First Animal Source Metagenome Assembly of Lawsonella clevelandensis from Canine External Otitis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for the opportunity to review the paper "First Animal Source Metagenome Assembly of Lawsonella clevelandensis from Canine External Otitis" by Toth et al. The study presents the first assembly of L. clevelandensis from an animal source. While the topic is interesting and the paper is well-written, it lacks sufficient depth to be considered a significant contribution to the literature. It is a very simple study that merely describes the genome of a single bacterium isolated from one dog using Nanopore sequencing. The study lacks originality, novelty, and scientific robustness. I acknowledge the authors' effort in assembling this metagenome, but I believe that additional data are needed to transform this into a meaningful contribution to the field. 

Author Response

Thank you for the opportunity to review the paper "First Animal Source Metagenome Assembly of Lawsonella clevelandensis from Canine External Otitis" by Toth et al. The study presents the first assembly of L. clevelandensis from an animal source. While the topic is interesting and the paper is well-written, it lacks sufficient depth to be considered a significant contribution to the literature. It is a very simple study that merely describes the genome of a single bacterium isolated from one dog using Nanopore sequencing. The study lacks originality, novelty, and scientific robustness. I acknowledge the authors' effort in assembling this metagenome, but I believe that additional data are needed to transform this into a meaningful contribution to the field. 

 

Thank You for the comment. To ensure increased robustness, further results were added to the manuscript that help to position the detected strain among other members of the Lawsonella clevelandensis species. Furthermore, the earlier version of the manuscript was upgraded with L. clevelandensis genomes that have recently been uploaded to NCBI. Considering the low number of available genomic information of this species, we believe that our results can contribute to understanding this pathogenic species more thoroughly.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors

Thanks for your work.

However, some comments should be addressed during revision;

1. 1. The word ‘’ First ‘’ in the title is not suitable, it usually replace by ‘’to our knowledge’’.
   2. A conclusion should be included at the end of the abstract, introduction, and discussion sections.
   3. The key words should be differ from those in the title.
   4. A paragraph about Lawsonella clevelandensis should be included in the introduction.
   5. Line 26, the abbreviation “PCR” should be written in full.
   6. A photo of the lesions sites where the samples were taken from could be added.
   7. The approval number of the Ethical Committee for Using Animals in Experimental Work should be included.
   8. Line 71, the bacterial abbreviation “L. clevelandensis” should be preceded by the full name of the bacterium.
   9. Line 90, and Peptoniphilus (1.2%).
   10. The genes names should be written in a correct form.
   11. The abbreviations should be revised all over the manuscript (AMR, MIC, NGS, etc.).
   12. Was there any link between the presence of this bacterium in the dog case and its owner?

   Best wishes

Author Response

 

1. The word ‘’ First ‘’ in the title is not suitable, it usually replace by ‘’to our knowledge’’.

   Thank You for the comment. All available assemblies of  Lawsonella clevelandensis were screened to make this statement. There are currently only 15 genomic assemblies of this species on NCBI, out of which 14 are reported to derive from humans. Due to the low number of available genomes, we did not find this statement over-exaggerating. However, to comply with the suggestion of the Reviewer, the statement was refined throughout the manuscript. Nevertheless, we did not find a way to change this word in the title and still keep the title robust and informative. We are open for any suggestions for a better title.

2. A conclusion should be included at the end of the abstract, introduction, and discussion sections.

   Thank You for the comment, the necessary changes were made.

3. The key words should be differ from those in the title.

   Thank You for the comment, the necessary changes were made.

4. A paragraph about Lawsonella clevelandensis should be included in the introduction.

   Thank You for the comment, the paragraph is now included.

5. Line 26, the abbreviation “PCR” should be written in full.

   Thank You for raising our attention to this, the necessary changes were made.

6. A photo of the lesions sites where the samples were taken from could be added.

   Even though, a photo of the lesion site would be interesting, no photos are added. The reason for this is that the aim of the study was not to explain the whole metagenomic profile of the given lesions. According to our opinion, L. clevelandensis might have contributed to the lesions, but further bacteria identified at the sampling site might have also played a role. Since the scope of the study did not include the analysis of the exact role of L. clevelandensis, nor the role of other microorganisms present at the sampling site, we would not like to indicate that L. clevelandensis caused the lesions alone and we have not got a clear picture of its role either. This aspect has been highlighted in the mauscript as well.

7. The approval number of the Ethical Committee for Using Animals in Experimental Work should be included.

   Thank You for pointing this out, however, the sampling has happened as a part of a routine diagnostic test. Furthermore, the approval form from the owners has been sent to MDPi during the submission process and got approved by MDPI.

8. Line 71, the bacterial abbreviation “L. clevelandensis” should be preceded by the full name of the bacterium.

   Thank You for the comment, the necessary changes were made.

9. Line 90, and Peptoniphilus (1.2%).

   Thank You for the comment, the necessary changes were made.

10. The genes names should be written in a correct form.

    Thank You for the comment. The gene names derive from CARD (Comprehensive Antimicrobial Resistance Database). We believe keeping them helps staying consistent and trackable to CARD.

11. The abbreviations should be revised all over the manuscript (AMR, MIC, NGS, etc.).

    Thank You for the comment, the necessary changes were made.

12. Was there any link between the presence of this bacterium in the dog case and its owner?

    Thank You for the question. While the question is very interesting, the owner of the dog was not sampled and the answer for this question is beyonf the scope of the manuscript. However, a novel study focusing on the One Health aspects would be of great interest.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript provides an accurate metagenomic and genomic characterization of Lawsonella clevelandensis from a dog, contributing in a very significant manner to our current understanding of its potential zoonotic relevance, genomic diversity, and clinical detectability. It is a well written article and very easy to follow through. I only have some suggestions to add. 

1. Please extend the discussions considering the fact that the AMR genes which were investigated were low in coverage, and this might affect the functional significance of these genes; Also, it should be discussed the potential for false positives or misannotations. There is also a lack of phenotypic resistance testing to validate these findings.

2. The comments should be focused also on the fact that it did not reveal the virulence factors which also weakens arguments about potential clinical relevance. Yet, the manuscript still speculates about its pathogenic impact, which creates a disconnect between findings and interpretation.

3. The study should add a more detailed statistical analysis, such as: Confidence in taxon abundance estimates. Significance of observed genome similarity. Read mapping precision. etc There is no coverage statistics or variability across genomic regions were shown to validate completeness or assembly accuracy.

Author Response

1. Please extend the discussions considering the fact that the AMR genes which were investigated were low in coverage, and this might affect the functional significance of these genes; Also, it should be discussed the potential for false positives or misannotations. There is also a lack of phenotypic resistance testing to validate these findings.

   Thank You for the comment. The above limitations of the study were involved in the manuscript according to the suggestions. As described above, the assembly of L. clevelandensis happened during a clinical metagenomic study, thus, unfortunately, the isolation and phenotypic antimicrobial susceptibility testing of L. clevelandensis was not performed. However, the routine bacteriological diagnostic testing of the sampled ear canal was performed. A result of Pasteurella multocida with phenotypic resistance to clindamycin and gentamycin and mixed anaerobic flora with no AMR testing was received. At the same time, a rich set of ARG was found in the genome of L. clevelandensis. While the expression of these genes is not granted, the potential of the expression or transfer of these genes is indisputable. This idea was also added to the manuscript.

    

2. The comments should be focused also on the fact that it did not reveal the virulence factors which also weakens arguments about potential clinical relevance. Yet, the manuscript still speculates about its pathogenic impact, which creates a disconnect between findings and interpretation.

   Thank You for the comment. Given that our information regarding the genomic properties of this species is very limited, and only few genomes are available at NCBI, the set of species-specific virulence genes that L. clevelandensis may harbour can also be unknown. While phenotypic findings prove the pathogen potential of the species, the genomic background is yet to be studied. This might have contributed to the above dissonance. The description of this limiting phenomenon has been added to the manuscript.

    

3. The study should add a more detailed statistical analysis, such as: Confidence in taxon abundance estimates. Significance of observed genome similarity. Read mapping precision. etc There is no coverage statistics or variability across genomic regions were shown to validate completeness or assembly accuracy.

   Thank You for the suggestion. The genome was reassembled and significant improvements have been added have been added to the manuscript. The necessitated statistical questions are now also answered in the manuscript.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have improved the manuscript by incorporating additional results and expanding the comparative genomic analysis using data from the NCBI database. Despite these efforts, I remain convinced that the study does not meet the standards or scope of the journal. The results are too limited in complexity, and the overall contribution to the literature remains minimal. While I acknowledge and appreciate the authors' work in revising the manuscript, in my opinion, the study is too simple to be considered a significant or impactful scientific contribution. Therefore, I do not recommend publication in its current form.

Author Response

Thank you to the Reviewer for taking the time to review our manuscript again. Unfortunately, apart from the overall assessment, the review does not provide detailed suggestions on how we could improve the manuscript. However, necessary updates were  made to address grammatical errors in the manuscript.