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IntroductionThe role of lipopolysaccharide binding protein (LBP), an inflammation marker of bacterial translocation from the gastrointestinal tract, in Alzheimer’s disease (AD) is not clearly understood.MethodsIn this study the concentrations of LBP were measured in n = 79 individuals: 20 apolipoprotein E (APOE)3/E3 carriers with and 20 without AD dementia, and 19 APOE3/E4 carriers with and 20 without AD dementia. LBP was found to be enriched in the 1.21–1.25 g/mL density fraction of plasma, which has previously been shown to be enriched in intestinally derived high-density lipoproteins (HDL). LBP concentrations were measured by ELISA.ResultsLBP was significantly increased within the 1.21–1.25 g/mL density fraction of plasma in APOE3/E3 AD patients compared to controls, but not APOE3/E4 patients. LBP was positively correlated with Clinical Dementia Rating (CDR) and exhibited an inverse relationship with Verbal Memory Score (VMS).DiscussionThese results underscore the potential contribution of gut permeability to bacterial toxins, measured as LBP, as an inflammatory mediator in the development of AD, particularly in individuals with the APOE3/E3 genotype, who are genetically at 4-12-fold lower risk of AD than individuals who express APOE4.

Abstract Glycosylation has been found to be altered in the brains of individuals with Alzheimer’s disease (AD). However, it is unknown which specific glycosylation-related pathways are altered in AD dementia. Using publicly available RNA-seq datasets covering seven brain regions and including 1724 samples, we identified glycosylation-related genes ubiquitously changed in individuals with AD. Several differentially expressed glycosyltransferases found by RNA-seq were confirmed by qPCR in a different set of human medial temporal cortex (MTC) samples (n = 20 AD vs. 20 controls). N-glycan-related changes predicted by expression changes in these glycosyltransferases were confirmed by mass spectrometry (MS)-based N-glycan analysis in the MTC (n = 9 AD vs. 6 controls). About 80% of glycosylation-related genes were differentially expressed in at least one brain region of AD participants (adjusted p-values

High-density lipoproteins (HDLs) are key regulators of cellular cholesterol homeostasis but are functionally altered in many chronic diseases. The factors that cause HDL functional loss in chronic disease are not fully understood. It is also unknown what roles antioxidant carotenoids play in protecting HDL against functional loss. The aim of this study was to measure how various disease-associated chemical factors including exposure to (1) Cu2+ ions, (2) hypochlorous acid (HOCL), (3) hydrogen peroxide (H2O2), (4) sialidase, (5) glycosidase, (6) high glucose, (7) high fructose, and (8) acidic pH, and the carotenoid antioxidants (9) lutein and (10) zeaxanthin affect HDL functionality. We hypothesized that some of the modifications would have stronger impacts on HDL particle structure and function than others and that lutein and zeaxanthin would improve HDL function. HDL samples were isolated from generally healthy human plasma and incubated with the corresponding treatments listed above. Cholesterol efflux capacity (CEC), lecithin–cholesterol acyl transferase (LCAT) activity, and paraoxonase-1 (PON1) activity were measured in order to determine changes in HDL functionality. Median HDL particle diameter was increased by acidic pH treatment and reduced by HOCl, high glucose, high fructose, N-glycosidase, and lutein treatments. Acidic pH, oxidation, and fructosylation all reduced HDL CEC, whereas lutein, zeaxanthin, and sialidase treatment improved HDL CEC. LCAT activity was reduced by acidic pH, oxidation, high fructose treatments, and lutein. PON1 activity was reduced by sialidase, glycosidase, H2O2, and fructose and improved by zeaxanthin and lutein treatment. These results show that exposure to oxidizing agents, high fructose, and low pH directly impairs HDL functionality related to cholesterol efflux and particle maturation, whereas deglycosylation impairs HDL antioxidant capacity. On the other hand, the antioxidants lutein and zeaxanthin improve or preserve both HDL cholesterol efflux and antioxidant activity but have no effect on particle maturation.

BackgroundProlonged fasting, characterized by restricting caloric intake for 24 h or more, has garnered attention as a nutritional approach to improve lifespan and support healthy aging. Previous research from our group showed that a single bout of 36-h water-only fasting in humans resulted in a distinct metabolomic signature in plasma and increased levels of bioactive metabolites, which improved macrophage function and lifespan in C. elegans.ObjectiveThis secondary outcome analysis aimed to investigate changes in the plasma lipidome associated with prolonged fasting and explore any potential links with markers of cardiometabolic health and aging.MethodWe conducted a controlled pilot study with 20 male and female participants (mean age, 27.5 ± 4.4 years; mean BMI, 24.3 ± 3.1 kg/m2) in four metabolic states: (1) overnight fasted (baseline), (2) 2-h postprandial fed state (fed), (3) 36-h fasted state (fasted), and (4) 2-h postprandial refed state 12 h after the 36-h fast (refed). Plasma lipidomic profiles were analyzed using liquid chromatography and electrospray ionization mass spectrometry.ResultsSeveral lipid classes, including lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylethanolamine, and triacylglycerol were significantly reduced in the 36-h fasted state, while free fatty acids, ceramides, and sphingomyelin were significantly increased compared to overnight fast and fed states (P

The function of high-density lipoprotein (HDL) particles has emerged as a promising therapeutic target and the measurement of HDL function is a promising diagnostic across several disease states. The vast majority of research on HDL functional biology has focused on adult participants with underlying chronic diseases, whereas limited research has investigated the role of HDL in childhood, pregnancy, and old age. Yet, it is apparent that functional HDL is essential at all life stages for maintaining health. In this review, we discuss current data regarding the role of HDL during childhood, pregnancy and in the elderly, how disturbances in HDL may lead to adverse health outcomes, and knowledge gaps in the role of HDL across these life stages.

Research has found that genes specific to microglia are among the strongest risk factors for Alzheimer’s disease (AD) and that microglia are critically involved in the etiology of AD. Thus, microglia are an important therapeutic target for novel approaches to the treatment of AD. High-throughput in vitro models to screen molecules for their effectiveness in reversing the pathogenic, pro-inflammatory microglia phenotype are needed. In this study, we used a multi-stimulant approach to test the usefulness of the human microglia cell 3 (HMC3) cell line, immortalized from a human fetal brain-derived primary microglia culture, in duplicating critical aspects of the dysfunctional microglia phenotype. HMC3 microglia were treated with cholesterol (Chol), amyloid beta oligomers (AβO), lipopolysaccharide (LPS), and fructose individually and in combination. HMC3 microglia demonstrated changes in morphology consistent with activation when treated with the combination of Chol + AβO + fructose + LPS. Multiple treatments increased the cellular content of Chol and cholesteryl esters (CE), but only the combination treatment of Chol + AβO + fructose + LPS increased mitochondrial Chol content. Microglia treated with combinations containing Chol + AβO had lower apolipoprotein E (ApoE) secretion, with the combination of Chol + AβO + fructose + LPS having the strongest effect. Combination treatment with Chol + AβO + fructose + LPS also induced APOE and TNF-α expression, reduced ATP production, increased reactive oxygen species (ROS) concentration, and reduced phagocytosis events. These findings suggest that HMC3 microglia treated with the combination of Chol + AβO + fructose + LPS may be a useful high-throughput screening model amenable to testing on 96-well plates to test potential therapeutics to improve microglial function in the context of AD.

Abstract High-density lipoprotein (HDL) particles have multiple beneficial and cardioprotective roles, yet our understanding of their full structural and functional repertoire is limited due to challenges in separating HDL particles from contaminating plasma proteins and other lipid-carrying particles that overlap HDL in size and/or density. Here we describe a method for isolating HDL particles using a combination of sequential flotation density ultracentrifugation and fast protein liquid chromatography with a size exclusion column. Purity was visualized by polyacrylamide gel electrophoresis and verified by proteomics, while size and structural integrity were confirmed by transmission electron microscopy. This HDL isolation method can be used to isolate a high yield of purified HDL from a low starting plasma volume for functional analyses. This method also enables investigators to select their specific HDL fraction of interest: from the least inclusive but highest purity HDL fraction eluting in the middle of the HDL peak, to pooling all of the fractions to capture the breadth of HDL particles in the original plasma sample. We show that certain proteins such as lecithin cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and clusterin (CLUS) are enriched in large HDL particles whereas proteins such as alpha-2HS-glycoprotein (A2HSG), alpha-1 antitrypsin (A1AT), and vitamin D binding protein (VDBP) are enriched or found exclusively in small HDL particles.

Summary: The extracellular RNA communication consortium (ERCC) is an NIH-funded program aiming to promote the development of new technologies, resources, and knowledge about exRNAs and their carriers. After Phase 1 (2013–2018), Phase 2 of the program (ERCC2, 2019–2023) aims to fill critical gaps in knowledge and technology to enable rigorous and reproducible methods for separation and characterization of both bulk populations of exRNA carriers and single EVs. ERCC2 investigators are also developing new bioinformatic pipelines to promote data integration through the exRNA atlas database. ERCC2 has established several Working Groups (Resource Sharing, Reagent Development, Data Analysis and Coordination, Technology Development, nomenclature, and Scientific Outreach) to promote collaboration between ERCC2 members and the broader scientific community. We expect that ERCC2’s current and future achievements will significantly improve our understanding of exRNA biology and the development of accurate and efficient exRNA-based diagnostic, prognostic, and theranostic biomarker assays.

Dietary fiber, a nutrient derived mainly from whole grains, vegetables, fruits, and legumes, is known to confer a number of health benefits, yet most Americans consume less than half of the daily recommended amount. Convenience and affordability are key factors determining the ability of individuals to incorporate fiber-rich foods into their diet, and many Americans struggle to access, afford, and prepare foods rich in fiber. The objective of this clinical study was to test the changes in microbial community composition, human metabolomics, and general health markers of a convenient, easy to use prebiotic supplement in generally healthy young participants consuming a diet low in fiber. Twenty healthy adults participated in this randomized, placebo-controlled, double-blind, crossover study which was registered at clinicaltrials.gov as NCT03785860. During the study participants consumed 12 g of a prebiotic fiber supplement and 12 g of placebo daily as a powder mixed with water as part of their habitual diet in randomized order for 4 weeks, with a 4-week washout between treatment arms. Fecal microbial DNA was extracted and sequenced by shallow shotgun sequencing on an Illumina NovaSeq. Plasma metabolites were detected using liquid chromatography–mass spectrometry with untargeted analysis. The phylum Actinobacteria, genus Bifidobacterium, and several Bifidobacterium species (B. bifidum, B. adolescentis, B. breve, B. catenulatum, and B. longum) significantly increased after prebiotic supplementation when compared to the placebo. The abundance of genes associated with the utilization of the prebiotic fiber ingredients (sacA, xfp, xpk) and the production of acetate (poxB, ackA) significantly changed with prebiotic supplementation. Additionally, the abundance of genes associated with the prebiotic utilization (xfp, xpk), acetate production (ackA), and choline to betaine oxidation (gbsB) were significantly correlated with changes in the abundance of the genus Bifidobacterium in the prebiotic group. Plasma concentrations of the bacterially produced metabolite indolepropionate significantly increased. The results of this study demonstrate that an easy to consume, low dose (12 g) of a prebiotic powder taken daily increases the abundance of beneficial bifidobacteria and the production of health-promoting bacteria-derived metabolites in healthy individuals with a habitual low-fiber diet.Clinical Trial Registrationwww.clinicaltrials.gov/, identifier: NCT03785860

High-density lipoprotein (HDL) particles, long known for their critical role in the prevention of cardiovascular disease (CVD), were recently identified to carry a wide array of glycosylated proteins, and the importance of this glycosylation in the structure, function and metabolism of HDL are starting to emerge. Early studies have demonstrated differential glycosylation of HDL-associated proteins in various pathological states, which may be key to understanding their etiological role in these diseases and may be important for diagnostic development. Given the vast array and specificity of glycosylation pathways, the study of HDL-associated glycosylation has the potential to uncover novel mechanisms and biomarkers of CVD. To date, no large studies examining the relationships between HDL glycosylation profiles and cardiovascular outcomes have been performed. However, small pilot studies provide promising preliminary evidence that such a relationship may exist. In this review article we discuss the current state of the evidence on the glycosylation of HDL-associated proteins, the potential for HDL glycosylation profiling in CVD diagnostics, how glycosylation affects HDL function, and the potential for modifying the glycosylation of HDL-associated proteins to confer therapeutic value.

Cholesterol is essential for brain function and structure, however altered cholesterol metabolism and transport are hallmarks of multiple neurodegenerative conditions, including Alzheimer’s disease (AD). The well-established link between apolipoprotein E (APOE) genotype and increased AD risk highlights the importance of cholesterol and lipid transport in AD etiology. Whereas more is known about the regulation and dysregulation of cholesterol metabolism and transport in neurons and astrocytes, less is known about how microglia, the immune cells of the brain, handle cholesterol, and the subsequent implications for the ability of microglia to perform their essential functions. Evidence is emerging that a high-cholesterol environment, particularly in the context of defects in the ability to transport cholesterol (e.g., expression of the high-risk APOE4 isoform), can lead to chronic activation, increased inflammatory signaling, and reduced phagocytic capacity, which have been associated with AD pathology. In this narrative review we describe how cholesterol regulates microglia phenotype and function, and discuss what is known about the effects of statins on microglia, as well as highlighting areas of future research to advance knowledge that can lead to the development of novel therapies for the prevention and treatment of AD.

High-density lipoproteins (HDL) play a critical role in cholesterol homeostasis. Apolipoprotein E (APOE), particularly the E4 allele, is a significant risk factor for Alzheimer’s disease but is also a key HDL-associated protein involved in lipid transport in both the periphery and central nervous systems. The objective was to determine the influence of the APOE genotype on HDL function and size in the context of Alzheimer’s disease. HDL from 194 participants (non-demented controls, mild cognitive impairment, and Alzheimer’s disease dementia) were isolated from the plasma. The HDL cholesterol efflux capacity (CEC), lecithin-cholesterol acyltransferase (LCAT) activity, and particle diameter were measured. Neuropsychological test scores, clinical dementia rating, and magnetic resonance imaging scores were used to determine if cognition is associated with HDL function and size. HDL CEC and LCAT activity were reduced in APOE3E4 carriers compared to APOE3E3 carriers, regardless of diagnosis. In APOE3E3 carriers, CEC and LCAT activity were lower in patients. In APOE3E4 patients, the average particle size was lower. HDL LCAT activity and particle size were positively correlated with the neuropsychological scores and negatively correlated with the clinical dementia rating. We provide evidence for the first time of APOE genotype-specific alterations in HDL particles in Alzheimer’s disease and an association between HDL function, size, and cognitive function.

Abstract We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single‐particle interferometric reflectance imaging sensing (SP‐IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP‐IRIS events could not be used to estimate particle concentrations. For sizing, SP‐IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP‐IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP‐IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single‐particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.

Intense recent interest in understanding how the human gut microbiome influences health has kindled a concomitant interest in linking dietary choices to microbiome variation. Diet is known to be a driver of microbiome variation, and yet the precise mechanisms by which certain dietary components modulate the microbiome, and by which the microbiome produces byproducts and secondary metabolites from dietary components, are not well-understood. Interestingly, despite the influence of diet on the gut microbiome, the majority of microbiome studies published to date contain little or no analysis of dietary intake. Although an increasing number of microbiome studies are now collecting some form of dietary data or even performing diet interventions, there are no clear standards in the microbiome field for how to collect diet data or how to design a diet-microbiome study. In this article, we review the current practices in diet-microbiome analysis and study design and make several recommendations for best practices to provoke broader discussion in the field. We recommend that microbiome studies include multiple consecutive microbiome samples per study timepoint or phase and multiple days of dietary history prior to each microbiome sample whenever feasible. We find evidence that direct effects of diet on the microbiome are likely to be observable within days, while the length of an intervention required for observing microbiome-mediated effects on the host phenotype or host biomarkers, depending on the outcome, may be much longer, on the order of weeks or months. Finally, recent studies demonstrating that diet-microbiome interactions are personalized suggest that diet-microbiome studies should either include longitudinal sampling within individuals to identify personalized responses, or should include an adequate number of participants spanning a range of microbiome types to identify generalized responses.

The gut microbiome has recently emerged as a critical modulator of brain function, with the so-called gut-brain axis having multiple links with a variety of neurodegenerative and mental health conditions, including Alzheimer’s Disease (AD). Various approaches for modulating the gut microbiome toward compositional and functional states that are consistent with improved cognitive health outcomes have been documented, including probiotics and prebiotics. While probiotics are live microorganisms that directly confer beneficial health effects, prebiotics are oligosaccharide and polysaccharide structures that can beneficially modulate the gut microbiome by enhancing the growth, survival, and/or function of gut microbes that in turn have beneficial effects on the human host. In this review, we discuss evidence showing the potential link between gut microbiome composition and AD onset or development, provide an overview of prebiotic types and their roles in altering gut microbial composition, discuss the effectiveness of prebiotics in regulating gut microbiome composition and microbially derived metabolites, and discuss the current evidence linking prebiotics with health outcomes related to AD in both animal models and human trials. Though there is a paucity of human clinical trials demonstrating the effectiveness of prebiotics in altering gut microbiome-mediated health outcomes in AD, current evidence highlights the potential of various prebiotic approaches for beneficially altering the gut microbiota or gut physiology by promoting the production of butyrate, indoles, and secondary bile acid profiles that further regulate gut immunity and mucosal homeostasis, which are associated with beneficial effects on the central immune system and brain functionality.

Abstract Background Inflammation is associated with increased bone resorption; the role of inflammation in postprandial bone turnover has not been explored. Consumption of milk fat globule membrane (MFGM) reduces inflammation in animal models. This study aimed to measure postprandial changes in bone turnover after intake of high saturated fat test meals, with- and without the anti-inflammatory ingredient MFGM. Methods Subjects (n = 36 adults) were obese (BMI 30–39.9 kg/m2) or overweight (BMI 25–29.9 kg/m2) with two traits of Metabolic Syndrome. Subjects consumed a different test meal on four occasions at random; blood draws were taken at baseline and 1, 3, and 6 h postprandial. Test meals included whipping cream (WC), WC + MFGM, palm oil (PO) and PO + MFGM. Biomarkers of bone turnover and inflammation were analyzed from all four time points. Results Test meal (treatment) by time interactions were significant for bone resorption marker C-telopeptide of type 1 collagen (CTX) (p

Mechanistic research suggests a unique evolutionary relationship between complex milk oligosaccharides and cognate bifidobacteria enriched in breast-fed infants. Bovine milk oligosaccharides (BMO) were recently identified as structurally and functionally similar to human milk oligosaccharides. The present single-blind three-way crossover study is the first to determine the safety and tolerability of BMO consumption by healthy human participants (n 12) and its effects on faecal microbiota and microbial metabolism. Participants consumed each supplement (placebo-control; low- and high-BMO doses) for eleven consecutive days, followed by a 2-week washout period prior to initiating the next supplement arm. Low and high BMO doses were consumed as 25 and 35 % of each individual's daily fibre intake, respectively. Safety and tolerability were measured using standardised questionnaires on gut and stomach discomfort and stool consistency. Faecal extracts were profiled for bacterial populations by next-generation sequencing (NGS) and bifidobacteria presence was confirmed using quantitative PCR. Urine was analysed for changes in microbial metabolism using nuclear magnetic resonance spectroscopy (1H-NMR). Consumption of both the low and high BMO doses was well tolerated and did not change stool consistency from baseline. Multivariate analysis of the NGS results demonstrated no change in faecal microbiota phyla among the placebo-control and BMO supplement groups. In conclusion, BMO supplementation was well tolerated in healthy adults and has the potential to shift faecal microbiota toward beneficial strains as part of a synbiotic treatment with probiotic cultures that selectively metabolise oligosaccharides.

Dietary recommendations suggest decreased consumption of SFA to minimise CVD risk; however, not all foods rich in SFA are equivalent. To evaluate the effects of SFA in a dairy food matrix, as Cheddar cheese, v. SFA from a vegan-alternative test meal on postprandial inflammatory markers, a randomised controlled cross-over trial was conducted in twenty overweight or obese adults with metabolic abnormalities. Individuals consumed two isoenergetic high-fat mixed meals separated by a 1- to 2-week washout period. Serum was collected at baseline, and at 1, 3 and 6 h postprandially and analysed for inflammatory markers (IL-6, IL-8, IL-10, IL-17, IL-18, TNFα, monocyte chemotactic protein-1 (MCP-1)), acute-phase proteins C-reactive protein (CRP) and serum amyloid-A (SAA), cellular adhesion molecules and blood lipids, glucose and insulin. Following both high-fat test meals, postprandial TAG concentrations rose steadily (P < 0·05) without a decrease by 6 h. The incremental AUC (iAUC) for CRP was significantly lower (P < 0·05) in response to the cheese compared with the vegan-alternative test meal. A treatment effect was not observed for any other inflammatory markers; however, for both test meals, multiple markers significantly changed from baseline over the 6 h postprandial period (IL-6, IL-8, IL-18, TNFα, MCP-1, SAA). Saturated fat in the form of a cheese matrix reduced the iAUC for CRP compared with a vegan-alternative test meal during the postprandial 6 h period. The study is registered at clinicaltrials.gov under NCT01803633.

Meals high in SFA, particularly palmitate, are associated with postprandial inflammation and insulin resistance. Milk fat globule membrane (MFGM) has anti-inflammatory properties that may attenuate the negative effects of SFA-rich meals. Our objective was to examine the postprandial metabolic and inflammatory response to a high-fat meal composed of palm oil (PO) compared with PO with an added dairy fraction rich in MFGM (PO+MFGM) in overweight and obese men and women (n 36) in a randomised, double-blinded, cross-over trial. Participants consumed two isoenergetic high-fat meals composed of a smoothie enriched with PO with v. without a cream-derived complex milk lipid fraction ( dairy fraction rich in MFGM) separated by a washout of 1–2 weeks. Serum cytokines, adhesion molecules, cortisol and markers of inflammation were measured at fasting, and at 1, 3 and 6 h postprandially. Glucose, insulin and lipid profiles were analysed in plasma. Consumption of the PO + MFGM v. PO meal resulted in lower total cholesterol (P = 0·021), LDL-cholesterol (P = 0·046), soluble intracellular adhesion molecule (P = 0·005) and insulin (P = 0·005) incremental AUC, and increased IL-10 (P = 0·013). Individuals with high baseline C-reactive protein (CRP) concentrations (≥3 mg/l, n 17) had higher (P = 0·030) insulin at 1 h after the PO meal than individuals with CRP concentrations

This article focuses on the role of omega-3 fatty acids as precursors for lipid signaling molecules known as oxylipins. Although omega-3 fatty acids are beneficial in autoimmune disorders, inflammatory diseases and heart disease, they are generally underrepresented in the American diet. A literature review confirms that the consumption of omega-3 fatty acids - whether in food sources such as walnuts, flax seeds and fatty fish (including salmon and sardines), or in supplements - is associated with decreased morbidity and mortality. This growing body of evidence, including the results of a recent study of patients with kidney disease, highlights the need to measure omega-3 fatty acids and their oxylipin products as markers of metabolic health and biomarkers of disease. In addition, there is substantial evidence of the need to increase the omega-3 fatty acid content of American diets to optimize metabolic health.

Far more attention has been paid to the microbes in our feces than the microbes in our food. Research efforts dedicated to the microbes that we eat have historically been focused on a fairly narrow range of species, namely those which cause disease and those which are thought to confer some “probiotic” health benefit. Little is known about the effects of ingested microbial communities that are present in typical American diets, and even the basic questions of which microbes, how many of them, and how much they vary from diet to diet and meal to meal, have not been answered.We characterized the microbiota of three different dietary patterns in order to estimate: the average total amount of daily microbes ingested via food and beverages, and their composition in three daily meal plans representing three different dietary patterns. The three dietary patterns analyzed were: (1) the Average American (AMERICAN): focused on convenience foods, (2) USDA recommended (USDA): emphasizing fruits and vegetables, lean meat, dairy, and whole grains, and (3) Vegan (VEGAN): excluding all animal products. Meals were prepared in a home kitchen or purchased at restaurants and blended, followed by microbial analysis including aerobic, anaerobic, yeast and mold plate counts as well as 16S rRNA PCR survey analysis.Based on plate counts, the USDA meal plan had the highest total amount of microbes at 1.3 × 109 CFU per day, followed by the VEGAN meal plan and the AMERICAN meal plan at 6 × 106 and 1.4 × 106 CFU per day respectively. There was no significant difference in diversity among the three dietary patterns. Individual meals clustered based on taxonomic composition independent of dietary pattern. For example, meals that were abundant in Lactic Acid Bacteria were from all three dietary patterns. Some taxonomic groups were correlated with the nutritional content of the meals. Predictive metagenome analysis using PICRUSt indicated differences in some functional KEGG categories across the three dietary patterns and for meals clustered based on whether they were raw or cooked.Further studies are needed to determine the impact of ingested microbes on the intestinal microbiota, the extent of variation across foods, meals and diets, and the extent to which dietary microbes may impact human health. The answers to these questions will reveal whether dietary microbes, beyond probiotics taken as supplements—i.e., ingested with food—are important contributors to the composition, inter-individual variation, and function of our gut microbiota.