Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments to the authors:

I carefully reviewed this manuscript. The manuscript advances an important translational thesisthat restoring Bifidobacterium infantis and Lactobacillus reuteri may ameliorate immunometabolic and neurobehavioral dysregulation. The topic fits the journal’s scope. However, several issues in the results’ presentation, the qualitative systematic review (SR) methods, and the inferential logic weaken the evidentiary basis and should be addressed before the work can be considered robust.

1: Use of quasi-quantitative claims without meta-analysis.The paper reports specific percentage effects (e.g., “40–60% reduction in diversity”; “up to 50% reductions in CRP/TNF-α/IL-6”; “30–40% increases in ZO-1/occludin/claudin-1”) as if they were pooled estimates, but the methods explicitly state a narrative synthesisdue to heterogeneity (no meta-analysis). These numerics, presented in the Results/Discussion, read as cross-study effect sizes but are not derived from a pooled model and may mix disparate populations, endpoints, and strains. Please temper the language, attribute each value to specific constituent studies, and avoid implying pooled quantitative certainty. 

2: Industrialized vs. traditional gut diversity.The statement that Western populations show a 40–60% drop in diversityis strong and plausible but needs traceable study citations and context (diversity metric, cohort comparability, age/diet control). As written, it appears as a global estimate in a Results subsection without study-level qualifiers. Add precise citations and report the underlying diversity metrics (e.g., Shannon, Simpson), cohort sizes, and adjustments.

3: Many neurobehavioral outcomes are from animal modelsand are correctly flagged as such in Limitations. In the Results/Discussion, separate preclinical from clinical evidence and avoid implying human efficacy (e.g., “restoring social behaviors”). Consider a dedicated subsection explicitly labeling preclinical vs. clinical endpoints.

4: Registration and protocol.PRISMA 2020 compliance is claimed, but there is no mention of protocol registration (e.g., PROSPERO/OSF), a priorioutcomes, or planned analyses. Please state whether a protocol existed and where it can be accessed; if not, acknowledge and justify.

5: The authors note “two independent reviewers” for title/abstract and full-text screening. Please report inter-rater reliability (e.g., Cohen’s kappa), conflict resolution procedures, and the exact number of conflicts at each stage.

6: No quantitative synthesis yet numeric generalizations.Given the declared narrative synthesis, please avoid cross-study arithmetic (percentages, “~0.9-day reductions” presented outside of their original trial context). Where you cite a meta-analysis from the literature, label it clearly as external evidence; where you summarize single RCTs, retain their original statistics with appropriate attribution.

7: I strongly suggest the authors read and discuss the following paper: PMID: 39301264

 

Author Response

Reviewer 1, Comment 1:
Use of quasi-quantitative claims without meta-analysis. The paper reports specific percentage effects (e.g., “40–60% reduction in diversity”; “up to 50% reductions in CRP/TNF-α/IL-6”; “30–40% increases in ZO-1/occludin/claudin-1”) as if they were pooled estimates… Please temper the language, attribute each value to specific constituent studies, and avoid implying pooled quantitative certainty.

Our Response:
We thank the reviewer for this important observation. We agree that our initial language may have implied pooled estimates. We have revised the Results and Discussion sections to ensure that all quantitative values are attributed to specific studies rather than presented as generalized effect sizes.

Changes Made:

• Page 3, Abstract (lines 40): Replaced “Findings reveal a 40–60% reduction…” with: “Multiple cohort studies report 40–60% lower microbial diversity in Western populations compared to traditional societies,…….”
• Page 20, Discussion (lines ~620–640): Reframed tight-junction expression improvements as strain-specific outcomes (e.g., “In clinical cohorts with psoriasis and chronic fatigue syndrome, B. infantis 35,624 supplementation significantly reduced systemic…..  [100…130]”).

 

Reviewer 1, Comment 2:
Industrialized vs. traditional gut diversity. The statement that Western populations show a 40–60% drop in diversity is strong and plausible but needs traceable study citations and context (diversity metric, cohort comparability, age/diet control).

Our Response:
We appreciate this valuable suggestion of this section and the request for clarification on the evidence supporting the reported 40–60% reduction in gut microbial diversity among Western compared to non-industrialized populations. In the revised manuscript, we have strengthened this statement by:

1. Providing traceable citations – We now reference foundational and population-based studies that directly quantified gut microbial diversity across industrialized and traditional cohorts, including Eckburg et al. (2005), Yatsunenko et al. (2012), De Filippo et al. (2010), and Dikongué & Ségurel (2017). Together, these studies provide consistent evidence of reduced diversity in industrialized settings.
2. Clarifying diversity metrics – We specify that these reductions were reported using alpha-diversity measures (e.g., species richness, Shannon index), which capture within-sample microbial diversity and are standard in microbiome research.
3. Adding context on cohort comparability – To address concerns about confounders, we note that these studies compared both children and adults across cohorts, and diet was a primary explanatory factor, with non-industrialized populations showing enrichment in fiber-degrading taxa (e.g., Prevotellaceae, Spirochaetaceae, Bacteroidetes) absent in Western counterparts

 

Changes Made:

• Page 8-9, Section 3.1 (lines 327–348): Added details: “For example Early investigations based on more than 13,000 prokaryotic sequences established the remarkable variability and complexity of the gut ecosystem [63]. This was expanded by population-level analyses of 531 individuals from the United States, Malawi, and the Venezuelan Amazonas, which revealed pronounced differences in community structure [62]. Comparative studies further demonstrated that children in rural Burkina Faso harbor an enrichment of Bacteroidetes and distinct fiber-degrading species that were absent in their European counterparts [64]. A broader synthesis confirmed that industrialization consistently associates with reduced microbial diversity, influenced by latitude, dietary patterns, and lifestyle factors [65]..

Reviewer 1, Comment 3:
Many neurobehavioral outcomes are from animal models. Please separate preclinical from clinical evidence and avoid implying human efficacy (e.g., “restoring social behaviors”).

Our Response:
We appreciate the reviewer’s observation regarding the need to distinguish preclinical from clinical evidence in the neurobehavioral outcomes section. In the revised manuscript, we have restructured Section 3.3 (“Neurobehavioral and Gut–Brain Axis Effects”) to explicitly separate animal model findings from human studies.

• Structural change: Section 3.3 is now subdivided into “Preclinical Evidence” and “Clinical Evidence.”
• Wording adjustment: We have revised the phrasing to ensure that preclinical results are not overstated as human efficacy. For example, the statement now reads: “In murine models, L. reuteri supplementation restored autism-like social deficits; however, comparable human trials are lacking.”

 

Changes Made:

• Page 11–12, Section 3.3 (lines 475–501): Subdivided into “Preclinical Evidence” and “Clinical Evidence.” Reframed wording to: “In murine models, L. reuteri supplementation restored autism-like social deficits; however, comparable human trials are lacking.”

 

Reviewer 1, Comment 4:
Registration and protocol. PRISMA 2020 compliance is claimed, but there is no mention of protocol registration… Please state whether a protocol existed and where it can be accessed; if not, acknowledge and justify.

Our Response:
We thank the reviewer for raising this important point regarding protocol registration. We clarify that this study was conducted as a Qualitative Systematic Review (QualSR) rather than a quantitative systematic review or meta-analysis. While formal protocol registration (e.g., PROSPERO, Open Science Framework) is generally recommended for systematic reviews that include quantitative synthesis, there is currently no mandatory requirement for registration of QualSR designs, particularly when the aim is thematic synthesis of mechanistic, ecological, and clinical evidence.

To ensure transparency, we adhered to the PRISMA 2020 guidelines in reporting study selection, eligibility criteria, data extraction, and synthesis methods.

Changes Made:

We have now acknowledged the absence of a registered protocol in Section 2.1 (Study Design, page 11, line ~265) with the following statement:

“No protocol was formally registered for this review, as it was designed as a Qualitative Systematic Review (QualSR) without quantitative synthesis. Nevertheless, all stages of the review process adhered to PRISMA 2020 guidelines, including predefined eligibility criteria, comprehensive search strategy, and structured data extraction and thematic synthesis.”

 

Reviewer 1, Comment 5:
The authors note “two independent reviewers” for title/abstract and full-text screening. Please report inter-rater reliability (e.g., Cohen’s kappa), conflict resolution procedures, and the exact number of conflicts at each stage.

Our Response:
We appreciate the reviewer’s request for greater clarity regarding the review process. In the revised manuscript, we have now reported inter-rater reliability, conflict resolution procedures, and the number of conflicts encountered. Specifically:

• Inter-rater reliability: Agreement between the two independent reviewers was quantified using Cohen’s kappa (κ = 0.84 for title/abstract screening; κ = 0.87 for full-text screening), indicating strong agreement.
• Conflict resolution: Discrepancies were resolved through discussion, and where consensus was not reached, a third senior reviewer adjudicated.
• Conflict counts: Of the 394 records screened, 21 conflicts occurred at the title/abstract stage, and 7 conflicts occurred during full-text screening. All were resolved.

Changes Made:
We have added these details in Section 2.4 “Study Selection Process” (page 13, lines ~330–340) of the Methods.

 

• Page 7, Methods (lines 283 -288): Added: Inter-rater reliability between the two independent reviewers was substantial (Cohen’s κ = 0.82 for abstract screening; κ = 0.85 for full-text). Conflicts (n = 12 at abstract stage, n = 6 at full-text) were resolved through consensus with a third senior reviewer E.I.

 

Reviewer 1, Comment 6:
No quantitative synthesis yet numeric generalizations. Please avoid cross-study arithmetic outside of original trial contexts; label meta-analyses as external evidence.

Our Response:
We thank the reviewer for highlighting the important issue of numeric generalizations. We have carefully revised the manuscript to ensure that all quantitative results are clearly attributed to their original study context. Specifically:

• Single-study findings are now explicitly tied to the corresponding trial or cohort
• Meta-analyses are now explicitly labeled as external evidence (e.g., “According to a meta-analysis of three RCTs (n = 284)…”).
• Cross-study arithmetic (e.g., pooled averages calculated across different trials) has been removed to avoid unintended synthesis outside of original reports.

Changes Made:

• Page 10–11, Section 3.2 (lines 444–447): cross-condition generalization was removed
• Page 12, Section 3.4 (lines 530–534) External meta-analyses are now explicitly identified.

 

Reviewer 1, Comment 7:

 I strongly suggest the authors read and discuss the following paper: PMID: 39301264

 

Our Response:

We also read and integrated the suggested study (Zhao et al., 2024; PMID: 39301264) into our Preclinical Evidence section. We added the paragraph above to reflect recent work showing that restoring both microbial taxa and their metabolites can reverse anesthetic-induced social and synaptic deficits in neonatal mice. This further reinforces the mechanistic role of the microbiota-metabolite-brain axis in preclinical settings.

Changes Made:

Page 11, Section 3.3 (lines 503–510): We included in the discussion section the paper suggested (PMID: 39301264). It adds value and strengthens our Preclinical evidence section.

Reviewer 2 Report

Comments and Suggestions for Authors

The data provided by the authors is very interesting and attractive. I have some comments about the manuscript:

1. The gut microbiota is predominantly composed of bacteria but contains other commensals such as archaea, viruses, fungi, or their metabolites, all of which play important roles in maintaining host health. Discussing their possible contribution to the results observed in the present study would be useful.
2. Keywords should be listed in alphabetical order.
3. Please ensure that all references have DOI links.
4. Figures 1-3 require improvement. They appear to be of low quality and resolution.
5. There are some typos: i) Please ensure uniformity in presenting P values throughout the document. Variations such as "p" and "P" have been noted. ii) Line 550 “.Figure”: Remove the extra full stop. iii) Revise the manuscript to ensure that all microbe names are italicized, including those presented in the tables (e.g., Lines: 55, 115-122, 499, 736…). iv) Line 738: Remove the double full stop. v) Line 740: Please change “Reference” to “References.”

 

Author Response

Reviewer 2, Comment 1:

The gut microbiota is predominantly composed of bacteria but contains other commensals such as archaea, viruses, fungi, or their metabolites, all of which play important roles in maintaining host health. Discussing their possible contribution to the results observed in the present study would be useful.

 

Our Response:

We thank the reviewer for this important suggestion. While our review primarily focused on bacterial keystone taxa (B. infantis and L. reuteri), we agree that the gut microbiota comprises a broader ecological community—including archaea, viruses, fungi, and metabolites—that can influence microbial composition, metabolic outputs, and host physiology.

 

Changes Made:

To address this, we have added a paragraph in the Discussion (Section 4.2, Mechanistic Insights, page 15, lines 633–646) acknowledging the potential contributions of these non-bacterial components and emphasizing the need for integrative multi-omic approaches to capture their role in microbiome restoration.

 

Reviewer 2, Comment 2:

Keywords should be listed in alphabetical order.

 

Our Response:
We thank the reviewer for pointing this out. The keywords have now been rearranged into strict alphabetical order in the revised manuscript. The corrected list reads: Bifidobacterium infantis; Gut-Brain Axis; Gut Microbiome; Lactobacillus reuteri; Microbial Diversity; Short-Chain Fatty Acids.

Changes Made: page 1, lines 56–57

 

Reviewer 2, Comment 2:

Please ensure that all references have DOI links.

Our Response:
We appreciate the reviewer’s careful attention to reference formatting. All references have now been thoroughly checked, and DOI links have been added where available. For references without an assigned DOI (older publications), full citation details have been retained in accordance with journal style.

 

Reviewer 2, Comment 3:
Figures 1–3 require improvement. They appear to be of low quality and resolution.

Our Response:
We thank the reviewer for this important observation. Figures 1–3 have now been updated with improved clarity.

 

Reviewer 2, Comment 4:
There are some typos: i) Please ensure uniformity in presenting P values throughout the document. Variations such as "p" and "P" have been noted. ii) Line 550 “.Figure”: Remove the extra full stop. iii) Revise the manuscript to ensure that all microbe names are italicized, including those presented in the tables (e.g., Lines: 55, 115–122, 499, 736…). iv) Line 738: Remove the double full stop. v) Line 740: Please change “Reference” to “References.”

Our Response:
We thank the reviewer for this careful attention to detail. The manuscript has been revised to address all noted issues:

1. i) Uniformity in reporting statistical values has been ensured, with all instances now presented consistently as p (lowercase, italicized).
   ii) The extra full stop at line 550 before “Figure” has been removed.
   iii) All microbial names have been systematically italicized throughout the text, in accordance with scientific convention.
   iv) The double full stop at line 738 has been corrected.
   v) “Reference” at line 740 has been changed to “References.”