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Quantifying technical confounders in microbiome studies.
Bartolomaeus, TUP, Birkner, T, Bartolomaeus, H, Löber, U, Avery, EG, Mähler, A, Weber, D, Kochlik, B, Balogh, A, Wilck, N, et al
Cardiovascular research. 2021;(3):863-875
Abstract
AIMS: Recent technical developments have allowed the study of the human microbiome to accelerate at an unprecedented pace. Methodological differences may have considerable impact on the results obtained. Thus, we investigated how different storage, isolation, and DNA extraction methods can influence the characterization of the intestinal microbiome, compared to the impact of true biological signals such as intraindividual variability, nutrition, health, and demographics. METHODS AND RESULTS An observative cohort study in 27 healthy subjects was performed. Participants were instructed to collect stool samples twice spaced by a week, using six different methods (naive and Zymo DNA/RNA Shield on dry ice, OMNIgene GUT, RNALater, 95% ethanol, Zymo DNA/RNA Shield at room temperature). DNA extraction from all samples was performed comparatively using QIAamp Power Fecal and ZymoBIOMICS DNA Kits. 16S rRNA sequencing of the gut microbiota as well as qPCRs were performed on the isolated DNA. Metrics included alpha diversity as well as multivariate and univariate comparisons of samples, controlling for covariate patterns computationally. Interindividual differences explained 7.4% of overall microbiome variability, whereas the choice of DNA extraction method explained a further 5.7%. At phylum level, the tested kits differed in their recovery of Gram-positive bacteria, which is reflected in a significantly skewed enterotype distribution. CONCLUSION DNA extraction methods had the highest impact on observed microbiome variability, and were comparable to interindividual differences, thus may spuriously mimic the microbiome signatures of various health and nutrition factors. Conversely, collection methods had a relatively small influence on microbiome composition. The present study provides necessary insight into the technical variables which can lead to divergent results from seemingly similar study designs. We anticipate that these results will contribute to future efforts towards standardization of microbiome quantification procedures in clinical research.
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Kinetics of Intestinal Presence of Spores Following Oral Administration of Bacillus clausii Formulations: Three Single-Centre, Crossover, Randomised, Open-Label Studies.
Navarra, P, Milleri, S, Perez Iii, M, Uboldi, MC, Pellegrino, P, Bois De Fer, B, Morelli, L
European journal of drug metabolism and pharmacokinetics. 2021;(3):375-384
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Abstract
UNLABELLED BACKGROUND AND OBJECTIVE Probiotics are live microorganisms that may provide benefits including the prevention of gastrointestinal disorders and other diseases. Enterogermina is a probiotic mix of spores from four strains of Bacillus clausii (O/C, T, N/R and SIN), available in several oral formulations. The objective of this analysis was to evaluate and compare the kinetic profiles of different formulations of Enterogermina-vial [E4 once daily (OD) and E2 twice daily (BID)], capsule [EC2 three times daily (TID)], oral powder for suspension (ES6 OD) and oral powder not requiring suspension (E6 OD) from two studies from 2012 (EUDRACT 2010-024497-19 and 2010-023187-41) and one study from 2016 (EUDRACT 2015-003330-27). METHODS B. clausii spores were counted in homogenised faecal samples (results expressed as counts per gram) or after culture at 37 °C for 24-36 h (results expressed as colony-forming units). Kinetics were assessed by area under the concentration-time curve (AUC), maximum concentration (Cmax), time to maximum concentration (Tmax) and spore presence/persistence. RESULTS In total, 22 subjects in each of the 2012 studies and 30 subjects in the 2016 study were randomised (mean age 25.0-33.8 years across studies). The mean (±SD) absolute faecal spore counts (in millions) expressed as AUC per hour were 270.7 ± 147.7 (E2 BID) and 213.8 ± 60.2 (E4 OD) in 2012 EGKINETIC4, 312.7 ± 218.0 (EC2 TID) and 319.0 ± 221.1 (ES6 OD) in 2012 EGKINETIC6, and 212.6 ± 118.0 (E6 OD) and 293.2 ± 247.2 (ES6 OD) in 2016 EGKINETIC6OP. The kinetic profiles of the different formulations of Enterogermina were similar, with superimposable AUC and daily curve profiles in each study up to the 8th day post dose. B. clausii spore presence/persistence in the intestine of healthy volunteers did not differ between the two formulations within each of the three studies. Enterogermina was well tolerated across all formulations and studies. CONCLUSION These results show different formulations of Enterogermina had similar kinetic profiles within each study; however, they also showed that probiotics could be associated with high variability. The European Medicines Agency guidelines are the current bioequivalence reference, although only the Tmax parameter is used for high variability drugs. Due to the specific kinetics of probiotics, new parameters of bioequivalence could be necessary, considering, for example, variability via a parameter such as AUC. TRIAL REGISTRATION EUDRACT 2010-024497-19, 2010-023187-41 and 2015-003330-27.
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Overweight and underweight status are linked to specific gut microbiota and intestinal tricarboxylic acid cycle intermediates.
Wan, Y, Yuan, J, Li, J, Li, H, Yin, K, Wang, F, Li, D
Clinical nutrition (Edinburgh, Scotland). 2020;(10):3189-3198
Abstract
BACKGROUND & AIMS Intestinal short-chain fatty acids have been demonstrated to modulate host energy metabolism and are elevated in overweight and obese individuals. We hypothesized that other intestinal energy products especially tricarboxylic acid (TCA) cycle intermediates might also related to overweight status. In addition, little information is available regarding to the potential relationship between gut microbiota and underweight status. Therefore, the aim of this study was to investigate whether gut microbiota and intestinal energy metabolites differ in underweight, normal weight, and overweight individuals, and their correlations with host cardiometabolic risk factors. SUBJECTS/METHODS Gut microbiome, intestinal energy metabolites, circulating cardiometabolic risk factors, and proinflammatory markers were determined in 29 underweight, 67 normal weight, and 67 overweight adults. RESULTS The fecal concentrations of succinic acid, fumaric acid, malic acid, propionic acid, and adipic acid were significantly increased in the overweight individuals in parallel with a higher relative abundance of Veillonellacea after adjusting for multiple comparisons (all p < 0.05). The intestinal concentration of TCA cycle intermediate succinic acid was positively associated with body weight (r = 0.28, p = 0.04), and malic acid were in positive association with circulating total cholesterol, low-density lipoprotein cholesterol, and interleukin-1β (all r > 0.25, p < 0.05). Compared with the normal weight individuals, the gut microbial α-diversity was lower in the overweight (p = 0.007 for Shannon index and p = 0.009 for Ace index) and underweight (p = 0.05 for Shannon index and p = 0.08 for Ace index) groups. However, no significant differences in the overall gut microbiota composition were observed among the three groups. CONCLUSIONS Our findings revealed that low gut microbiota diversity was associated with both overweight and underweight status. Intestinal TCA cycle intermediates were associated with overweight development and might be potential markers for future studies related to gut microbiota and host cardiometabolic health.
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Intestinal phase-II metabolism of quercetin in HT29 cells, 3D human intestinal tissues and in healthy volunteers: a qualitative comparison using LC-IMS-MS and LC-HRMS.
Chalet, C, Hollebrands, B, Duchateau, GS, Augustijns, P
Xenobiotica; the fate of foreign compounds in biological systems. 2019;(8):945-952
Abstract
Flavonoids are a large class of dietary molecules, among which quercetin is the most ubiquitous, which undergo an extensive intestinal phase-II metabolism. We compared the in vivo metabolism of quercetin in healthy volunteers with two in vitro models, HT29 cells and 3 D human intestinal tissues. Supernatants of the in vitro experiments and the human intestinal fluids (HIF) were analyzed by LC-IMS-MS and LC-HRMS in a qualitative way. Quercetin glucuronides, sulfates and their methyl conjugates were detected in all three systems. The metabolic profiles were found to be different, both in terms of the metabolites produced and their relative proportions. In particular, quercetin sulfates were almost absent in supernatants from HT29 cells incubations while they were a major metabolite in HIF and also found in 3 D intestinal tissues incubations. IMS provided structural information as well as a third dimension of characterization, while HRMS brought increased sensitivity and MS/MS confirmation. HT29 cells are a useful tool to generate phase-II metabolites but do not represent the in vivo situation. 3 D intestinal tissues appear as a more relevant tool to study the intestinal phase-II metabolism of flavonoids.
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Coeliac disease: beyond genetic susceptibility and gluten. A narrative review.
Pes, GM, Bibbò, S, Dore, MP
Annals of medicine. 2019;(1):1-16
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Abstract
Coeliac disease (CD) is an immune-mediated disorder triggered by the ingestion of gluten in genetically susceptible individuals. However, only a small proportion of subjects harbouring CD-related genetic risk develop the disease. Among the environmental factors that may influence CD risk, pre- and perinatal factors, delivery methods, parental lifestyle, infant feeding practices, seasonality, dietary factors, drug use, childhood infections and variability in gut microbiota are those most widely studied regarding the risk to develop CD. Although for many of these external factors the exact mechanism of action is unknown, most of them are thought to act by disrupting the intestinal barrier, facilitating contact between potential antigens and the immune system effector cells. Management of CD is relatively easy in patients with a definite diagnosis and requires a strict, lifelong, gluten-free diet. Better knowledge of environmental exposures apart from gluten can facilitate understanding of the pathogenesis of the disorder and the wide heterogeneity of its clinical spectrum. The purpose of this review is to discuss current knowledge on environmental CD risk factors, as well as possible interaction between them, on the grounds of the reliable scientific evidence available. Key messages The risk of developing CD is influenced not only by gluten ingestion but also by a number of environmental factors including childhood infections and variability in gut microbiota, pre- and perinatal factors, infant feeding practices, delivery methods, parental lifestyle, seasonality, dietary factors and drug use, acting mainly by disrupting intestinal permeability. Better knowledge of exposure to these factors can facilitate their identification, and subsequent elimination, in the individual patient.
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High intake of dairy during energy restriction does not affect energy balance or the intestinal microflora compared with low dairy intake in overweight individuals in a randomized controlled trial.
Bendtsen, LQ, Blædel, T, Holm, JB, Lorenzen, JK, Mark, AB, Kiilerich, P, Kristiansen, K, Astrup, A, Larsen, LH
Applied physiology, nutrition, and metabolism = Physiologie appliquee, nutrition et metabolisme. 2018;(1):1-10
Abstract
During weight loss, dairy calcium is proposed to accelerate weight and fat-mass loss through increased fecal fat excretion. The primary objective was to investigate if a high-dairy energy-restricted diet is superior to low dairy in terms of changes in body weight, body composition, and fecal fat excretion over 24 weeks. Secondary objectives included fecal energy and calcium excretion, resting energy expenditure, blood pressure, lipid metabolism, and gut microbiota. In a randomized, parallel-arm intervention study, 11 men and 69 women (body mass index, 30.6 ± 0.3 kg/m2; age, 44 ± 1 years) were allocated to a 500-kcal (2100 kJ) -deficit diet that was either high (HD: 1500 mg calcium/day) or low (LD: 600 mg calcium/day) in dairy products for 24 weeks. Habitual calcium intake was ∼1000 mg/day. Body weight loss (HD: -6.6 ± 1.3 kg, LD: -7.9 ± 1.5 kg, P = 0.73), fat-mass loss (HD: -7.8% ± 1.3%, LD: -8.5% ± 1.1%, P = 0.76), changes in fecal fat excretion (HD: -0.57 ± 0.76 g, LD: 0.46 ± 0.70 g, P = 0.12), and microbiota composition were similar for the groups over 24 weeks. However, total fat-mass loss was positively associated with relative abundance of Papillibacter (P = 0.017) independent of diet group. Consumption of a high-dairy diet did not increase fecal fat or accelerate weight and fat-mass loss beyond energy restriction over 24 weeks in overweight and obese adults with a habitual calcium intake of ∼1000 mg/day. However, this study indicates that Papillibacter is involved in body compositional changes.
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Gut permeability is related to body weight, fatty liver disease, and insulin resistance in obese individuals undergoing weight reduction.
Damms-Machado, A, Louis, S, Schnitzer, A, Volynets, V, Rings, A, Basrai, M, Bischoff, SC
The American journal of clinical nutrition. 2017;(1):127-135
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BACKGROUND Obesity and associated metabolic disorders are related to impairments of the intestinal barrier. OBJECTIVE We examined lactulose:mannitol (Lac:Man) permeability in obese individuals with and without liver steatosis undergoing a weight-reduction program to test whether an effective weight-loss program improves gut barrier function and whether obese patients with or without liver steatosis differ in this function. DESIGN Twenty-seven adult, nondiabetic individuals [mean ± SD body mass index (BMI; in kg/m2): 43.7 ± 5.2; 78% with moderate or severe liver steatosis] were included in the follow-up intervention study (n = 13 by month 12). All patients reduced their weight to a mean ± SD BMI of 36.4 ± 5.1 within 12 mo. We assessed barrier functions by the oral Lac:Man and the fecal zonulin tests. Insulin resistance was assessed by the homeostatic model assessment index (HOMA), and liver steatosis by sonography and the fatty liver index (FLI). RESULTS The Lac:Man ratio and circulating interleukin (IL) 6 concentration decreased during intervention from 0.080 (95% CI: 0.073, 0.093) to 0.027 (95% CI: 0.024, 0.034; P < 0.001) and from 4.2 ± 1.4 to 2.8 ± 1.6 pg/mL (P < 0.01), respectively. At study start, the Lac:Man ratio was higher in patients with moderate or severe steatosis than in those without any steatosis (P < 0.001). The Lac:Man ratio tended to correlate with HOMA (ρ = 0.55, P = 0.052), which correlated with FLI (ρ = 0.75, P < 0.01). A multiple-regression analysis led to a final model explaining FLI best through BMI, waist circumference, and the Lac:Man ratio. CONCLUSIONS Intestinal permeability is increased in obese patients with steatosis compared with obese patients without. The increased permeability fell to within the previously reported normal range after weight reduction. The data suggest that a leaky gut barrier is linked with liver steatosis and could be a new target for future steatosis therapies. This trial was registered at clinicaltrials.gov as NCT01344525.
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Changes in intestinal microbiota composition and metabolism coincide with increased intestinal permeability in young adults under prolonged physiological stress.
Karl, JP, Margolis, LM, Madslien, EH, Murphy, NE, Castellani, JW, Gundersen, Y, Hoke, AV, Levangie, MW, Kumar, R, Chakraborty, N, et al
American journal of physiology. Gastrointestinal and liver physiology. 2017;(6):G559-G571
Abstract
The magnitude, temporal dynamics, and physiological effects of intestinal microbiome responses to physiological stress are poorly characterized. This study used a systems biology approach and a multiple-stressor military training environment to determine the effects of physiological stress on intestinal microbiota composition and metabolic activity, as well as intestinal permeability (IP). Soldiers (n = 73) were provided three rations per day with or without protein- or carbohydrate-based supplements during a 4-day cross-country ski-march (STRESS). IP was measured before and during STRESS. Blood and stool samples were collected before and after STRESS to measure inflammation, stool microbiota, and stool and plasma global metabolite profiles. IP increased 62 ± 57% (mean ± SD, P < 0.001) during STRESS independent of diet group and was associated with increased inflammation. Intestinal microbiota responses were characterized by increased α-diversity and changes in the relative abundance of >50% of identified genera, including increased abundance of less dominant taxa at the expense of more dominant taxa such as Bacteroides Changes in intestinal microbiota composition were linked to 23% of metabolites that were significantly altered in stool after STRESS. Together, pre-STRESS Actinobacteria relative abundance and changes in serum IL-6 and stool cysteine concentrations accounted for 84% of the variability in the change in IP. Findings demonstrate that a multiple-stressor military training environment induced increases in IP that were associated with alterations in markers of inflammation and with intestinal microbiota composition and metabolism. Associations between IP, the pre-STRESS microbiota, and microbiota metabolites suggest that targeting the intestinal microbiota could provide novel strategies for preserving IP during physiological stress.NEW & NOTEWORTHY Military training, a unique model for studying temporal dynamics of intestinal barrier and intestinal microbiota responses to stress, resulted in increased intestinal permeability concomitant with changes in intestinal microbiota composition and metabolism. Prestress intestinal microbiota composition and changes in fecal concentrations of metabolites linked to the microbiota were associated with increased intestinal permeability. Findings suggest that targeting the intestinal microbiota could provide novel strategies for mitigating increases in intestinal permeability during stress.
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Effects of the adjunctive probiotic VSL#3 on portal haemodynamics in patients with cirrhosis and large varices: a randomized trial.
Gupta, N, Kumar, A, Sharma, P, Garg, V, Sharma, BC, Sarin, SK
Liver international : official journal of the International Association for the Study of the Liver. 2013;(8):1148-57
Abstract
BACKGROUND Probiotics, by altering gut flora, may favourably alter portal haemodynamics in patients with cirrhosis. AIM: To investigate the effect of probiotics on portal pressure in patients with cirrhosis. METHODS Randomized double-blind placebo-controlled trial conducted in G.B. Pant Hospital, New Delhi. A total of 94 cirrhotic patients having large oesophageal varices without history of variceal bleeding were randomized to three treatment groups and given 2 months' treatment with propranolol plus placebo, propranolol plus antibiotics (norfloxacin 400 mg BD) or propranolol plus probiotic (VSL#3, 900 billion/day) randomly assigned in 1:1:1 ratio. Outcome measures were change in Hepatic venous pressure gradient (HVPG): Response rate (Percentage of patients having a decrease from baseline of ≥20% or to ≤12 mm Hg) and changes from baseline; biochemical markers of inflammation: changes from baseline. RESULTS Adjunctive probiotics increased the response rate compared with propranolol alone (58% vs. 31%, P = 0.046), similar to adjunctive antibiotics (54%). The mean fall in HVPG was greater with either adjunctive probiotics (3.7 mm Hg vs. 2.1 mm Hg, P = 0.061) or adjunctive antibiotics (3.4 mm Hg) than with propranolol alone. Both adjunctive therapies were associated with greater decreases in TNF-α levels (in both peripheral and hepatic venous blood) that resulted from propranolol-only treatment. No clinically relevant between-group differences were observed in the type or frequency of adverse events. CONCLUSIONS Adjunctive probiotic (VSL#3) improved the response rate to propranolol therapy and was safe and well tolerated in patients with cirrhosis. Adjunctive probiotic therapy merits further study for reduction in portal pressure.
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Effect of high β-palmitate content in infant formula on the intestinal microbiota of term infants.
Yaron, S, Shachar, D, Abramas, L, Riskin, A, Bader, D, Litmanovitz, I, Bar-Yoseph, F, Cohen, T, Levi, L, Lifshitz, Y, et al
Journal of pediatric gastroenterology and nutrition. 2013;(4):376-81
Abstract
OBJECTIVES Palmitic acid (PA) constitutes 17% to 25% of the human milk fatty acids, and ~70% is esterified in the sn-2 position of triglycerides (β-palmitate). In the sn-2 position, PA is not hydrolyzed and thus is efficiently absorbed. The PA in palm oils, commonly used in infant formulas, is esterified in the sn-1 and sn-3 positions. In these positions, PA is hydrolyzed and forms poorly absorbed calcium complexes. The present study assessed whether high β-palmitate in infant formulas affects the intestinal flora. METHODS Thirty-six term infants were enrolled: 14 breast-fed (BF group) and 22 formula-fed infants who were randomly assigned to receive formula containing high β-palmitate (HBP group, n=14), or low β-palmitate (LBP group, n=8), where 44% and 14% of the PA was β-palmitate, respectively. The total amount of PA in the formulas was 19% and 22% in the LBP and HBP groups, respectively. Neither formula contained pre- or probiotics. Stool samples were collected at enrollment and at 6 weeks for the quantification of bacteria. RESULTS At 6 weeks, the HBP and BF groups had higher Lactobacillus and bifidobacteria counts than the LBP group (P<0.01). The Lactobacillus counts at 6 weeks were not significantly different between the HBP and BF groups. Lactobacillus counts were 1.2×10¹⁰, 1.2×10¹¹, and 5.6×10¹⁰ CFU/g for LBP, HBP, and BF groups, respectively. Bifidobacteria counts were 5.1×10⁹, 1.2×10¹¹, and 3.9×10¹⁰ CFU/g for LBP, HBP, and BF groups, respectively. CONCLUSIONS HBP formula beneficially affected infant gut microbiota by increasing the Lactobacillus and bifidobacteria counts in fecal stools.