Responses of Ileal and Fecal Microbiota to Withdrawal of Pancreatic Enzyme Replacement Therapy in a Porcine Model of Exocrine Pancreatic Insufficiency.

Little is known regarding the interplay between microbiota and pancreas functions in humans as investigations are usually limited to distal sites, namely the analyses of fecal samples. The aim of this study was to investigate both ileal and fecal microbiota in response to pancreatic enzyme replacement therapy (PERT) in a porcine model of exocrine pancreatic insufficiency (EPI). PERT was stopped for ten days in ileo-cecal fistulated minipigs with experimentally induced EPI (n = 8) and ileal digesta as well as fecal samples were obtained before withdrawal, during withdrawal and after the reintroduction of PERT. Profound community changes occurred three days after enzyme omission and were maintained throughout the withdrawal phase. A reduction in α-diversity together with relative abundance changes in several taxa, in particular increases in Bifidobacteria (at both sites) and Lactobacilli (only feces) were observed. Overall, dysbiosis events from the ileum had accumulating effects in distal parts of the gastrointestinal tract with additional alterations occurring only in the colon. Changes were reversible after continuing PERT, and one week later, bacterial communities resembled those at baseline. Our study demonstrates the rapid and profound impacts of enzyme withdrawal in bacterial communities, contributing to our understanding of the interplay between pancreas function and microbiota.

A simplified LC-MS/MS method for the quantification of the cardiovascular disease biomarker trimethylamine-N-oxide and its precursors.

Trimethylamine-N-oxide (TMAO) has emerged as a potential biomarker for atherosclerosis and the development of cardiovascular diseases (CVDs). Although several clinical studies have shown striking associations of TMAO levels with atherosclerosis and CVDs, TMAO determinations are not clinical routine yet. The current methodology relies on isotope-labeled internal standards, which adds to pre-analytical complexity and costs for the quantification of TMAO and its precursors carnitine, betaine or choline. Here, we report a liquid chromatography-tandem mass spectrometry based method that is fast (throughput up to 240 samples/day), consumes low sample volumes (e.g., from a finger prick), and does not require isotope-labeled standards. We circumvented the analytical problem posed by the presence of endogenous TMAO and its precursors in human plasma by using an artificial plasma matrix for calibration. We cross-validated the results obtained using an artificial matrix with those using mouse plasma matrix and demonstrated that TMAO, carnitine, betaine and choline were accurately quantified in 'real-life' human plasma samples from healthy volunteers, obtained either from a finger prick or from venous puncture. Additionally, we assessed the stability of samples stored at -20 °C and room temperature. Whereas all metabolites were stable at -20 °C, increasing concentrations of choline were determined when stored at room temperature. Our method will facilitate the establishment of TMAO as a routine clinical biomarker in hematology in order to assess the risk for CVDs development, or to monitor disease progression and intervention effects.

Higher Trimethylamine-N-Oxide Plasma Levels with Increasing Age Are Mediated by Diet and Trimethylamine-Forming Bacteria.

The gut microbiota-dependent metabolite trimethylamine-N-oxide (TMAO) is linked to an increased risk for cardiovascular diseases. Trimethylamine (TMA), which is subsequently oxidized to TMAO in the liver, is formed by intestinal bacteria via distinct biochemical routes from dietary precursors that are enriched in animal product-based foods. To get a full picture of the entire process of the diet > gut microbiota > TMAO axis, we quantified potential TMA-forming gut bacteria and plasma metabolites using gene-targeted assays and targeted metabolomics on a subsample (n = 425) of a German population-based cohort study. We specifically compared persons reporting daily meat intake with those that rarely or never consume meat. While meat intake did not predict TMAO plasma levels in our study, two major bacterial TMA-forming pathways were linked to the metabolite's concentration. Furthermore, advancing age was strongly associated with TMAO. Construction of a structural equation model allowed us to disentangle the different routes that promote higher TMAO levels with increasing age, demonstrating, for the first time, a functional role of gut microbiota in the process, where specific food items augmented abundances of TMA-forming bacteria that were associated with higher TMAO plasma concentrations. Analyses stratified by age showed an association between carotid intima-media thickness and TMAO only in individuals >65 of age, indicating that this group is particularly affected by the metabolite. IMPORTANCE Many cohort studies have investigated the link between diet and plasma TMAO levels, reporting incongruent results, while gut microbiota were only recently included into analyses. In these studies, taxonomic data were recorded that are not a good proxy for TMA formation, as specific members of various taxa exhibit genes catalyzing this reaction, demanding function-based technologies for accurate quantification of TMA-synthesizing bacteria. Using this approach, we demonstrated that abundances of the main components leading to TMAO formation, i.e., TMA precursors and TMA-forming bacteria, are uncoupled and not governed by the same (dietary) factors. Results emphasize that all levels leading to TMA(O) formation should be considered for accurate risk assessment, rejecting the simple view that diets rich in TMA precursors directly lead to increased plasma levels of this hazardous compound. The results can assist in developing strategies to reduce TMAO levels, specifically in the elderly, who are prone to TMAO-associated diseases.

A simplified LC-MS/MS method for the quantification of the cardiovascular disease biomarker trimethylamine-N-oxide and its precursors / 药物分析学报

Trimethylamine-N-oxide (TMAO) has emerged as a potential biomarker for atherosclerosis and the development of cardiovascular diseases (CVDs).Although several clinical studies have shown striking associations of TMAO levels with atherosclerosis and CVDs,TMAO determinations are not clinical routine yet.The current methodology relies on isotope-labeled internal standards,which adds to pre-analytical complexity and costs for the quantification of TMAO and its precursors carnitine,betaine or choline.Here,we report a liquid chromatography-tandem mass spectrometry based method that is fast(throughput up to 240 samples/day),consumes low sample volumes (e.g.,from a finger prick),and does not require isotope-labeled standards.We circumvented the analytical problem posed by the presence of endogenous TMAO and its precursors in human plasma by using an artificial plasma matrix for cali-bration.We cross-validated the results obtained using an artificial matrix with those using mouse plasma matrix and demonstrated that TMAO,carnitine,betaine and choline were accurately quantified in 'real-life'human plasma samples from healthy volunteers,obtained either from a finger prick or from venous puncture.Additionally,we assessed the stability of samples stored at-20 ℃ and room temperature.Whereas all metabolites were stable at-20 ℃,increasing concentrations of choline were determined when stored at room temperature.Our method will facilitate the establishment of TMAO as a routine clinical biomarker in hematology in order to assess the risk for CVDs development,or to monitor disease progression and intervention effects.

Diversity of Bacteria Exhibiting Bile Acid-inducible 7α-dehydroxylation Genes in the Human Gut.

The secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), formed by gut microbiota from primary bile acids via a multi-step 7α-dehydroxylation reaction, have wide-ranging effects on host metabolism and play an important role in health and disease. A few 7α-dehydroxylating strains have been isolated, where bile acid-inducible (bai) genes were organized in a gene cluster and encoded major enzymes involved. However, only little is known on diversity and abundance of intestinal bacteria catalysing DCA/LCA formation in the human gut in situ. In this study, we took the opportunity to screen metagenome-assembled genomes (MAGs) from sequence data of stool samples provided by two recent studies along with newly available gut-derived isolates for the presence of the bai gene cluster. We revealed in total 765 and 620 MAGs encoding the potential to form DCA/LCA that grouped into 21 and 26 metagenomic species, respectively. The majority of MAGs (92.4 and 90.3%) were associated with a Ruminococcaceae clade that still lacks an isolate, whereas less MAGs belonged to Lachnospiraceae along with eight new isolates (n total = 11) that contained the bai genes. Only a few MAGs were linked to Peptostreptococcaceae. Signatures for horizontal transfer of bai genes were observed. This study gives a comprehensive overview of the diversity of bai-exhibiting bacteria in the human gut highlighting the application of metagenomics to unravel potential functions hidden from current isolates. Eventually, isolates of the identified main MAG clade are required in order to prove their capability of 7α-dehydroxylating primary bile acids.

Potential TMA-Producing Bacteria Are Ubiquitously Found in Mammalia.

Human gut bacteria metabolize dietary components such as choline and carnitine to trimethylamine (TMA) that is subsequently oxidized to trimethylamine-N-oxide (TMAO) by hepatic enzymes. Increased plasma levels of TMAO are associated with the development of cardiovascular and renal disease. In this study, we applied gene-targeted assays in order to quantify (qPCR) and characterize (MiSeq) bacterial genes encoding enzymes responsible for TMA production, namely choline-TMA lyase (CutC), carnitine oxygenase (CntA) and betaine reductase (GrdH) in 89 fecal samples derived from various mammals spanning three dietary groups (carnivores, omnivores and herbivores) and four host orders (Carnivora, Primates, Artiodactyla and Perissodactyla). All samples contained potential TMA-producing bacteria, however, at low abundances (<1.2% of total community). The cutC gene was more abundant in omnivores and carnivores compared with herbivores. CntA was almost absent from herbivores and grdH showed lowest average abundance of all three genes. Bacteria harboring cutC and grdH displayed high diversities where sequence types affiliated with various taxa within Firmicutes dominated, whereas cntA comprised sequences primarily linked to Escherichia. Composition of TMA-forming communities was strongly influenced by diet and host taxonomy and despite their high correlation, both factors contributed uniquely to community structure. Furthermore, Random Forest (RF) models could differentiate between groups at high accuracies. This study gives a comprehensive overview of potential TMA-producing bacteria in the mammalian gut demonstrating that both diet and host taxonomy govern their abundance and composition. It highlights the role of functional redundancy sustaining potential TMA formation in distinct gut environments.

Pathogenic functions of host microbiota.

BACKGROUND: It is becoming evident that certain features of human microbiota, encoded by distinct autochthonous taxa, promote disease. As a result, borders between the so-called opportunistic pathogens, pathobionts, and commensals are increasingly blurred, and specific targets for manipulating microbiota to improve host health are becoming elusive. RESULTS: In this study, we focus on the functions of host bacterial communities that have the potential to cause disease, proposing the term "pathogenic function (pathofunction)". The concept is presented via three distinct examples, namely, the formation of (i) trimethylamine, (ii) secondary bile acids, and (iii) hydrogen sulfide, which represent metabolites of the gut microbiota linked to the development of non-communicable diseases. Using publicly available metagenomic and metatranscriptomic data (n = 2975), we quantified those pathofunctions in health and disease and exposed the key players. Pathofunctions were ubiquitously present with increased abundances in patient groups. Overall, the three pathofunctions were detected at low mean concentrations (< 1% of total bacteria carried respective genes) and encompassed various taxa, including uncultured members. CONCLUSIONS: We outline how this function-centric approach, where all members of a community exhibiting a particular pathofunction are redundant, can contribute to risk assessment and the development of precision treatment directing gut microbiota to increase host health.

Uncovering the trimethylamine-producing bacteria of the human gut microbiota.

BACKGROUND: Trimethylamine (TMA), produced by the gut microbiota from dietary quaternary amines (mainly choline and carnitine), is associated with atherosclerosis and severe cardiovascular disease. Currently, little information on the composition of TMA producers in the gut is available due to their low abundance and the requirement of specific functional-based detection methods as many taxa show disparate abilities to produce that compound. RESULTS: In order to examine the TMA-forming potential of microbial communities, we established databases for the key genes of the main TMA-synthesis pathways, encoding choline TMA-lyase (cutC) and carnitine oxygenase (cntA), using a multi-level screening approach on 67,134 genomes revealing 1107 and 6738 candidates to exhibit cutC and cntA, respectively. Gene-targeted assays enumerating the TMA-producing community by quantitative PCR and characterizing its composition via Illumina sequencing were developed and applied on human fecal samples (n = 50) where all samples contained potential TMA producers (cutC was detected in all individuals, whereas only 26% harbored cntA) constituting, however, only a minor part of the total community (below 1% in most samples). Obtained cutC amplicons were associated with various taxa, in particular with Clostridium XIVa strains and Eubacterium sp. strain AB3007, though a bulk of sequences displayed low nucleotide identities to references (average 86% ± 7%) indicating that key human TMA producers are yet to be isolated. Co-occurrence analysis revealed specific groups governing the community structure of cutC-exhibiting taxa across samples. CntA amplicons displayed high identities (~99%) to Gammaproteobacteria-derived references, primarily from Escherichia coli. Metagenomic analysis of samples provided by the Human Microbiome Project (n = 154) confirmed the abundance patterns as well as overall taxonomic compositions obtained with our assays, though at much lower resolution, whereas 16S ribosomal RNA gene sequence analysis could not adequately uncover the TMA-producing potential. CONCLUSIONS: In this study, we developed a diagnostic framework that enabled the quantification and comprehensive characterization of the TMA-producing potential in human fecal samples. The key players were identified, and together with predictions on their environmental niches using functional genomics on most closely related reference strains, we provide crucial information for the development of specific treatment strategies to restrain TMA producers and limit their proliferation.