Fecal microbiota transplantation (FMT) is effective for treating recurrent Clostridioides difficile infection (CDI), but there are concerns about its long-term safety. Understanding the mechanisms of ...the effects of FMT could help us design safer, targeted therapies. We aimed to identify microbial metabolites that are important for C difficile growth.
We used a CDI chemostat model as a tool to study the effects of FMT in vitro. The following analyses were performed: C difficile plate counts, 16S rRNA gene sequencing, proton nuclear magnetic resonance spectroscopy, and ultra-performance liquid chromatography and mass spectrometry bile acid profiling. FMT mixtures were prepared using fresh fecal samples provided by donors enrolled in an FMT program in the United Kingdom. Results from chemostat experiments were validated using human stool samples, C difficile batch cultures, and C57BL/6 mice with CDI. Human stool samples were collected from 16 patients with recurrent CDI and healthy donors (n = 5) participating in an FMT trial in Canada.
In the CDI chemostat model, clindamycin decreased valerate and deoxycholic acid concentrations and increased C difficile total viable counts and valerate precursors, taurocholic acid, and succinate concentrations. After we stopped adding clindamycin, levels of bile acids and succinate recovered, whereas levels of valerate and valerate precursors did not. In the CDI chemostat model, FMT increased valerate concentrations and decreased C difficile total viable counts (94% decrease), spore counts (86% decrease), and valerate precursor concentrations; concentrations of bile acids were unchanged. In stool samples from patients with CDI, valerate was depleted before FMT but restored after FMT. Clostridioides difficile batch cultures confirmed that valerate decreased vegetative growth, and that taurocholic acid was required for germination but had no effect on vegetative growth. Clostridioides difficile total viable counts were decreased by 95% in mice with CDI given glycerol trivalerate compared with phosphate buffered saline.
We identified valerate as a metabolite that is depleted with clindamycin and only recovered with FMT. Valerate is a target for a rationally designed recurrent CDI therapy.
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Bile acids are important end products of cholesterol metabolism. While they have been identified as key factors in lipid emulsification and absorption due to their detergent properties, bile acids ...have also been shown to act as signaling molecules and intermediates between the host and the gut microbiota. To further the investigation of bile acid functions in humans, an advanced platform for high throughput analysis is essential. Herein, we describe the development and application of a 15 min UPLC procedure for the separation of bile acid species from human biofluid samples requiring minimal sample preparation. High resolution time-of-flight mass spectrometry was applied for profiling applications, elucidating rich bile acid profiles in both normal and disease state plasma. In parallel, a second mode of detection was developed utilizing tandem mass spectrometry for sensitive and quantitative targeted analysis of 145 bile acid (BA) species including primary, secondary, and tertiary bile acids. The latter system was validated by testing the linearity (lower limit of quantification, LLOQ, 0.25–10 nM and upper limit of quantification, ULOQ, 2.5–5 μM), precision (≈6.5%), and accuracy (81.2–118.9%) on inter- and intraday analysis achieving good recovery of bile acids (serum/plasma 88% and urine 93%). The ultra performance liquid chromatography–mass spectrometry (UPLC-MS)/MS targeted method was successfully applied to plasma, serum, and urine samples in order to compare the bile acid pool compositional difference between preprandial and postprandial states, demonstrating the utility of such analysis on human biofluids.
Microbiota and bile acids in the gastrointestinal tract profoundly alter systemic metabolic processes. In obese subjects, gradual weight loss ameliorates adipose tissue inflammation and related ...systemic changes. We assessed how rapid weight loss due to a very low calorie diet (VLCD) affects the fecal microbiome and fecal bile acid composition, and their interactions with the plasma metabolome and subcutaneous adipose tissue inflammation in obesity.
We performed a prospective cohort study of VLCD-induced weight loss of 10% in ten grades 2-3 obese postmenopausal women in a metabolic unit. Baseline and post weight loss evaluation included fasting plasma analyzed by mass spectrometry, adipose tissue transcription by RNA sequencing, stool 16S rRNA sequencing for fecal microbiota, fecal bile acids by mass spectrometry, and urinary metabolic phenotyping by
H-NMR spectroscopy. Outcome measures included mixed model correlations between changes in fecal microbiota and bile acid composition with changes in plasma metabolite and adipose tissue gene expression pathways.
Alterations in the urinary metabolic phenotype following VLCD-induced weight loss were consistent with starvation ketosis, protein sparing, and disruptions to the functional status of the gut microbiota. We show that the core microbiome was preserved during VLCD-induced weight loss, but with changes in several groups of bacterial taxa with functional implications. UniFrac analysis showed overall parallel shifts in community structure, corresponding to reduced abundance of the genus Roseburia and increased Christensenellaceae;g__ (unknown genus). Imputed microbial functions showed changes in fat and carbohydrate metabolism. A significant fall in fecal total bile acid concentration and reduced deconjugation and 7-α-dihydroxylation were accompanied by significant changes in several bacterial taxa. Individual bile acids in feces correlated with amino acid, purine, and lipid metabolic pathways in plasma. Furthermore, several fecal bile acids and bacterial species correlated with altered gene expression pathways in adipose tissue.
VLCD dietary intervention in obese women changed the composition of several fecal microbial populations while preserving the core fecal microbiome. Changes in individual microbial taxa and their functions correlated with variations in the plasma metabolome, fecal bile acid composition, and adipose tissue transcriptome. Trial Registration ClinicalTrials.gov NCT01699906, 4-Oct-2012, Retrospectively registered. URL- https://clinicaltrials.gov/ct2/show/NCT01699906.
Biomarker research across the health-to-disease continuum is being increasingly applied. We applied blood-based metabolomics in order to identify patient clusters with a first demyelinating episode, ...and explored the prognostic potential of the method by thoroughly characterizing each cluster in terms of clinical, laboratory and MRI markers of established prognostic potential for Multiple Sclerosis (MS). Recruitment consisted of 11 patients with Clinically Isolated Syndrome (CIS), 37 patients with a first demyelinating episode in the context of Relapsing-Remitting MS (RRMS) and 11 control participants. Blood-based metabolomics and hierarchical clustering analysis (HCL) were applied. Constructed OPLS-DA models illustrated a discrimination between patients with CIS and the controls (p = 0.0014), as well as between patients with RRMS and the controls (p = 1 × 10−5). Hierarchical clustering analysis (HCL) for patients with RRMS identified three clusters. RRMS-patients-cluster-3 exhibited higher mean cell numbers in the Cerebro-spinal Fluid (CSF) compared to patients with CIS (18.17 ± 6.3 vs. 1.09 ± 0.41, p = 0.004). Mean glucose CSF/serum ratio and infratentorial lesion burden significantly differed across CIS- and HCL-derived RRMS-patient clusters (F = 14.95, p < 0.001 and F = 6.087, p = 0.002, respectively), mainly due to increased mean values for patients with RRMS-cluster-3. HCL discriminated a cluster of patients with a first demyelinating episode in the context of RRMS with increased disability, laboratory findings linked with increased pathology burden and MRI markers of poor prognosis.
Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease with ursodeoxycholic acid (UDCA) as first-line treatment. Poor response to UDCA is associated with a higher risk of ...progressing to cirrhosis, but the underlying mechanisms are unclear. UDCA modulates the composition of primary and bacterial-derived bile acids (BAs). We characterized the phenotypic response to UDCA based on BA and bacterial profiles of PBC patients treated with UDCA. Patients from the UK-PBC cohort (n = 419) treated with UDCA for a minimum of 12-months were assessed using the Barcelona dynamic response criteria. BAs from serum, urine, and feces were analyzed using Ultra-High-Performance Liquid Chromatography-Mass Spectrometry and fecal bacterial composition measured using 16S rRNA gene sequencing. We identified 191 non-responders, 212 responders, and a subgroup of responders with persistently elevated liver biomarkers (n = 16). Responders had higher fecal secondary and tertiary BAs than non-responders and lower urinary bile acid abundances, with the exception of 12-dehydrocholic acid, which was higher in responders. The sub-group of responders with poor liver function showed lower alpha-diversity evenness, lower abundance of fecal secondary and tertiary BAs than the other groups and lower levels of phyla with BA-deconjugation capacity (Actinobacteriota/Actinomycetota, Desulfobacterota, Verrucomicrobiota) compared to responders. UDCA dynamic response was associated with an increased capacity to generate oxo-/epimerized secondary BAs. 12-dehydrocholic acid is a potential biomarker of treatment response. Lower alpha-diversity and lower abundance of bacteria with BA deconjugation capacity might be associated with an incomplete response to treatment in some patients.
Metabonomics is an established strategy in the exploration of the effects of various stimuli on the metabolic fingerprint of biofluids. Here, we present an application of 1H NMR-based metabonomics on ...the field of exercise biochemistry. Fourteen men were assigned to either of two training programs, which lasted 8 weeks and involved sets of 80-m maximal runs separated by either 10 s or 1 min of rest. Analysis of pre- and postexercise serum samples, both at the beginning and end of training, by 1H NMR spectroscopy and subsequent multivariate statistical techniques revealed alterations in the levels of 18 metabolites. Validated O-PLS models could classify the samples in regard to exercise, the separation being mainly due to lactate, pyruvate, alanine, leucine, valine, isoleucine, arginine/lysine, glycoprotein acetyls, and an unidentified metabolite resonating at 8.17 ppm. Samples were also classified safely with respect to training, the separation being mainly due to lactate, pyruvate, methylguanidine, citrate, glucose, valine, taurine, trimethylamine N-oxide, choline-containing compounds, histidines, acetoacetate/acetone, glycoprotein acetyls, and lipids. Samples could not be classified according to the duration of the rest interval between sprints. Our findings underline the power of metabonomics to offer new insights into the short- and long-term impact of exercise on metabolism.
Acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy. Despite high cure rates, several questions remain regarding predisposition, response to treatment, and prognosis of the ...disease. The role of intermediary metabolism in the individualized mechanistic pathways of the disease is unclear. We have hypothesized that children with any (sub)type of ALL have a distinct metabolomic fingerprint at diagnosis when compared: (i) to a control group; (ii) to children with a different (sub)type of ALL; (iii) to the end of the induction treatment.
In this prospective case-control study (NCT03035344), plasma and urinary metabolites were analyzed in 34 children with ALL before the beginning (D0) and at the end of the induction treatment (D33). Their metabolic fingerprint was defined by targeted analysis of 106 metabolites and compared to that of an equal number of matched controls. Multivariate and univariate statistical analyses were performed using SIMCAP and scripts under the R programming language.
Metabolomic analysis showed distinct changes in patients with ALL compared to controls on both D0 and D33. The metabolomic fingerprint within the patient group differed significantly between common B-ALL and pre-B ALL and between D0 and D33, reflecting the effect of treatment. We have further identified the major components of this metabolic dysregulation, indicating shifts in fatty acid synthesis, transfer and oxidation, in amino acid and glycerophospholipid metabolism, and in the glutaminolysis/TCA cycle.
The disease type and time point-specific metabolic alterations observed in pediatric ALL are of particular interest as they may offer potential for the discovery of new prognostic biomarkers and therapeutic targets.
The national infrastructure FoodOmicsGR_RI coordinates research efforts from eight Greek Universities and Research Centers in a network aiming to support research and development (R&D) in the ...agri-food sector. The goals of FoodOmicsGR_RI are the comprehensive in-depth characterization of foods using cutting-edge omics technologies and the support of dietary/nutrition studies. The network combines strong omics expertise with expert field/application scientists (food/nutrition sciences, plant protection/plant growth, animal husbandry, apiculture and 10 other fields). Human resources involve more than 60 staff scientists and more than 30 recruits. State-of-the-art technologies and instrumentation is available for the comprehensive mapping of the food composition and available genetic resources, the assessment of the distinct value of foods, and the effect of nutritional intervention on the metabolic profile of biological samples of consumers and animal models. The consortium has the know-how and expertise that covers the breadth of the Greek agri-food sector. Metabolomics teams have developed and implemented a variety of methods for profiling and quantitative analysis. The implementation plan includes the following research axes: development of a detailed database of Greek food constituents; exploitation of "omics" technologies to assess domestic agricultural biodiversity aiding authenticity-traceability control/certification of geographical/genetic origin; highlighting unique characteristics of Greek products with an emphasis on quality, sustainability and food safety; assessment of diet's effect on health and well-being; creating added value from agri-food waste. FoodOmicsGR_RI develops new tools to evaluate the nutritional value of Greek foods, study the role of traditional foods and Greek functional foods in the prevention of chronic diseases and support health claims of Greek traditional products. FoodOmicsGR_RI provides access to state-of-the-art facilities, unique, well-characterised sample sets, obtained from precision/experimental farming/breeding (milk, honey, meat, olive oil and so forth) along with more than 20 complementary scientific disciplines. FoodOmicsGR_RI is open for collaboration with national and international stakeholders.
Aspartame is a commonly used intense artificial sweetener, being approximately 200 times sweeter than sucrose. There have been concerns over aspartame since approval in the 1980s including a large ...anecdotal database reporting severe symptoms. The objective of this study was to compare the acute symptom effects of aspartame to a control preparation.
This was a double-blind randomized cross over study conducted in a clinical research unit in United Kingdom. Forty-eight individual who has self reported sensitivity to aspartame were compared to 48 age and gender matched aspartame non-sensitive individuals. They were given aspartame (100mg)-containing or control snack bars randomly at least 7 days apart. The main outcome measures were acute effects of aspartame measured using repeated ratings of 14 symptoms, biochemistry and metabonomics.
Aspartame sensitive and non-sensitive participants differed psychologically at baseline in handling feelings and perceived stress. Sensitive participants had higher triglycerides (2.05 ± 1.44 vs. 1.26 ± 0.84mmol/L; p value 0.008) and lower HDL-C (1.16 ± 0.34 vs. 1.35 ± 0.54 mmol/L; p value 0.04), reflected in 1H NMR serum analysis that showed differences in the baseline lipid content between the two groups. Urine metabonomic studies showed no significant differences. None of the rated symptoms differed between aspartame and control bars, or between sensitive and control participants. However, aspartame sensitive participants rated more symptoms particularly in the first test session, whether this was placebo or control. Aspartame and control bars affected GLP-1, GIP, tyrosine and phenylalanine levels equally in both aspartame sensitive and non-sensitive subjects.
Using a comprehensive battery of psychological tests, biochemistry and state of the art metabonomics there was no evidence of any acute adverse responses to aspartame. This independent study gives reassurance to both regulatory bodies and the public that acute ingestion of aspartame does not have any detectable psychological or metabolic effects in humans.
ISRCTN Registry ISRCTN39650237.
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