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Fast Identification Method for Screening Bacteria from Faecal Samples Using Oxford Nanopore Technologies MinION Sequencing.
Borges, ASG, Basu, M, Brinks, E, Bang, C, Cho, GS, Baines, JF, Franke, A, Franz, CMAP
Current microbiology. 2023;(3):101
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Abstract
Most bacterial identification methods require extensive culturing, strain purification and DNA extraction protocols. This leads to additional expenses and time lags when isolating specific bacteria from complex microbiological ecosystems. This study aimed to develop a fast and robust method for identification of lactobacilli, bifidobacteria and Bacteroides in human faecal samples. Bacteria from faecal samples were cultured anaerobically on selective media. Sonication-based DNA extraction was performed, followed by almost complete 16S rRNA gene polymerase chain reaction amplification and MinION sequencing with the Flongle adapter. Sequence analysis was performed using NanoCLUST, while RStudio was used for graphics. For 110 of the 125 colonies investigated, 100% of reads were attributed to a single species, while the remaining 15 colonies consisted of mixtures of up to three different species. The proposed bacterial identification method is advantageous for isolating particular bacteria for which there are no exclusively selective media, as it avoids lengthy colony purification and DNA purification methods, and yields a quick colony identification with high accuracy. Therefore, this method can be used for directly screening for pure cultures of target microorganisms and is suitable for the identification of bacteria in culturomics studies.
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A dietary carbohydrate - gut Parasutterella - human fatty acid biosynthesis metabolic axis in obesity and type 2 diabetes.
Henneke, L, Schlicht, K, Andreani, NA, Hollstein, T, Demetrowitsch, T, Knappe, C, Hartmann, K, Jensen-Kroll, J, Rohmann, N, Pohlschneider, D, et al
Gut microbes. 2022;(1):2057778
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Abstract
Recent rodent microbiome experiments suggest that besides Akkermansia, Parasutterella sp. are important in type 2 diabetes and obesity development. In the present translational human study, we aimed to characterize Parasutterella in our European cross-sectional FoCus cohort (n = 1,544) followed by validation of the major results in an independent Canadian cohort (n = 438). In addition, we examined Parasutterella abundance in response to a weight loss intervention (n = 55). Parasutterella was positively associated with BMI and type 2 diabetes independently of the reduced microbiome α/β diversity and low-grade inflammation commonly found in obesity. Nutritional analysis revealed a positive association with the dietary intake of carbohydrates but not with fat or protein consumption. Out of 126 serum metabolites differentially detectable by untargeted HPLC-based MS-metabolomics, L-cysteine showed the strongest reduction in subjects with high Parasutterella abundance. This is of interest, since Parasutterella is a known high L-cysteine consumer and L-cysteine is known to improve blood glucose levels in rodents. Furthermore, metabolic network enrichment analysis identified an association of high Parasutterella abundance with the activation of the human fatty acid biosynthesis pathway suggesting a mechanism for body weight gain. This is supported by a significant reduction of the Parasutterella abundance during our weight loss intervention. Together, these data indicate a role for Parasutterella in human type 2 diabetes and obesity, whereby the link to L-cysteine might be relevant in type 2 diabetes development and the link to the fatty acid biosynthesis pathway for body weight gain in response to a carbohydrate-rich diet in obesity development.
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The effects of probiotics administration on the gut microbiome in adolescents with anorexia nervosa-A study protocol for a longitudinal, double-blind, randomized, placebo-controlled trial.
Gröbner, EM, Zeiler, M, Fischmeister, FPS, Kollndorfer, K, Schmelz, S, Schneider, A, Haid-Stecher, N, Sevecke, K, Wagner, G, Keller, L, et al
European eating disorders review : the journal of the Eating Disorders Association. 2022;(1):61-74
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Abstract
OBJECTIVE Knowledge on gut-brain interaction might help to develop new therapies for patients with anorexia nervosa (AN), as severe starvation-induced changes of the microbiome (MI) do not normalise with weight gain. We examine the effects of probiotics supplementation on the gut MI in patients with AN. METHOD This is a study protocol for a two-centre double-blind randomized-controlled trial comparing the clinical efficacy of multistrain probiotic administration in addition to treatment-as-usual compared to placebo in 60 patients with AN (13-19 years). Moreover, 60 sex- and age-matched healthy controls are included in order to record development-related changes. Assessments are conducted at baseline, discharge, 6 and 12 months after baseline. Assessments include measures of body mass index, psychopathology (including eating-disorder-related psychopathology, depression and anxiety), neuropsychological measures, serum and stool analyses. We hypothesise that probiotic administration will have positive effects on the gut microbiota and the treatment of AN by improvement of weight gain, gastrointestinal complaints and psychopathology, and reduction of inflammatory processes compared to placebo. CONCLUSIONS If probiotics could help to normalise the MI composition, reduce inflammation and gastrointestinal discomfort and increase body weight, its administration would be a readily applicable additional component of multi-modal AN treatment.
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Genome-wide association study in 8,956 German individuals identifies influence of ABO histo-blood groups on gut microbiome.
Rühlemann, MC, Hermes, BM, Bang, C, Doms, S, Moitinho-Silva, L, Thingholm, LB, Frost, F, Degenhardt, F, Wittig, M, Kässens, J, et al
Nature genetics. 2021;(2):147-155
Abstract
The intestinal microbiome is implicated as an important modulating factor in multiple inflammatory1,2, neurologic3 and neoplastic diseases4. Recent genome-wide association studies yielded inconsistent, underpowered and rarely replicated results such that the role of human host genetics as a contributing factor to microbiome assembly and structure remains uncertain5-11. Nevertheless, twin studies clearly suggest host genetics as a driver of microbiome composition11. In a genome-wide association analysis of 8,956 German individuals, we identified 38 genetic loci to be associated with single bacteria and overall microbiome composition. Further analyses confirm the identified associations of ABO histo-blood groups and FUT2 secretor status with Bacteroides and Faecalibacterium spp. Mendelian randomization analysis suggests causative and protective effects of gut microbes, with clade-specific effects on inflammatory bowel disease. This holistic investigative approach of the host, its genetics and its associated microbial communities as a 'metaorganism' broaden our understanding of disease etiology, and emphasize the potential for implementing microbiota in disease treatment and management.
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The Impact of Starvation on the Microbiome and Gut-Brain Interaction in Anorexia Nervosa.
Seitz, J, Belheouane, M, Schulz, N, Dempfle, A, Baines, JF, Herpertz-Dahlmann, B
Frontiers in endocrinology. 2019;:41
Abstract
Interactions between the gut microbiome and the brain are of increasing interest to both researchers and clinicians. Evidence is mounting on the causal role of an altered gut microbiome in inflammatory diseases such as arthritis, inflammatory bowel disease, obesity and diabetes, and psychiatric diseases like anxiety and depression. Mechanisms include altered energy harvest from food, hormonal changes, increased gut permeability, inflammation, immune response, and a direct influence on the brain and behavior. Anorexia nervosa (AN) is the third most common disease in adolescence and exacts a high burden on patients and caregivers. It often becomes chronic and has the highest mortality of all psychiatric diseases. As AN is characterized by nutritional restrictions, weight loss, and severe behavioral symptoms including weight phobia, comorbid anxiety and depression, accompanied by endocrine alterations, increased inflammation, and immune response, exploring the role of the gut microbiome is crucial. Here, we present an overview of the potential mechanisms of interaction between the gut microbiome, the host and particularly the brain in AN and summarize the initial findings of microbiome research on AN. We conclude by identifying future research directions and potential therapeutic approaches, including nutritional interventions, probiotics, prebiotics and food supplements, that could become important additions to current AN therapy.
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Application of the distance-based F test in an mGWAS investigating β diversity of intestinal microbiota identifies variants in SLC9A8 (NHE8) and 3 other loci.
Rühlemann, MC, Degenhardt, F, Thingholm, LB, Wang, J, Skiecevičienė, J, Rausch, P, Hov, JR, Lieb, W, Karlsen, TH, Laudes, M, et al
Gut microbes. 2018;(1):68-75
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Abstract
Factors shaping the human intestinal microbiota range from environmental influences, like smoking and exercise, over dietary patterns and disease to the host's genetic variation. Recently, we could show in a microbiome genome-wide association study (mGWAS) targeting genetic variation influencing the β diversity of gut microbial communities, that approximately 10% of the overall gut microbiome variation can be explained by host genetics. Here, we report on the application of a new method for genotype-β-diversity association testing, the distance-based F (DBF) test. With this we identified 4 loci with genome-wide significant associations, harboring the genes CBEP4, SLC9A8, TNFSF4, and SP140, respectively. Our findings highlight the utility of the high-performance DBF test in β diversity GWAS and emphasize the important role of host genetics and immunity in shaping the human intestinal microbiota.
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Functional variants in the sucrase-isomaltase gene associate with increased risk of irritable bowel syndrome.
Henström, M, Diekmann, L, Bonfiglio, F, Hadizadeh, F, Kuech, EM, von Köckritz-Blickwede, M, Thingholm, LB, Zheng, T, Assadi, G, Dierks, C, et al
Gut. 2018;67(2):263-270
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Plain language summary
Congenital sucrase-isomaltase deficiency (CSID) is a genetic disorder which results in a lower ability to digest certain sugars, resulting in diarrhoea, abdominal pain and bloating, which are also common symptoms of Irritable Bowel Syndrome (IBS). The objective of this study was to test sucrase-isomaltase (SI) gene variants for their potential relevance in IBS. The researchers looked at genetics in several populations with and without IBS. The researchers found that genetic mutations are associated with a 35% reduction in the activity of the SI enzymes. CSID mutations were almost twice as common in IBS patients than healthy controls. The genetic variant 15Phe was associated with diarrhoea, stool frequency and changes in the gut bacteria. The authors concluded that people with SI gene variants associated with reduced enzyme activity are more at risk of IBS. Genetic screening could help to identify individuals at increased risk of IBS, and may lead to more targeted treatment for some people with IBS.
Abstract
OBJECTIVE IBS is a common gut disorder of uncertain pathogenesis. Among other factors, genetics and certain foods are proposed to contribute. Congenital sucrase-isomaltase deficiency (CSID) is a rare genetic form of disaccharide malabsorption characterised by diarrhoea, abdominal pain and bloating, which are features common to IBS. We tested sucrase-isomaltase (SI) gene variants for their potential relevance in IBS. DESIGN We sequenced SI exons in seven familial cases, and screened four CSID mutations (p.Val557Gly, p.Gly1073Asp, p.Arg1124Ter and p.Phe1745Cys) and a common SI coding polymorphism (p.Val15Phe) in a multicentre cohort of 1887 cases and controls. We studied the effect of the 15Val to 15Phe substitution on SI function in vitro. We analysed p.Val15Phe genotype in relation to IBS status, stool frequency and faecal microbiota composition in 250 individuals from the general population. RESULTS CSID mutations were more common in patients than asymptomatic controls (p=0.074; OR=1.84) and Exome Aggregation Consortium reference sequenced individuals (p=0.020; OR=1.57). 15Phe was detected in 6/7 sequenced familial cases, and increased IBS risk in case-control and population-based cohorts, with best evidence for diarrhoea phenotypes (combined p=0.00012; OR=1.36). In the population-based sample, 15Phe allele dosage correlated with stool frequency (p=0.026) and Parabacteroides faecal microbiota abundance (p=0.0024). The SI protein with 15Phe exhibited 35% reduced enzymatic activity in vitro compared with 15Val (p<0.05). CONCLUSIONS SI gene variants coding for disaccharidases with defective or reduced enzymatic activity predispose to IBS. This may help the identification of individuals at risk, and contribute to personalising treatment options in a subset of patients.
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Faecal microbiota composition associates with abdominal pain in the general population.
Hadizadeh, F, Bonfiglio, F, Belheouane, M, Vallier, M, Sauer, S, Bang, C, Bujanda, L, Andreasson, A, Agreus, L, Engstrand, L, et al
Gut. 2018;(4):778-779
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Stool frequency is associated with gut microbiota composition.
Hadizadeh, F, Walter, S, Belheouane, M, Bonfiglio, F, Heinsen, FA, Andreasson, A, Agreus, L, Engstrand, L, Baines, JF, Rafter, J, et al
Gut. 2017;(3):559-560
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Beneficial Effects of a Dietary Weight Loss Intervention on Human Gut Microbiome Diversity and Metabolism Are Not Sustained during Weight Maintenance.
Heinsen, FA, Fangmann, D, Müller, N, Schulte, DM, Rühlemann, MC, Türk, K, Settgast, U, Lieb, W, Baines, JF, Schreiber, S, et al
Obesity facts. 2016;9(6):379-391
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Plain language summary
Recent findings have suggested that the composition of the gut microbiome plays an important role in energy balance and metabolism. The aim of this study was to examine if weight loss altered the gut micriobiota diversity and to determine whether these changes were sustained during a weight maintenance period. Dietary intervention included a very low calorie diet (800 kcal/day) for three months followed by a weight maintenance period for three months. This study consisted of three groups – an intervention of 18 obese adults, a control of 13 lean adults and a second control of 13 obese adults. This study found that a very low calorie diet intervention in obese humans over a three-month period resulted in a significant change in microbiome diversity and metabolism, however these changes diminished during the weight maintenance period. Based on these findings, the authors suggest that nutritional components may exert a stronger effect on the human gut microbiome than changes in body weight or metabolism.
Abstract
OBJECTIVE In the present study, we examined the effect of a very low-calorie diet(VLCD)-based obesity program on human gut microbiome diversity and metabolism during weight loss and weight maintenance. METHODS Obese subjects underwent 3 months of VLCD followed by 3 months of weight maintenance. A lean and an obese control group were included. The microbiome was characterized by performing high-throughput dual-indexed 16S rDNA amplicon sequencing. RESULTS At baseline, a significant difference in the Firmicutes/Bacteroidetes ratio between the lean and obese individuals was observed (p = 0.047). The VLCD resulted in significant alterations in gut microbiome diversity from baseline to 3 months (p = 0.0053). Acinetobacter represented an indicator species for the observed effect (indicator value = 0.998, p = 0.006). Metabolic analyses revealed alterations of the bacterial riboflavin pathway from baseline to 3 months (pnom = 0.0078). These changes in diversity and bacterial metabolism induced by VLCD diminished during the weight maintenance phase, despite sustained reductions in body weight and sustained improvements of insulin sensitivity. CONCLUSION The present data show that a VLCD is able to beneficially alter both gut microbiome diversity and metabolism in obese humans, but that these changes are not sustained during weight maintenance. This finding might suggest that the microbiome should be targeted during obesity programs.