Uncovering the trimethylamine-producing bacteria of the human gut microbiota

The quote only partly states this claim.

Multi-level screening of 67,134 genomes identified 6738 candidate genomes exhibiting cntA.

“Putative cntA genes were identified in 6738 genomes (491 dereplicated protein sequences)”

The quote only partly states this claim.

Multi-level screening of 67,134 genomes identified 1107 candidate genomes exhibiting cutC.

“Putative cutC genes were detected in a total of 1107 genomes (454 dereplicated protein sequences)”

May combine two claims.

Putative cutC genes belong to Proteobacteria, particularly Gamma- and Deltaproteobacteria, and Firmicutes, mainly Clostridia and some Bacilli, as well as a few Actinobacteria.

“Putative cutC genes were detected in a total of 1107 genomes (454 dereplicated protein sequences) that belong to Proteobacteria (in particular Gamma- and Deltaproteobacteria) and Firmicutes (mainly Clostridia and some Bacilli), as well as a few Actinobacteria”

May combine two claims.

Strains are predicted to contain 101 ± 79 glycoside hydrolases, 41 ± 13 glycosyltransferases, 25 ± 17 carbohydrate-binding modules, 26 ± 13 carbohydrate esterases, and 4 ± 4 polysaccharide lyases.

“Overall, strains are predicted to contain 101 ± 79 glycoside hydrolases (GHs), 41 ± 13 glycosyltransferases (GTs), 25 ± 17 carbohydrate-binding modules (CBMs), 26 ± 13 carbohydrate esterases (CEs), and 4 ± 4 polysaccharide lyases (PLs).”

May combine two claims.

At higher cutoffs of 99 and 98% identity, several thousand cntA clusters were detected and richness drastically declined with decreasing identity cutoffs; only 55 and 14 total clusters were detected at 95 and 90% cutoff values, respectively.

“At higher cutoffs (99 and 98% identity), several thousand clusters were detected and richness drastically declined with decreasing identity cutoffs. For example, only 55 and 14 total clusters were detected at 95 and 90% cutoff values, respectively.”

• AssumptionRests on an assumption: The TMA-producing communities in fecal samples are representative of the colonic TMA-producing communities in humans.

May combine two claims.

All cntA sequences obtained were similar to Gammaproteobacteria, particularly Escherichia/Shigella; closest match analysis revealed 98.1% being closely related to sequences from E. coli.

“In contrast, all cntA sequences obtained were similar to those previously observed in Gammaproteobacteria, in particular from Escherichia/Shigella (Fig. 2c); closest match analysis revealed 98.1% being closely related to sequences from E. coli (data not shown).”

• AssumptionRests on an assumption: The TMA-producing communities in fecal samples are representative of the colonic TMA-producing communities in humans.

May combine two claims.

Screening of HMP contigs for cutC/D yielded similar results: only two samples contained contigs with decent sequence stretches indicating the presence of cutC/D, and those were nearly identical, >99% identity, to Clostridium sp. AT5 sequences.

“Screening of contigs provided by the Human Microbiome Project (HMP) for cutC/D sequences yielded similar results. Only two samples contained contigs with decent sequence stretches indicating the presence of cutC/D, and those were nearly identical (>99% identity) to Clostridium sp. AT5 sequences (data not shown).”

May combine two claims.

On average, 76 ± 18 clusters per sample at 90% nucleotide identity were observed, with the majority similar to Firmicutes, particularly Clostridium XIVa strains and Eubacterium sp. strain AB3007.

“On average, 76 ± 18 clusters per sample (90% nucleotide identity) were observed, where the majority was similar to genes previously observed in Firmicutes, in particular to those of Clostridium XIVa strains and Eubacterium sp. strain AB3007 (Fig. 2b).”

• AssumptionRests on an assumption: The TMA-producing communities in fecal samples are representative of the colonic TMA-producing communities in humans.

May combine two claims.

CntA was quantified in 13 samples, with abundances ranging from 0.03 to 2.17% of bacteria possessing cntA, and only three samples had an abundance ≥0.5%.

“CntA was quantified in 13 samples displaying large abundance differences ranging from 0.03 to 2.17% of bacteria possessing cntA, with only three samples displaying an abundance ≥0.5%.”

• AssumptionRests on an assumption: The TMA-producing communities in fecal samples are representative of the colonic TMA-producing communities in humans.

May combine two claims.

All revealed cutC candidates contain a single copy of cutC, except for seven genomes that display double entries.

“All revealed candidates contain a single copy of cutC, except for seven genomes that display double entries.”

May combine two claims.

A major part of cutC sequences, especially clusters a-k, were only distantly related to reference sequences and formed separate clades; overall nucleotide similarity of cutC amplicons to references was low, 86% ± 7%.

“However, a major part of sequences, especially those of clusters a–k (Fig. 2b), were only distantly related to reference sequences and formed separate clades on the tree. Consequently, overall nucleotide similarity of cutC amplicons to reference sequences was low (86% ± 7%).”

• AssumptionRests on an assumption: The TMA-producing communities in fecal samples are representative of the colonic TMA-producing communities in humans.

May combine two claims.

Putative cntA genes were identified in 6738 genomes, with the majority in Proteobacteria, especially Gammaproteobacteria mostly derived from Escherichia and Acinetobacter, some in Betaproteobacteria and Epsilonproteobacteria, and two sequences from Enterococcus sp.

“Putative cntA genes were identified in 6738 genomes (491 dereplicated protein sequences) where the majority was observed in Proteobacteria, especially Gammaproteobacteria (mostly derived from Escherichia and Acinetobacter), and some entries belonging to Betaproteobacteria and Epsilonproteobacteria (Fig. 1b, Additional file 1B, Additional file 2B). Two sequences were obtained from Enterococcus sp.”

May combine two claims.

Global feature analysis based on FIGfams largely reflected 16S rRNA gene phylogeny, where cutC-containing strains span the entire dendrogram and are dominated by three separate groups.

“Results of global feature analysis based on FIGfams largely reflected 16S rRNA gene phylogeny where cutC-containing strains span the entire dendrogram and are dominated by three separate groups (Additional file 10A).”

May combine two claims. The quote only partly states this claim.

Of those cntA clusters, a few, nine and one at 95 and 90% cutoffs respectively, were observed in >90% of all samples and comprised the majority of obtained sequences.

“Of those, a few clusters (nine and one, respectively) were observed in >90% of all samples comprising the majority of obtained sequences (Additional file 6B).”

• AssumptionRests on an assumption: The TMA-producing communities in fecal samples are representative of the colonic TMA-producing communities in humans.

May combine two claims.

Overall identity of cntA amplicons to reference sequences was high, 99.5% ± 0.5%, and 3 ± 2 clusters per sample at 90% nucleotide identity were detected.

“Overall, the identity of amplicons to reference sequences was high (99.5% ± 0.5%), and with 3 ± 2 clusters (90% nucleotide identity) detected per sample, the gene diversity was much lower compared to that observed for cutC.”

• AssumptionRests on an assumption: The TMA-producing communities in fecal samples are representative of the colonic TMA-producing communities in humans.

May combine two claims.

Reanalysis of metagenomes from Karlsson and colleagues indicated increased abundances of bacteria exhibiting the potential to produce TMA by about a factor of 2 in symptomatic atherosclerosis patients (n = 12) compared to healthy controls (n = 13), with median values of 0.39 and 0.11% in patients versus 0.22 and 0% in controls for cutC and cntA, respectively.

“Our results indicated increased abundances of bacteria exhibiting the potential to produce TMA (by about a factor of 2) in the patient group where the abundances of key genes of both pathways were elevated (median values of 0.39 and 0.11% (patient group) versus 0.22 and 0% (controls) for cutC and cntA, respectively, as a percentage of bacteria calculated to exhibit the genes) (Additional file 8).”

• cites [12] Karlsson FH, Fåk F, Nookaew I, Tremaroli V, Fagerberg B, Petranovic D, et al. Symptomatic atherosclerosis is associated with an altered gut metagenome. Nat Commun. 2012;3:1245. sourceNot checked yet

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May combine two claims.

The established databases present a comprehensive update of previous cutC and cntA gene sequence collections, adding a multitude of new gene sequences.

“The established databases present a comprehensive update of previous cutC [9] and cntA [7] gene sequence collections adding a multitude of new gene sequences.”

• cites [9] Campo AM, Bodea S, Hamer H. Characterization and detection of a widely distributed gene cluster that predicts anaerobic choline utilization by human gut bacteria. MBio. 2015;6:e00042–15.Not checked yet

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• cites [7] Zhu Y, Jameson E, Crosatti M, Schäfer H, Rajakumar K, Bugg TDH, et al. Carnitine metabolism to trimethylamine by an unusual Rieske-type oxygenase from human microbiota. Proc Natl Acad Sci U S A. 2014;111:4268–73. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Samples enriched in other clusters linked to clade I form another group, whereas a few distant samples displayed high abundances of clusters tightly related with Clostridium XIVa strains.

“Samples enriched in other clusters linked to clade I form another group on the plot, whereas a few distant samples displayed high abundances of clusters tightly related with Clostridium XIVa strains.”

• AssumptionRests on an assumption: The TMA-producing communities in fecal samples are representative of the colonic TMA-producing communities in humans.

May combine two claims.

Some sequences in the databases are not expected to be amplified: the cntA forward primer does not match a few Betaproteobacteria, and a few cutC sequences observed in Streptococcus show ≥2 mismatches with the reverse primer.

“Nevertheless, some sequences comprised in our databases are not expected to be amplified, as the cntA forward primer does not match a few Betaproteobacteria, and in the case of cutC, a few sequences observed in Streptococcus show ≥2 mismatches with the reverse primer.”

May combine two claims.

Most sequence reads displayed an identity of only 92.3% ± 7.1% for cutC and 96% ± 6.4% for cutD.

“However, most sequence reads displayed an identity of only 92.3% ± 7.1% and 96% ± 6.4% for cutC and cutD, respectively.”

May combine two claims.

C. phytofermentans, Clostridium sp. AT5, and Dorea sp. 5-2 cluster apart displaying ~55% functional similarity with members of the three main groups, while Eubacterium sp. strain AB3007, together with three additional strains, forms the outgroup sharing less than half of FIGfams with other strains.

“C. phytofermentans, Clostridium sp. AT5, and Dorea sp. 5-2 are clustering apart displaying ~55% functional similarity with members of the three main groups, while Eubacterium sp. strain AB3007, together with three additional strains, forms the outgroup sharing less than half of FIGfams with other strains.”

May combine two claims.

For cutC/D, the majority of contigs from those samples were gene fragments, and only four assembled sequences, two for each gene, with length >50% of median reference gene lengths were obtained from two distinct samples.

“For cutC/D, the majority of contigs from those samples were gene fragments and only four assembled sequences (two for each gene) with a length >50% of median reference gene lengths were obtained (from two distinct samples).”

May combine two claims.

Gene-targeted assembly yielded sequences for cutC/D and cntA/B in only six and three samples, respectively, though at similar abundances as through BLAST search.

“Application of a gene-targeted assembly method yielded sequences for cutC/D and cntA/B in only six and three samples, respectively, though at similar abundances as through our BLAST search (Fig. 4).”

May combine two claims.

Even at the low identity cutoff of 90%, the vast amount of cutC clusters was only detected in a minority of samples, with many being sample specific.

“Even at the low identity cutoffs of 90%, the vast amount of clusters was only detected in a minority of samples with many being sample specific.”

• AssumptionRests on an assumption: The TMA-producing communities in fecal samples are representative of the colonic TMA-producing communities in humans.

May combine two claims.

At a 95% cutoff, 1310 distinct cutC sequence types could be differentiated; only 41 were detected in the majority of samples, but they comprised ≥67% of obtained sequences.

“At a cutoff level of 95% 1310 distinct sequence types could be differentiated. Only a few of them (41) were detected in the majority of samples; however, they comprised the bulk of obtained sequences (≥67%).”

• AssumptionRests on an assumption: The TMA-producing communities in fecal samples are representative of the colonic TMA-producing communities in humans.

May combine two claims.

CutC was enumerated in all 50 human fecal samples, with an average abundance of 0.16% ± 0.15% of the total community calculated to contain the gene.

“CutC was enumerated in all samples, and an average abundance of 0.16% ± 0.15% bacteria of the total community was calculated to contain the gene.”

• AssumptionRests on an assumption: The TMA-producing communities in fecal samples are representative of the colonic TMA-producing communities in humans.

May combine two claims.

Members of clade I, defined by clusters a-d, are separated where main clusters a and b are not connected to each other nor to other phylogenetically related clusters, whereas members from clades II (g) and III (j) are located in the same module linked to other clusters from those clades.

“Members of clade I (defined by clusters a–d from Fig. 2b) are separated where the main clusters a and b are not connected to each other nor to other phylogenetically related clusters, whereas members from clades II (g) and III (j) are located in the same module linked to other clusters from those clades.”

• AssumptionRests on an assumption: The TMA-producing communities in fecal samples are representative of the colonic TMA-producing communities in humans.

May combine two claims.

Reads indicating the presence of cntA/B were nearly identical to references, 99.6% ± 1.1% and 99.8% ± 0.8% for cntA and cntB, respectively, and predominantly related to Gammaproteobacteria, especially Escherichia/Shigella and Klebsiella.

“In contrast, reads indicating the presence of cntA/B were nearly identical to references (99.6% ± 1.1% and 99.8% ± 0.8% for cntA and cntB, respectively) and predominantly related to Gammaproteobacteria, in particular to Escherichia/Shigella and Klebsiella.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

On average, 98.2% (cutC) and 92.5% (cntA) of all merged sequences passed the initial quality steps.

“On average, 98.2% (cutC) and 92.5% (cntA) of all merged sequences passed these initial steps”

Probably not a substantial claim: may not pass S3 (load-bearing).

Colonization of the mouse gut with a specific consortium of CutC-encoding human isolates results in TMA synthesis and subsequent TMAO accumulation in serum.

“Another recent study [10] demonstrated that colonization of the mouse gut with a specific consortium of CutC-encoding human isolates results in TMA synthesis and subsequent TMAO accumulation in the serum of animals”

• cites [10] Romano KA, Vivas EI, Amador-Noguez D, Rey FE. Intestinal microbiota composition modulates choline bioavailability. MBio. 2015;6:e02481–14. sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Functionality of CntA/B and YeaX/Y has been shown for Acinetobacter baumannii and Escherichia coli, where both genes cntA/yeaX and cntB/yeaW are required for TMA formation.

“Functionality has been shown for Acinetobacter baumannii and Escherichia coli where both genes cntA/yeaX and cntB/yeaW are required for the formation of TMA.”

Probably not a substantial claim: may not pass S3 (load-bearing).

TMAO promotes atherogenesis through formation of foam cells and reduction of reverse cholesterol transport from atherosclerotic plaque.

“A mechanistic model has been formulated proposing that TMAO promotes atherogenesis through the formation of foam cells (lipid-laden macrophages) and a reduction of the reverse cholesterol transport from the atherosclerotic plaque [5].”

• cites [5] Koeth R, Wang Z, Levison BS, Buffa J, Org E, Sheehy BT, et al. Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. Nat Med. 2013;19:576–85. sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim. May combine two claims.

Two major TMA-synthesis pathways have been described: CutC/CutD takes choline as substrate, and CntA/B acts on carnitine and gamma-butyrobetaine.

“Two major TMA-synthesis pathways have been described with a specific glycyl radical enzyme, the choline TMA-lyase (CutC) and its activator CutD [6], that takes choline as a substrate and a two-component Rieske-type oxygenase/reductase (CntA/B) [7], which acts on carnitine and its derivative gamma-butyrobetaine, as the key enzymes.”

• cites [6] Craciun S, Balskus EP. Microbial conversion of choline to trimethylamine requires a glycyl radical enzyme. Proc Natl Acad Sci U S A. 2012;109:21307–12. sourceNot checked yet

  Not checked in the free run.

• cites [7] Zhu Y, Jameson E, Crosatti M, Schäfer H, Rajakumar K, Bugg TDH, et al. Carnitine metabolism to trimethylamine by an unusual Rieske-type oxygenase from human microbiota. Proc Natl Acad Sci U S A. 2014;111:4268–73. sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

The choline utilization cluster including cutC/D is widely but discontinuously distributed across Firmicutes, Actinobacteria, and Proteobacteria.

“Campo and colleagues have shown that the so-called choline utilization cluster, which includes cutC/D among other genes involved in anaerobic choline metabolism, is widely, but discontinuously, distributed across various taxa belonging to Firmicutes, Actinobacteria, and Proteobacteria [9].”

• cites [9] Campo AM, Bodea S, Hamer H. Characterization and detection of a widely distributed gene cluster that predicts anaerobic choline utilization by human gut bacteria. MBio. 2015;6:e00042–15.Not checked yet

  Not checked in the free run.

May combine two claims.

In silico analysis suggested that the developed primers target 93% of unique cutC genes and 96% of unique cntA genes, allowing one mismatch in each primer.

“In silico analysis on coverage of developed primers suggested that a broad range of cutC and cntA gene sequences are targeted, namely 93 and 96% of all unique cutC and cntA genes, respectively (allowing for one mismatch in each primer).”

May combine two claims.

For both genes, metagenomic analysis suggested that sequences not targeted by the assays do not play an important role in the colonic TMA-producing communities, as not a single read was related to any of those reference sequences.

“However, for both genes, metagenomic analysis suggested that sequences not targeted by our assays do not play an important role in the colonic TMA-producing communities as not a single read was related to any of those reference sequences.”

May combine two claims.

The yeaW gene displays close sequence identity, 74%, to the cntA model sequence from A. baumannii and is arranged in similar operons that additionally encode a reductase (yeaX and cntB) as well as a betaine/carnitine/choline transporter.

“However, the yeaW gene displays close sequence identity (74%) to the cntA model sequence from A. baumannii and is arranged in similar operons that additionally encode a reductase (yeaX and cntB, respectively) as well as a betaine/carnitine/choline transporter”

• cites [8] Koeth RA, Levison BS, Culley MK, Buffa JA, Wang Z, Gregory JC, et al. γ-Butyrobetaine is a proantherogenic intermediate in gut microbial metabolism of L-carnitine to TMAO. Cell Metab. 2014;20:799–812. sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

For both enzymes, promiscuity to transform carnitine and butyrobetaine at similar rates was shown.

“and for both enzymes, promiscuity to transform carnitine and butyrobetaine at similar rates was shown (yeaW/X was reported to act on choline and to a lesser extent on betaine as well).”

May combine two claims.

The gene-targeted assays encompass key genes of the two major TMA-synthesis pathways and target a broad range of distinct taxa.

“Our gene-targeted assays encompass key genes of the two major TMA-synthesis pathways and target a broad range of distinct taxa.”

May combine two claims.

Primer coverage is thorough, and amplification as well as quantification of numerous distinct target sequences at similar levels was confirmed.

“Primer coverage is thorough, and amplification as well as quantification of numerous distinct target sequences at similar levels was confirmed.”

May combine two claims.

qPCR on one-third diluted samples indicated quantities of 40.6% ± 12.2% and 39.9% ± 22.2% of undiluted samples for cutC and cntA, respectively, evidencing that PCR mixtures were devoid of interfering ingredients.

“The qPCR on one third diluted samples indicated quantities of 40.6% ± 12.2% and 39.9% ± 22.2% of undiluted samples for cutC and cntA (five samples were on the borderline of detection), respectively, evidencing that the PCR mixtures were devoid of any interfering ingredients.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

For cutC qPCR, one major band with the expected size was detected in all samples; for a few samples, some very faint unspecific bands were additionally visible.

“For cutC, one major band with the expected size was detected in all samples; for a few samples, some very faint unspecific bands were additionally visible.”

Classified as probably not a substantial claim. May combine two claims.

A cutoff of 906.4, the median HMM score of the two sequences framing the drop, was set, and entries above that cutoff were considered true cutC sequences.

“Hence, a cutoff of 906.4, representing the median HMM score of the two sequences that frame that drop, was set, and all entries above that cutoff were considered as true cutC sequences.”

May combine two claims.

TMA producers constitute members of the core gut community, though at very low abundances.

“suggesting that TMA producers constitute members of the core gut community, though at very low abundances”

May combine two claims.

Sequencing depth in the Karlsson datasets was roughly 1 order of magnitude lower compared to HMP datasets, hampering accurate analyses.

“Sequencing depth was roughly 1 order of magnitude lower compared to HMP datasets hampering accurate analyses.”

May combine two claims.

The chosen HMM similarity cutoff for cntA was set at an obvious HMM score drop, which correlated less strongly with other parameters than the cutC analysis.

“Similar to cutC, the chosen HMM similarity cutoff was set at an obvious HMM score drop that, however, correlated less strongly with other parameters investigated in comparison with the cutC analysis shown above.”

May combine two claims.

Thirty-four lower-scoring sequences from Bacilli were not considered true cntA because they are neither closely phylogenetically related to top-scoring sequences nor exhibit syntenic cntB sequences.

“Specifically, 34 lower-scoring sequences from Bacilli are neither in close phylogenetic relation to the top-scoring sequences nor do they exhibit syntenic cntB sequences and were, hence, not considered as true cntA”

May combine two claims.

Overall, Clostridium XIVa cutC protein sequences form a monophyletic clade, where phylogenetic relations, with a few exceptions, resemble those derived from 16S rRNA gene sequence analysis, suggesting a close linkage between host phylogeny and cutC gene evolution.

“Overall, Clostridium XIVa cutC protein sequences form a monophyletic clade (Additional file 1), where phylogenetic relations, with a few exceptions such as Clostridium phytofermentans, Clostridium sp. AT5, and Dorea sp. 5-2, resemble those derived from 16S rRNA gene sequence analysis (Additional file 9B) suggesting a close linkage between host phylogeny and cutC gene evolution.”

May combine two claims.

A quantification limit of 5 × 103 genome copies per reaction for both genes was determined; below those concentrations, primer dimers prevented accurate target enumeration.

“Based on genomic sequences that were used as standards in the developed assays (see the “Methods” section), a quantification limit of 5 × 103 genome copies per reaction for both genes was determined. Below those concentrations, the formation of primer dimers did not allow for accurate target enumeration (Additional file 3A, B).”

May combine two claims.

For both genes, all tested synthetic sequences were amplified and, with a few exceptions, quantified at similar abundance levels.

“For both genes, all tested synthetic sequences were amplified and, with a few exceptions, quantified at similar abundance levels (Additional file 3C, D ).”

May combine two claims.

Given that 16S rRNA gene sequence analysis cannot adequately predict the TMA-producing potential and metagenomic data lack depth to satisfactorily resolve this low-abundance functional community, the assays developed here represent the most proper technique to investigate intestinal TMA-producing communities.

“Given that 16S rRNA gene sequence analysis cannot adequately predict the TMA-producing potential (Additional file 7) and metagenomic data lack depth to satisfactorily resolve this low abundant functional community, the assays developed here represent the most proper technique to investigate intestinal TMA-producing communities.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In the cntA qPCR, two samples (10 and 19) showed a single unspecific band and were not considered positive for the gene; one sample displayed two bands.

“In the cntA qPCR, two samples (10 and 19) showed a single unspecific band (sequencing confirmed them as non-target (data not shown)) and samples were, hence, not considered as positive for the gene; one sample displayed two bands.”

May combine two claims.

Due to the low abundance of bacteria encoding a TMA-producing potential, sequencing depth was not enough to ensure adequate coverage for assembly; BLAST searches yielded on average only 56 and 13 reads for cutC and cutD, respectively, even for HMP datasets with on average >108 reads.

“Thus, due to the low abundance of bacteria encoding a TMA-producing potential, sequencing depth was not enough to ensure adequate coverage for assembly (the BLAST searches yielded on average only 56 and 13 reads for cutC and cutD, respectively), even for HMP datasets where an ample amount of reads (on average >108) is provided.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Our developed assays are applicable in high throughput.

“Our developed assays are applicable in high throughput”

May combine two claims.

A nutrition rich in animal-derived products delivers both essential amino acids required for growth and high concentrations of the main TMA precursors choline and carnitine.

“a nutrition that is rich in animal-derived products delivers both essential amino acids required for growth and high concentrations of the main TMA precursors choline and carnitine”

May combine two claims.

The metagenomic methodology used in the reanalysis provided only limited insights and together with a low sample size did not allow for extraction of statistically significant results.

“though the methodology used, i.e., metagenomics, provided only limited insights and together with a low sample size it did not allow for extraction of statistically significant results (Additional file 8)”

Probably not a substantial claim: may not pass S2 (checkable).

The results may assist the design of subject-specific therapies that prevent TMA synthesis and the development of disease.

“may assist the design of subject-specific therapies that prevent TMA synthesis and the development of disease”

May combine two claims.

The diversity of the sample population allowed insights into the TMA-producing community from a diverse population but hindered revealing specific host factors that govern its proliferation.

“which allowed insights into the TMA-producing community from a diverse population, but hindered revealing specific host factors that govern its proliferation”

Probably not a substantial claim: may not pass S3 (load-bearing).

Patients suffering from trimethylaminuria are often treated by adapting to a vegan diet in order to limit the intake of dietary animal products that promote TMA production such as egg yolk and various meats.

“patients suffering from trimethylaminuria, where TMA accumulates in various body fluids, are often treated by adapting to a vegan diet in order to limit the intake of dietary animal products that promote TMA production such as egg yolk and various meats [15]”

• cites [15] Christodoulou J. Trimethylaminuria: an under-recognised and socially debilitating metabolic disorder. J Paediatr Child Health. 2012;48:2011–3. sourceNot checked yet

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Classified as probably not a substantial claim.

Only samples displaying both genes of a pathway were considered for analysis.

“Only samples displaying both genes of a pathway were considered for analysis”

Probably not a substantial claim: may not pass S3 (load-bearing).

Results are expressed as averages of three independent experiments.

“Results are expressed as averages of three independent experiments”

Probably not a substantial claim: may not pass S2 (checkable).

Results of the developed assays together with presented functional predictions of major taxa involved provide crucial information for uncovering factors limiting growth of TMA producers in the gut.

“Results of the developed assays together with presented functional predictions of major taxa involved provide crucial information for uncovering factors limiting growth of TMA producers in the gut”

Classified as probably not a substantial claim.

A modified forward primer was used to ensure adequate amplification, including taxa previously underrepresented using conventional primers.

“To ensure adequate amplification, including taxa that were previously underrepresented using conventional primers that target the V1-2 region such as Bifidobacteria [29], a modified forward primer was used”

• cites [29] Walker AW, Martin JC, Scott P, Parkhill J, Flint HJ, Scott KP. 16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice. Microbiome. 2015;3:26. sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

A correlation between dietary habits and production of TMAO was previously established.

“a correlation between dietary habits and production of TMAO was previously established”

• cites [5] Koeth R, Wang Z, Levison BS, Buffa J, Org E, Sheehy BT, et al. Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. Nat Med. 2013;19:576–85. sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Only clusters that exhibit ≥5 sequence counts were considered, and the data was rarified to equal depth.

“Only clusters that exhibit ≥5 sequence counts were considered, and the data was rarified to equal depth”

Probably not a substantial claim: may not pass S3 (load-bearing).

The database construction cycle was repeated three times to ensure construction of robust models.

“this cycle was repeated three times to ensure construction of robust models”

Probably not a substantial claim: may not pass S3 (load-bearing).

For cntA, sample 3 was omitted as >99% of obtained sequences failed FrameBot analysis.

“For cntA, sample 3 was omitted as >99% of obtained sequences (from 13,095) failed FrameBot analysis”

Classified as probably not a substantial claim. May combine two claims.

Top hits that covered ≥70 bp and displayed ≥70% identity to a reference were considered for analysis where both genes of each pathway had to be detected for a positive result.

“Top hits (based on e-value) that covered ≥70 bp and displayed ≥70% identity to a reference were considered for analysis where both genes of each pathway had to be detected for a positive result”

May combine two claims.

The study only targeted the TMA-producing potential by quantifying respective gene abundances, and no investigations on gene expression/TMA production were performed.

“we only targeted the TMA-producing potential by quantifying respective gene abundances, and no investigations on gene expression/TMA production were performed”

Classified as probably not a substantial claim.

Only significant correlations (p < 0.01) displaying a ρ ≥ 0.5 and a false-discovery correction q-value <0.01 between clusters that were detected in at least 50% of samples were considered for analysis.

“Only significant correlations (p < 0.01) displaying a ρ ≥ 0.5 and a false-discovery correction q-value <0.01 between clusters that were detected in at least 50% of samples were considered for analysis”