterium were enriched in individuals in the lowest versus the highest TMAO tertile. TMAO was positively associated with α-diversity and compositional differences were identified between groups. TMAO was not associated with classic cardiovascular risk factors in the healthy cohort. Similarly, endothelial function was not related to fasting TMAO, whereas the inflammatory marker TNF-α was significantly associated. Fasting plasma TMAO may not be a metabolite of concern in generally healthy adults unmedicated for chronic disease. Prospective studies in healthy individuals are necessary.

Tree-Based Analysis of Dietary Diversity Captures Associations Between Fiber Intake and Gut Microbiota Composition in a Healthy US Adult Cohort.

BACKGROUND: Diet patterns are a significant and modifiable contributing factor to the composition of the human gut microbiota. OBJECTIVES: We set out to identify reproducible relationships between diet and gut microbial community composition in a diverse, healthy US adult cohort. METHODS: We collected 2 to 3 automated self-administered 24-hour dietary recalls over 10-14 days, together with a single stool sample, from 343 healthy adults in a cross-sectional phenotyping study. This study examined a multi-ethnic cohort balanced for age (18-65 years), sex, and BMI (18.5-45 kg/m2). Dietary data were edited to a tree format according to published methods. The tree structure was annotated with the average total grams of dry weight, fat, protein, carbohydrate, or fiber from each food item reported. The alpha and beta diversity measurements, calculated using the tree structure, were analyzed relative to the microbial community diversity, determined by a Quantitative Insights Into Microbial Ecology (QIIME) 2 analysis of the bacterial 16S ribosomal RNA V4 region, sequenced from stool samples. K-means clustering was used to form groups of individuals consuming similar diets, and gut microbial communities were compared among groups using differential expression analysis for sequence count data. RESULTS: The alpha diversity of diet dry weight was significantly correlated with the gut microbial community alpha diversity (r = 0.171). The correlation improved when diet was characterized using grams of carbohydrates (r = 0.186) or fiber (r = 0.213). Bifidobacterium was enriched with diets containing higher levels of total carbohydrate from cooked grains. Lachnospira, was enriched with diet patterns containing high consumption of fiber from fruits excluding berries. CONCLUSIONS: The tree structure, annotated with grams of carbohydrate, is a robust analysis method for comparing self-reported diet to the gut microbial community composition. This method identified consumption of fiber from fruit robustly associated with an abundance of pectinolytic bacterial genus, Lachnospira, in the guts of healthy adults. This trial was registered at clinicaltrials.gov as NCT02367287.

Resistant Starch Type 2 from Wheat Reduces Postprandial Glycemic Response with Concurrent Alterations in Gut Microbiota Composition.

The majority of research on the physiological effects of dietary resistant starch type 2 (RS2) has focused on sources derived from high-amylose maize. In this study, we conduct a double-blind, randomized, placebo-controlled, crossover trial investigating the effects of RS2 from wheat on glycemic response, an important indicator of metabolic health, and the gut microbiota. Overall, consumption of RS2-enriched wheat rolls for one week resulted in reduced postprandial glucose and insulin responses relative to conventional wheat when participants were provided with a standard breakfast meal containing the respective treatment rolls (RS2-enriched or conventional wheat). This was accompanied by an increase in the proportions of bacterial taxa Ruminococcus and Gemmiger in the fecal contents, reflecting the composition in the distal intestine. Additionally, fasting breath hydrogen and methane were increased during RS2-enriched wheat consumption. However, although changes in fecal short-chain fatty acid (SCFA) concentrations were not significant between control and RS-enriched wheat roll consumption, butyrate and total SCFAs were positively correlated with relative abundance of Faecalibacterium, Ruminoccocus, Roseburia, and Barnesiellaceae. These effects show that RS2-enriched wheat consumption results in a reduction in postprandial glycemia, altered gut microbial composition, and increased fermentation activity relative to wild-type wheat.

Microbiota Assessments for the Identification and Confirmation of Slit Defect-Causing Bacteria in Milk and Cheddar Cheese.

Validated methods are needed to detect spoilage microbes present in low numbers in foods and ingredients prior to defect onset. We applied propidium monoazide combined with 16S rRNA gene sequencing, qPCR, isolate identification, and pilot-scale cheese making to identify the microorganisms that cause slit defects in industrially produced Cheddar cheese. To investigate milk as the source of spoilage microbes, bacterial composition in milk was measured immediately before and after high-temperature, short-time (HTST) pasteurization over 10-h periods on 10 days and in the resulting cheese blocks. Besides HTST pasteurization-induced changes to milk microbiota composition, a significant increase in numbers of viable bacteria was observed over the 10-h run times of the pasteurizer, including 68-fold-higher numbers of the genus Thermus However, Thermus was not associated with slit development. Milk used to make cheese which developed slits instead contained a lower number of total bacteria, higher alpha diversity, and higher proportions of Lactobacillus, Bacillus, Brevibacillus, and Clostridium Only Lactobacillus proportions were significantly increased during cheese aging, and Limosilactobacillus (Lactobacillus) fermentum, in particular, was enriched in slit-containing cheeses and the pre- and post-HTST-pasteurization milk used to make them. Pilot-scale cheeses developed slits when inoculated with strains of L. fermentum, other heterofermentative lactic acid bacteria, or uncultured bacterial consortia from slit-associated pasteurized milk, thereby confirming that low-abundance taxa in milk can negatively affect cheese quality. The likelihood that certain microorganisms in milk cause slit defects can be predicted based on comparisons of the bacteria present in the milk used for cheese manufacture.IMPORTANCE Food production involves numerous control points for microorganisms to ensure quality and safety. These control points (e.g., pasteurization) are difficult to develop for fermented foods wherein some microbial contaminants are also expected to provide positive contributions to the final product and spoilage microbes may constitute only a small proportion of all microorganisms present. We showed that microbial composition assessments with 16S rRNA marker gene DNA sequencing are sufficiently robust to detect very-low-abundance bacterial taxa responsible for a major but sporadic Cheddar cheese spoilage defect. Bacterial composition in the (pasteurized) milk and cheese was associated with slit defect development. The application of Koch's postulates showed that individual bacterial isolates as well as uncultured bacterial consortia were sufficient to cause slits, even when present in very low numbers. This approach may be useful for detection and control of low-abundance spoilage microorganisms present in other foods.

The Znt7-null mutation has sex dependent effects on the gut microbiota and goblet cell population in the mouse colon.

Cellular homeostasis of zinc, an essential element for living organisms, is tightly regulated by a family of zinc transporters. The zinc transporter 7, ZnT7, is highly expressed on the membrane of the Golgi complex of intestinal epithelial cells and goblet cells. It has previously been shown that Znt7 knockout leads to zinc deficiency and decreased weight gain in C57BL/6 mice on a defined diet. However, effects within the colon are unknown. Given the expression profile of Znt7, we set out to analyze the changes in mucin density and gut microbial composition in the mouse large intestine induced by Znt7 knockout. We fed a semi-purified diet containing 30 mg Zn/kg to Znt7-/- mice with their heterozygous and wild type littermates and found a sex specific effect on colonic mucin density, goblet cell number, and microbiome composition. In male mice Znt7 knockout led to increased goblet cell number and mucin density but had little effect on gut microbiome composition. However, in female mice Znt7 knockout was associated with decreased goblet cell number and mucin density, with increased proportions of the microbial taxa, Allobaculum, relative to wild type. The gut microbial composition was correlated with mucin density in both sexes. These findings suggest that a sex-specific relationship exists between zinc homeostasis, mucin production and the microbial community composition within the colon.

Infant gut microbiota characteristics generally do not modify effects of lipid-based nutrient supplementation on growth or inflammation: secondary analysis of a randomized controlled trial in Malawi.

An unhealthy gut microbial community may act as a barrier to improvement in growth and health outcomes in response to nutritional interventions. The objective of this analysis was to determine whether the infant microbiota modified the effects of a randomized controlled trial of lipid-based nutrient supplements (LNS) in Malawi on growth and inflammation at 12 and 18 months, respectively. We characterized baseline microbiota composition of fecal samples at 6 months of age (n = 506, prior to infant supplementation, which extended to 18 months) using 16S rRNA gene sequencing of the V4 region. Features of the gut microbiota previously identified as being involved in fatty acid or micronutrient metabolism or in outcomes relating to growth and inflammation, especially in children, were investigated. Prior to correction for multiple hypothesis testing, the effects of LNS on growth appeared to be modified by Clostridium (p-for-interaction = 0.02), Ruminococcus (p-for-interaction = 0.007), and Firmicutes (p-for-interaction = 0.04) and effects on inflammation appeared to be modified by Faecalibacterium (p-for-interaction = 0.03) and Streptococcus (p-for-interaction = 0.004). However, after correction for multiple hypothesis testing these findings were not statistically significant, suggesting that the gut microbiota did not alter the effect of LNS on infant growth and inflammation in this cohort.

The Role of the Gut Microbiome in Predicting Response to Diet and the Development of Precision Nutrition Models. Part II: Results.

The gut microbiota is increasingly implicated in the health and metabolism of its human host. The host's diet is a major component influencing the composition and function of the gut microbiota, and mounting evidence suggests that the composition and function of the gut microbiota influence the host's metabolic response to diet. This effect of the gut microbiota on personalized dietary response is a growing focus of precision nutrition research and may inform the effort to tailor dietary advice to the individual. Because the gut microbiota has been shown to be malleable to some extent, it may also allow for therapeutic alterations of the gut microbiota in order to alter response to certain dietary components. This article is the second in a 2-part review of the current research in the field of precision nutrition incorporating the gut microbiota into studies investigating interindividual variability in response to diet. Part I reviews the methods used by researchers to design and carry out such studies as well as analyze the results subsequently obtained. Part II reviews the findings of these studies and discusses the gaps in our current knowledge and directions for future research. The studies reviewed provide the current understanding in this field of research and a foundation from which we may build, utilizing and expanding upon the methods and results they present to inform future studies.

The Role of the Gut Microbiome in Predicting Response to Diet and the Development of Precision Nutrition Models-Part I: Overview of Current Methods.

Health care is increasingly focused on health at the individual level. In the rapidly evolving field of precision nutrition, researchers aim to identify how genetics, epigenetics, and the microbiome interact to shape an individual's response to diet. With this understanding, personalized responses can be predicted and dietary advice can be tailored to the individual. With the integration of these complex sources of data, an important aspect of precision nutrition research is the methodology used for studying interindividual variability in response to diet. This article stands as the first in a 2-part review of current research investigating the contribution of the gut microbiota to interindividual variability in response to diet. Part I reviews the methods used by researchers to design and carry out such studies as well as the statistical and bioinformatic methods used to analyze results. Part II reviews the findings of these studies, discusses gaps in our current knowledge, and summarizes directions for future research. Taken together, these reviews summarize the current state of knowledge and provide a foundation for future research on the role of the gut microbiome in precision nutrition.

Viable and Total Bacterial Populations Undergo Equipment- and Time-Dependent Shifts during Milk Processing.

We set out to identify the viable and total bacterial content in milk as it passes through a large-scale, dairy product manufacturing plant for pasteurization, concentration, separation, blending, and storage prior to cheese manufacture. A total of 142 milk samples were collected from up to 10 pieces of equipment for a period spanning 21 h on two collection dates in the spring and late summer of 2014. Bacterial composition in the milk was determined by 16S rRNA marker gene, high-throughput DNA sequencing. Milk samples from the late summer were paired such that half were treated with propidium monoazide (PMA) to enrich for viable cells prior to quantification by PCR and identification by DNA sequence analysis. Streptococcus had the highest median relative abundance across all sampling sites within the facility on both sampling dates. The proportions of Anoxybacillus, Thermus, Lactococcus, Lactobacillus, Micrococcaceae, and Pseudomonas were also elevated in some samples. Viable cells detected by PMA treatment showed that Turicibacter was enriched after high-temperature short-time pasteurization, whereas proportions of Staphylococcus were significantly reduced. Using clean-in-place (CIP) times as a reference point, Bacillus, Pseudomonas, and Anoxybacillus were found in high relative proportions in several recently cleaned silos (<19 h since CIP). At later times (>19 h after CIP), 10 of 11 silos containing elevated viable cell numbers were enriched in Acinetobacter and/or Lactococcus These results show the tremendous point-to-point and sample-dependent variations in bacterial composition in milk during processing.IMPORTANCE Milk undergoes sustained contact with the built environment during processing into finished dairy products. This contact has the potential to influence the introduction, viability, and growth of microorganisms within the milk. Currently, the population dynamics of bacteria in milk undergoing processing are not well understood. Therefore, we measured for total and viable bacterial composition and cell numbers in milk over time and at different processing points in a cheese manufacturing facility in California. Our results provide new perspectives on the dramatic variations in microbial populations in milk during processing even over short amounts of time. Although some of the changes in the milk microbiota were predictable (e.g., reduced viable cell numbers after pasteurization), other findings could not be easily foreseen based on knowledge of bacteria contained in raw milk or when the equipment was last cleaned. This information is important for predicting and controlling microbial spoilage contaminants in dairy products.

Fecal metatranscriptomics of macaques with idiopathic chronic diarrhea reveals altered mucin degradation and fucose utilization.

BACKGROUND: Idiopathic chronic diarrhea (ICD) is a common cause of morbidity and mortality among juvenile rhesus macaques. Characterized by chronic inflammation of the colon and repeated bouts of diarrhea, ICD is largely unresponsive to medical interventions, including corticosteroid, antiparasitic, and antibiotic treatments. Although ICD is accompanied by large disruptions in the composition of the commensal gut microbiome, no single pathogen has been concretely identified as responsible for the onset and continuation of the disease. RESULTS: Fecal samples were collected from 12 ICD-diagnosed macaques and 12 age- and sex-matched controls. RNA was extracted for metatranscriptomic analysis of organisms and functional annotations associated with the gut microbiome. Bacterial, fungal, archaeal, protozoan, and macaque (host) transcripts were simultaneously assessed. ICD-afflicted animals were characterized by increased expression of host-derived genes involved in inflammation and increased transcripts from bacterial pathogens such as Campylobacter and Helicobacter and the protozoan Trichomonas. Transcripts associated with known mucin-degrading organisms and mucin-degrading enzymes were elevated in the fecal microbiomes of ICD-afflicted animals. Assessment of colon sections using immunohistochemistry and of the host transcriptome suggests differential fucosylation of mucins between control and ICD-afflicted animals. Interrogation of the metatranscriptome for fucose utilization genes reveals possible mechanisms by which opportunists persist in ICD. Bacteroides sp. potentially cross-fed fucose to Haemophilus whereas Campylobacter expressed a mucosa-associated transcriptome with increased expression of adherence genes. CONCLUSIONS: The simultaneous profiling of bacterial, fungal, archaeal, protozoan, and macaque transcripts from stool samples reveals that ICD of rhesus macaques is associated with increased gene expression by pathogens, increased mucin degradation, and altered fucose utilization. The data suggest that the ICD-afflicted host produces fucosylated mucins that are leveraged by potentially pathogenic microbes as a carbon source or as adhesion sites.

Impact of DNA Sequencing and Analysis Methods on 16S rRNA Gene Bacterial Community Analysis of Dairy Products.

DNA sequencing and analysis methods were compared for 16S rRNA V4 PCR amplicon and genomic DNA (gDNA) mock communities encompassing nine bacterial species commonly found in milk and dairy products. The two communities comprised strain-specific DNA that was pooled before (gDNA) or after (PCR amplicon) the PCR step. The communities were sequenced on the Illumina MiSeq and Ion Torrent PGM platforms and then analyzed using the QIIME 1 (UCLUST) and Divisive Amplicon Denoising Algorithm 2 (DADA2) analysis pipelines with taxonomic comparisons to the Greengenes and Ribosomal Database Project (RDP) databases. Examination of the PCR amplicon mock community with these methods resulted in operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) that ranged from 13 to 118 and were dependent on the DNA sequencing method and read assembly steps. The additional 4 to 109 OTUs/ASVs (from 9 OTUs/ASVs) included assignments to spurious taxa and sequence variants of the 9 species included in the mock community. Comparisons between the gDNA and PCR amplicon mock communities showed that combining gDNAs from the different strains prior to PCR resulted in up to 8.9-fold greater numbers of spurious OTUs/ASVs. However, the DNA sequencing method and paired-end read assembly steps conferred the largest effects on predictions of bacterial diversity, with effect sizes of 0.88 (Bray-Curtis) and 0.32 (weighted Unifrac), independent of the mock community type. Overall, DNA sequencing performed with the Ion Torrent PGM and analyzed with DADA2 and the Greengenes database resulted in the most accurate predictions of the mock community phylogeny, taxonomy, and diversity.IMPORTANCE Validated methods are urgently needed to improve DNA sequence-based assessments of complex bacterial communities. In this study, we used 16S rRNA PCR amplicon and gDNA mock community standards, consisting of nine, dairy-associated bacterial species, to evaluate the most commonly applied 16S rRNA marker gene DNA sequencing and analysis platforms used in evaluating dairy and other bacterial habitats. Our results show that bacterial metataxonomic assessments are largely dependent on the DNA sequencing platform and read curation method used. DADA2 improved sequence annotation compared with QIIME 1, and when combined with the Ion Torrent PGM DNA sequencing platform and the Greengenes database for taxonomic assignment, the most accurate representation of the dairy mock community standards was reached. This approach will be useful for validating sample collection and DNA extraction methods and ultimately investigating bacterial population dynamics in milk- and dairy-associated environments.