Review for 10.1186/s12876-025-04061-0 (2025)

Title: Unveiling the diagnostic and pro-inflammatory role of Crohn’s disease: insights from
16S-guided discovery and species-specific validation
Version: 3 Date: 30 Mar 2025
Reviewer's report:
This revised manuscript presents a multidisciplinary study combining clinical data, microbial
sequencing, machine learning, and in vivo modeling to explore the diagnostic and inflammatory
role of Ruminococcus gnavus in Crohn’s disease (CD). The study is both scientifically
relevant and timely, addressing critical gaps in understanding CD pathogenesis and microbial
biomarkers.
Scientific Strengths
Innovative Integration: The combination of 16S rRNA sequencing, qPCR validation, random
forest modeling, and murine DSS colitis models demonstrates a robust, multi-modal research
design.
Clinical Relevance: The identification of R. gnavus and Agathobacter as potential diagnostic
and mechanistic biomarkers could enhance non-invasive screening strategies for CD.
Validation Efforts: Use of three independent cohorts (discovery, validation, serum) strengthens
the reliability of findings and improves external validity.
Mechanistic Insight: The in vivo experiments confirm a pro-inflammatory role for R. gnavus,
supporting the translational importance of the microbial signal.
Points for Revision Before Acceptance
Language and Grammar
While the manuscript has been substantially improved, there are still numerous minor
grammatical and syntactic issues. A final professional English language edit is strongly advised.
Examples:
"pseudonym-free mice" should be corrected to "specific-pathogen-free (SPF) mice".
Some figure legends and in-text citations (e.g., “CD. I” vs. “CD.I”) lack consistent formatting.
Methodological Clarifications
Please specify statistical power calculations or justify sample sizes, especially for the animal
experiments and serum cytokine measurements.
Provide additional details about random forest hyperparameter tuning—e.g., how many
permutations were tested, and which metric minimized the classification error?
Include a short paragraph under Methods explaining the rationale for selecting the six final
genera for the diagnostic model.
Figures and Supplementary Material
Figures must be submitted in high-resolution, publication-ready format (#300 dpi).
Please ensure all supplementary tables (particularly Supplementary Table 5) and figures (S1,
S2) are properly cited and described in the main text.
The results from the qPCR validation and histopathological scoring should be supported by
graphical data and statistical annotations in the supplementary material if not already provided.
Statistical Reporting
Clearly distinguish between p-values and adjusted q-values across the Results section.
The authors mention Benjamini-Hochberg correction, but it is inconsistently applied in tables
and figure legends—please harmonize.
Ensure that correlation coefficients include both r and p-values for transparency.
Ethical and Experimental Rigor
The animal experimental protocol is well described, but please mention the number of animals
per group and the humane endpoints defined for euthanasia.
Clarify whether the pathologist evaluating histological sections was blinded to treatment group.
Discussion Enhancements
The Discussion would benefit from further contextualization of the findings in light of recent
microbiome studies (e.g., multi-omics, functional metagenomics).
Consider including potential therapeutic implications, such as microbiome modulation or
bacteriotherapy.
Expand on the limitations of 16S sequencing resolution and how it affects strain-level
classification of R. gnavus.
Conclusion and Recommendation
This study provides valuable scientific and clinical insights into the microbiota-associated
mechanisms underlying Crohn’s disease, with implications for diagnosis and pathophysiology.
With minor but necessary revisions focusing on methodological transparency, language clarity,
and consistency in data reporting, the manuscript will be suitable for publication.
Recommendation: Accept after minor revisions.