Antimicrobial Compounds from Anaerobic Microorganisms: A Review of an Untapped Reservoir

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear colleagues!

I would like to note the importance of such research.

But I have the following comments:

1. “Antimicrobial Compounds from Anaerobic Microorganisms” — did you mean obligate or facultative?

If both, the material should be expanded. If only obligate, then shortened.

Or better think about how to rename the article

2. Please pay more attention to the structure of the article, for example: there is a paragraph 2.1. and then it should be 2.1.1., 2.1.2.

3. I would like to make some changes to the structure of the article - all clostridia should be put in a separate subsection

4. I did not see the materials and methods section. Where did you get the information from and how did you “filter” it?

5. 139 Clostridium species →Clostridium spp.

6. 149 Clostridium →Clostridium spp. or Clostridium sp.

7. 188 “This research represents the first characterization of a bacteriocin from toxigenic group I C. botulinum, offering insights into its genetics and potential use against clostridial pathogens.” - What is “this study”? Add a link

8. The links placed in the text partially do not correspond to those indicated in the reference, for example:

“Three strains of C. beijerinckii, known for industrial solvent production, were found to produce new congeners of sattazolin and a novel acyloin named clostrocyloin. The sattazolin compounds exhibited antibacterial activity against Mycobacteria and Pseudomonads with minimal cytotoxicity, suggesting potential as antimicrobial agents [37].” →

37. Wang T, Fu J, Xiao X1 et al. CBP22, a Novel Bacteriocin Isolated from Clostridium butyricum ZJU-F1, Protects against LPS-546 Induced Intestinal Injury through Maintaining the Tight Junction Complex. Hindawi Mediators of Inflammation. 2021. doi: 547 10.1155/2021/8032125

у статті йдеться про “....CBP22 showed activity against E. col K88, E. coli ATCC25922, and S. aureus ATCC26923.”

9. Almost half of the sources used are outdated — more than 10 years old — this is unacceptable.

10. Check the correctness of the reference design according to the journal's requirements.

Best regards,

Reviewer

Author Response

Dear colleagues!

I would like to note the importance of such research.

Thank you very much for your thoughtful and constructive feedback. We appreciate your recognition of the importance and timeliness of our review on antimicrobial compounds from anaerobic microorganisms.

But I have the following comments:

1. “Antimicrobial Compounds from Anaerobic Microorganisms” — did you mean obligate or facultative?

If both, the material should be expanded. If only obligate, then shortened.

Or better think about how to rename the article

Response: We thank the reviewer for this helpful suggestion. Based on this comment, we have revised the manuscript to focus exclusively on antimicrobial compounds derived from obligate anaerobes. Accordingly, we have removed the previous subsection on antimicrobial peptides from lactic acid bacteria (facultative anaerobes).

2. Please pay more attention to the structure of the article, for example: there is a paragraph 2.1. and then it should be 2.1.1., 2.1.2.

Response: We thank the reviewer for pointing this out. We have revised and standardized the section and subsection numbering throughout the manuscript to ensure a clear and consistent hierarchical structure.

3. I would like to make some changes to the structure of the article - all clostridia should be put in a separate subsection

Response: Thank you for this helpful suggestion. In response, we have created a dedicated subsection titled “4.1 Bacteriocins or AMPs from Clostridium species” to consolidate and clearly present all antimicrobial peptides and bacteriocins derived from Clostridium spp.

4. I did not see the materials and methods section. Where did you get the information from and how did you “filter” it?

Response: Thank you for raising this point. We have now incorporated a clear description of our literature search strategy in the revised text. Specifically, the manuscript now includes the following at line no. 91:

“We conducted a non-systematic literature review using databases such as PubMed, Scopus, and Google Scholar. No specific time restriction was applied; studies were included regardless of publication date, provided they offered relevant insights into antimicrobial compounds produced by anaerobic bacteria. Keywords used during the search included ‘anaerobic bacteria,’ ‘antibiotic,’ ‘bacteriocins,’ ‘secondary metabolites,’ ‘biosynthetic gene clusters (BGCs),’ and ‘antimicrobial peptides.’ Only peer-reviewed publications in English were considered. Priority was given to studies that included experimental validation (e.g., antimicrobial activity assays), genomic or bioinformatic characterization of BGCs.”

 

5. 139 Clostridium species →Clostridiumspp.

Response: Thank you for pointing this out. We have thoroughly reviewed the manuscript and corrected any naming inconsistencies in accordance with taxonomic conventions.

6. 149 Clostridium→Clostridium spp. or Clostridium sp.

Thank you for pointing this out. We have thoroughly reviewed the manuscript and corrected any naming inconsistencies in accordance with taxonomic conventions.

7. 188 “This research represents the first characterization of a bacteriocin from toxigenic group I C. botulinum, offering insights into its genetics and potential use against clostridial pathogens.” - What is “this study”? Add a link

Response: We thank the reviewer for pointing this out. In the revised manuscript, we have significantly condensed and combined the sections discussing Boticin B and P to improve clarity and avoid redundancy. As a result, the specific sentence in question has been removed. We believe the updated version presents the relevant information more clearly without loss of essential content.

8. The links placed in the text partially do not correspond to those indicated in the reference, for example:

“Three strains of C. beijerinckii, known for industrial solvent production, were found to produce new congeners of sattazolin and a novel acyloin named clostrocyloin. The sattazolin compounds exhibited antibacterial activity against Mycobacteria and Pseudomonads with minimal cytotoxicity, suggesting potential as antimicrobial agents [37].” →

37. Wang T, Fu J, Xiao X1 et al. CBP22, a Novel Bacteriocin Isolated from Clostridium butyricumZJU-F1, Protects against LPS-546 Induced Intestinal Injury through Maintaining the Tight Junction Complex. Hindawi Mediators of Inflammation. 2021. doi: 547 10.1155/2021/8032125

у статті йдеться про “....CBP22 showed activity against E. col K88, E. coli ATCC25922, and S. aureus ATCC26923.”

Response: Thank you for pointing out this inconsistency. We have carefully reviewed the reference and replaced it with the correct citation that accurately corresponds to the content described in the text.

9. Almost half of the sources used are outdated — more than 10 years old — this is unacceptable.

Response: We appreciate the reviewer’s concern regarding the age of some references. Antimicrobial discovery from anaerobic microorganisms is a relatively underexplored and neglected area, and many of the foundational studies in this field were published over a decade ago. These earlier works remain relevant due to the limited number of follow-up studies and the lack of newer alternatives. That said, we have carefully reviewed and updated the reference list to include recent publications wherever possible, particularly for genome mining tools, biosynthetic gene cluster (BGC) characterization, and advances in heterologous expression systems.

 

10. Check the correctness of the reference design according to the journal's requirements.

Response: Thank you for this reminder. We have carefully reviewed and revised the reference formatting throughout the manuscript to ensure it complies with the journal’s guidelines.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Summary

This comprehensive review examines antimicrobial compounds produced by anaerobic microorganisms, addressing an important and timely topic given the growing antimicrobial resistance crisis. While the authors provide extensive coverage of anaerobic-derived antimicrobials, the manuscript would benefit from improved organization, more critical analysis, and enhanced presentation quality.

Comments

1. The authors did not provide sufficient critical evaluation of the antimicrobial compounds discussed. I suggest that the authors include comparative analyses of efficacy, discuss limitations of each compound class, and provide more balanced coverage of both successes and failures in development.
2. The authors did not adequately address the translational aspects of these compounds from laboratory to clinical applications. I recommend that the authors discuss regulatory hurdles, clinical trial status, and commercialization challenges for anaerobic-derived antimicrobials.
3. The authors did not provide systematic comparison of antimicrobial activities across different compound classes. I suggest that the authors include comparative tables showing MIC values, spectrum of activity, and other quantitative parameters to help readers assess relative efficacy.
4. The authors did not provide specific, actionable recommendations for future research directions. I suggest that the authors include a dedicated section outlining key research gaps, technological needs, and strategic priorities for the field.
5. The authors did not maintain consistent organizational structure throughout the review. I recommend that the authors restructure sections to follow a more logical flow, perhaps organizing by mechanism of action or therapeutic potential rather than by producing organism.
6. The authors did not maintain consistent naming conventions for bacterial species and compounds. I suggest that the authors standardize nomenclature throughout the manuscript according to current taxonomic standards.
7. The authors did not adequately reference relevant databases and bioinformatics resources. I suggest that the authors include more discussion of available genomic databases, BGC prediction tools, and compound databases relevant to anaerobic antimicrobials.

Author Response

This comprehensive review examines antimicrobial compounds produced by anaerobic microorganisms, addressing an important and timely topic given the growing antimicrobial resistance crisis. While the authors provide extensive coverage of anaerobic-derived antimicrobials, the manuscript would benefit from improved organization, more critical analysis, and enhanced presentation quality.

Response: Thank you very much for your thoughtful and constructive feedback. We appreciate your recognition of the importance and timeliness of our review.

Comments

1. The authors did not provide sufficient critical evaluation of the antimicrobial compounds discussed. I suggest that the authors include comparative analyses of efficacy, discuss limitations of each compound class, and provide more balanced coverage of both successes and failures in development.

Response: Thank you for this valuable suggestion. In the revised manuscript, we have included a basic introduction to each class of antimicrobial compounds where we discuss their limitations and challenges. Additionally, we have added comprehensive tables (Table 2 and 3) that provides a comparative analysis of their efficacy and other key attributes.

1. The authors did not adequately address the translational aspects of these compounds from laboratory to clinical applications. I recommend that the authors discuss regulatory hurdles, clinical trial status, and commercialization challenges for anaerobic-derived antimicrobials.

Response: Thank you for this important comment. To better address the translational aspects of anaerobic-derived antimicrobials, we have added a new dedicated section titled “8.0 Translational Challenges in Clinical Development of Anaerobic-Derived Antimicrobials.”

1. The authors did not provide systematic comparison of antimicrobial activities across different compound classes. I suggest that the authors include comparative tables showing MIC values, spectrum of activity, and other quantitative parameters to help readers assess relative efficacy.

Response: Thank you for this helpful suggestion. In response, we have included a comprehensive comparative tables (Table 2 and 3)  that systematically presents key quantitative parameters such as MIC values, spectrum of activity, and other relevant efficacy metrics across different classes of antimicrobial compounds.

1. The authors did not provide specific, actionable recommendations for future research directions. I suggest that the authors include a dedicated section outlining key research gaps, technological needs, and strategic priorities for the field.

Response: Thank you for this valuable recommendation. We have improved the introduction to better highlight existing research gaps in the field. Additionally, we have added a dedicated section, “8.0 Translational Challenges in Clinical Development of Anaerobic-Derived Antimicrobials,” to discuss key hurdles and needs. Finally, we have expanded the conclusions to include a clear “Future Research Directions” that outlines specific, actionable recommendations addressing critical gaps, technological needs, and strategic priorities.

1. The authors did not maintain consistent organizational structure throughout the review. I recommend that the authors restructure sections to follow a more logical flow, perhaps organizing by mechanism of action or therapeutic potential rather than by producing organism.

Response: Thank you for this valuable suggestion. In response, we have restructured the manuscript to organize antimicrobial compounds into distinct sections based on their structural classes, rather than by producing organisms. The revised sections now include: antibiotics, bacteriocins, thiopeptides, glycopeptides, and lipopeptides. This structure improves clarity and facilitates comparative analysis across compound types in terms of their biosynthetic pathways, mechanisms of action, and potential therapeutic applications.

1. The authors did not maintain consistent naming conventions for bacterial species and compounds. I suggest that the authors standardize nomenclature throughout the manuscript according to current taxonomic standards.

Response: Thank you for pointing this out. We have thoroughly reviewed the manuscript and corrected any naming inconsistencies in accordance with taxonomic conventions.

 

1. The authors did not adequately reference relevant databases and bioinformatics resources. I suggest that the authors include more discussion of available genomic databases, BGC prediction tools, and compound databases relevant to anaerobic antimicrobials.

Response: Thank you for this helpful comment. In response, we have added a dedicated section titled “2.2 Genome Mining and Bioinformatic Tools” in the revised manuscript. This section discusses key tools such as antiSMASH, BAGEL4, and PRISM, as well as curated repositories including MIBiG, NPASS, NPAtlas, CAMP, and APD. These resources are now referenced and described in the context of predicting and characterizing biosynthetic gene clusters (BGCs) and antimicrobial compounds from anaerobic microorganisms.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The proposed work constitutes a clear and readable review of new bioactive compounds present in anaerobic microorganisms. The novelty of the review could be key for the development of new antimicrobial drugs and the implementation of targeted therapeutic strategies, as the authors mention in a couple of examples.

While this review includes specific examples of compounds with these properties, the reader may find this information somewhat scattered throughout the manuscript. This reviewer strongly suggests the use of at least one Table where all of this information is assembled, and includes the name of each agent, its molecular mass (when known), the nature of each biomolecule (peptide, lipopeptide, glycopeptide, etc.), antagonistic activity against specific pathogens, possible mechanisms of action (when known from the literature), possible toxic or undesirable effects, etc.

On the other hand, the interest of these bioactive compounds is in itself of great relevance from a genetic perspective, since these biocompounds are found forming biosynthetic gene clusters (BGCs), however, the genomic organization of these in specific examples is not clear. Although Figures 2, 3, 4, and 5 attempt to give a general idea of this genomic information, it is only schematic. The authors could perhaps access sequence banks for specific BGCs and indicate in a little more detail the organization and expression of these clusters for the various compounds mentioned in the text. This would allow access to genomic information of great value to the readers.

Finally, would it be possible to include a brief section on the efforts that have been made to maintain anaerobic microorganisms in culture in the laboratory? Although this is surely complicated, I am sure that some effort has also been made to directly obtain such compounds; this should be mentioned somewhere in this work.

 

Author Response

The proposed work constitutes a clear and readable review of new bioactive compounds present in anaerobic microorganisms. The novelty of the review could be key for the development of new antimicrobial drugs and the implementation of targeted therapeutic strategies, as the authors mention in a couple of examples.

Response: We sincerely thank the reviewer for the positive feedback and thoughtful evaluation. We are pleased that the novelty and clarity of the review were appreciated.

1. While this review includes specific examples of compounds with these properties, the reader may find this information somewhat scattered throughout the manuscript. This reviewer strongly suggests the use of at least one Table where all of this information is assembled, and includes the name of each agent, its molecular mass (when known), the nature of each biomolecule (peptide, lipopeptide, glycopeptide, etc.), antagonistic activity against specific pathogens, possible mechanisms of action (when known from the literature), possible toxic or undesirable effects, etc.

Response: Thank you for this helpful suggestion. In response, we have included a comprehensive comparative table (Table 2 and 3) that systematically presents key quantitative parameters such as MIC values, spectrum of activity, and other relevant efficacy metrics across different classes of antimicrobial compounds.

 

2. On the other hand, the interest of these bioactive compounds is in itself of great relevance from a genetic perspective, since these biocompounds are found forming biosynthetic gene clusters (BGCs), however, the genomic organization of these in specific examples is not clear. Although Figures 2, 3, 4, and 5 attempt to give a general idea of this genomic information, it is only schematic. The authors could perhaps access sequence banks for specific BGCs and indicate in a little more detail the organization and expression of these clusters for the various compounds mentioned in the text. This would allow access to genomic information of great value to the readers.

Response: We thank the reviewer for this thoughtful and constructive suggestion. In response, we have thoroughly revised the manuscript to include genomic and biosynthetic details for all compounds where such information is currently available. For key examples, we now provide descriptions of BGC size, gene arrangement, major biosynthetic and regulatory genes, and expression mechanisms (also summarized in Table 1). Where applicable, we have included MIBiG accession numbers and cited relevant genomic or functional studies. To make this information easily accessible, we have also added a summary table listing these details for each compound.

 

3. Finally, would it be possible to include a brief section on the efforts that have been made to maintain anaerobic microorganisms in culture in the laboratory? Although this is surely complicated, I am sure that some effort has also been made to directly obtain such compounds; this should be mentioned somewhere in this work.

 Response: We thank the reviewer for this important and thoughtful suggestion. In response, we have added a new subsection titled “2.1 Cultivation Strategies for Anaerobic Bacteria” in the revised manuscript. This section outlines traditional and emerging methods for cultivating obligate anaerobes in the laboratory. In addition, we have expanded the surrounding sections to describe modern genome mining tools (Section 2.2) and heterologous expression platforms (Section 2.3) that complement cultivation-based strategies for accessing cryptic biosynthetic gene clusters. These additions provide a more complete picture of how both cultivation and molecular approaches are being used to obtain antimicrobial compounds from anaerobic microbes.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear colleagues!

You must have done a good job improving the article, but I find it difficult to assess.

Please format the document and send it in a readable format. I cannot read it in its current form.

Best regards,

Reviewer

Comments for author File: Comments.odt

Author Response

Dear colleagues!

You must have done a good job improving the article, but I find it difficult to assess.

Please format the document and send it in a readable format. I cannot read it in its current form.

Response: Thank you for your message. We are sorry to hear that the file was unreadable. To ensure you have no trouble accessing it, the correctly formatted version is reattached.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

Dear colleagues!

It should be noted that the authors put a lot of effort into writing this article. However, I still note the somewhat unusual format of presenting the material, but if it is acceptable for the journal, then everything is OK.

Best regards,

Reviewer

Author Response

Comment: It should be noted that the authors put a lot of effort into writing this article. However, I still note the somewhat unusual format of presenting the material, but if it is acceptable for the journal, then everything is OK.

Response: We sincerely thank the reviewer for their kind feedback and appreciation of our efforts. Regarding the comment on the format, we acknowledge that the structure is somewhat layered due to the diversity of antimicrobial classes and producing organisms discussed. We aimed to present the content in a structured and accessible manner that reflects the diversity and complexity of anaerobe-derived antimicrobials. The format was further refined based on earlier reviewer suggestions. We are pleased that the current format is acceptable and remain open to any further guidance from the editorial team.