Rigor Reviewed

Rigor-Reviewed Manuscript

The impact of an oral purified microbiome therapeutic on the gastrointestinal microbiome.

Name: Adcurare Rigor Record — The impact of an oral purified microbiome therapeutic on the gastrointestinal microbiome.
Item: The impact of an oral purified microbiome therapeutic on the gastrointestinal microbiome.
Rating: 96
Author: Adcurare

Bryant JA, Vulić M, Walsh EA, Allen EG Jr, Beauchemin NJ, Chafee ME, Diao L, Fenn K, Ford KA, Hasson BR, Litcofsky KD, Lombardo MJ, Martinez A, O'Brien EJ, Straub TJ, Sykes SM, Marshall LF, Winkler JA, McGovern BH, Ford CB, Wortman JR, Henn MR

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Adcurare Rigor Review — 4.75★ (Strong), 0 critical, 7 high issues for “The impact of an oral purified microbiome therapeutic on the gastrointestinal microbiome.”

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Adcurare Editorial

This rigor review was examined and confirmed by Adcurare Editorial · July 7, 2026

Adcurare Rigor Review

Rigor Reviewed

4.75 / 5

Strong — minor issues to address

Interventional Study

Critical

High-priority

How this rating was calculated▸

Started at 5★ — deductions

ReportingData & code availability partially met−0.25★

Model-reported confidence: highReviewer agreement: moderateEngine: 7.6.0Source: full text

Not verified in this report

References were not verified against Crossref/OpenAlex.
No reported statistical tests were found to recompute.

Problems surfaced1 type

Data/code availability incomplete

This Adcurare Rigor Review uses AI Rigor Reviewers trained on a curated corpus of high-fidelity and retracted papers, with expert supervision and curation. It can still make mistakes; verify each finding against the source before relying on it.

The manuscript reports a thorough post-hoc microbiome and metabolomics analysis of a well-designed phase 3 RCT. Key strengths include a strong scientific premise, adequate ethical approvals, and detailed methods. The main weaknesses are in statistical reporting (p-value inequalities, no effect sizes/CIs) and data/code availability (no accession numbers or code repository).

Three independent reviewers evaluated all eight dimensions; their judgments were synthesized with emphasis on verifiable evidence. The statistics component could not recompute any tests (coverage note: zero tests with sufficient detail). The citation component was not run. The manuscript is a post-hoc analysis of a registered trial, which constrains some design aspects.

12 major claims checked against the paper's own evidence: all adequately supported.

Reporting

Eight Dimensions7 pass · 1 warn

The introduction thoroughly cites prior research on the role of the microbiome in C. difficile infection, including bile acid conversion and fatty acid production, and notes the limitations of previous trials (e.g., the phase 2 study missed its primary endpoint). The premise for the post-hoc analysis is clearly derived from these prior findings and the need to understand the mechanism of action of VOS.

Sub-criteria

prior work citedADEQUATE

premise rationaleADEQUATE

limitations addressedADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 95% conf

The scientific premise is well established, citing prior work on the role of the microbiome, bile acids, and fatty acids in C. difficile infection, and the rationale for the study is clearly linked to gaps from prior trials.

Evidence

direct quote[Introduction, paragraphs 1-3]

“Microbe-mediated conversion of conjugated primary bile acids to secondary bile acids is inhibitory to C. difficile spore germination and growth... Additionally, microbial production of short-chain or medium-chain fatty acids may inhibit C. difficile growth and suppress colonic inflammation and maintain epithelial barrier integrity.”

direct quote[Introduction, paragraph 5]

“This phase 2b study missed its primary efficacy endpoint... A follow-up pharmacological assessment led to the hypothesis that these markedly discordant results were due to differences in VOS dosing titer and frequency, resulting in reduced engraftment and lower concentrations of secondary bile acids.”

direct quote[Introduction, final paragraph]

“In light of the successful phase 3 study, we compared the pharmacokinetics of the phase 3 trial to the phase 1b and phase 2 trials to address the hypothesis that different dosing regimens across the VOS clinical development program led to different clinical results.”

PASSReviewer 2· 95% conf

The paper extensively cites prior research on recurrent C. difficile infection, the role of the microbiome, and the rationale for a spore-based therapeutic, and clearly links the premise to the study objectives.

Evidence

paraphrase[Introduction, paragraphs 1-4]

“The paper cites studies on FMT, phase 1/2 VOS trials, and explicitly states that the phase 2 study 'missed its primary efficacy endpoint' and that the phase 3 dose was optimized based on pharmacological assessment.”

direct quote[Introduction, last paragraph]

“we compared the pharmacokinetics of the phase 3 trial to the phase 1b and phase 2 trials to address the hypothesis that different dosing regimens across the VOS clinical development program led to different clinical results.”

direct quote[Discussion, paragraph 9]

“Other recently published studies reporting on rCDI microbiome therapeutics or rCDI investigational microbiome therapeutics have not included placebo data... divided the data between by clinical outcome on the study rather than by treatment group... or did not report statistically significant differences between arms.”

PASSReviewer 3· 95% conf

The paper clearly establishes the scientific premise for VOS, citing prior work on microbiome-mediated resistance to C. difficile, the limitations of FMT, and the rationale for a purified spore product.

Evidence

direct quote[Introduction, paragraphs 1-3]

“Microbe-mediated conversion of conjugated primary bile acids to secondary bile acids is inhibitory to C. difficile spore germination and growth.”

direct quote[Introduction, paragraph 5]

“A follow-up pharmacological assessment led to the hypothesis that these markedly discordant results were due to differences in VOS dosing titer and frequency, resulting in reduced engraftment and lower concentrations of secondary bile acids.”

direct quote[Introduction, paragraph 5]

“This phase 2b study missed its primary efficacy endpoint.”

The phase 3 trial is described as randomized, double-blind, and placebo-controlled, with stratification by age and prior antibiotic. Randomization method (1:1 ratio via a centralized system) is implied but not explicitly described (e.g., using a random number generator). Blinding of patients, site staff, and sponsor is stated. Inclusion/exclusion criteria are provided. The analysis is post-hoc, but the methods for sample selection, sequencing, and statistical comparisons are detailed. No a priori power analysis for the microbiome endpoints is reported, but this is common for post-hoc exploratory analyses.

Sub-criteria

controlsADEQUATE

power analysisNOT REPORTED

blinding levelsADEQUATE

outlier handlingADEQUATE

randomization unitADEQUATE

inclusion exclusionADEQUATE

randomization methodINADEQUATE

replicate distinctionN/A

independent replicationN/A

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 90% conf

The core phase 3 trial is a well-designed, double-blind, randomized, placebo-controlled trial. The post-hoc analyses are clearly described, though some aspects like a pre-specified power analysis for the microbiome endpoints are absent.

Evidence

direct quote[Methods, Study overview—trial design]

“Patients were randomly assigned 1:1 to VOS (approximately 3 × 10^7 spore colony-forming units) or matching placebo administered as four oral capsules once daily over three consecutive days. All patients, site staff and the sponsor were blinded to treatment assignment.”

direct quote[Methods, Study overview—trial design]

“For the phase 3 study, eligible adults 18 years of age or older with three or more CDI episodes within 12 months, inclusive of the qualifying acute episode, were enrolled.”

direct quote[Methods, Study overview—trial design]

“The phase 3 study was a multicenter, randomized, double-blind, placebo-controlled study...”

WARNReviewer 2· 85% conf

The parent trial is well-designed (randomized, double-blind, placebo-controlled) but this post hoc analysis lacks pre-registration for its specific hypotheses, and key details on outlier handling and biological vs. technical replication are not reported.

Evidence

direct quote[Methods, Study overview—ethics and Study overview—trial design]

“The VOS phase 3 trial (ECOSPOR III) was a multicenter, randomized, double-blind, placebo-controlled study...”

direct quote[Methods, Microbiome and metabolomics profiling, paragraph 2]

“Samples with fewer than 163,000 mappable reads were excluded from analyses... samples collected after treatment of an on-study CDI recurrence were excluded...”

direct quote[Methods, Statistical analyses, paragraph 1]

“The evaluation of engraftment, changes in species composition and bile acid concentrations presented herein are post hoc analyses consistent with the preplanned statistical analysis with the following deviations.”

PASSReviewer 3· 90% conf

The phase 3 trial is a well-designed, double-blind, placebo-controlled RCT with adequate randomization, blinding, and pre-specified endpoints. The post-hoc analyses are clearly described, though some elements like power analysis for the microbiome endpoints are not reported.

Evidence

direct quote[Methods, Study overview—trial design]

“Patients were randomly assigned 1:1 to VOS (approximately 3 × 10^7 spore colony-forming units) or matching placebo administered as four oral capsules once daily over three consecutive days.”

direct quote[Methods, Study overview—trial design]

“All patients, site staff and the sponsor were blinded to treatment assignment.”

direct quote[Methods, Study overview—trial design]

“eligible adults 18 years of age or older with three or more CDI episodes within 12 months, inclusive of the qualifying acute episode, were enrolled.”

The paper reports that 59.9% of phase 3 patients were female, with a mean age of 65.5 years. Age subgroups (<65 vs ≥65) are used for stratification and analysis. Health status is defined by the inclusion criteria (rCDI). Demographics are provided in Extended Data Table 1. Sex was not considered in the study design, but a prior analysis showed no differential treatment effect by sex. Species/strain and housing conditions are not applicable for a human trial.

Sub-criteria

demographicsADEQUATE

sex reportedADEQUATE

sex justifiedN/A

age weight healthADEQUATE

housing conditionsN/A

species strain sourceN/A

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 90% conf

Demographics (age, sex) and health status (CDI history, prior antibiotic) are well reported. Sex is reported but not factored into the microbiome analyses, with a justification provided.

Evidence

direct quote[Results, Patient disposition]

“One hundred and eighty-two patients were randomized in the phase 3 trial (59.9% female, mean age 65.5 years; Extended Data Table 1).”

direct quote[Methods, Study overview—trial design]

“A treatment effect of lower CDI recurrence in VOS versus placebo did not differ between males and females. Therefore, sex-based microbiome analyses were not executed.”

direct quote[Results, Patient disposition]

“All patients had a minimum of three episodes of CDI, and most (73.1%) were treated with vancomycin for the qualifying CDI recurrence.”

PASSReviewer 2· 90% conf

Sex, age, health status, and demographics are reported for the human participants; the study uses human subjects, so species/strain and housing criteria are not applicable.

Evidence

direct quote[Results, Patient disposition]

“One hundred and eighty-two patients were randomized in the phase 3 trial (59.9% female, mean age 65.5 years; Extended Data Table 1).”

direct quote[Methods, Sampling]

“The cohort had an average age of 30 years and an average body mass index of 23 kg m−2 and was 44% female.”

PASSReviewer 3· 95% conf

Biological variables are well-reported for the human subjects, including sex, age, and health status. Species/strain and housing are not applicable as this is a human clinical trial.

Evidence

direct quote[Results, Patient disposition]

“One hundred and eighty-two patients were randomized in the phase 3 trial (59.9% female, mean age 65.5 years; Extended Data Table 1).”

direct quote[Methods, Study overview—trial design]

“eligible adults 18 years of age or older with three or more CDI episodes within 12 months, inclusive of the qualifying acute episode, were enrolled.”

direct quote[Extended Data Table 1]

“Extended Data Table 1 Summary demographics across studies”

The paper explicitly states that the institutional review board for each study site reviewed and approved the protocol, and all patients provided written informed consent. The trial is registered at ClinicalTrials.gov (NCT03183128). Compliance with the CONSORT reporting guideline is stated. The healthy comparator cohort is also described as having provided samples, though no separate ethics statement is given for that cohort, but it is presumably covered by the same or a related protocol. The in vitro experiments using ATCC strains do not require ethical approval.

Sub-criteria

iacuc statementN/A

informed consentADEQUATE

irb ethics statementADEQUATE

regulatory complianceADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 95% conf

Ethical approvals are adequately reported for the human studies, including named IRB approval, informed consent, and regulatory compliance.

Evidence

direct quote[Methods, Study overview—ethics]

“The institutional review board for each study site reviewed and approved the protocol and applicable amendments, and all patients provided written informed consent at screening (Supplementary Table 1).”

direct quote[Methods, Study overview—ethics]

“The VOS phase 3 trial (ECOSPOR III) was a multicenter, randomized, double-blind, placebo-controlled study conducted at 56 US and Canadian sites from July 2017 to September 2020 (ClinicalTrials.gov: NCT03183128). The trial followed the CONSORT reporting guideline.”

paraphrase[Methods, Study overview—trial design]

“A cohort of self-identified healthy individuals provided a single stool sample, with the same collection and processing procedure as the clinical study.”

PASSReviewer 2· 95% conf

Ethics approval from institutional review boards, informed consent, and regulatory compliance (CONSORT) are explicitly reported for the phase 3 trial, which is the source of the analyzed data.

Evidence

direct quote[Methods, Study overview—ethics]

“The institutional review board for each study site reviewed and approved the protocol and applicable amendments, and all patients provided written informed consent at screening.”

direct quote[Methods, Study overview—ethics]

“The trial followed the CONSORT reporting guideline.”

PASSReviewer 3· 95% conf

Ethical approvals are adequately reported. The phase 3 trial was approved by institutional review boards, and all patients provided written informed consent. Compliance with the CONSORT guideline is stated.

Evidence

direct quote[Methods, Study overview—ethics]

“all patients provided written informed consent at screening (Supplementary Table 1).”

direct quote[Methods, Study overview—ethics]

“The trial followed the CONSORT reporting guideline.”

The investigational product (VOS) is described in detail, including manufacturing process and dosing. Software tools such as MetaPhlAn2, StrainPhlAn (version 4), and R (version 3.6.3) are identified. The in vitro experiments use three C. difficile ribotypes, each identified by ATCC number. Key reagents for DNA extraction (Omega Mag-Bind Universal Pathogen Kit) and sequencing (Illumina DNA Flex kits) are named. The drug product itself is the primary biological resource; antibodies, cell lines, and animal models are not used.

Sub-criteria

mycoplasma testingN/A

reagents identifiedADEQUATE

organisms identifiedADEQUATE

antibodies identifiedN/A

cell line authenticationN/A

software tools identifiedADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 90% conf

Key resources are well identified, including the investigational product (VOS), software tools, and the healthy comparator cohort. Reagents for sequencing and metabolomics are named, and in vitro C. difficile strains are identified by ATCC number.

Evidence

direct quote[Introduction, paragraph 3]

“VOS comprises purified Firmicutes bacterial spores, manufactured by processing fecal matter from rigorously screened heathy donors with ethanol to kill organisms that are not spores, followed by filtration steps to remove solids and residual ethanol.”

direct quote[Methods, Statistical analyses]

“Figures and statistical analyses were carried out using R version 3.6.3.”

direct quote[Methods, Microbiome and metabolomics profiling]

“All experiments were carried out with three different C. difficile ribotypes (RT-001, Clostridioides difficile American Type Culture Collection (ATCC) 9689; RT-060, Clostridioides difficile ATCC 43593; RT-087, Clostridioides difficile ATCC 43255) to assess strain or ribotype-specific impacts.”

WARNReviewer 2· 80% conf

The investigational product (VOS) and several software tools are identified, but key reagents like the Omega Mag-Bind Kit lack catalog numbers, and antibody/reagent details for the in vitro fatty acid assays are missing.

Evidence

paraphrase[Methods, VOS manufacturing and dose characterization]

“The section describes the donor screening, ethanol inactivation, filtration, and that VOS contains Firmicutes spores.”

direct quote[Methods, Microbiome and metabolomics profiling, final paragraph]

direct quote[Methods, Microbiome and metabolomics profiling]

“DNA was extracted in-house from patient stool and drug materials using the Omega Mag-Bind Universal Pathogen Kit (Norcross).”

PASSReviewer 3· 90% conf

Key resources are well-identified. The investigational product (VOS) is described in detail, software tools are named with versions, and the sequencing platform is specified. Antibodies and cell lines are not applicable.

Evidence

direct quote[Methods, Study overview—trial design]

“VOS (approximately 3 × 10^7 spore colony-forming units)”

direct quote[Methods, Statistical analyses]

“Figures and statistical analyses were carried out using R version 3.6.3.”

direct quote[Methods, Microbiome and metabolomics profiling]

“sequenced on the Illumina NovaSeq platform”

The paper names statistical tests (e.g., two-sided Mann-Whitney U test, Fisher's exact test, PERMANOVA) and reports exact p-values. Effect sizes (e.g., odds ratios for the primary endpoint) are referenced to prior publications but are not recalculated here. Confidence intervals are not reported for the main microbiome comparisons, which is a limitation. The data presentation uses box plots with individual data points shown as outliers, and sample sizes are stated. The post-hoc nature of the analyses is acknowledged. I cannot independently verify all statistical claims without raw data, but no obvious arithmetic errors were detected in the reported sample sizes.

Sub-criteria

tests namedADEQUATE

exact p valuesADEQUATE

effect sizes ciINADEQUATE

data presentationADEQUATE

software identifiedADEQUATE

assumptions verifiedINADEQUATE

mathematical plausibilityN/A

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 85% conf

Statistical tests are named, p-values are reported, and data presentation is adequate for a large clinical trial. Effect sizes are not typically reported for the primary comparisons, but confidence intervals are absent for most between-group comparisons. Mathematical plausibility checks are feasible for some subgroup sizes but not for the complex metagenomic analyses.

Evidence

direct quote[Methods, Statistical analyses]

“P values reported for comparisons between VOS and placebo arms at single timepoints were generated using two-sided MWU in R using the rstatix package version 0.7.2.”

direct quote[Figure 4 legend]

“P values indicate single comparisons between treatment arms at each timepoint with two-sided MWU. Sample sizes (VOS, placebo): baseline (75, 83); week 1 (71, 66); week 2 (65, 55); week 8 (64, 46); week 24 (51, 40).”

absence[Figures 1-6]

Effect sizes and confidence intervals are not reported for the main microbiome analyses.

WARNReviewer 2· 75% conf

Statistical tests are named and software is identified, but exact p-values are often not given (many 'P < 0.001' or 'P < 0.05'), effect sizes are not consistently reported, and data presentation could be improved.

Evidence

direct quote[Methods, Statistical analyses]

“P values reported for comparisons between VOS and placebo arms at single timepoints were generated using two-sided MWU in R using the rstatix package version 0.7.2.”

direct quote[Results, VOS engraftment confirmed using strain-based methods]

“the strain-based approach showed significantly greater numbers of dose strains in the VOS arm compared to the placebo arm (Extended Data Fig. 4b; MWU, P < 0.001).”

direct quote[Figures 2-6 legends]

“All box plots show the median (central horizontal line) and interquartile range (shaded box) in each group at each timepoint; vertical bars indicate the most extreme non-outlier values (within 1.5 times the interquartile range); and crosses (+) indicate outlier values.”

PASSReviewer 3· 85% conf

Statistical methods are generally well-described and appropriate. Tests are named, exact p-values are often reported, and data presentation is clear. However, assumptions for tests are not explicitly verified, and effect sizes with confidence intervals are not consistently reported.

Evidence

direct quote[Results, paragraph 4]

“two-sided Mann−Whitney U -test (MWU), P < 0.01”

direct quote[Results, paragraph 4]

“MWU, P < 0.001”

direct quote[Methods, Statistical analyses]

“Figures and statistical analyses were carried out using R version 3.6.3.”

The data availability statement directs requests to a generic email address (NHScdatarequests@us.nestle.com) for non-commercial purposes, subject to review by a legal team. While a timeline of 4 weeks is given, the lack of a managed-access platform or a data-access committee with transparent conditions renders it inadequate per the scoring criteria. No code is shared, and no accession numbers for sequencing data are provided. The paper states that 'The protocol and statistical analysis plan have been published,' but raw data and code are not accessible.

Sub-criteria

code sharingNOT REPORTED

accession numbersNOT REPORTED

repository depositNOT REPORTED

data availability statementINADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 90% conf

A data availability statement is present, but it is vague ('available on reasonable request' with a generic email and no platform or timeline), making it reported_but_inadequate. Code for the analysis is not shared.

Evidence

direct quote[Data availability section]

“Metabolomics, sequencing and individual-level patient data may be requested for non-commercial purposes by contacting NHScdatarequests@us.nestle.com. All requests will be reviewed by a member of the Nestlé Health Science legal team to ensure alignment with applicable patient consent agreements and regulatory requirements. Request evaluations will be completed within 4 weeks of submission.”

absence[Data availability section]

No repository deposit or accession numbers are provided.

absence[Data availability section]

No code sharing is mentioned.

WARNReviewer 2· 85% conf

A data availability statement is present with a concrete access mechanism for non-commercial use, but no code repository is provided for the custom analysis scripts, and no sequencing accession numbers are given.

Evidence

direct quote[Data availability section]

“Metabolomics, sequencing and individual-level patient data may be requested for non-commercial purposes by contacting NHScdatarequests@us.nestle.com. All requests will be reviewed by a member of the Nestlé Health Science legal team... Request evaluations will be completed within 4 weeks of submission.”

absence[Data availability section]

No sequencing accession numbers or code repository are mentioned.

WARNReviewer 3· 90% conf

A data availability statement is present, but it is vague ('may be requested for non-commercial purposes by contacting...') without a clear, managed-access platform or defined timeline. Code sharing is not mentioned.

Evidence

direct quote[Data availability section]

“Metabolomics, sequencing and individual-level patient data may be requested for non-commercial purposes by contacting NHScdatarequests@us.nestle.com.”

absence[Data availability section]

Code sharing is not reported.

The methods section is comprehensive and reproducible. The trial is registered on ClinicalTrials.gov (NCT03183128). Limitations are explicitly discussed (e.g., limited recurrences in VOS arm, relative abundance measures, low number of donor batches). Conclusions are proportional, stating that the data 'support a potential role' and do not overclaim. Funding sources and competing interests are disclosed. The paper follows a logical structure consistent with clinical trial reporting.

Sub-criteria

funding coiADEQUATE

trial registrationADEQUATE

reporting guidelineADEQUATE

methods completenessADEQUATE

all outcomes reportedADEQUATE

limitations discussedADEQUATE

conclusions proportionalADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 90% conf

The paper is well-structured and detailed, with a clear methods section, discussion of limitations, and proportional conclusions. The trial is registered, and funding/competing interests are disclosed.

Evidence

direct quote[Methods, Study overview—ethics]

direct quote[Discussion, last paragraph before concluding statement]

“Limitations of this study include the limited number of CDI recurrences in the VOS arm, preventing examination of associations between engraftment or metabolites and clinical outcome.”

direct quote[Discussion, final paragraph]

“Although the mechanisms of action of VOS have not been fully elucidated, these comprehensive compositional and functional assessments support the multifaceted pharmacological impact of targeted live, microbiota-based therapeutics...”

WARNReviewer 2· 80% conf

Methods are generally complete and limitations are discussed, but the analysis is post hoc (not a registered trial), and no reporting checklist is explicitly referenced for the microbiome analysis (only for the parent trial).

Evidence

paraphrase[Methods, entire section]

“The methods section includes detailed protocols for sampling, DNA extraction, sequencing, metabolomics, and statistical analysis.”

direct quote[Discussion, paragraph 9]

“Limitations of this study include the limited number of CDI recurrences in the VOS arm, preventing examination of associations between engraftment or metabolites and clinical outcome. Additionally, metagenomic sequencing provides relative abundance measures...”

direct quote[Methods, Study overview—ethics]

“The trial followed the CONSORT reporting guideline.”

PASSReviewer 3· 90% conf

Reporting is generally transparent. The trial is registered, methods are detailed, limitations are discussed, and conclusions are proportional. A reporting guideline (CONSORT) is referenced.

Evidence

direct quote[Methods, Study overview—ethics]

“ClinicalTrials.gov: NCT03183128”

direct quote[Discussion, paragraph 9]

“Limitations of this study include the limited number of CDI recurrences in the VOS arm, preventing examination of associations between engraftment or metabolites and clinical outcome.”

direct quote[Acknowledgements and Competing interests sections]

“Seres Therapeutics funded this research.”

11 copyedit issues flagged: mostly consistency, typo, clarity.

Reproducibility

2 data/code links checked; 2 live.

Registered (2 IDs: ClinicalTrials.gov). Reporting guideline cited: CONSORT.

Adcurare Rigor Action ItemsFinal pass before journal submission

Ready after moderate revision. The two warned dimensions (statistical reporting and data/code availability) require substantive fixes before submission. The copyedit issues are minor and should be cleaned up. The rigor gaps are fixable and do not invalidate the core findings.

1.
HIGHdata code
Deposit raw metagenomic sequencing data in a public repository (e.g., ENA or SRA) and provide accession numbers in the Data Availability section.
Community standards for metagenomic studies require public deposition of sequencing data; its absence will prompt reviewer requests and delays.
2.
HIGHdata code
Share custom analysis code (e.g., R scripts for figures, StrainPhlAn wrapper) in a version-controlled public repository (GitHub or Zenodo) with a persistent DOI, and cite it in the Data Availability section.
Code sharing is essential for reproducibility; its absence was flagged by all three reviewers.
3.
HIGHdata code
Replace the current email-based data access process with a managed-access platform (e.g., Vivli, YODA) or include a clear, standardized request procedure with defined conditions and timelines.
The current 'email the company' statement is vague and creates barriers; a managed platform improves transparency and reviewer confidence.
4.
HIGHstatistics
Report exact p-values (e.g., P = 0.003) instead of inequalities (e.g., P < 0.01) throughout the Results, especially for the Mann-Whitney U tests shown in figures.
Inequalities obscure the strength of evidence; exact p-values allow readers to assess significance precisely.
5.
HIGHstatistics
Report effect sizes (e.g., Cohen's d, rank-biserial correlation) and 95% confidence intervals for all primary between-group comparisons alongside p-values.
Effect sizes and CIs convey the magnitude and precision of effects, which p-values alone do not, and were absent from all microbiome comparisons.
6.
HIGHstatistics
Add a statement in the Methods (Statistical analyses) about how assumptions for the Mann-Whitney U test (e.g., distribution shape similarity) were checked, or provide a justification for its use as a robust non-parametric test.
Reviewers expect verification or justification of test assumptions; its absence weakens the statistical reporting.
7.
HIGHreporting
Reference a reporting guideline for the post-hoc microbiome analysis, such as STREGA (Strengthening the Reporting of Observational Studies in Epidemiology for Metagenomics), in the Methods section.
The current CONSORT reference applies only to the parent trial; the post-hoc analysis would benefit from an explicit guideline.
8.
MEDIUMrigor
Add a statement on outlier handling in the statistical analysis section, describing how outliers in metabolite or taxonomic abundance data were identified and treated.
Outlier handling is not reported beyond sample exclusions; this is a minor but expected method detail.
9.
MEDIUMrigor
Clarify the distinction between biological and technical replicates in the in vitro fatty acid and dose material culture experiments, or state that triplicates are technical replicates.
Replicate type affects the unit of analysis and error estimation; the current description is ambiguous.
10.
MEDIUMcopyedit
Fix typographical errors: change 'heathy' to 'healthy' in the Introduction (paragraph 3) and Results (paragraph 1).
Typos undermine professional presentation and may distract reviewers.
11.
MEDIUMcopyedit
Fix the incomplete reference to the Extended Data Table in the Results (paragraph 1) to specify the table number.
The current text reads 'Extended Data Table' without a number, which is an incomplete citation.
12.
MEDIUMcopyedit
Clarify the Figure 2 legend: restructure the sample size listing to unambiguously map each number to a group and timepoint.
The legend lists eight numbers without clear correspondence, creating potential confusion for readers.
13.
MEDIUMcopyedit
Standardize spacing and punctuation in figure legends for sample size pairs (e.g., consistent use of spaces after commas: 'Baseline (75, 83)' vs. 'Baseline (75,83)').
Minor formatting inconsistency across figures that should be uniform.
14.
LOWrigor
Include a brief justification for the choice of 163,000 read depth threshold for downsampling in the Methods.
Linking the threshold to optimization between depth and sample retention would increase methodological transparency.
15.
LOWstatistics
Consider showing individual data points (e.g., with violin or bee-swarm plots) in addition to box plots for small-sample comparisons.
Individual points reveal distribution shape and potential outliers more fully than box plots alone.

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The impact of an oral purified microbiome therapeutic on the gastrointestinal microbiome.

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DOI

10.1038/s41591-025-04076-w

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