From Culture to Metagenomics: How Methodological Advances Reshape Our Understanding of the Oral Microbiota of Venomous Snakes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review addresses an important and underrepresented topic in microbiology: the oral microbiota of venomous snakes and its clinical relevance. The manuscript is valuable for its clear historical overview, integration of ecological and clinical perspectives, identification of research gaps, and discussion of future opportunities.

A few suggestions for improvement:

1. Language and Style: Some sentences are long or repetitive. Shortening them and varying connectors (e.g., “however”, “furthermore”) would improve clarity and flow.

2. Redundancy: Certain phrases could be simplified to avoid repetition.

3. Terminology: Minor revisions could enhance precision (e.g., replace “describes the rendrance of snakebite envenomation” with “highlights the burden of snakebite envenomation”).

4. Figures and Workflows: The figures are helpful; a summary figure showing the transition from culture-based to metagenomic approaches with clinical implications would add clarity.

Author Response

We would like to sincerely thank the reviewers for the time and effort invested in evaluating our manuscript, as well as for the constructive comments and valuable suggestions that have greatly helped us improve the quality and clarity of the article.

 1. Language and Style: Some sentences were long or repetitive. Shortening them and varying connectors (e.g., “however”, “furthermore”) would improve clarity and flow.

In accordance with these suggestions, we revised the manuscript to improve clarity and flow by shortening overly long sentences and varying the use of connectors such as “however” and “furthermore.”

2. Redundancy: Certain phrases could be simplified to avoid repetition.

We also addressed redundancies by simplifying certain phrases to avoid repetition and ensure more concise expression of ideas.

3. Terminology: Minor revisions could enhance precision (e.g., replace “describes the rendrance of snakebite envenomation” with “highlights the burden of snakebite envenomation”).

As recommended, we revised terminology for greater precision, including replacing the phrase “describes the rendrance of snakebite envenomation” with “highlights the burden of snakebite envenomation.”

4. A comparative methodology table was created and added to the introduction.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Major Comments

1. The logic and focus of the Introduction should be strengthened.
   The current introduction covers a broad range of topics, but the logical flow could be improved. The discussion on the psychological impacts of snakebite (lines 70-81), while relevant to overall patient care, is not directly related to the core theme of methodological evolution. It is suggested to remove or significantly condense this part to maintain a tighter focus. Furthermore, the transition between the introduction of culture-independent methods as a broader trend (lines 63-69) and the subsequent text is somewhat disrupted. It is recommended to reorganize the narrative to clearly establish the limitations of traditional methods and the consequent necessity of molecular approaches, culminating in a clear rationale for the pivotal role of sequencing technologies, thereby better introducing the content of Section 4.
2. Claims about pathogenic bacteria require explicit literature support.
   The statement (lines 110-113) that Enterobacter, Proteus, Morganella morganii, and Aeromonas hydrophilaare frequently found in infected wounds and imply their clinical significance should be directly supported by citations demonstrating their pathogenicity and association with severe outcomes. If strong supportive evidence is lacking, the phrasing should be moderated to reflect uncertainty, or this section could be merged with the subsequent paragraph.
3. A transitional paragraph is needed between Sections 3 and 4.
   The shift from Section 3 (Early Studies) to Section 4 (Transition to Molecular Techniques) is somewhat abrupt. It is recommended to add a concise transitional paragraph at the end of Section 3 or the beginning of Section 4. This paragraph should summarize the key limitations of culture-based methods and logically introduce how molecular techniques overcome these limitations to provide a more comprehensive understanding of microbial diversity.
4. A dedicated section on clinical translation is recommended.
   The potential clinical implications of microbiome research (e.g., guiding antibiotic therapy, antivenom development) are mentioned scattered throughout the manuscript (e.g., Introduction, Complications, Conclusion). To deepen the discussion, it is suggested to add a new section entitled "Clinical Translation and Therapeutic Implications" before Section 7 (Research Gaps). This section should cohesively discuss how sequencing data can be translated into practice, for instance, by building regional databases of microbial profiles and antimicrobial resistance genes to inform empirical antibiotic guidelines, and by exploring microbiome-venom interactions for antivenom improvement.
5. The Conclusion should be reframed to synthesize findings beyond methodological recap.
   The current conclusion primarily recaps the chronological evolution of the technologies used. It should be revised to provide a higher-level synthesis of the key scientific insights gained (e.g., characteristics of the snake oral microbiome, complexity of microbiota-venom-host interactions). The first paragraph should summarize main findings; the second should elaborate on their ecological, clinical, and bioprospecting significance; the final paragraph can outline future interdisciplinary research directions. The technological evolution should serve as the context, not the focus.

 

Minor Comments

6. Repetitive use of the word "complex" should be avoided.
   The term "complex" is used repeatedly in the Abstract and Introduction (e.g., "complex secondary infections," "complex interactions"). Consider using synonyms such as "severe," "intricate," "multifaceted," or "heterogeneous" to enhance linguistic variety.
7. Logical connection between sentences can be strengthened.
   In lines 158-161, the sentence beginning "This selectivity will lead..." appears slightly abrupt. The connection to the previous sentence discussing the bias for aerobic and fast-growing bacteria can be improved by adding a logical connector like "Consequently," or "As a result," at the beginning.
8. Figure labels should be formatted correctly.
   In Figure 2, Change "1500pb" to "1500 bp"，Change "460pb" to "460 bp".
9. Tense should be consistent when describing established research findings.
   In lines 310-316, a mix of present perfect ("has been found") and past tense ("concluded", "was") is used to describe research findings. When stating generally accepted conclusions, the present tense is preferred: E.g., "Research finds that captivity has... and concludes that the intestinal bacterial community is...".

Author Response

Dear Reviewer,

Thank you very much for your valuable comments and suggestions. All text highlighted in blue has been revised and adjusted in accordance with the recommendations to enhance the manuscript’s clarity, focus, and scientific rigor.

The logic and focus of the Introduction should be strengthened.

• The Introduction was tightened by removing the section on psychological impacts and reorganized to first highlight the limitations of traditional methods and then justify the adoption of molecular approaches, providing a smooth transition into Section 4.

Claims about pathogenic bacteria require explicit literature support.
The statement (lines 110-113) that Enterobacter, Proteus, Morganella morganii, and Aeromonas hydrophilaare frequently found in infected wounds and imply their clinical significance should be directly supported by citations demonstrating their pathogenicity and association with severe outcomes. If strong supportive evidence is lacking, the phrasing should be moderated to reflect uncertainty, or this section could be merged with the subsequent paragraph.

• Specific citations have been added to support the pathogenic roles of Enterobacter, Proteus, Morganella morganii, and Aeromonas hydrophila in snakebite wounds, ensuring rigor behind those clinical claims.

A transitional paragraph is needed between Sections 3 and 4.
The shift from Section 3 (Early Studies) to Section 4 (Transition to Molecular Techniques) is somewhat abrupt. It is recommended to add a concise transitional paragraph at the end of Section 3 or the beginning of Section 4. This paragraph should summarize the key limitations of culture-based methods and logically introduce how molecular techniques overcome these limitations to provide a more comprehensive understanding of microbial diversity.

• A transitional paragraph was inserted at the end of Section 3 summarizing key deficiencies of culture-based methods and introducing molecular techniques as a solution for a more comprehensive view of microbial diversity.

A dedicated section on clinical translation is recommended.
The potential clinical implications of microbiome research (e.g., guiding antibiotic therapy, antivenom development) are mentioned scattered throughout the manuscript (e.g., Introduction, Complications, Conclusion). To deepen the discussion, it is suggested to add a new section entitled "Clinical Translation and Therapeutic Implications" before Section 7 (Research Gaps). This section should cohesively discuss how sequencing data can be translated into practice, for instance, by building regional databases of microbial profiles and antimicrobial resistance genes to inform empirical antibiotic guidelines, and by exploring microbiome-venom interactions for antivenom improvement.

• A new section, “Clinical Translation and Therapeutic Implications,” was added

The Conclusion should be reframed to synthesize findings beyond methodological recap.
The current conclusion primarily recaps the chronological evolution of the technologies used. It should be revised to provide a higher-level synthesis of the key scientific insights gained (e.g., characteristics of the snake oral microbiome, complexity of microbiota-venom-host interactions). The first paragraph should summarize main findings; the second should elaborate on their ecological, clinical, and bioprospecting significance; the final paragraph can outline future interdisciplinary research directions. The technological evolution should serve as the context, not the focus.

• The Conclusion was completely rewritten to synthesize the main scientific insights into snake oral microbiomes, emphasize their ecological, clinical, and bioprospecting significance, and outline future interdisciplinary research directions—using methodological evolution only as contextual background.

Minor Comments

Repetitive use of the word "complex" should be avoided.
The term "complex" is used repeatedly in the Abstract and Introduction (e.g., "complex secondary infections," "complex interactions"). Consider using synonyms such as "severe," "intricate," "multifaceted," or "heterogeneous" to enhance linguistic variety.

• The repeated use of “complex” was replaced with synonyms such as “severe,” “intricate,” “multifaceted,” and “heterogeneous” to improve lexical variety.

Figure labels should be formatted correctly.
In Figure 2, Change "1500pb" to "1500 bp"，Change "460pb" to "460 bp".

• Figure 2 labels “1500pb” and “460pb” were corrected to “1500 bp” and “460 bp.”

Tense should be consistent when describing established research findings.
In lines 310-316, a mix of present perfect ("has been found") and past tense ("concluded", "was") is used to describe research findings. When stating generally accepted conclusions, the present tense is preferred: E.g., "Research finds that captivity has... and concludes that the intestinal bacterial community is...".

• Verb tenses were standardized to the present tense when describing established findings, ensuring temporal consistency throughout the manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Please see my comments in the attached file. Thank you.

 

Comments for author File: Comments.pdf

Author Response

Dear reviewer,

Thank you for your insightful comments. We have carefully addressed each suggestion as follows:

• We included a clear definition of ASVs and OTUs, explaining how ASVs provide single-nucleotide resolution and OTUs cluster sequences by similarity thresholds.
• We corrected the text in lines 241-242 to remove any reference to venom detection, clarifying that these techniques identify microbial DNA only.
• We enriched with detailed mechanisms by which venom components select for resistant microbial strains, citing studies on venom antimicrobial activity, pH effects, and proteolytic enzyme interactions.

• All instances of “16S rRNA sequencing” have been replaced with “16S rRNA gene sequencing” to ensure precise terminology throughout the manuscript.

You can see in blue text all the paragraphs that were revised and to which relevant information was added to complement the manuscript.

Author Response File: Author Response.docx