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165 results found
Hoyles L, Fernández-Real J-M, Federici M, et al., 2018, Molecular phenomics and metagenomics of hepatic steatosis in non-diabetic obese women., Nature medicine, Vol: 24, Pages: 1070-1080, ISSN: 1078-8956
Hepatic steatosis is a multifactorial condition that is often observed in obese patients and is a prelude to non-alcoholic fatty liver disease. Here, we combine shotgun sequencing of fecal metagenomes with molecular phenomics (hepatic transcriptome and plasma and urine metabolomes) in two well-characterized cohorts of morbidly obese women recruited to the FLORINASH study. We reveal molecular networks linking the gut microbiome and the host phenome to hepatic steatosis. Patients with steatosis have low microbial gene richness and increased genetic potential for the processing of dietary lipids and endotoxin biosynthesis (notably from Proteobacteria), hepatic inflammation and dysregulation of aromatic and branched-chain amino acid metabolism. We demonstrated that fecal microbiota transplants and chronic treatment with phenylacetic acid, a microbial product of aromatic amino acid metabolism, successfully trigger steatosis and branched-chain amino acid metabolism. Molecular phenomic signatures were predictive (area under the curve = 87%) and consistent with the gut microbiome having an effect on the steatosis phenome (>75% shared variation) and, therefore, actionable via microbiome-based therapies.
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Alexander JL, Scott A, Poynter LR, et al., 2018, Sa1840 - The colorectal cancer mucosal microbiome is defined by disease stage and the tumour metabonome, Digestive Disease Week 2018, Publisher: Elsevier, Pages: S415-S415, ISSN: 0016-5085
Alexander JL, Scott A, Poynter LR, et al., 2018, THE COLORECTAL CANCER MUCOSAL MICROBIOME IS DEFINED BY DISEASE STAGE AND THE TUMOUR METABONOME, Annual Meeting of the American-Society-for-Gastrointestinal-Endoscopy / Digestive Disease Week, Publisher: W B SAUNDERS CO-ELSEVIER INC, Pages: S415-S415, ISSN: 0016-5085
Hoyles L, Jiménez-Pranteda MJ, Chilloux J, et al., 2018, Metabolic retroconversion of trimethylamine N-oxide and the gut microbiota, Microbiome, Vol: 6, ISSN: 2049-2618
Background:The dietary methylamines choline, carnitine, and phosphatidylcholine are used by the gut microbiota to produce a range of metabolites, including trimethylamine (TMA). However, little is known about the use of trimethylamine N-oxide (TMAO) by this consortium of microbes.Results:A feeding study using deuterated TMAO in C57BL6/J mice demonstrated microbial conversion of TMAO to TMA, with uptake of TMA into the bloodstream and its conversion to TMAO. Microbial activity necessary to convert TMAO to TMA was suppressed in antibiotic-treated mice, with deuterated TMAO being taken up directly into the bloodstream. In batch-culture fermentation systems inoculated with human faeces, growth of Enterobacteriaceae was stimulated in the presence of TMAO. Human-derived faecal and caecal bacteria (n = 66 isolates) were screened on solid and liquid media for their ability to use TMAO, with metabolites in spent media analysed by 1H-NMR. As with the in vitro fermentation experiments, TMAO stimulated the growth of Enterobacteriaceae; these bacteria produced most TMA from TMAO. Caecal/small intestinal isolates of Escherichia coli produced more TMA from TMAO than their faecal counterparts. Lactic acid bacteria produced increased amounts of lactate when grown in the presence of TMAO but did not produce large amounts of TMA. Clostridia (sensu stricto), bifidobacteria, and coriobacteria were significantly correlated with TMA production in the mixed fermentation system but did not produce notable quantities of TMA from TMAO in pure culture.Conclusions:Reduction of TMAO by the gut microbiota (predominantly Enterobacteriaceae) to TMA followed by host uptake of TMA into the bloodstream from the intestine and its conversion back to TMAO by host hepatic enzymes is an example of metabolic retroconversion. TMAO influences microbial metabolism depending on isolation source and taxon of gut bacterium. Correlation of metabolomic and abundance data from mixed microbiota fermenta
Hoyles L, Snelling T, Umlai UK, et al., 2018, Microbiome–host systems interactions: protective effects of propionate upon the blood–brain barrier, Microbiome, Vol: 6, ISSN: 2049-2618
Background: Gut microbiota composition and function are symbiotically linked with host health, and altered in metabolic, inflammatory and neurodegenerative disorders. Three recognized mechanisms exist by which the microbiome influences the gut--brain axis: modification of autonomic/sensorimotor connections, immune activation, and neuroendocrine pathway regulation. We hypothesized interactions between circulating gut-derived microbial metabolites and the blood--brain barrier (BBB) also contribute to the gut--brain axis. Propionate, produced from dietary substrates by colonic bacteria, stimulates intestinal gluconeogenesis and is associated with reduced stress behaviours, but its potential endocrine role has not been addressed. Results: After demonstrating expression of the propionate receptor FFAR3 on human brain endothelium, we examined the impact of a physiologically relevant propionate concentration (1 μM) on BBB properties in vitro. Propionate inhibited pathways associated with non-specific microbial infections via a CD14-dependent mechanism, suppressed expression of LRP-1 and protected the BBB from oxidative stress via NRF2 (NFE2L2) signaling. Conclusions: Together, these results suggest gut-derived microbial metabolites interact with the BBB, representing a fourth facet of the gut--brain axis that warrants further attention.
Hoyles L, Snelling T, Umlai U-K, et al., 2018, Propionate has protective and anti-inflammatory effects on the blood–brain barrier, Alzheimer's Research UK Research Conference 2018
Propionate is a short-chain fatty acid (SCFA) produced by the human gut microbiota from dietary substrates, and is biologically active via the G protein coupled receptors FFAR2 and FFAR3. It is taken up from the gut and reaches systemic circulation in micromolar quantities. The blood–brain barrier (BBB) is the major interface between the circulation and central nervous system. FFAR3 is expressed on the vascular endothelium and a likely target for propionate in the BBB. We hypothesized exposure of the BBB to propionate influences barrier integrity and function.Methods and materialsWe investigated the in vitro effects of a physiologically relevant concentration (1 μM) of propionate upon the human immortalised cerebromicrovascular endothelial cell line hCMEC/D3. FFAR3 was present on these cells. We, therefore, performed an unbiased transcriptomic analysis of confluent hCMEC/D3 monolayers treated or not for 24 h with 1 μM propionate, supported by in vitro validation of key findings and assessment of functional endothelial permeability barrier properties.ResultsPropionate treatment had a significant (PFDR < 0.1) effect on the expression of 1136 genes. It inhibited several inflammation-associated pathways: TLR-specific signalling, NFkappaB signalling, and cytosolic DNA-sensing. Functional validation of these findings confirmed the down-regulation of TLR signalling by propionate, achieved primarily through down-regulation of endothelial CD14 expression. Accordingly, propionate prevented LPS-induced increases in paracellular permeability to 70 kDa FITC-dextran and loss of transendothelial electrical resistance. Propionate activated the NFE2L2 (NRF2)-driven protective response against oxidative stress. Confirming these data, propionate limited free reactive oxygen species induction by the mitochondrial respiratory inhibitor rotenone. ConclusionsOur data strongly suggest the SCFA propionate contributes to maintaining BBB integrity and protecting against inflamm
McArthur S, Carvalho A, Fonseca S, et al., 2018, Effects of gut-derived trimethylamines on the blood–brain barrier, Alzheimer's Research UK Research Conference 2018
Introduction: The gut microbiota and its metabolites exert significant effects on host health, with disturbances to composition and function associated with conditions including obesity, type II diabetes and, more recently, Alzheimer’s disease (AD). Communication between microbes and the host can take a number of forms, but central to all of them is a role for gut-derived microbial metabolites, with trimethylamine N-oxide (TMAO) and its precursor trimethylamine (TMA) being important examples. TMA produced by gut bacteria is converted to TMAO in the liver by flavin monooxygenases whereupon it enters the circulation. TMAO was recently identified as potentially important in genetic pathways associated with AD, and has been shown to influence peripheral vascular function. Given these links, the key position of the cerebral vasculature as the major interface between circulating molecules and the brain, and evidence that deficits in blood–brain barrier (BBB) function occur early in AD, we investigated the effects of TMAO and TMA on key BBB properties in vitro and in vivo.Materials and Methods: Male C57Bl/6 mice (n=4-5) were used to examine the effect of TMAO treatment (1.8 mg/kg, 2 h, dose equivalent to circulating human concentrations) upon BBB permeability in vivo, assessed by Evans’ blue dye extravasation. TMA was not investigated as the average mouse plasma concentration of this methylamine is substantially greater than that seen in humans (TMAO-to-TMA ratio 1:10 in mice, 10:1 in humans).Human hCMEC/D3 cerebromicrovascular cells were used as an in vitro model of the BBB to investigate the effects of 24 h treatment with human physiologically relevant doses of TMAO (40 μM) and TMA (0.4 μM), studying (i) functional barrier properties of cell monolayers and (ii) gene expression. Results: Administration of TMAO to mice enhanced BBB integrity above baseline after 2 h treatment (p<0.05). Similarly, in vitro exposure of hCMEC/D3 cells to TMAO enhanc
Hoyles L, Snelling T, Umlai U-K, et al., 2018, Microbiome–host interactions: protective effects of propionate upon the blood–brain barrier, Publisher: biorixiv
Breakdown of foodstuffs by the gut microbiota results in the production of the short-chain fatty acids (SCFAs) acetate, propionate and butyrate. SFCAs are potent bioactive molecules, providing energy for intestinal cells, enhancing satiety and positively influencing metabolic health. They also influence the gut–brain axis. The gut microbiota and/or its bioactive molecules contribute to maintaining the integrity of the blood–brain barrier (BBB), the primary defensive structure of the brain. Propionate is produced by the gut microbiota from the breakdown of glucans found in whole grains, mushrooms and yeast products. It is found in the blood at ≤1 μM. At this physiologically relevant concentration, propionate enhances BBB integrity, mitigating against deleterious inflammatory and oxidative stimuli known to contribute to neurological and psychological diseases. Therefore, there is the potential that dietary supplementation with glucan-containing products may offer protection of the brain against detrimental stimuli.
Dehghan A, 2018, Linking metabolic phenotyping and genomic information, The Handbook of Metabolic Phenotyping, Pages: 561-569, ISBN: 9780128122945
Metabolomics is one of the “omics” that has recently become available in epidemiologic studies. Other omics, including genomics, transcriptomics, and proteomics, are also applied at population level to study complex traits and disorders. However, these approaches are mainly used in isolation. Each of these omics, in fact, pertains to only one layer of the cellular information. It is known that none of these omics capture the totality of the cellular information. Therefore, multi-omics studies are designed to enhance our understanding of the molecular interactions by adding up more layers of information. Linking to genomics is one of the first multi-omics approaches that have been put in practice for metabolites. Recent advances in genomic approaches have made it possible to search for genetics of metabolites in large scale. In this chapter, we review a number of studies that have applied genome-wide association studies (GWAS) to identify genetic determinants of metabolites measured by various metabolic assays. We show that despite their discoveries, the exact mechanisms that link the genes to metabolites are yet unknown. Moreover, we briefly review the technical complexity of the approaches and challenges that are either tackled or are still challenging such studies.
Hoyles L, Jiménez-Pranteda ML, Chilloux J, et al., 2017, Metabolic retroconversion of trimethylamine <i>N</i>-oxide and the gut microbiota
<jats:title>ABSTRACT</jats:title><jats:sec><jats:title>BACKGROUND</jats:title><jats:p>The dietary methylamines choline, carnitine and phosphatidylcholine are used by the gut microbiota to produce a range of metabolites, including trimethylamine (TMA). However, little is known about the use of trimethylamine <jats:italic>N</jats:italic>-oxide (TMAO) by this consortium of microbes.</jats:p></jats:sec><jats:sec><jats:title>RESULTS</jats:title><jats:p>A feeding study using deuterated TMAO in C57BL6/J mice demonstrated microbial conversion of TMAO to TMA, with uptake of TMA into the bloodstream and its conversion to TMAO. Microbial activity necessary to convert TMAO to TMA was suppressed in antibiotic-treated mice, with deuterated TMAO being taken up directly into the bloodstream. In batch-culture fermentation systems inoculated with human faeces, growth of <jats:italic>Enterobacteriaceae</jats:italic> was stimulated in the presence of TMAO. Human-derived faecal and caecal bacteria (<jats:italic>n</jats:italic> = 66 isolates) were screened on solid and liquid media for their ability to use TMAO, with metabolites in spent media analysed by <jats:sup>1</jats:sup>H-NMR. As with the <jats:italic>in vitro</jats:italic> fermentation experiments, TMAO stimulated the growth of <jats:italic>Enterobacteriaceae</jats:italic>; these bacteria produced most TMA from TMAO. Caecal/small intestinal isolates of <jats:italic>Escherichia coli</jats:italic> produced more TMA from TMAO than their faecal counterparts. Lactic acid bacteria produced increased amounts of lactate when grown in the presence of TMAO, but did not produce large amounts of TMA. Clostridia (<jats:italic>sensu stricto</jats:italic>), bifidobacteria and coriobacteria were significantly correlated with TMA production in the mixed fermentation system but did
Hoyles L, Fernández-Real JM, Federici M, et al., 2017, Integrated systems biology to study the contribution of the gut microbiome to steatosis in obese women, Exploring Human Host-Microbiome Interactions in Health and Disease
Non-alcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease, increasing in worldwide prevalence as a result of the obesity epidemic. It manifests in hepatic cells as steatosis with or without lobular inflammation and/or ballooning. Animal and human studies have suggested the gut microbiome contributes to steatosis/NAFLD. The aim of this study was to use an integrated approach with various -omics and clinical data to evaluate the contribution of the gut microbiome to the molecular phenome (hepatic transcriptome, metabonome) of steatosis. Metagenomic (faecal microbiome), transcriptomic (liver biopsy), metabonomic (plasma and urine, 1H-NMR) and clinical data were collected for 56 morbidly obese (BMI >35) women from Italy (n = 31) and Spain (n = 25) who elected for bariatric surgery. Confounder analyses of clinical data were done using linear modelling. Histological examination of liver biopsies was used to grade steatosis. Faecal metagenomes were generated and analysed using the SCalable Automated Metagenomics Pipeline (SCAMP). Differentially expressed genes were identified in hepatic transcriptomes, and analysed using a range of different bioinformatics tools. 1H-NMR data were generated for plasma and urinary metabonomes. Clinical, metagenomic, transcriptomic and metabonomic data were integrated in the context of steatosis using partial Spearman's correlation, taking confounders (age, body mass index and cohort) into account. Steatosis was anti-correlated with microbial gene richness, and correlated with abundance of Proteobacteria. KEGG analyses of metagenomic data suggested increased microbial processing of dietary lipids and amino acids, as well as endotoxin-related processes related to Proteobacteria. Steatosis-associated hepatic transcriptomes were associated with branched-chain amino acid (BCAA) metabolism, endoplasmic reticulum/phagosome, and immune responses associated with non-specific microbial infections. Metabonom
Hoyles L, Snelling T, Umlai UK, et al., 2017, Propionate has protective and anti-inflammatory effects on the blood–brain barrier, Exploring Human Host-Microbiome Interactions in Health and Disease
Production of short-chain fatty acids (SCFAs) from dietary substrates by the gut microbiota is associated with health, with these metabolites influencing the host via the ‘gut–brain axis’. Micromolar quantities of microbially derived SCFAs are taken up from the gut and reach systemic circulation, where they can influence host gene expression through a variety of largely unknown mechanisms. The blood–brain barrier (BBB) is the major interface between the circulation and central nervous system, and is critically involved in the pathogenesis of neuroinflammatory disorders such as stroke and vascular dementia. We hypothesized exposure of the BBB to SCFAs influences barrier integrity and function.To test our hypothesis, we investigated the in vitro effects of a physiologically relevant concentration (1 μM) of propionate upon the human immortalised cerebromicrovascular endothelial cell line hCMEC/D3. Propionate is produced by the microbiota from dietary glucans, and is biologically active via the G protein coupled receptors FFAR2 and FFAR3. It is a highly potent FFAR2 agonist (agonist activity 3.99) and has close to optimal ligand efficiency (-ΔG=1.19 kcal mol-1 atom-1) for this receptor. Notably, FFAR3 is expressed on the vascular endothelium and a likely target for propionate in the BBB.After confirming the presence of FFAR3 on hCMEC/D3 cells, we undertook an unbiased transcriptomic analysis of confluent hCMEC/D3 monolayers treated or not for 24 h with 1 μM propionate, supported by in vitro validation of key findings and assessment of functional endothelial permeability barrier properties.Propionate treatment had a significant (PFDR < 0.1) effect on the expression of 1136 genes: 553 upregulated, 583 downregulated. Propionate inhibited several inflammation-associated pathways: namely, TLR-specific signalling, NFkappaB signalling, and cytosolic DNA-sensing. Functional validation of these findings confirmed the down-regulation of TLR
Carding SR, Davis N, Hoyles L, 2017, Review article: The human intestinal virome in health and disease, Alimentary Pharmacology and Therapeutics, Vol: 46, Pages: 800-815, ISSN: 1365-2036
Background: The human virome consists of animal-cell viruses causing transient infections, bacteriophage (phage) predators of bacteria and archaea, endogenous retroviruses, and viruses causing persistent and latent infections. High-throughput, inexpensive, sensitive sequencing methods and metagenomics have made it possible to study the contribution dsDNA, ssDNA and RNA virus-like particles make to the human virome, and in particular the intestinal virome. Aim: To review and evaluate the pioneering studies that have attempted to characterise the human virome and generated an increased interest in understanding how the intestinal virome might contribute to maintaining health, and the pathogenesis of chronic diseases. Methods: Relevant virome-related articles were selected for review following extensive language- and date-unrestricted, electronic searches of the literature.Results: The human intestinal virome is personalized and stable, and dominated by phages. It develops soon after birth in parallel with prokaryotic communities of the microbiota, becoming established during the first few years of life. By infecting specific populations of bacteria, phages can alter microbiota structure by killing host cells or altering their phenotype, enabling phages to contribute to maintaining intestinal homeostasis or to dysbiosis and the development of chronic infectious and autoimmune diseases including HIV infection and Crohn’s disease, respectively. Conclusions: Our understanding of the intestinal virome is fragmented and requires standardized methods for virus isolation and sequencing to provide a more complete picture of the virome, which is key to explaining the basis of virome–disease associations, and how enteric viruses can contribute to disease aetiologies and be rationalized as targets for interventions in disease states.
Dumas M, Rothwell AR, Hoyles L, et al., 2017, Microbial-host co-metabolites are prodromal markers predicting phenotypic heterogeneity in behavior, obesity and impaired glucose tolerance, Cell Reports, Vol: 20, Pages: 136-148, ISSN: 2211-1247
The influence of the gut microbiome on metabolic and behavioral traits is now widely accepted, though the microbiome-derived metabolites involved remain unclear. We carried out untargeted urine 1H NMR spectroscopy-based metabolic phenotyping in an isogenic C57BL/6J mouse population (n=50) and show that microbial-host co-metabolites are prodromal (i.e., early) markers predicting future divergence in metabolic (obesity and glucose homeostasis) and behaviorial (anxiety and activity) outcomes with 94-100% accuracy. Some of these metabolites also modulate disease phenotypes, best illustrated by trimethylamine-N-oxide (TMAO), a product of microbial-host co-metabolism predicting future obesity, impaired glucose tolerance (IGT) and behavior, whilst reducing endoplasmic reticulum stress and lipogenesis in 3T3-L1 adipocytes. Chronic in vivo TMAO treatment limits IGT in HFD-fed mice and isolated pancreatic islets by increasing insulin secretion. We highlight the prodromal potential of microbial metabolites to predict disease outcomes and their potential in shaping mammalian phenotypic heterogeneity.
Hoyles L, Fernández-Real JM, Federici M, et al., 2017, Integrated systems biology to study non-alcoholic fatty liver disease in obese women, International Scientific Association for Probiotics and Prebiotics
Metagenomic (faecal microbiome), transcriptomic (liver biopsy), metabonomic (plasma and urine, 1H-NMR) and clinical (28 variables) data were collected for 56 morbidly obese (BMI >35) women from Italy (n = 31) and Spain (n = 25) who elected for bariatric surgery. Data were integrated to evaluate the contribution of the gut microbiome to the molecular phenome (hepatic transcriptome, plasma and urine metabonome) of NAFLD independent of clinical confounders (age, BMI, cohort) using partial Spearman’s correlation. NAFLD activity score (NAS) was anti-correlated with microbial gene richness, and correlated with abundance of Proteobacteria. KEGG analyses of metagenomic data suggested increased microbial processing of dietary lipids and amino acids, as well as endotoxin-related processes related to Proteobacteria. Metabonomic profiles highlighted imbalances in choline metabolism, branched-chain amino acid (BCAA) metabolism and gut-derived microbial metabolites resulting from metabolism of amino acids. NAFLD-associated hepatic transcriptomes were associated with BCAA metabolism, endoplasmic reticulum/phagosome, and immune responses associated with non-specific microbial infections. Molecular phenomic signatures were stable and predictive regardless of sample size, and consistent with the microbiome making a significant contribution to the NAFLD phenome. There is disruption of the gut– liver axis in NAFLD, which can be seen in the gut microbiome, hepatic transcriptome and urinary and plasma metabonomes. Consistency of phenome signatures strongly supports a relationship between microbial amino acid metabolism and microbial gene richness, hepatic gene expression and biofluid metabonomes, and ultimately NAS.
Hoyles L, Fernandez-Real JM, Federici M, et al., 2017, Integrated systems biology to study non-alcoholic fatty liver disease in obese women, Tranlsational Bioinformatics
Non-alcoholic fatty liver disease (NAFLD) is a multifactorial condition and one of the most common causes of chronic liver disease, with increasing worldwide prevalence. Microbiome-associated lipopolysaccharides (LPS) are associated with NAFLD in rodent models, but their relevance in human liver disease is not understood. In addition, microbiome-driven degradation of dietary choline – and its subsequent removal from host-associated metabolic processes – is thought to contribute to development of NAFLD. The FLORINASH study set out to determine the contribution of the gut microbiome to the NAFLD-associated molecular phenome (transcriptome, metabonome) independent of clinical confounders.Morbidly obese women [body mass index (BMI) >35] from Italy (n = 31) and Spain (n = 25) who elected for bariatric surgery were recruited to the study. Clinical data (28 variables) were recorded. Faecal samples, liver biopsies, blood and urine samples were collected. Faecal metagenomes were analysed using an in-house metagenomics pipeline (SCaleble Automated Metagenomics Pipeline). NAFLD activity score (NAS; 0, 1, 2, 3) was determined by histological examination of liver biopsies. Differentially expressed genes in hepatic transcriptomes were identified, and analysed using several complementary tools. 1H-NMR data were generated for plasma and urinary metabonomes. Clinical, metagenomic, transcriptomic and metabonomic data were integrated using partial Spearman’s correlation, taking identified confounders (age, BMI and cohort) into account.NAS was anti-correlated with microbial gene richness, and correlated with abundance of Gram-negative Proteobacteria. KEGG analyses of metagenomic data suggested increased microbial processing of dietary lipids and amino acids, as well as LPS-related processes associated with Proteobacteria in NAFLD. Activation of immune responses associated with Gram-negative (LPS-associated) microbial infections was correlated with NAS in hepatic tr
McArthur S, Umlai UK, Snelling T, et al., 2017, Effects of gut-derived methylamines on the blood–brain barrier, 2017 Alzheimer's Research UK Conference
Introduction: Composition and functions of the gut microbiota are inextricably linked with host health, and altered in conditions such as obesity and type II diabetes. Central to microbe–host crosstalk are gut-derived microbial metabolites, of which trimethylamine N-oxide (TMAO) and its precursor trimethylamine (TMA) are of particular importance. TMA produced by intestinal microbes is converted to TMAO in the liver by flavin monooxygenases with circulating TMAO being associated with cardiovascular disease and insulin resistance. TMAO was also recently identified as potentially important in genetic pathways associated with Alzheimer’s disease (AD). In considering that deficits in blood–brain barrier (BBB) function occur early in AD, and its position as the major interface between circulating metabolites and the brain, we investigated the effects of TMAO and TMA on key BBB properties in vitro.Materials and Methods: Human hCMEC/D3 cerebromicrovascular cells were used as an in vitro model of the BBB to investigate the effects of 24 h treatment with physiologically relevant doses of TMAO and TMA, studying (i) functional barrier properties of cell monolayers, (ii) Aβ efflux transporters and (iii) gene expression.Results: Exposure of hCMEC/D3 cells to TMAO (40 μM) reinforced barrier integrity by enhancing transendothelial electrical resistance (P <0.001) and reducing paracellular permeability to a 70 kDa dextran tracer (P <0.001). In contrast, while TMA (0.4 μM) enhanced electrical resistance (P <0.001), it significantly increased tracer paracellular permeability (P <0.05), consistent with compromised barrier function. Transporter activity analysis showed TMAO inhibited p-glycoprotein function (P <0.001), which was not seen with TMA; neither metabolite affected BCRP function. Human-genome transcriptomic data are currently being analysed.Conclusions: TMAO and TMA affect BBB function in a metabolite-specific manner, regulating barr
Hoyles L, Fernández-Real JM, Federici M, et al., 2017, Integrated systems biology to study non-alcoholic fatty liver disease in obese women, Gut Microbiota for Health World Summit 2017
Objectives: To integrate metagenomic (faecal microbiome), transcriptomic, metabonomic and clinical data to evaluate the contribution of the gut microbiome to the molecular phenome (hepatic transcriptome, plasma and urine metabonome) of non-alcoholic fatty liver disease (NAFLD) independent of clinical confounders in morbidly obese women recruited to the FLORINASH study.Methods: Faecal, liver biopsy, blood and urine samples and data for 28 clinical variables were collected for 56 obese [body mass index (BMI) >35] women from Italy (n = 31) and Spain (n = 25) who elected for bariatric surgery. Confounder analyses of clinical data were done using linear modeling. Histological examination of liver biopsies was used to grade NAFLD (NAFLD activity score: 0, 1, 2, 3). Faecal metagenomes were generated and analysed using the Imperial Metagenomics Pipeline. Differentially expressed genes were identified in hepatic transcriptomes, and analysed using Enrichr, network analyses and Signaling Pathway Impact Analysis. 1H-NMR data were generated for plasma and urinary metabonomes. Clinical, metagenomic, transcriptomic and metabonomic data were integrated using partial Spearman’s correlation, taking confounders (age, body mass index and cohort) into account.Results: NAFLD activity score was anti-correlated with microbial gene richness, and correlated with abundance of Proteobacteria. KEGG analyses of metagenomic data suggested increased microbial processing of dietary lipids and amino acids, as well as endotoxin-related processes related to Proteobacteria. Metabonomic profiles highlighted imbalances in choline metabolism, branched-chain amino acid metabolism and gut-derived microbial metabolites resulting from metabolism of amino acids. NAFLD-associated hepatic transcriptomes were associated with branched-chain amino acid metabolism, endoplasmic reticulum/phagosome, and immune responses associated with microbial infections. Molecular phenomic signatures were stable and predic
Noble A, Durant L, Hoyles L, et al., 2017, Dysregulation of cellular vs humoral immunity to the intestinal microbiota in inflammatory bowel disease, JOURNAL OF CROHNS & COLITIS, Vol: 11, Pages: S123-S124, ISSN: 1873-9946
Hoyles L, Fernández-Real JM, Federici M, et al., 2017, Integrated systems biology to study non-alcoholic fatty liver disease in obese women, MRC-PHE Centre for Environment & Health - Centre Training Programme Annual Meeting
Hoyles L, Jimenez-Pranteda ML, Chilloux J, et al., 2016, Reduction of trimethylamine N-oxide to trimethylamine by the human gut microbiota: supporting evidence for 'metabolic retroversion', Exploring Human Host-Microbiome Interactions in Health and Disease
Dietary methylamines [choline, trimethylamine N-oxide (TMAO), phosphatidylcholine, carnitine] are present in meat, fish, nuts and eggs. Gut bacteria are able to use choline and carnitine in a fermentation-like process, with trimethylamine (TMA) among the main end-products. TMA is transported from the intestine via the hepatic vein to hepatocytes, then converted to TMAO by hepatic flavin-containing monooxygenases. TMAO present in urine and plasma is currently considered a biomarker for non-alcoholic fatty liver disease, insulin resistance and cardiovascular disease. However, circulating TMAO may play roles in protection from hyperammonemia, and glutamate neurotoxicity. Little is known about the reduction of TMAO (predominantly from fish) to TMA and other compounds by the gut microbiota. We screened 66 strains of human-associated gut bacteria on solid and liquid media for their ability to use TMAO, with metabolites in spent media analysed by 1H-NMR. Enterobacteriaceae produced most TMA from TMAO, with caecal/small intestinal isolates of Escherichia coli producing more TMA than their faecal counterparts. Lactic acid bacteria produced increased amounts of lactate and biomass when grown in the presence of TMAO, but did not appear to use TMAO as an alternative electron acceptor. Stimulation of the growth of gut Enterobacteriaceae in the presence of TMAO was confirmed in faeces-inoculated, anaerobic, stirred, pH-controlled fermentation systems. Feeding deuterated TMAO to C57BL6/J mice demonstrated microbial conversion of TMAO to TMA, with uptake of TMA into the bloodstream and its conversion to TMAO. Antibiotic-treated mice lacked microbial activity necessary to convert TMAO to TMA, instead taking up TMAO into the bloodstream by an unknown mechanism. This study demonstrates microbial reduction of TMAO to TMA followed by host-mediated oxidation of TMA to regenerate TMAO, i.e. metabolic retroversion.
Brook TC, Tijani T, Shibu P, et al., 2016, Characterization of lytic bacteriophages of Klebsiella pneumoniae with capsular depolymerases, Bacteriophage in Medicine, Food & Biotechnology – Phages 2016
Klebsiella pneumoniae is a Gram-negative opportunistic pathogen responsible for a wide range of nosocomial and community-acquired infections. The bacterium is increasingly difficult to treat and K. pneumoniae is considered a reservoir of antimicrobial resistance genes, including those encoding resistance to carbapenems and colistin. K. pneumoniae forms biofilms that further inhibit the uptake and efficacy of current antibiotics. Treatment difficulties are compounded by phenotypic diversity among K. pneumoniae isolates, with the bacterium having 78 recognized capsular (K) types. Alternative treatment options for K. pneumoniae infections are vital, and interest in the potential of bacteriophage therapies that target this bacterium is increasing. A hypervirulent intestinal isolate of K. pneumoniae, L4-FAA5 (K2:O1, ST380), has been used as a host strain to isolate lytic bacteriophages from mixed-liquor sewage samples. Three bacteriophages have been identified as having unique DNA restriction enzyme profiles, and their DNA has been submitted for whole-genome sequencing. Each bacteriophage appears to encode a depolymerase, as evidenced by formation of haloes around plaques in double-agar overlays seeded with L4-FAA5. When tested against 13 clinical isolates of K. pneumoniae (including K2 and unknown K, NDM, OXA-48 and KPC types), none of the bacteriophages (along with KLPN1) lysed any of the isolates; however, suspected depolymerase activity was observed for 10 of the 13 isolates tested. To this end, the project seeks to confirm the presence of depolymerases in the genomes of the three new bacteriophages, and to assess their efficacy as biofilm-degrading enzymes useful as adjuncts to antibiotic or lysin therapy.
Brook TC, Hoyles L, Markiv A, 2016, Characterising bacteriophages with lytic activity against Klebsiella pneumoniae, University of Westminster Postgraduate Fair
This project focuses on the identification and classification of bacteriophages used to target the opportunistic pathogen Klebsiella pneumoniae, a Gram-negative pathogen responsible for causing a wide range of community-acquired and nosocomial infections, most notably in immunocompromised patients. There is an increasing incidence of extended-spectrum-beta-lactamase and carbapenemase-producing strains of K. pneumoniae; therefore, the need for alternatives to antibiotic treatment for this pathogen is becoming more apparent. Bacteriophage therapy may provide a vital clinical alternative to antibiotics to treat multi-resistant strains of K. pneumoniae. Such phage therapies have been previously shown to be effective against K. pneumoniae infections in several animal models with differing infection types and phage administration methods. The overall aims of this project are to isolate and characterize a number of bacteriophages active against K. pneumoniae, and to assess their use in a bacteriophage cocktail in vitro.To date, we have identified four potentially novel lytic phages with activity against K. pneumoniae subsp. pneumoniae L4-FAA5, a hypermucoviscous and virulent strain isolated from the human gut. The whole-genome sequence of L4-FAA5 has been determined, and is currently being annotated. Growth characteristics of the host strain have been determined; phenotypic characteristics (titre, one step growth curves, burst-size, pH and chloroform sensitivity) of the phages are currently being determined. Whole-genome sequences of the phages’ DNA will be determined in the coming months. Future research utilising these phages will focus on an assessment of their activity against a wide range of clinical strains of K. pneumoniae with antibiotic resistance.
Bell ME, Lasker BA, Klenk HP, et al., 2016, Kroppenstedtia pulmonis sp. nov. and Kroppenstedtia sanguinis sp. nov., isolated from human patients, Antonie van Leeuwenhoek, Vol: 109, Pages: 603-610, ISSN: 0003-6072
Three human clinical strains (W9323T, X0209T and X0394) isolated from a lung biopsy, blood and cerebral spinal fluid, respectively, were characterised using a polyphasic taxonomic approach. Comparative analysis of the 16S rRNA gene sequences showed the three strains belong to two novel branches within the genus Kroppenstedtia: 16S rRNA gene sequence analysis of W9323T showed close sequence similarity to Kroppenstedtia eburnea JFMB-ATET (95.3 %), Kroppenstedtia guangzhouensis GD02T (94.7 %) and strain X0209T (94.6 %); sequence analysis of strain X0209T showed close sequence similarity to K. eburnea JFMB-ATET (96.4 %) and K. guangzhouensis GD02T (96.0 %). Strains X0209T and X0394 were 99.9 % similar to each other by 16S rRNA gene sequence analysis. The DNA–DNA relatedness was 94.6 %, confirming that X0209T and X0394 belong to the same species. Chemotaxonomic data for strains W9323T and X0209T were consistent with those described for the members of the genus Kroppenstedtia: the peptidoglycan was found to contain LL-diaminopimelic acid; the major cellular fatty acids were identified as iso-C15 and anteiso-C15; and the major menaquinone was identified as MK-7. Differences in endospore morphology, carbon source utilisation profiles, and cell wall sugar patterns of strains W9323T and X0209T, supported by phylogenetic analysis, enabled us to conclude that the strains each represent a new species within the genus Kroppenstedtia, for which the names Kroppenstedtia pulmonis sp. nov. (type strain W9323T = DSM 45752T = CCUG 68107T) and Kroppenstedtia sanguinis sp. nov. (type strain X0209T = DSM 45749T = CCUG 38657T) are proposed.
Dao MC, Everard A, Aron-Wisnewsky J, et al., 2016, Akkermansia muciniphila and improved metabolic health during a dietary intervention in obesity: relationship with gut microbiome richness and ecology, Gut, Vol: 65, Pages: 426-436, ISSN: 1468-3288
Objective Individuals with obesity and type 2 diabetes differ from lean and healthy individuals in their abundance of certain gut microbial species and microbial gene richness. Abundance of Akkermansia muciniphila, a mucin-degrading bacterium, has been inversely associated with body fat mass and glucose intolerance in mice, but more evidence is needed in humans. The impact of diet and weight loss on this bacterial species is unknown. Our objective was to evaluate the association between faecal A. muciniphila abundance, faecal microbiome gene richness, diet, host characteristics, and their changes after calorie restriction (CR).Design The intervention consisted of a 6-week CR period followed by a 6-week weight stabilisation diet in overweight and obese adults (N=49, including 41 women). Faecal A. muciniphila abundance, faecal microbial gene richness, diet and bioclinical parameters were measured at baseline and after CR and weight stabilisation.Results At baseline A. muciniphila was inversely related to fasting glucose, waist-to-hip ratio and subcutaneous adipocyte diameter. Subjects with higher gene richness and A. muciniphila abundance exhibited the healthiest metabolic status, particularly in fasting plasma glucose, plasma triglycerides and body fat distribution. Individuals with higher baseline A. muciniphila displayed greater improvement in insulin sensitivity markers and other clinical parameters after CR. These participants also experienced a reduction in A. muciniphila abundance, but it remained significantly higher than in individuals with lower baseline abundance. A. muciniphila was associated with microbial species known to be related to health.Conclusions A. muciniphila is associated with a healthier metabolic status and better clinical outcomes after CR in overweight/obese adults. The interaction between gut microbiota ecology and A. muciniphila warrants further investigation.
Shoaie S, Ghaffari P, Kovatcheva-Datchary P, et al., 2015, Quantifying Diet-Induced Metabolic Changes of the Human Gut Microbiome, Cell Metabolism, Vol: 22, Pages: 320-331, ISSN: 1932-7420
The human gut microbiome is known to be associated with various human disorders, but a major challenge is to go beyond association studies and elucidate causalities. Mathematical modeling of the human gut microbiome at a genome scale is a useful tool to decipher microbe-microbe, diet-microbe and microbe-host interactions. Here, we describe the CASINO (Community And Systems-level INteractive Optimization) toolbox, a comprehensive computational platform for analysis of microbial communities through metabolic modeling. We first validated the toolbox by simulating and testing the performance of single bacteria and whole communities in vitro. Focusing on metabolic interactions between the diet, gut microbiota, and host metabolism, we demonstrated the predictive power of the toolbox in a diet-intervention study of 45 obese and overweight individuals and validated our predictions by fecal and blood metabolomics data. Thus, modeling could quantitatively describe altered fecal and serum amino acid levels in response to diet intervention.
Hoyles L, Murphy J, Neve H, et al., 2015, Klebsiella pneumoniae subsp. pneumoniae – bacteriophagecombination from the caecal effluent of a healthy woman, PeerJ, Vol: 3, ISSN: 2167-8359
A sample of caecal effluent was obtained from a female patient who had undergone a routine colonoscopic examination. Bacteria were isolated anaerobically from the sample, and screened against the remaining filtered caecal effluent in an attempt to isolate bacteriophages (phages). A lytic phage, named KLPN1, was isolated on a strain identified as Klebsiella pneumoniae subsp. pneumoniae (capsular type K2, rmpA+). This Siphoviridae phage presents a rosette-like tail tip and exhibits depolymerase activity, as demonstrated by the formation of plaque-surrounding haloes that increased in size over the course of incubation. When screened against a panel of clinical isolates of K. pneumoniae subsp. pneumoniae, phage KLPN1 was shown to infect and lyse capsular type K2 strains, though it did not exhibit depolymerase activity on such hosts. The genome of KLPN1 was determined to be 49,037 bp (50.53 %GC) in length, encompassing 73 predicted ORFs, of which 23 represented genes associated with structure, host recognition, packaging, DNA replication and cell lysis. On the basis of sequence analyses, phages KLPN1 (GenBank: KR262148) and 1513 (a member of the family Siphoviridae, GenBank: KP658157) were found to be two new members of the genus “Kp36likevirus.”
Hoyles L, Murphy J, Neve H, et al., 2015, Klebsiella pneumoniae subsp. pneumoniae–bacteriophage combinationfrom the caecal effluent of a healthy woman, PeerJ, Vol: 3, ISSN: 2167-8359
A sample of caecal effluent was obtained from a female patient who had undergonea routine colonoscopic examination. Bacteria were isolated anaerobically from thesample, and screened against the remaining filtered caecal effluent in an attemptto isolate bacteriophages (phages). A lytic phage, named KLPN1, was isolatedon a strain identified as Klebsiella pneumoniae subsp. pneumoniae (capsular typeK2, rmpA+). This Siphoviridae phage presents a rosette-like tail tip and exhibitsdepolymerase activity, as demonstrated by the formation of plaque-surroundinghaloes that increased in size over the course of incubation. When screened againsta panel of clinical isolates of K. pneumoniae subsp. pneumoniae, phage KLPN1was shown to infect and lyse capsular type K2 strains, though it did not exhibitdepolymerase activity on such hosts. The genome of KLPN1 was determined tobe 49,037 bp (50.53 %GC) in length, encompassing 73 predicted ORFs, of which23 represented genes associated with structure, host recognition, packaging,DNA replication and cell lysis. On the basis of sequence analyses, phages KLPN1(GenBank: KR262148) and 1513 (a member of the family Siphoviridae, GenBank:KP658157) were found to be two new members of the genus “Kp36likevirus.”
McCartney AL, Hoyles L, Jiménez-Pranteda ML, et al., 2015, Reduction of trimethylamine N-oxide to trimethylamine by the human gut microbiota: supporting evidence for ‘metabolic retroversion’, Exploring Human Host-Microbiome Interactions in Health and Disease
Dietary sources of methylamines such as choline, trimethylamine (TMA), trimethylamine N-oxide (TMAO), phosphatidylcholine (PC) and carnitine are present in a number of foodstuffs, including meat, fish, nuts and eggs. It is recognized that the gut microbiota is able to convert choline to TMA in a fermentation-like process. Similarly, PC and carnitine are converted to TMA by the gut microbiota. It has been suggested that TMAO is subject to ‘metabolic retroversion’ in the gut (i.e. it is reduced to TMA by the gut microbiota, with this TMA being oxidized to produce TMAO in the liver). Sixty-six strains of human faecal and caecal bacteria were screened on solid and liquid media for their ability to utilize trimethylamine N-oxide (TMAO), with metabolites in spent media profiled by Proton Nuclear Magnetic Resonance (1H NMR) spectroscopy. Enterobacteriaceae produced mostly TMA from TMAO, with caecal/small intestinal isolates of Escherichia coli producing more TMA than their faecal counterparts. Lactic acid bacteria (enterococci, streptococci, bifidobacteria) produced increased amounts of lactate when grown in the presence of TMAO, but did not produce large amounts of TMA from TMAO. The presence of TMAO in media increased the growth rate of Enterobacteriaceae; while it did not affect the growth rate of lactic acid bacteria, TMAO increased the biomass of these bacteria. The positive influence of TMAO on Enterobacteriaceae was confirmed in anaerobic, stirred, pH-controlled batch culture fermentation systems inoculated with human faeces, where this was the only bacterial population whose growth was significantly stimulated by the presence of TMAO in the medium. We hypothesize that dietary TMAO is used as an alternative electron acceptor by the gut microbiota in the small intestine/proximal colon, and contributes to microbial population dynamics upon its utilization and retroversion to TMA, prior to absorption and secondary conversion to TMAO by hepatic flavin-contai
Hoyles L, Abbott JC, Holmes E, et al., 2015, IMP: Imperial Metagenomics Pipeline for high-throughput sequence data, Exploring Human Host-Microbiome Interactions in Health and Disease
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