Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I have read the review by Ciesielska-Markowska and collaborators with great interest. Overall, the article is well-organized and follows a rational flow. However, there are some issues that should be addressed: 

1) This seems to be a narrative review, although it is not clearly stated in the manuscript. Nonetheless, I'd suggest expanding on the literature search strategy adopted for this manuscript, as in its current state it is inadequately described. Particularly, what kind of articles were included? How did you manage the assessment of study quality, the evaluation of biases and potential disagreement among the authors? 

2) In its current form, the manuscript is largely descriptive and does not sufficiently integrate or critically evaluate the cited studies. In particular, inconsistencies across findings (e.g., regarding alpha diversity or taxa abundance in COPD) are noted but not explored in depth. A more analytical discussion addressing potential sources of heterogeneity, including differences in sampling methods, sequencing techniques, and patient populations would be highly beneficial for the reader.

3) Several conclusions imply causal relationships between microbiome composition and COPD progression or exacerbations, whereas the cited evidence is predominantly associative. The discussion should be revised to better reflect the limitations of current evidence. 

4) The manuscript does not sufficiently address key limitations of the existing literature, including small sample sizes, methodological variability, and contamination risks inherent to respiratory microbiome sampling. I'd suggest expanding on them and also including a column about limitations in Table 2. 

5) A paragraph on omic sciences could improve the overall discussion. I'd suggest looking at the following recent articles: DOI: 10.3390/biology15090684; DOI: 10.1088/1752-7163/ae2f95.

Author Response

Note from Authors: We thank the Editor and the Reviewers for the time and effort dedicated to the evaluation of our manuscript entitled “Characteristics of the respiratory microbiome in COPD – a literature review” (Manuscript ID: ARM-4228526). We greatly appreciate the insightful and constructive comments, which have helped us improve the clarity, structure, and overall quality of the manuscript. 

Comment 1: This seems to be a narrative review, although it is not clearly stated in the manuscript. Nonetheless, I'd suggest expanding on the literature search strategy adopted for this manuscript, as in its current state it is inadequately described. Particularly, what kind of articles were included? How did you manage the assessment of study quality, the evaluation of biases and potential disagreement among the authors?

Response 1: We are grateful to the Reviewer for pointing this out. The manuscript was intended as a narrative review rather than a systematic review, although this was not sufficiently emphasized in the original version. We chose the narrative review approach deliberately in order to provide an interdisciplinary overview of the respiratory microbiome, including not only biological and clinical aspects but also technical considerations related to sampling methods and their substantial methodological heterogeneity.
In response to the Reviewer’s suggestion, we have clarified the narrative nature of the review in the revised manuscript. Additionally, to avoid creating the impression of a systematic review methodology, we removed the subsection entitled “Data sources,” which could have been misleading. As the aim of the manuscript was to provide a broad expert overview rather than a systematic synthesis of evidence, formal assessment of study quality, risk of bias and inter-author disagreement resolution procedures were not performed.

Comment 2: In its current form, the manuscript is largely descriptive and does not sufficiently integrate or critically evaluate the cited studies. In particular, inconsistencies across findings (e.g., regarding alpha diversity or taxa abundance in COPD) are noted but not explored in depth. A more analytical discussion addressing potential sources of heterogeneity, including differences in sampling methods, sequencing techniques, and patient populations would be highly beneficial for the reader.

Response 2: We thank the Reviewer for this insightful comment. We agree that the heterogeneity of findings across studies on the respiratory microbiome in COPD is an important issue that warrants careful consideration. Differences in sampling strategies, sequencing approaches, bioinformatic processing and patient populations likely contribute to variability in reported outcomes, including alpha diversity and taxa abundance.
In the revised manuscript, we have further clarified and emphasized these sources of heterogeneity in the relevant sections to better guide the reader in interpreting the existing evidence. At the same time, given the broad and interdisciplinary scope of the review, we aimed to maintain a balanced focus on both methodological aspects and clinical relevance, avoiding overly extensive methodological digressions that could detract from the main narrative of the manuscript.

Comment 3: Several conclusions imply causal relationships between microbiome composition and COPD progression or exacerbations, whereas the cited evidence is predominantly associative. The discussion should be revised to better reflect the limitations of current evidence. 

Response 3: We would like to thank the Reviewer for this insightful comment. We agree that the currently available evidence regarding the relationship between respiratory microbiome composition and COPD progression or exacerbations is predominantly associative rather than causal. In the original version of the manuscript, some statements may have implied cause-and-effect relationships too strongly.
In response to this comment, we revised the relevant sections of the manuscript to use more cautious and precise language, emphasizing the associative nature of the available evidence and acknowledging the current limitations in establishing causality.

Comment 4:  The manuscript does not sufficiently address key limitations of the existing literature, including small sample sizes, methodological variability, and contamination risks inherent to respiratory microbiome sampling. I'd suggest expanding on them and also including a column about limitations in Table 2. 

Response 4: We are grateful for this important comment. We agree that limitations of the existing literature, including small sample sizes, methodological variability and the risk of contamination in respiratory microbiome sampling, are critical factors that influence interpretation of current findings.
In response to this suggestion, we have added a dedicated subsection (Section 2.3, “Microbiome methodology limitations”), in which these issues are now discussed in greater detail to better highlight their impact on study comparability and interpretation.
Regarding Table 2, we carefully considered the suggestion to include an additional column on limitations. However, given the already comprehensive nature and considerable length of the table, we aimed to preserve its readability and usability for the reader. Instead, we chose to consolidate and expand the discussion of methodological limitations within the dedicated subsection, which allows for a more coherent and contextualized presentation of these issues.

Comment 5: A paragraph on omic sciences could improve the overall discussion. I'd suggest looking at the following recent articles: DOI: 10.3390/biology15090684; DOI: 10.1088/1752-7163/ae2f95

Response 5: Thank you for this valuable suggestion. We agree that multi-omics approaches represent an important and rapidly developing direction in respiratory microbiome research, particularly in the context of COPD pathophysiology, biomarker discovery and personalized therapeutic strategies.
The Reviewer’s comment inspired us to further expand this aspect of the manuscript and contributed to the development of Chapter 5 (“Clinical implications for COPD and future directions in microbiome research”), where we added a dedicated discussion on omics sciences and multi-omics integration. We also included and discussed the suggested references, as they provide relevant insights into emerging methodological and translational aspects of the field.

Reviewer 2 Report

Comments and Suggestions for Authors

Peer Review Report

Manuscript Title:

Characteristics of respiratory microbiome in COPD – a literature review

1. General Assessment

This manuscript presents a narrative review of the respiratory microbiome in chronic obstructive pulmonary disease (COPD), with emphasis on sampling methodologies, microbial composition across airway regions, and associations with disease severity and exacerbations.

The topic is timely and clinically relevant, given the growing interest in host–microbiome interactions in respiratory diseases. The manuscript demonstrates a broad literature search and covers key domains including sampling techniques, regional microbiome differences, and clinical implications.

However, the manuscript requires moderate  revisions before it is suitable for publication. The main concerns relate to clarity of structure, methodological rigor (as a review), language quality, and critical synthesis of the literature.

2. Major Comments

2.1. Type of Review and Methodology

• The manuscript is presented as a “literature review,” but the methodology lacks sufficient rigor and transparency.
• The search strategy is insufficiently detailed:

• No inclusion/exclusion criteria
• No PRISMA framework or flow diagram
• No description of study selection process
• No quality assessment of included studies

Recommendation:

• Clarify whether this is a narrative review or systematic review.
• If systematic:

• Add PRISMA-compliant methodology
• Include a flowchart and selection criteria
  • If narrative:
• Explicitly state this and justify the approach

2.2. Lack of Critical Analysis

• The manuscript is largely descriptive rather than analytical.
• Many sections summarize findings without:

• Comparing conflicting studies
• Discussing limitations of evidence
• Identifying gaps in knowledge

Recommendation:

• Strengthen critical appraisal:

• Why do studies differ? (sampling, population, sequencing methods)
• What are the limitations of current microbiome research in COPD?
• What are the strongest vs weakest evidence areas?

2.3. Overly Long and Imbalanced Sections

• Section 3 (sampling methods) is very detailed and lengthy, while:

• Clinical implications (Section 5.4) are relatively brief
  • The manuscript reads more like a methods compendiumthan a clinically oriented review

Recommendation:

• Condense technical details of sampling
• Expand:

• Clinical relevance
• Translational implications
• Future directions

2.4. Redundancy and Repetition

• Several ideas are repeated across sections:

• Microbiome varies by airway region
• Sampling method influences results
• Reduced diversity linked to disease severity

Recommendation:

• Reduce redundancy and improve flow
• Merge overlapping concepts

2.5. Language and Grammar Issues

There are some grammatical and stylistic errors.

Recommendation:

• Thorough English editing is required

2.6. Figures and Tables

• Figure 1is referenced but not sufficiently described in the text
• Tables are useful but:

• Table 2 is overly dense and difficult to interpret
• Some abbreviations are repeated unnecessarily

Recommendation:

• Improve the quality of figure1  
• Simplify Table 2 or split into smaller tables

2.7. Novelty and Contribution

• The manuscript compiles existing knowledge but lacks a clear novel perspective
• No strong conceptual framework or model is proposed

Recommendation:

• Add a synthesis section, e.g.:

• Proposed model of COPD–microbiome interaction
• Clinical decision pathway based on microbiome
• Key research priorities

3. Minor Comments

3.1. Formatting Issues

• Inconsistent spacing and punctuation

3.2. Terminology

• Use consistent terminology:

• “microbiome” vs “microbial flora”
  • Avoid outdated terms like “flora”

3.3. Abbreviations

• Some abbreviations are introduced late or inconsistently used
• Ensure all abbreviations are defined at first use

4. Strengths

• Comprehensive coverage of:

• Sampling methodologies
• Regional microbiome differences
  • Clinically relevant topic with translational potential
  • Inclusion of both stable and exacerbation states
  • Broad and relatively up-to-date reference list

5. Weaknesses

• Lack of methodological rigor for a review
• Limited critical synthesis
• Language quality issues
• Imbalance between technical and clinical content
• Limited novelty

6. Recommendation

Decision: Moderate Revision

7. Specific Suggestions for Improvement
8. Clarify review type and strengthen methodology
9. Improve critical discussion and reduce descriptive repetition
10. Balance structure (reduce methods, expand clinical insights)
11. Revise language thoroughly
12. Enhance figures and tables clarity

Comments on the Quality of English Language

Language and Grammar Issues

There are some grammatical and stylistic errors.

Recommendation:

• Thorough English editing is required

Author Response

Note from Authors: We thank the Editor and the Reviewers for the time and effort dedicated to the evaluation of our manuscript entitled “Characteristics of the respiratory microbiome in COPD – a literature review” (Manuscript ID: ARM-4228526). We greatly appreciate the insightful and constructive comments, which have helped us improve the clarity, structure, and overall quality of the manuscript. 

Comment 2.1:  The manuscript is presented as a “literature review,” but the methodology lacks sufficient rigor and transparency.
The search strategy is insufficiently detailed:
No inclusion/exclusion criteria
No PRISMA framework or flow diagram
No description of study selection process
No quality assessment of included studies
Recommendation:

Clarify whether this is a narrative review or systematic review.
If systematic:
Add PRISMA-compliant methodology
Include a flowchart and selection criteria
If narrative:
Explicitly state this and justify the approach

Response 2.1: We thank the Reviewer for this important comment. We would like to clarify that the manuscript is a narrative review rather than a systematic review. This approach was chosen intentionally to allow for a broad, interdisciplinary synthesis of the respiratory microbiome literature in COPD, including biological, clinical, and methodological aspects, particularly given the substantial heterogeneity of available studies.
In line with this design, a formal systematic methodology (including PRISMA framework, predefined inclusion/exclusion criteria, study selection flow diagram, and risk-of-bias or quality assessment) was not applied, as these elements are not typically required for narrative reviews.
To improve transparency, we have revised the manuscript to explicitly state its narrative nature and to clarify the scope and methodology of the literature selection process.

Comment 2.2:  The manuscript is largely descriptive rather than analytical.
Many sections summarize findings without:
Comparing conflicting studies
Discussing limitations of evidence
Identifying gaps in knowledge
Recommendation:

Strengthen critical appraisal:
Why do studies differ? (sampling, population, sequencing methods)
What are the limitations of current microbiome research in COPD?
What are the strongest vs weakest evidence areas?

Response 2.2: We thank the Reviewer for this insightful comment. We agree that strengthening the critical synthesis of the literature is essential in narrative reviews. In the revised manuscript, we have enhanced the analytical depth by explicitly discussing sources of heterogeneity across studies, including differences in sampling methods, study populations, and sequencing approaches, which may contribute to variability in reported findings.
In addition, we have expanded the discussion of limitations in a dedicated subsection (Section 2.3, “Microbiome methodology limitations”), and further emphasized areas of stronger and weaker evidence within the clinical and translational discussion (Section 5, “Clinical implications for COPD and future directions in microbiome research”).
We also clarified the interpretation of comparative study data presented in Table 2 by highlighting methodological differences that may underlie observed inconsistencies between studies.

Comment 2.3: Section 3 (sampling methods) is very detailed and lengthy, while:
Clinical implications (Section 5.4) are relatively brief
The manuscript reads more like a methods compendiumthan a clinically oriented review
Recommendation:

Condense technical details of sampling
Expand:
Clinical relevance
Translational implications
Future directions

Response 2.3: We thank the Reviewer for this valuable comment. We agree that an appropriate balance between methodological detail and clinical interpretation is essential for readability and translational value.
In response to this suggestion, we have revised the manuscript to improve this balance. Specifically, we have condensed the section on microbiome sampling methodologies (Section 2), focusing on the most essential aspects relevant to interpretation of the literature, while reducing overly detailed technical descriptions.
At the same time, we have expanded the clinical and translational aspects of the manuscript. In particular, we have strengthened Section 4 and further developed the discussion of factors influencing microbiome composition in COPD, including lifestyle factors and exacerbations. Moreover, we have added a new Section 5 (“Clinical implications for COPD and future directions in microbiome research”), which provides a more comprehensive overview of translational perspectives and future research directions, as well as… (grafikę podsumowującą)
We believe these revisions have improved the overall balance between methodological and clinical content, in line with the Reviewer’s recommendation.

Comment 2.4: Several ideas are repeated across sections:
Microbiome varies by airway region
Sampling method influences results
Reduced diversity linked to disease severity
Recommendation:

Reduce redundancy and improve flow
Merge overlapping concepts

Response 2.4:  We thank the Reviewer for this helpful observation. We agree that some key concepts, including regional variability of the respiratory microbiome, the influence of sampling methodology on study outcomes, and the association between reduced microbial diversity and disease severity, were reiterated across different sections of the manuscript.
In the revised version, we have carefully reviewed the manuscript to reduce redundancy and improve the overall flow of the narrative. Repeated statements have been consolidated where appropriate, and overlapping content has been merged or streamlined to ensure that each concept is introduced and discussed in a single, more structured context, with subsequent sections referring back to the relevant discussion where needed.
We believe these revisions have improved the coherence and readability of the manuscript while preserving all key scientific content.

Comment 2.5: There are some grammatical and stylistic errors.

Recommendation:

Thorough English editing is required

Response 2.5: In response, the manuscript has undergone thorough language revision, including careful correction of grammatical and stylistic issues and refinement of sentence structure throughout the text to improve clarity and readability. We believe these revisions have enhanced the overall linguistic quality of the manuscript. 

Comment 2.6: Figure 1 is referenced but not sufficiently described in the text
Tables are useful but:
Table 2 is overly dense and difficult to interpret
Some abbreviations are repeated unnecessarily
Recommendation:

Improve the quality of figure1  
Simplify Table 2 or split into smaller tables

Response 2.6: We thank the Reviewer for this helpful comment. We have improved the presentation and description of Figure 1 by adding a clearer reference and explanatory text in the manuscript, explicitly introducing the figure and summarizing that it illustrates the most commonly reported bacterial taxa across different regions of the respiratory tract. We believe this has improved the clarity and interpretability of the figure.
Regarding Table 2, we agree that it contains a large amount of detailed information, as it is intended to provide a comprehensive overview of included studies. In the revised manuscript, we have carefully reviewed the table to improve clarity and ensured consistent presentation. At the same time, we aimed to preserve its comprehensiveness and utility as a reference resource for readers, and therefore did not further subdivide the table.

Comment 2.7: The manuscript compiles existing knowledge but lacks a clear novel perspective
No strong conceptual framework or model is proposed
Recommendation:

Add a synthesis section, e.g.:
Proposed model of COPD–microbiome interaction
Clinical decision pathway based on microbiome
Key research priorities

Response 2.7: We thank the Reviewer for this insightful comment. We agree that narrative reviews benefit from providing a clear synthesis and forward-looking perspective to guide future research and clinical translation.
In the revised manuscript, we have strengthened the synthesis aspect of the work by further emphasizing key research priorities and future directions in Section 5 (“Clinical implications for COPD and future directions in microbiome research”), where we consolidate current evidence gaps and highlight priority areas for future investigation.
At the same time, we respectfully note that the aim of this review was to provide a comprehensive and evidence-based overview of current knowledge on the respiratory microbiome in COPD rather than to propose a formal predictive model or clinical decision algorithm, which would require a level of standardization and validation beyond the scope of the available literature. We therefore focused on synthesizing existing evidence and identifying research priorities within the current state of the field.

Comments 3.1, 3.2, 3.3: 

• 3.1. Formatting Issues

Inconsistent spacing and punctuation

• 3.2. Terminology

Use consistent terminology:
“microbiome” vs “microbial flora”
Avoid outdated terms like “flora”

• 3.3. Abbreviations

Some abbreviations are introduced late or inconsistently used
Ensure all abbreviations are defined at first use

Responses 3.1, 3.2, 3.3: We thank the Reviewer for these technical comments. We have carefully revised the manuscript to address all formatting and language consistency issues. Specifically, we have corrected spacing and punctuation throughout the text, standardized terminology by consistently using “microbiome” and removing outdated terms such as “microbial flora,” and reviewed all abbreviations to ensure they are defined at first use and used consistently across the manuscript. These revisions have improved the overall consistency and readability of the manuscript. 

Comments 4, 5:

• Comment 4:

Comprehensive coverage of:
Sampling methodologies
Regional microbiome differences
Clinically relevant topic with translational potential
Inclusion of both stable and exacerbation states
Broad and relatively up-to-date reference list

• Comment 5:

Lack of methodological rigor for a review
Limited critical synthesis
Language quality issues
Imbalance between technical and clinical content
Limited novelty

Response 4, 5: We thank the Reviewer for the overall evaluation of our manuscript and for highlighting both its strengths and areas requiring improvement. We appreciate the recognition of the comprehensive scope of the review, including coverage of sampling methodologies, regional microbiome differences, clinical relevance, inclusion of both stable and exacerbation states, and the breadth of the literature base.
We also acknowledge the concerns raised regarding methodological clarity, critical synthesis, balance between technical and clinical content, language quality, and novelty. All of these points have been carefully addressed during revision, as detailed in our point-by-point responses above. In particular, we have clarified the narrative nature of the review and its methodological scope, improved the balance between methodological and clinical sections, strengthened the critical synthesis, reduced redundancy, and substantially revised the language for clarity and consistency. Figures and tables have also been reviewed to improve readability and presentation.
We believe that these revisions have significantly improved the clarity, structure, and overall quality of the manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revision has much improved the paper. It is now suitable for publication. 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have adequately addressed all my comments, significantly improving the quality of the manuscript.