Society: AGA

Background:
We and others have demonstrated a tight coupling between dietary intake, gut microbial ecology, and near-term alterations in cardiometabolic biomarkers in the ZOE PREDICT-1 study (Zeevi 2015; Asnicar & Berry 2021). However, it is unknown whether adherence to a de novo data-driven microbially-informed diet derived from the PREDICT-1 is associated with long-term weight change and its complications.
Methods:
To evaluate the impact of a “Metabolic Microbiome Score” (MMS) on long-term weight change, we pooled data from 3 ongoing U.S.-based prospective cohorts with lifestyle and dietary data collected through biennial questionnaires and validated food frequency questionnaires. MMS was calculated by summing reported food intake multiplied by food quality scores derived from the PREDICT-1, while normalizing for the servings of foods consumed. To validate prior findings, we assessed the association between MMS, food groups, and gut microbial features using partial Spearman correlations adjusted for age, sex, and body mass index. We used a multivariable generalized linear regression model to examine the association between 4-year changes in MMS and 4-year weight change after adjusting for relevant clinical and lifestyle confounders. We fitted Cox proportional hazards models to examine the associations between MMS and incident diabetes, cardiovascular disease (CVD), and mortality.
Results:
Among 186,661 participants with more than 20 years of follow-up, we observed that compared to the lowest quartile, those in the highest quartile of MMS were associated with 1.36 kg less weight gain (95% CI, -1.40 to -1.32; Table). A 1-SD increase in MMS was associated with 0.54 kg less weight gain over 4-year study intervals (95% CI, -0.55 to -0.52). There was a modest correlation between MMS and other established dietary patterns, such as the Western (Spearman rho = -0.43) and prudent diets (rho = 0.54). We also confirmed previously observed links between MMS, higher quality foods, and the relative enrichment of health-associated gut microbial features, such as Eubacterium eligens (Fig. A). The link between MMS and weight change appeared to differ according to age, sex, and body mass index, suggesting that those younger, female, or with obesity would derive greater benefit from adhering to diet high in MMS (P for interactions <0.05). In addition, adherence to MMS diet was associated with statistically-significant reductions in obesity-related complications including type 2 diabetes, CVD, and mortality (Fig. B).
Conclusion:
A data-driven dietary score corresponding with better near-term cardiometabolic markers was associated with less long-term weight gain and reduced risk of obesity-related complications in 3 independent U.S. cohorts, suggesting a role for population-wide public health initiatives geared towards personalized improvement of diet quality.

<b>Table. Weight change over 4-year periods according to per 1-SD increase and quartiles of change in Metabolic Microbiome Score </b><br /> Abbreviations: SD, standard deviation; MV, multivariable<br /> <sup>a</sup>Additionally adjusted for sex, questionnaire cycle (4-year intervals), White (yes/no), height (continuous), body mass index (continuous), smoking pack-years (continuous), physical activity (continuous), change in physical activity (continuous), and post-menopausal hormone use (yes/no [for women])<br /> <sup>b</sup>Additionally adjusted for total energy intake (continuous)

Table. Weight change over 4-year periods according to per 1-SD increase and quartiles of change in Metabolic Microbiome Score
Abbreviations: SD, standard deviation; MV, multivariable
^aAdditionally adjusted for sex, questionnaire cycle (4-year intervals), White (yes/no), height (continuous), body mass index (continuous), smoking pack-years (continuous), physical activity (continuous), change in physical activity (continuous), and post-menopausal hormone use (yes/no [for women])
^bAdditionally adjusted for total energy intake (continuous)

<b>Figure A. Metabolic Microbiome Score (MMS) validation. </b>Partial Spearman correlation heatmap for food groups and microbiome adjusted for sex, age, and body mass index. Red color represents positive correlation while blue color represents negative correlation. Asterisks represent significance. ***: p<sub>FDR </sub>≤ 0.001; **: 0.001 < p<sub>FDR </sub>≤ 0.01; *: 0.01 < p<sub>FDR </sub>≤ 0.05.<br /> <b>Figure B. Metabolic Microbiome Score and risk of obesity-related complications.</b> Adjusted for age, sex, White (yes/no), body mass index (continuous), smoking pack-years (continuous), physical activity (continuous), alcohol intake (continuous), post-menopausal hormone use (yes/no [for women]), and disease-specific confounders.

Figure A. Metabolic Microbiome Score (MMS) validation. Partial Spearman correlation heatmap for food groups and microbiome adjusted for sex, age, and body mass index. Red color represents positive correlation while blue color represents negative correlation. Asterisks represent significance. ***: p_FDR≤ 0.001; **: 0.001 < p_FDR≤ 0.01; *: 0.01 < p_FDR≤ 0.05.
Figure B. Metabolic Microbiome Score and risk of obesity-related complications. Adjusted for age, sex, White (yes/no), body mass index (continuous), smoking pack-years (continuous), physical activity (continuous), alcohol intake (continuous), post-menopausal hormone use (yes/no [for women]), and disease-specific confounders.

Background: Coffee intake has been associated with lower risks of many chronic diseases and with features of the gut microbiome. However, no study has systematically examined the interplay between coffee intake and the gut microbiome in relation to host metabolome.
Methods: We collected detailed dietary information every 4 years since 1986 from 51,529 men in the Health Professionals Follow-up Study (HPFS). In 2012-2013, a subcohort of 307 healthy HPFS men provided up to 2 pairs of stool and 2 blood samples. Intakes of overall coffee and caffeinated, decaffeinated, filtered, instant, and espresso coffee were used as exposures. We profiled 236 plasma metabolomes as compared with 925 stool metagenomes, for which microbial species, functional pathways (MetaCyc), enzymes (EC), and gene family (UniRef90) profiles were generated. We validated our findings in a subcohort of 220 healthy women from the Nurses’ Health Study II.
Results: Higher coffee intake was strongly associated with increased abundances of several microbial species, particularly Clostridium phoceensis (FDR-adjusted p=5*10^-11) as previously observed (prev. Lawsonibacter asaccharolyticus). These associations were similar for caffeinated and decaffeinated coffee. A plasma metabolomic signature of coffee intake developed using elastic net regression was also strongly positively correlated with the abundance of C. phoceensis (p<0.001) (Fig.1). Although a variety of small molecular metabolites are jointly correlated both with coffee intake and with C. phoceensis abundance – primarily those known to be derived from coffee itself – the strongest individual correlation was between C. phoceensis and quinic acid (FDR-adjusted p=1*10^-6), a metabolite of chlorogenic acid, one of the most abundant polyphenols in coffee. Stratified analyses of pathways, enzymes, and gene families contributed by C. phoceensis also revealed a particularly strong positive association with plasma levels of quinic acid (FDR-adjusted p<0.005). In contrast, none of the metabolic pathways or enzymes of caffeine metabolism were significant in the paired multi-omic analysis (concordant with the microbial correlation with decaffeinated coffee). Also, coffee intake was associated with a reduced plasma level of high-sensitivity C-reactive protein (p=0.008). Notably, this inverse association was only observed in individuals with a higher C. phoceensis level (p = 0.004), but not in those with a lower C. phoceensis level (p=0.79) (p-interaction=0.009) (Fig. 2). We validated the strong association of coffee intake with abundance of C. phoceensis in the women’s cohort.
Conclusions: We reproduced a strong association of coffee intake with a specific gut microbial species, C. phoceensis, and showed that C. phoceensis might be involved in the metabolism of coffee polyphenols and modify the anti-inflammatory effect of coffee intake in the host.

<b>Figure 1. Associations of coffee intake with gut microbial species and plasma metabolites abundances.</b> <b>a,</b> Significant associations of coffee intake measures with microbial species (FDR-adjusted <i>p</i> < 0.25) by MaAsLin analysis. <b>b</b>-<b>d</b>, Associations of total coffee intake with the species of <i>Clostridium phoceensis</i>,<i> SGB14966</i>, and<i> SGB4777</i>. <b>e</b>-<b>f</b>, Taxonomy and spearman correlation between <i>C. phoceensis</i>,<i> SGB14966</i>, and<i> SGB4777</i>. <b>g</b>, Significant spearman partial correlations of total coffee intake with all known plasma metabolites (<i>n</i>=373). <b>h</b>, Metabolomic signature of total coffee by elastic net regression. <b>i</b>, Association of metabolomic signature of coffee intake with <i>C. phoceensis</i>.

Figure 1. Associations of coffee intake with gut microbial species and plasma metabolites abundances. a, Significant associations of coffee intake measures with microbial species (FDR-adjusted p < 0.25) by MaAsLin analysis. b-d, Associations of total coffee intake with the species of Clostridium phoceensis, SGB14966, and SGB4777. e-f, Taxonomy and spearman correlation between C. phoceensis, SGB14966, and SGB4777. g, Significant spearman partial correlations of total coffee intake with all known plasma metabolites (n=373). h, Metabolomic signature of total coffee by elastic net regression. i, Association of metabolomic signature of coffee intake with C. phoceensis.

<b>Figure 2. Interplay between coffee intake and the microbial species <i>Clostridium phoceensis</i> in relation to plasma metabolome. </b><i>Clostridium phoceensis</i> may be involved in the metabolism of polyphenols in coffee. <b>a, </b>HAllA (high-sensitivity pattern discovery in large, paired multi-omic datasets) heatmap showing significant clusters between all microbial species (<i>n</i> = 333) and all known metabolites (<i>n</i> = 375). <b>b</b>, The metabolism of the significant <i>Clostridium phoceensis</i>-correlated clusters of coffee component metabolites. <b>c</b>, HAllA heatmap between <i>Clostridium phoceensis</i>-stratified EC enzymes (<i>n</i> = 277) and known metabolites (<i>n</i> = 375). <b>d</b>, the modification effect of <i>Clostridium phoceensis</i> on the association between coffee intake and a reduced plasma level of high-sensitivity C-reactive protein (CRP).

Figure 2. Interplay between coffee intake and the microbial species Clostridium phoceensis in relation to plasma metabolome. Clostridium phoceensis may be involved in the metabolism of polyphenols in coffee. a, HAllA (high-sensitivity pattern discovery in large, paired multi-omic datasets) heatmap showing significant clusters between all microbial species (n = 333) and all known metabolites (n = 375). b, The metabolism of the significant Clostridium phoceensis-correlated clusters of coffee component metabolites. c, HAllA heatmap between Clostridium phoceensis-stratified EC enzymes (n = 277) and known metabolites (n = 375). d, the modification effect of Clostridium phoceensis on the association between coffee intake and a reduced plasma level of high-sensitivity C-reactive protein (CRP).

Introduction: Despite concerted efforts Alaska Native (AN) people continue to have the highest recorded incidence of colon cancer in the world. There is convincing evidence that diet drives cancer risk. Our pilot study with AN people suggested that whilst the diet contained high quality meat and fish from wild sources, it was deficient in fiber rich foods with a fiber content of only 10-15g/d vs the recommended >30g/d. Population studies suggest that a total fiber intake of >50g/d is associated with low cancer risk (O’Keefe, Lancet GI 2016).
Hypothesis: Excess risk in AN is explained by the combined effects of low fiber, suppressing the microbial production of antineoplastic butyrate, and high fat consumption, increasing colonic microbial production of secondary bile acids, whose carcinogenicity may be enhanced by butyrate deficiency.
Methods: We conducted a randomized controlled trial supplementing the AN people’s usual diet for 4 weeks with a daily 40g drink containing either 40g digestible starch (DS), amylopectin, a naturally occurring branched glucose polymer; or isocaloric 40g resistant starch (RS), a food starch extracted from high amylose corn. Recruitment was from screening colonoscopy (SC) which included sigmoid biopsies for proliferative (primary endpoint) and inflammatory biomarkers of cancer risk, measured by immunohistochemistry: Ki67+ staining of epithelial proliferative cells, CD3+ intraepithelial lymphocytes and CD68+ lamina propria macrophages. To investigate potential mechanisms, we measured fecal butyrate, secondary bile acids, and the global microbiome by 16SrRNA approach. All these tests were repeated after 4 weeks of supplementation. Compliance was assured by direct observation in-person or via mobile phone video app.
Results: A total of 48 healthy 40-75 y.o. AN adults completed the study; 43% had polyps removed at SC. Fiber supplementation was associated with a significant suppression (p=0.036) of epithelial proliferation (Fig 1). CD68+ decreased in both groups. Possible mechanisms for proliferation suppression include, increased colonic microbial butyrogenesis (p=0.029), suppressed secondary bile acids (Table 1) and decreases in fecal Blautia, Faecalibacterium, Agathobacter. Lachnospiraceae_ge, and Dorea. Interestingly, subjects with polyps showed higher responses to RS.
Conclusions: High fiber supplements have the potential to suppress the extreme risk of colon cancer in AN people by suppressing colonic mucosal proliferation through modulation of the colonic microbiota, with increased colonic butyrogenesis and suppressed secondary bile acids. Our results provide the scientific framework for a long-term population-based study to determine whether high fiber foods can suppress development of colonic polyps and cancer, thereby reducing the remarkably high risk of colon cancer in AN people.

Background: Gut dysfunction following antibiotic-treated C. difficile infection (CDI) is common and may be severe. We established a humanized mouse model in which germ-free (GF) mice colonized with microbiota from patients with severe constipation following antibiotic-treated CDI developed slow colonic transit due to macrophage mediated damage to the Interstitial cells of Cajal (ICC) network. These changes were reversed after fecal microbiota transplantation from healthy donor mice, thus implicating the microbiota. These findings prompted us to evaluate the therapeutic potential of microbiota-directed dietary therapies to normalize gut dysfunction in this model.

Aims: 1. To investigate whether dietary psyllium, pectin and flavonoids can rescue the development of gut dysfunction in our humanized mouse model, and 2. To explore mechanisms underlying beneficial interventions.

Methods: GF mice were colonized with microbiota from the post-CDI patients or sex and age matched healthy controls (HC) for 3-weeks. Following colonization post-CDI mice were fed 4 separate diets containing 15% psyllium, 10% pectin, 0.05% quercetin or a control diet for 4-5 weeks. Colonic motility was assessed before and after the dietary interventions. Fecal samples were collected for microbial profiling and metabolomic analysis. Gene expression, innate immune activation and the ICC network were examined in colonic tissue.

Results: Post-CDI microbiota induced a slow colonic transit phenotype in ex-GF mice compared to mice with HC microbiota and this was accompanied by macrophage infiltration and disruption of the ICC network. Psyllium and pectin each reversed slow colonic transit, macrophage infiltration, and the loss of integrity of the ICC network. These results were accompanied by changes in immune and neural-related gene expression in the colonic muscle layers including CD11b, NOS, GFAP, Myd88, Mapk1 and NF-κB. In addition, psyllium or pectin altered bacterial composition and metabolic profiles in post-CDI mice. Specifically, short chain fatty acids (acetic and propionic acid) were increased, while branched chain fatty acids (isobutyric and isovaleric acid) were decreased following psyllium treatment. These metabolite profiles were paralleled by changes in specific bacteria, including Butyricimonas, and Phascolarctobacterium. In contrast, the flavonoid quercetin had no effect in this model.

Conclusions: Our results indicate that severe gut dysfunction following antibiotic-treated CDI can be reversed by dietary psyllium or pectin that may be therapeutically useful in patients with severe constipation following antibiotic-treated CDI. The beneficial effects of these interventions are due to alteration of microbiota composition and metabolism, resulting in attenuation of innate immune activation and restoration of ICC integrity and colonic transit.

Background: Dietary fiber has strong mechanistic rationale for use in irritable bowel syndrome (IBS) due to its impact on whole gut transit time (WGTT) and gut microbiome, despite the potential for side-effects from colonic gas production. Co-administration of different fibers may optimize these advantages whilst limiting these side-effects. The aim was to investigate the co-administration of dietary fibers in extracted or native form on clinical, physiological and microbiological outcomes in IBS. Methods: People with IBS were recruited to a randomized, double-blind, controlled trial from clinics in United Kingdom and Mexico. Inclusion criteria were adults with IBS-D, IBS-C or IBS-M (Rome IV criteria). Exclusion criteria included recent antibiotics, fiber, prebiotic or probiotics. Participants were randomized to co-administration of: (i) extracted fibers (inulin/psyllium); (ii) native fibers (nopal cactus fiber); or (iii) placebo (dextrose) for 8-weeks, resulting in 15 g/d of fiber in the intervention groups. Clinical outcomes included ‘adequate relief of symptoms’ (primary outcome), IBS severity scoring system (IBS-SSS), and stool and symptom diary. Gut transit time and colonic pH (wireless motility capsule), gut microbiota composition (16S rRNA sequencing) and stool short-chain fatty acids (gas-liquid chromatography) were measured at baseline and endpoint. Results: 133 patients were randomized to inulin/psyllium (n=47), nopal (n=44) and placebo (n=42). There were 5 (10.6%) withdrawals from inulin/psyllium and 8 (18.2%) from nopal (P=0.017). Following 8-weeks, there were no differences in ‘adequate relief of IBS symptoms’ between inulin/psyllium (59.6%), nopal (52.3%) and placebo (52.4%) (P=0.724), nor were there differences in other key clinical outcomes (Table). There were no differences in stool frequency, however there were lower proportions of hard stools for inulin/psyllium (4.5%) than for nopal (15.1%, P=0.026) and placebo (15.6%, P=0.02). Inulin/psyllium reduced the incidence and severity of constipation, while nopal increased loose stool. There were no differences in regional or WGTT or colonic pH between interventions. Shannon index was lower following inulin/psyllium (4.0) compared with both nopal (4.4, P=0.024) and placebo (4.5, P=0.016). Inulin/psyllium resulted in lower abundance of Ruminococcus (P=0.0009), Dorea (P=0.0162) and Coprococcus (P=0.0112) and higher Bifidobacteria (P=0.0546), however, there were no differences in short-chain fatty acids between groups. Conclusions: Co-administration of fibers in extracted (inulin/psyllium) or native (nopal) form did not result in clinical response in IBS. Co-administration with psyllium enabled inulin to be supplemented without exacerbating IBS symptoms. Inulin/psyllium improved constipation, consistent with previous evidence for psyllium alone, while also modifying the gut microbiome.

Background: The mucins produced by the goblet cells of the gut epithelial layer have an important role in maintaining barrier integrity and preventing pathogen attachment and invasion. Notch signaling, influenced by the mTORC1 pathway in response to altered nutritional status, could modulate goblet cell differentiation. In addition, the transcription factor Kruppel-like factor 4 (KLF4) has been shown to govern the terminal differentiation of goblet cells. Goblet cell differentiation from intestinal stem cells is altered in various diseases, including inflammatory bowel disease and obesity, causing defective mucin production and compromising barrier integrity. On the other hand, gut microbial metabolites of dietary tryptophan (Trp) have been shown to augment epithelial barrier function via stimulation of goblet cell differentiation. However, the underlying mechanisms are not fully known. Hypothesis: Gut microbial metabolites of dietary tryptophan stimulate goblet cell differentiation via modulation of Notch signaling and expression of KLF4. Aim: To elucidate the mechanism of stimulation of goblet cell differentiation by gut microbial metabolites of tryptophan. Methods: Jejunal mucosal biopsies of histologically confirmed normal small intestinal tissues obtained during endoscopic procedures (under an approved IRB protocol) were used to generate human intestinal organoid cultures. Goblet cell differentiation in organoids in response to either the Trp metabolite indole (0.1-2.0 mM), or the culture supernatant of Lactobacillus acidophilus (LA) grown overnight in the presence or absence of 5 mM Trp, was measured as mRNA levels of mucin-2 (MUC2), the most abundant mucin in goblet cells. KLF4 mRNA and protein levels were measured by QRT-PCR and Western blot analysis, respectively. Phospho-S6 representing mTORC1 activity, which in turn activates Notch signaling, was measured by Western blot analysis. Results: Indole treatment of human intestinal organoids increased MUC2 mRNA levels dose-dependently, with the maximum effect being shown with 0.5 mM indole. Indole also increased KLF4 mRNA and protein levels. Culture supernatant of LA, either grown in the presence or absence of Trp, significantly increased MUC2 mRNA, although the effect was more marked when LA was grown with Trp (supernatant containing Trp metabolites). Further, phospho-S6 protein levels were significantly reduced when organoids were treated with LA supernatant with Trp metabolites, suggesting inhibition of mTORC1 phosphorylation, and in turn, inhibition of Notch signaling by Trp metabolites. Conclusion: Our studies define a novel mechanism of stimulation of goblet cell differentiation in human intestinal organoids by gut microbial metabolites of tryptophan via increasing KLF4 expression and inhibiting mTORC1-mediated Notch activation.

Background: It is recognized that the gut microbiome can play a role in both health and disease in mammalian hosts, including in the pathogenesis of inflammatory bowel disease (IBD). One feature of the altered microbiome associated with IBD, known as dysbiosis, is an increased abundance of bacteria within the family Enterobacteriaceae, including Klebsiella pneumoniae. Colonization of inflammation-susceptible mice with a dysbiotic microbiota induces colitis, suggesting that targeted resolution of dysbiosis may help induce or sustain remission in patients with IBD. Dietary factors have been shown to correlate with dysbiosis, though the underlying mechanisms remain poorly understood. As bacterial growth is dependent upon sources of carbon and nitrogen, we hypothesized that diet directly affects the replication of Enterobacteriaceae by altering nutrient composition of the gut. Herein, we test the impact of nitrogen and carbon availability on the colonization of K. pneumoniae in the intestinal tract through modulation of bacterial and dietary factors.
Methods and Results: We find that K. pneumoniae predominates in gut microbiota of healthy human subjects consuming an exclusive enteral nutrition diet upon recovery after antibiotic treatment. Metabolomic analysis revealed that the abundance of K. pneumoniae was associated with a shift in fecal nitrogen composition with an increase in amino acids and ammonia, but with a decrease in urea. We hypothesized that urease, an enzyme capable of degrading host-produced urea into ammonia, encoded by K. pneumoniae, alters intestinal nitrogen, and may allow for increased growth of this organism in the context of nitrogen limitation. Colonization of both antibiotic-treated conventional mice and germ-free mice showed that K. pneumoniae urease depletes luminal urea and produces ammonia. However, urease deficient K. pneumoniae had no colonization defect. Moreover, deletion of the critical nitrogen scavenging regulator ntrC also had no effect on intestinal engraftment, even in the context of a low-protein diet, suggesting that nitrogen is not a limiting resource for K. pneumoniae in the gut under our model conditions. By contrast, although the addition of nitrogen had no effect on K. pneumoniae growth, in vitro and ex-vivo modeling revealed that the addition of simple carbohydrates enhanced growth. Indeed, the supplementation of diet with simple carbohydrates significantly increased K. pneunomiae gut colonization, demonstrating carbon limitation for this organism in the mammalian gut.
Conclusions: These results indicate that colonization with K. pneumoniae is dependent upon diet, which subsequently alters the nitrogen composition of the gut. We demonstrate that the intestinal lumen is a nitrogen-rich and carbon limited environment for K. pneumoniae, with dietary supplementation increasing growth and dysbiosis.

Aaron Hecht

Penn Medicine

Appearances