Gut Microbiota and Symptom Expression and Severity in Neonatal Abstinence Syndrome.

Problem: Neonatal abstinence syndrome (NAS) affecting neonates with fetal exposure to opioids, is defined by expression and severity of symptoms. The pathophysiology behind symptoms variability is lacking. The study aims were to examine (a) differences in gut microbiota of neonates with and without NAS, (b) the relationships between gut microbiota and symptom expression and NAS severity, and (c) the changes in the neonate gut microbiota diversity during the course of NAS treatment. Methods: A cross-sectional observational design was used to examine differences in microbiota and a longitudinal, repeated measures approach was used to determine relationships between gut microbiota and NAS symptoms. Symptom data were collected using the Finnegan Neonatal Abstinence Scoring Tool and the Neonatal Pain Agitation and Sedation Scale. Stool samples were collected for microbiome analyses with 16S rRNA microbiome sequencing. Results: Differences in alpha and beta diversity between neonates with and without NAS were seen. Relative abundance results revealed 18 taxa were different in neonates with NAS compared to neonates without NAS. No differences were found in alpha or beta diversity in neonates with NAS between enrollment and hospital discharge. There was increased abundance of Escherichia-Shigella and Bacteriodes genera related to higher symptom scores. Discussion: Differences in alpha and beta diversity between neonates with and without NAS may be due to differences in birth mode and type of feeding. The findings of specific increased bacteria related to increased symptoms in the neonates with NAS may also be influenced by birth mode and type of feeding.

Gut microbiome responds to alteration in female sex hormone status and exacerbates metabolic dysfunction.

Women are at significantly greater risk of metabolic dysfunction after menopause, which subsequently leads to numerous chronic illnesses. The gut microbiome is associated with obesity and metabolic dysfunction, but its interaction with female sex hormone status and the resulting impact on host metabolism remains unclear. Herein, we characterized inflammatory and metabolic phenotypes as well as the gut microbiome associated with ovariectomy and high-fat diet feeding, compared to gonadal intact and low-fat diet controls. We then performed fecal microbiota transplantation (FMT) using gnotobiotic mice to identify the impact of ovariectomy-associated gut microbiome on inflammatory and metabolic outcomes. We demonstrated that ovariectomy led to greater gastrointestinal permeability and inflammation of the gut and metabolic organs, and that a high-fat diet exacerbated these phenotypes. Ovariectomy also led to alteration of the gut microbiome, including greater fecal ß-glucuronidase activity. However, differential changes in the gut microbiome only occurred when fed a low-fat diet, not the high-fat diet. Gnotobiotic mice that received the gut microbiome from ovariectomized mice fed the low-fat diet had greater weight gain and hepatic gene expression related to metabolic dysfunction and inflammation than those that received intact sham control-associated microbiome. These results indicate that the gut microbiome responds to alterations in female sex hormone status and contributes to metabolic dysfunction. Identifying and developing gut microbiome-targeted modulators to regulate sex hormones may be useful therapeutically in remediating menopause-related diseases.

Effect of Diets Varying in Iron and Saturated Fat on the Gut Microbiota and Intestinal Inflammation: A Crossover Feeding Study among Older Females with Obesity.

Obesity is considered an independent risk factor for colorectal cancer (CRC). Altered nutrient metabolism, particularly changes to digestion and intestinal absorption, may play an important role in the development of CRC. Iron can promote the formation of tissue-damaging and immune-modulating reactive oxygen species. We conducted a crossover, controlled feeding study to examine the effect of three, 3-week diets varying in iron and saturated fat content on the colonic milieu and systemic markers among older females with obesity. Anthropometrics, fasting venous blood and stool were collected before and after each diet. There was a minimum 3-week washout period between diets. Eighteen participants consumed the three diets (72% Black; mean age 60.4 years; mean body mass index 35.7 kg/m2). Results showed no effect of the diets on intestinal inflammation (fecal calprotectin) or circulating iron, inflammation, and metabolic markers. Pairwise comparisons revealed less community diversity between samples (beta diversity, calculated from 16S rRNA amplicon sequences) among participants when consuming a diet low in iron and high in saturated fat vs. when consuming a diet high in iron and saturated fat. More studies are needed to investigate if dietary iron represents a salient target for CRC prevention among individuals with obesity.

Facilitating a high-quality dietary pattern induces shared microbial responses linking diet quality, blood pressure, and microbial sterol metabolism in caregiver-child dyads.

Low-resource individuals are at increased risk of obesity and cardiovascular disease (CVD), partially attributable to poor dietary patterns and dysfunctional microbiota. Dietary patterns in childhood play critical roles in physiological development and are shaped by caregivers, making caregiver-child dyads attractive targets for dietary interventions to reduce metabolic disease risk. Herein, we targeted low-resource caregiver-child dyads for a 10-week, randomized, controlled, multifaceted lifestyle intervention including: nutrition and physical activity education, produce harvesting, cooking demonstrations, nutrition counseling, and kinetic activites; to evaluate its effects on dietary patterns, CVD risk factors, and microbiome composition. Subjects in the lifestyle intervention group improved total diet quality, increased whole grain intake, decreased energy intake, and enhanced fecal elimination of the microbe-derived metabolite lithocholic acid (LCA) in contrast to control subjects. Microbiomes were highly personalized, similar within dyads, and altered by lifestyle intervention. Differential modeling of microbiome composition identified taxa associated with total diet quality, whole grain intake, and LCA elimination including recognized fiber-degrading bacteria such as Subdoligranulum, and bile acid metabolizing organisms like Bifidobacterium. Inclusion of taxa identified in diet and metabolite modeling within blood pressure models improved prediction accuracy of microbiome-blood pressure associations. Importantly, microbiota-blood pressure relationships were shared between dyads, implying shared host-microbiota responses to lifestyle intervention. Overall, these outcomes provide insight into mechanisms by which dietary interventions impact the gut-cardiovascular axis to reduce future CVD risk. Registered at clinicaltrials.gov: NCT05367674.

Collection of biospecimens from parent-child dyads in a community garden-based nutrition intervention: protocol and feasibility.

BACKGROUND: Non-invasive human biospecimens, including stool, urine, and hair, are important in understanding the relationship between diet and changes in human physiologic processes that affect chronic disease outcomes. However, biospecimen collection can be difficult when collecting samples for research studies that occur away from a centralized location. We describe the protocol and feasibility in collecting stool, urine, and hair biospecimens from parents and their children at a remote location as a part of a summer community garden-based intervention. METHODS: Stool, urine, and hair were collected as a part of the Summer Harvest Adventure (SHA) study, a randomized controlled, community garden-based intervention targeting children (ages 8-11 years) and their parents from low-resource neighborhoods. Biospecimens were collected from willing children and/or their parent/adult caregivers at baseline and post-intervention for evaluation of microbiome, metabolomics, and hair analyses among both intervention and control groups at a location distant from the academic laboratories conducting the analysis. The protocol used to assemble, deliver, collect, and process biospecimens are presented along with the frequencies with which specimens were successfully obtained. RESULTS: One hundred forty six participants (73 parent-child dyads) were part of the larger SHA study and thus eligible to provide a biospecimen. A total of 126 participants, 115 participants, and 127 participants consented to provide their hair, stool and urine samples, respectively. Of the participants that consented to provide a sample, 44 children (69.8%) and 38 parents (60.3%) provided at least one hair sample, 27 children (48.2%) and 37 parents (62.7%) provided at least one stool sample, and 36 children (57.1%) and 42 parents (65.6%) provided at least one urine sample. Sample collection at the offsite location, transport, and handling at the academic center were successful and all biospecimens were deemed adequate for analyses. DNA and metabolomics yield on a subset of stool samples obtained provided excellent results in terms of an abundance of species and metabolities, as would be predicted. Urine and hair analyses are underway. CONCLUSION: Our work is one of the first to describe the feasibility of collecting human biospecimens, specifically stool, urine, and hair, from both parents and their children from low-resourced neighborhoods in a non-traditional garden research setting. Future work will report findings related to mechanisms between diet, microbiome, metabolites, and clinical outcomes.

Mammary tumors alter the fecal bacteriome and permit enteric bacterial translocation.

BACKGROUND: Cancer patients experience gastrointestinal and behavioral symptoms, and are at increased risk of systemic infection and inflammation. These conditions are a major source of morbidity and decreased quality of life prior to cancer treatment, but poorly defined etiologies impede successful treatment. The gastrointestinal microbiota shape inflammation, influence cancer progression and treatment, and colonize tumors. However, research has not directly determined if peripheral tumors influence the microbiome and intestinal physiology, thus influencing gastrointestinal and behavioral symptoms. Therefore, the purpose of this study was to examine consequences of orthotopic, syngeneic mammary tumor implantation, growth, and resection on fecal bacteriome composition and intestinal barrier function in relation to systemic inflammation and enteric bacterial translocation in mice. METHODS: Female mice were randomized to 3 experimental groups: sham surgical control, tumor recipients, and tumor recipients later receiving tumor-resection. Mice were sacrificed three weeks after tumor implantation or resection for collection of stool, colon, spleen, and brain tissue and analysis. RESULTS: Tumor-bearing mice exhibited several markers of colonic barrier disruption, including dampened expression of tight junction proteins (Cldn1 and Ocln) and elevated circulating lipopolysaccharide binding protein (LBP). Compromised colonic barrier integrity was associated with altered fecal bacterial profiles in tumor-mice, including lower relative abundance of Lactobacillus, but higher Bacteroides. Consistent with colonic barrier disruption and altered microbiomes, tumor-mice displayed markers of systemic inflammation including splenomegaly, higher splenic bacterial load, and elevated splenic and brain pro-inflammatory cytokines. Several  bacteria cultured from spleens had 16S rRNA gene amplicons matching those in fecal samples, suggesting they were of intestinal origin. Fecal Lactobacillus was highly-interrelated to physiological parameters disrupted by tumors via correlation network analysis. Tumor resection ameliorated circulating LBP, splenomegaly, and splenic cytokines, but not other parameters associated with loss of colonic barrier integrity and bacterial translocation. CONCLUSIONS: Orthotopic mammary tumors alter the microbiome, reduce intestinal barrier function, increase translocation of enteric bacteria, and alter systemic inflammation. This provides insight into how tumors commence gastrointestinal and behavioral symptoms prior to treatment, and identify targets for future therapeutics, such as probiotic Lactobacillus supplementation.

Stressor-Induced Reduction in Cognitive Behavior is Associated with Impaired Colonic Mucus Layer Integrity and is Dependent Upon the LPS-Binding Protein Receptor CD14.

Purpose: Commensal microbes are impacted by stressor exposure and are known contributors to cognitive and social behaviors, but the pathways through which gut microbes influence stressor-induced behavioral changes are mostly unknown. A murine social stressor was used to determine whether host-microbe interactions are necessary for stressor-induced inflammation, including neuroinflammation, that leads to reduced cognitive and social behavior. Methods: C57BL/6 male mice were exposed to a paired fighting social stressor over a 1 hr period for 6 consecutive days. Y-maze and social interaction behaviors were tested following the last day of the stressor. Serum cytokines and lipopolysaccharide binding protein (LBP) were measured and the number and morphology of hippocampal microglia determined via immunohistochemistry. Intestinal mucous thickness and antimicrobial peptide expression were determined via fluorescent staining and real-time PCR (respectively) and microbial community composition was assessed using 16S rRNA gene amplicon sequencing. To determine whether the microbiota or the LBP receptor (CD14) are necessary for stressor-induced behavioral changes, experiments were performed in mice treated with a broad-spectrum antibiotic cocktail or in CD14-/- mice. Results: The stressor reduced Y-maze spontaneous alternations, which was accompanied by increased microglia in the hippocampus, increased circulating cytokines (eg, IL-6, TNF-α) and LBP, and reduced intestinal mucus thickness while increasing antimicrobial peptides and cytokines. These stressor-induced changes were largely prevented in mice given broad-spectrum antibiotics and in CD14-/- mice. In contrast, social stressor-induced alterations of social behavior were not microbe-dependent. Conclusion: Stressor-induced cognitive deficits involve enhanced bacterial interaction with the intestine, leading to low-grade, CD14-dependent, inflammation.

Psychological stress disrupts intestinal epithelial cell function and mucosal integrity through microbe and host-directed processes.

Psychological stress alters the gut microbiota and predisposes individuals to increased risk for enteric infections and chronic bowel conditions. Intestinal epithelial cells (IECs) are responsible for maintaining homeostatic interactions between the gut microbiota and its host. In this study, we hypothesized that disruption to colonic IECs is a key factor underlying stress-induced disturbances to intestinal homeostasis. Conventionally raised (CONV-R) and germ-free (GF) mice were exposed to a social disruption stressor (Str) to ascertain how stress modifies colonic IECs, the mucosal layer, and the gut microbiota. RNA sequencing of IECs isolated from CONV-R mice revealed a robust pro-inflammatory (Saa1, Il18), pro-oxidative (Duox2, Nos2), and antimicrobial (Reg3b/g) transcriptional profile as a result of Str. This response occurred concomitant to mucus layer thinning and signs of microbial translocation. In contrast to their CONV-R counterparts, IECs from GF mice or mice treated with broad spectrum antibiotics exhibited no detectable transcriptional changes in response to Str. Nevertheless, IECs from Str-exposed GF mice exhibited an altered response to ex vivo bacterial challenge (increased dual Oxidase-2 [Duox2] and nitric oxide synthase-2 (Nos2)), indicating that STR primes host IEC pro-oxidative responses. In CONV-R mice stress-induced increases in colonic Duox2 and Nos2 (ROS generating enzymes) strongly paralleled changes to microbiome composition and function, evidencing Str-mediated ROS production as a primary factor mediating gut-microbiota dysbiosis. In conclusion, a mouse model of social stress disrupts colonic epithelial and mucosal integrity, a response dependent on an intact microbiota and host stress signals. Together these preclinical findings may provide new insight into mechanisms of stress-associated bowel pathologies in humans.

Stressor-Induced Increases in Circulating, but Not Colonic, Cytokines Are Related to Anxiety-like Behavior and Hippocampal Inflammation in a Murine Colitis Model.

Stressor exposure increases colonic inflammation. Because inflammation leads to anxiety-like behavior, we tested whether stressor exposure in mice recovering from dextran-sulfate-sodium (DSS)-induced colitis enhances anxiety-like behavior. Mice received 2% DSS for five consecutive days prior to being exposed to a social-disruption (SDR) stressor (or being left undisturbed). After stressor exposure, their behavior was tested and colitis was assessed via histopathology and via inflammatory-cytokine measurement in the serum and colon. Cytokine and chemokine mRNA levels in the colon, mesenteric lymph nodes (MLNs), hippocampus, and amygdala were measured with RT-PCR. SDR increased anxiety-like behaviors, which correlated with serum and hippocampal IL-17A. The stressor also reduced IL-1ß, CCL2, and iNOS in the colonic tissue, but increased iNOS, IFNγ, IL-17A, and TNFα in the MLNs. A network analysis indicated that reductions in colonic iNOS were related to elevated MLN iNOS and IFNγ. These inflammatory markers were related to serum and hippocampal IL-17A and associated with anxiety-like behavior. Our data suggest that iNOS may protect against extra-colonic inflammation, and when suppressed during stress it is associated with elevated MLN IFNγ, which may coordinate gut-to-brain inflammation. Our data point to hippocampal IL-17A as a key correlate of anxiety-like behavior.

Age and environmental exposures influence the fecal bacteriome of young children with cystic fibrosis.

BACKGROUND: Mechanisms that facilitate early infection and inflammation in cystic fibrosis (CF) are unclear. We previously showed that young CF children with secondhand smoke exposure (SHSe) have increased susceptibility to respiratory infections. We aimed to define the impact of SHSe and other external factors upon the fecal bacteriome in early CF. METHODS: Twenty CF infants and children were enrolled, clinical data recorded, and hair nicotine measured as an objective surrogate of SHSe. Fecal samples were collected at clinic visits and bacteriome 16S rRNA gene sequencing performed. RESULTS: SHSe was associated with increased alpha diversity and increased relative abundance of Acinetobacter and Akkermansia, along with decreased Bifidobacterium and Lactobacillus. Recent antibiotic exposure predicted bacterial population structure in children less than 2 years of age and was associated with decreased Bacteroides relative abundance. Age was the strongest predictor of overall fecal bacterial composition and positively associated with Blautia and Parabacteroides. Weight for length was negatively associated with Staphylococcus relative abundance. CONCLUSIONS: SHSe and other external factors such as antibiotics appear to alter fecal bacterial composition in young CF children, but the strongest predictor of overall composition was age. These findings have implications for understanding the intestinal microbiome in young CF children.

Dietary Oligosaccharides Attenuate Stress-Induced Disruptions in Immune Reactivity and Microbial B-Vitamin Metabolism.

Background: Exposure to stressful stimuli dysregulates inflammatory processes and alters the gut microbiota. Prebiotics, including long-chain fermentable fibers and milk oligosaccharides, have the potential to limit inflammation through modulation of the gut microbiota. To determine whether prebiotics attenuate stress-induced inflammation and microbiota perturbations, mice were fed either a control diet or a diet supplemented with galactooligosaccharides, polydextrose and sialyllactose (GOS+PDX+SL) or sialyllactose (SL) for 2 weeks prior to and during a 6-day exposure to a social disruption stressor. Spleens were collected for immunoreactivity assays. Colon contents were examined for stressor- and diet- induced changes in the gut microbiome and metabolome through 16S rRNA gene sequencing, shotgun metagenomic sequencing and UPLC-MS/MS. Results: Stress increased circulating IL-6 and enhanced splenocyte immunoreactivity to an ex vivo LPS challenge. Diets containing GOS+PDX+SL or SL alone attenuated these responses. Stress exposure resulted in large changes to the gut metabolome, including robust shifts in amino acids, peptides, nucleotides/nucleosides, tryptophan metabolites, and B vitamins. Multiple B vitamins were inversely associated with IL-6 and were augmented in mice fed either GOS+PDX+SL or SL diets. Stressed mice exhibited distinct microbial communities with lower abundances of Lactobacillus spp. and higher abundances of Bacteroides spp. Diet supplementation with GOS+PDX+SL, but not SL alone, orthogonally altered the microbiome and enhanced the growth of Bifidobacterium spp. Metagenome-assembled genomes (MAGs) from mice fed the GOS+PDX+SL diet unveiled genes in a Bifidobacterium MAG for de novo B vitamin synthesis. B vitamers directly attenuated the stressor-induced exacerbation of cytokine production in LPS-stimulated splenocytes. Conclusions: Overall, these data indicate that colonic metabolites, including B vitamins, are responsive to psychosocial stress. Dietary prebiotics reestablish colonic B vitamins and limit stress-induced inflammation.

Specialized High-Protein Oral Nutrition Supplement Improves Home Nutrient Intake of Malnourished Older Adults Without Decreasing Usual Food Intake.

BACKGROUND: Reduced nutrient intake is common in patients after hospitalization, contributing to increased risk for readmission and mortality. Oral nutrition supplements can improve nutrition status and clinical outcomes, but intake of food is prioritized by clinicians. This study examines the impact of a high-protein oral nutrition supplement (S-ONS) on nutrient intake post discharge. METHODS: In a subset of patients (14 S-ONS and 16 placebo) from the NOURISH (Nutrition effect On Unplanned ReadmIssions and Survival in Hospitalized patients) trial, 24-hour dietary recalls were conducted on 3 randomly selected days during the weeks of 30, 60, and 90 days post discharge. Nutrient intake was estimated using Nutrition Data System for Research software. Adequate energy and protein intake were defined as 30 kcal/kg/d and 1.2 g/kg/d, respectively. Dietary Reference Intakes (DRIs) were used for other nutrients. RESULTS: Less than half of patients met the requirements for energy, protein, and 12 micronutrients from food intake alone during the study. Energy and protein intakes from food were not diminished relative to placebo. Considering nutrient intake from both food and S-ONS, 50% and 71% of patients receiving S-ONSs met energy and protein goals respectively at 90 days (compared with 29% and 36%, in the placebo group), and 100% met the DRI for total carbohydrate, iron, phosphorus, copper, selenium, thiamin, and riboflavin at all time points, all of which were consumed at higher amounts vs placebo. CONCLUSION: Three months of S-ONS consumption increases intake of numerous nutrients without decreasing nutrient intake from food in older malnourished adults post discharge.

Prebiotic and probiotic treatment of nonalcoholic fatty liver disease: a systematic review and meta-analysis.

Context: Nonalcoholic fatty liver disease (NAFLD) is a highly prevalent and underdiagnosed comorbidity of many chronic diseases that is associated with altered intestinal bacterial communities. This association has prompted research into alternative treatments aimed at modulating intestinal microbiota. Given the novelty of these treatments, scarce evidence regarding their effectiveness in clinical populations exists. Objective: This meta-analysis sought to systemically review and quantitatively synthesize evidence on prebiotic, probiotic, and synbiotic therapies for patients with NAFLD in randomized controlled trials. Data sources: PRISMA guidelines ensured transparent reporting of evidence. PICOS criteria defined the research question for the systematic review. A systematic keyword search in PubMed and EMBASE identified 25 studies: 9 assessed prebiotic, 11 assessed probiotic, and 7 assessed symbiotic therapies for a total of 1309 patients. Data extraction: Basic population characteristics, the primary variables of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (utilized for NAFLD diagnosis), and the secondary variables of body mass index (BMI), gamma-glutamyl transferase (γ-GT), tumor necrosis factor alpha (TNF-α), C-reactive protein (CRP), total cholesterol, high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), and triglyceridges (TAG) were extracted. Pooled effect sizes of these variables were calculated by meta-analysis. No publication bias was identified using Begg's and Egger's tests or Cochrane bias assessment tool. Results: Meta-analysis indicated that microbial therapies significantly reduced BMI (-0.37 kg/m2; 95% confidence interval [CI], -0.46 to -0.28; P < 0.001), hepatic enzymes (ALT, -6.9 U/L [95%CI, -9.4 to -4.3]; AST, -4.6 U/L [95%CI, -6.6 to -2.7]; γ-GT, -7.9 U/L [95%CI, -11.4 to -4.4]; P < 0.001), serum cholesterol (-10.1 mg/dL 95%CI, -13.6 to -6.6; P < 0.001), LDL-c (-4.5 mg/dL; 95%CI, -8.9 to -0.17; P < 0.001), and TAG (-10.1 mg/dL; 95%CI, -18.0 to -2.3; P < 0.001), but not inflammation (TNF-α, -2.0 ng/mL; [95%CI, -4.7 to 0.61]; CRP, -0.74 mg/L [95%CI, -1.9 to 0.37]). Subgroup analysis by treatment category indicated similar effects of prebiotics and probiotics on BMI and liver enzymes but not total cholesterol, HDL-c, and LDL-c. Conclusion: This meta-analysis supports the potential use of microbial therapies in the treatment of NAFLD and sheds light on their potential mode of action. Further research into these treatments should consider the limitations of biomarkers currently used for the diagnosis and progression of NAFLD, in addition to the inherent challenges of personalized microbial-based therapies.

Gut microbiota-immune-brain interactions in chemotherapy-associated behavioral comorbidities.

Increasing scientific attention is focused on the gut-brain axis, including the ability of the gastrointestinal (GI) tract to modulate central nervous system function. Changes in the intestinal microbiome can influence affective-like behavior, cognitive performance, fatigue, and sleep in rodents and humans. Patients with cancer who are receiving chemotherapy experience similar negative behavioral changes and concurrent GI symptoms. These chemotherapy comorbidities can be long-lasting and may reduce patients' quality of life and motivation to comply with treatment. This review summarizes the clinical and preclinical evidence supporting a role for the intestinal microbiome in mediating behavioral comorbidities through peripheral immune activation in patients with cancer who are receiving chemotherapy. In addition, evidence suggesting that targeted modification of the intestinal microbiome during cancer treatment could ameliorate associated behavioral comorbidities is reviewed.

Developing a digital photography-based method for dietary analysis in self-serve dining settings.

Current population-based methods for assessing dietary intake, including food frequency questionnaires, food diaries, and 24-h dietary recall, are limited in their ability to objectively measure food intake. Digital photography has been identified as a promising addition to these techniques but has rarely been assessed in self-serve settings. We utilized digital photography to examine university students' food choices and consumption in a self-serve dining hall setting. Research assistants took pre- and post-photos of students' plates during lunch and dinner to assess selection (presence), servings, and consumption of MyPlate food groups. Four coders rated the same set of approximately 180 meals for inter-rater reliability analyses; approximately 50 additional meals were coded twice by each coder to assess intra-rater agreement. Inter-rater agreement on the selection, servings, and consumption of food groups was high at 93.5%; intra-rater agreement was similarly high with an average of 95.6% agreement. Coders achieved the highest rates of agreement in assessing if a food group was present on the plate (95-99% inter-rater agreement, depending on food group) and estimating the servings of food selected (81-98% inter-rater agreement). Estimating consumption, particularly for items such as beans and cheese that were often in mixed dishes, was more challenging (77-94% inter-rater agreement). Results suggest that the digital photography method presented is feasible for large studies in real-world environments and can provide an objective measure of food selection, servings, and consumption with a high degree of agreement between coders; however, to make accurate claims about the state of dietary intake in all-you-can-eat, self-serve settings, researchers will need to account for the possibility of diners taking multiple trips through the serving line.