Identification and Genomic Characterization of Aeromonas dhakensis from a Human Sample
, Alexander Tristancho-Baró 1,2, Juan Manuel GarcÃa-Lechuz 1,2, Natalia Burillo-Navarrete 1,2, Sara Sanz-Sanz 1,2, Ana MarÃa Milagro-Beamonte 1,2, Ana Isabel López-Calleja 1,2 and Antonio Rezusta-López 1,2
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsI find the manuscript interesting and the data presented relevant.
However, I have a few questions for the authors:
- If the genome has been deposited, I would need the corresponding accession number. If it has not yet been deposited, its submission would be essential before proceeding with the review of the manuscript.
- In one section, the authors state:
“The resistance study revealed the presence of an OXA-726, TRU-1 and imiH using the RGI tool y OXA-724 y imiH7 using abricate. The alignment determined that there is no evidence of the presence of these resistance genes due to their low percentage of identity: 95.47% for OXA-724, 95.97% for OXA-726, 92.81% for imiH and 92.16% for imiH7.”
However, later they state:
“In our case, A. dhakensis was isolated, which, in addition to the TRU β-lactamase typical of the Aeromonas genus—conferring the ability to hydrolyze penicillins and broad-spectrum cephalosporins—also exhibited the class D β-lactamase OXA-726 and ImiH carbapenemase, being the latter responsible for the carbapenem resistance observed in our isolate.”
This appears contradictory.
It is likely that the authors are dealing with a novel OXA-type β-lactamase, which would require biochemical and kinetic characterization to determine whether it is indeed responsible for the observed resistance, as well as with a new imiH allele. It would be important to reanalyze the genome, and if this is the case, deposit the novel β-lactamase sequence.
With regard to species identification, I believe the authors have appropriately addressed all available identification strategies.
Minor revisions:
- Line 54: Replace hydrophyla with A. hydrophila. Please check the rest of the manuscript for consistency.
- Line 120: The phrase “based on single nucleotide polymorphism based” contains a redundancy. Please correct it to: “phylogeny based on single nucleotide polymorphisms.”
- Line 271: Replace “Our patient's clinical course was favourable following five days of treatment with cefepime and fluconazole” with “The clinical course was favorable after five days of treatment.”
Author Response
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Response to Reviewer 1 Comments
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1. Summary |
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Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted in the re-submitted files.
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2. Point-by-point response to Comments and Suggestions for Authors |
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Comments 1: If the genome has been deposited, I would need the corresponding accession number. If it has not yet been deposited, its submission would be essential before proceeding with the review of the manuscript.
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Response 1: Thank you for pointing this out. The genome was uploaded to NCBI on June 5, 2025, under BioSample ID SAMN48761348.
We have added the Data Availability statement. |
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Comments 2: “The resistance study revealed the presence of an OXA-726, TRU-1 and imiH using the RGI tool y OXA-724 y imiH7 using abricate. The alignment determined that there is no evidence of the presence of these resistance genes due to their low percentage of identity: 95.47% for OXA-724, 95.97% for OXA-726, 92.81% for imiH and 92.16% for imiH7.” “In our case, A. dhakensis was isolated, which, in addition to the TRU β-lactamase typical of the Aeromonas genus—conferring the ability to hydrolyze penicillins and broad-spectrum cephalosporins—also exhibited the class D β-lactamase OXA-726 and ImiH carbapenemase, being the latter responsible for the carbapenem resistance observed in our isolate.” It is likely that the authors are dealing with a novel OXA-type β-lactamase, which would require biochemical and kinetic characterization to determine whether it is indeed responsible for the observed resistance, as well as with a new imiH allele. It would be important to reanalyze the genome, and if this is the case, deposit the novel β-lactamase sequence.
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Response 2: Thank you for pointing this out. We have uploaded the resistance gene sequences to NCBI, and we have added the submission IDs for each gene to the Data Availability section. |
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Comments 3: Line 54: Replace hydrophyla with A. hydrophila. Please check the rest of the manuscript for consistency. |
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Response 3: Thank you for pointing this out. This change has already been taken into account
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Comments 4: Line 120: The phrase “based on single nucleotide polymorphism based” contains a redundancy. Please correct it to: “phylogeny based on single nucleotide polymorphisms.”
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Response 4: Thank you for pointing this out. This change has already been taken into account |
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Comments 5: Line 271: Replace “Our patient's clinical course was favourable following five days of treatment with cefepime and fluconazole” with “The clinical course was favorable after five days of treatment.” |
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Response to Reviewer 1 Comments
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1. Summary |
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Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted in the re-submitted files.
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2. Point-by-point response to Comments and Suggestions for Authors |
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Comments 1: If the genome has been deposited, I would need the corresponding accession number. If it has not yet been deposited, its submission would be essential before proceeding with the review of the manuscript.
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Response 1: Thank you for pointing this out. The genome was uploaded to NCBI on June 5, 2025, under BioSample ID SAMN48761348.
We have added the Data Availability statement. |
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Comments 2: “The resistance study revealed the presence of an OXA-726, TRU-1 and imiH using the RGI tool y OXA-724 y imiH7 using abricate. The alignment determined that there is no evidence of the presence of these resistance genes due to their low percentage of identity: 95.47% for OXA-724, 95.97% for OXA-726, 92.81% for imiH and 92.16% for imiH7.” “In our case, A. dhakensis was isolated, which, in addition to the TRU β-lactamase typical of the Aeromonas genus—conferring the ability to hydrolyze penicillins and broad-spectrum cephalosporins—also exhibited the class D β-lactamase OXA-726 and ImiH carbapenemase, being the latter responsible for the carbapenem resistance observed in our isolate.” It is likely that the authors are dealing with a novel OXA-type β-lactamase, which would require biochemical and kinetic characterization to determine whether it is indeed responsible for the observed resistance, as well as with a new imiH allele. It would be important to reanalyze the genome, and if this is the case, deposit the novel β-lactamase sequence.
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Response 2: Thank you for pointing this out. We have uploaded the resistance gene sequences to NCBI, and we have added the submission IDs for each gene to the Data Availability section. |
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Comments 3: Line 54: Replace hydrophyla with A. hydrophila. Please check the rest of the manuscript for consistency. |
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Response 3: Thank you for pointing this out. This change has already been taken into account
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Comments 4: Line 120: The phrase “based on single nucleotide polymorphism based” contains a redundancy. Please correct it to: “phylogeny based on single nucleotide polymorphisms.”
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Response 4: Thank you for pointing this out. This change has already been taken into account |
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Comments 5: Line 271: Replace “Our patient's clinical course was favourable following five days of treatment with cefepime and fluconazole” with “The clinical course was favorable after five days of treatment.” |
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Response 5: Thank you for pointing this out. This change has already been taken into account |
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Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsDear Authors,
Your paper entitled "Identification and genomic characterization of Aeromonas dhakensis" has been carefully reviewed.
The paper is very important from bacteriological point of view, since it highlights on the importance of molecular identification of specific bacterial species (such Aeromonas dhakensis in the present study). Many bacterial species are very close on phenotypical level, therefore an additional molecular studies must be done to differentiate between these species for a better epidemiological and treatment perspectives.
The article is well written in English, well Designed and simple for readers.
Kindly find below the list of my comments regarding this work:
01- First I suggest to publish this work as a Clinical Case Report, since the present paper is limited to one case.
02- I invite you to add a list of keywords to this work.
03- The First two sentences, in the introduction (Lines 30-32) Lack a reference, I invite you to add a reference.
04- In Line 52, Kindly replace "batalactamases" by "bata-lactamases"
05- In invite you to talk about Culturomics as an important recent technique that may also help in identifying new bacterial species, which is very important from clinical and environmental points of view.
Best Regards,
Author Response
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Response to Reviewer 2 Comments
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1. Summary |
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Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.
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3. Point-by-point response to Comments and Suggestions for Authors |
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Comments 1: First I suggest to publish this work as a Clinical Case Report, since the present paper is limited to one case.
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Response 1: Thank you for pointing this out. But after speaking with all the authors, we decided that it should be a scientific article and not a clinical case, since the case information is very brief and focuses primarily on sequencing. |
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Comments 2: I invite you to add a list of keywords to this work. |
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Response 2: Thanks for the comment, we have added the keywords Aeromonas dhakensis, Carbapenem resistance, Whole genome sequencing, MALDI-TOF misidentification
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Comments 3: The First two sentences, in the introduction (Lines 30-32) Lack a reference, I invite you to add a reference. |
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Response 3: Thanks for the comment. We wrote the first sentences by looking at the following paper: 10.3390/microorganisms8010129
We put the reference at the end of the paragraph, but I added it too at the end of the line.
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Comments 4 In Line 52, Kindly replace "batalactamases" by "bata-lactamases"
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Response 4: Thank you for pointing this out. This change has already been taken into account
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Comments 5: In invite you to talk about Culturomics as an important recent technique that may also help in identifying new bacterial species, which is very important from clinical and environmental points of view. |
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Response to Reviewer 2 Comments
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1. Summary |
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Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.
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3. Point-by-point response to Comments and Suggestions for Authors |
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Comments 1: First I suggest to publish this work as a Clinical Case Report, since the present paper is limited to one case.
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Response 1: Thank you for pointing this out. But after speaking with all the authors, we decided that it should be a scientific article and not a clinical case, since the case information is very brief and focuses primarily on sequencing. |
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Comments 2: I invite you to add a list of keywords to this work. |
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Response 2: Thanks for the comment, we have added the keywords Aeromonas dhakensis, Carbapenem resistance, Whole genome sequencing, MALDI-TOF misidentification
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Comments 3: The First two sentences, in the introduction (Lines 30-32) Lack a reference, I invite you to add a reference. |
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Response 3: Thanks for the comment. We wrote the first sentences by looking at the following paper: 10.3390/microorganisms8010129
We put the reference at the end of the paragraph, but I added it too at the end of the line.
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Comments 4 In Line 52, Kindly replace "batalactamases" by "bata-lactamases"
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Response 4: Thank you for pointing this out. This change has already been taken into account
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Comments 5: In invite you to talk about Culturomics as an important recent technique that may also help in identifying new bacterial species, which is very important from clinical and environmental points of view. |
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Response 5: Thank you for pointing this out. Between lines 318 and 337 |
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Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript entitled "Identification and Genomic Characterization of Aeromonas dhakensis" publishes the results of a study aimed at accurate species identification of the clinically obtained isolate of Aeromonas bacteria. The authors use several different identification methods, in comparison they show the limitations of each of them. At the same time, there is criticism of mass spectrometry as a method that has limitations in this case. The research article is completely methodological, and an extremely small number of samples are analyzed, so there can be no question of statistical processing. The study is of scientific and practical interest, since the identification of clinically significant bacteria is an important task and it is necessary to improve such methods. The article is written competently, the narrative is consistent, the conclusions are confirmed by the results. There is quite a bit of content in the article, the research was carried out locally. The graphic design is satisfactory. It is recommended to accept the article after minor changes.
1) Lines 44-45. «Aeromonas … characterized watery or dysenteric diarrhoea». Dysentery is caused by other pathogens, such as shigella or amoebas.
2) Lines 240-242. "The literature contains only one reported case detecting OXA-726 in A. dhakensis isolated from an edible snail, exerting the remarkable characteristics of this case." It is not entirely clear what idea the authors wanted to express in the second part of the sentence. It is advisable to be clearer and more specific. For example, that this case is unique. Or the thought is about an exotic animal that is not often eaten.
3) Considering that the correct identification of A. dhakensis is difficult, the authors should keep in mind that the same cases of incorrect identification of bacteria are possible in literary sources. This suggests that some of the data may have been mistakenly attributed to other Aeromonas species. In the article, it is necessary to raise the issue of the reliability of information, dividing the literature sources by methods, emphasizing the most likely reliable data.
4) As for the applied goals, it is not entirely clear how the authors propose that doctors act. It is difficult to imagine that a complex set of molecular methods will be used every time. How necessary is accurate species identification? Mass spectrometry makes it possible to determine the genus of bacteria, and the most important information is data on the resistance of an isolated strain to antibiotics.
Author Response
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Response to Reviewer 3 Comments
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1. Summary |
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Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.
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3. Point-by-point response to Comments and Suggestions for Authors |
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Comments 1: Lines 44-45. «Aeromonas … characterized watery or dysenteric diarrhoea». Dysentery is caused by other pathogens, such as shigella or amoebas. |
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Response 1: Thank you for pointing this out. It has been eliminated that it can cause dysenteric diarrhea |
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Comments 2: Lines 240-242. "The literature contains only one reported case detecting OXA-726 in A. dhakensis isolated from an edible snail, exerting the remarkable characteristics of this case." It is not entirely clear what idea the authors wanted to express in the second part of the sentence. It is advisable to be clearer and more specific. For example, that this case is unique. Or the thought is about an exotic animal that is not often eaten. |
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Response 2: Thank you for pointing this out. I've specified that this case is unique. I've modified the paragraph to be more specific. The literature contains only one reported case of A. dhakensis detection, isolated from an edible snail, which underscores the uniqueness and clinical relevance of our isolate. Lines 268-270. |
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Comments 3: Considering that the correct identification of A. dhakensis is difficult, the authors should keep in mind that the same cases of incorrect identification of bacteria are possible in literary sources. This suggests that some of the data may have been mistakenly attributed to other Aeromonas species. In the article, it is necessary to raise the issue of the reliability of information, dividing the literature sources by methods, emphasizing the most likely reliable data.
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Response 3: Thank you for pointing this out. We have added a paragraph in the discussion commenting on this. Lines 281-288
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Comments 4: As for the applied goals, it is not entirely clear how the authors propose that doctors act. It is difficult to imagine that a complex set of molecular methods will be used every time. How necessary is accurate species identification? Mass spectrometry makes it possible to determine the genus of bacteria, and the most important information is data on the resistance of an isolated strain to antibiotics.
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Response 4: Thank you for pointing this out. We have added a paragraph to the discussion referring to this, found between lines 303 and 317. |
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Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsBadenas-Alzugaray et al. presented a study devoted to the comprehensive analysis of the Aeromonas dhakensis strain isolated from a patient with an intra-abdominal infection. The authors demonstrate that using whole-genome data is more robust against misidentification based on MALDI-TOF, a method frequently employed in clinical practice. The search for efficient methods to distinguish pathogenic species from their non-pathogenic counterparts is a crucial issue that extends beyond the scope of public health; therefore, the presented study is of importance to the field. The spectrum of genomic methods applied in the study is valid and fits the current standards. Therefore, the study surely merits to be considered. Nevertheless, the research certainly requires refinement due to the vague structure, lack of comparative analysis, and the absence of necessary information. Below, please find the reviewer’s point-to-point comments and suggestions.
- The title of the article seems to be somewhat misleading. I recommend stating the name of the strain since the current version implies the characterization of the whole species, while the research is focused on one isolate.
- Both results and methods lack a structured narrative, making it hard for the reader to follow the text. Please divide the logical parts into sections with subheadings containing the key findings/groups of methods.
- The reviewer failed to find any identifier of the genome in the NCBI databases, which is necessary for genomic studies. Please provide the accession number.
- Please provide the photographs of the colonies.
- Figure legends lack proper descriptions.
- In some parts of the results (e.g., description of platforms to check for resistance to antibiotics, etc.), the text repeats methods. Please carefully check the manuscript and amend these instances.
- Certain points in the methods section lack the required details, namely: 1) filters specified using trimmonatic (if the default mode was used, please specify), 2) the used genomic dataset (RefSeq or GenBank genomes), 3) methods for retrieving 16S rRNA sequences (Barrnap or manual extraction).
- For a better understanding of the genomic relationships, the reviewer recommends providing a table (probably in the Supplementary material) with the calculated ANI values coupled with accession numbers of the compared genomes.
- Please check italics for gene names throughout the text (line 203, etc.).
- Using full genus names after the first mention is unnecessary and could be omitted in the text.
- Please fix the typo error: 16S ARN (line 177).
- When characterizing the resistome, it is intriguing to reveal the source of resistance genes. To this end, the reviewer recommends searching for mobile genetic elements and revealing the associations with resistome elements.
- The genome comparisons lack depth, hiding potential insights. Revealing the resistome components in other Aeromonas genomes would certainly provide a better understanding of the distribution of antibiotic resistance capacity within the species in general and the isolated strain as well.
Author Response
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Response to Reviewer 4 Comments
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1. Summary |
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Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.
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2. Point-by-point response to Comments and Suggestions for Authors |
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Comments 1: The title of the article seems to be somewhat misleading. I recommend stating the name of the strain since the current version implies the characterization of the whole species, while the research is focused on one isolate.
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Response 1: I Thanks for pointing this out. Identification and Genomic Characterization of Aeromonas dhakensis from a human sample. |
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Comments 2: Both results and methods lack a structured narrative, making it hard for the reader to follow the text. Please divide the logical parts into sections with subheadings containing the key findings/groups of methods. |
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Response 2: We have added subsections within material/methods and results
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Comments 3: The reviewer failed to find any identifier of the genome in the NCBI databases, which is necessary for genomic studies. Please provide the accession number.
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Response 3: Thank you for pointing this out. The genome was uploaded to NCBI on June 5, 2025, under BioSample ID SAMN48761348.
We have added the Data Availability statement. |
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Comments 4: Please provide the photographs of the colonies.
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Response 4: Thanks for pointing this out. We have added the image of the colony
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Comments 5: Figure legends lack proper descriptions.
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Response 5: Thanks for pointing this out. We have refined the image captions |
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Comments 6: In some parts of the results (e.g., description of platforms to check for resistance to antibiotics, etc.), the text repeats methods. Please carefully check the manuscript and amend these instances.
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Response 6: Thanks for pointing this out. We have added this text to the methodology, removing it from the results: “Susceptibility testing ruled out the presence of plasmid-mediated carbapenemases, including the IMP, IMI, GES, and GIM families. However, the Carbapenem Inactivation Method (CIM) test with meropenem yielded a positive result, indicating carbapenemase activity.”
In the resistome study section, we did not remove the flagging tools from the results to clarify that abricate and RGI provided different results. |
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Comments 7: Certain points in the methods section lack the required details, namely: 1) filters specified using trimmonatic (if the default mode was used, please specify), 2) the used genomic dataset (RefSeq or GenBank genomes), 3) methods for retrieving 16S rRNA sequences (Barrnap or manual extraction).
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Response 7: These modifications appear in section 2.2 Genomic sequencing analysis |
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Comments 8: For a better understanding of the genomic relationships, the reviewer recommends providing a table (probably in the Supplementary material) with the calculated ANI values coupled with accession numbers of the compared genomes.
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Response 8: Thank you for pointing this out. We added an image with the ANI matrix and a table with the NCBI IDs of the genomes |
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Comments 9: Please check italics for gene names throughout the text (line 203, etc.).
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Response 9: Thank you for pointing this out. These modifications have been applied |
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Comments 10: Using full genus names after the first mention is unnecessary and could be omitted in the text.
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Response 10: Thank you for pointing this out. |
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Comments 11: Please fix the typo error: 16S ARN (line 177).
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Response 11: Thank you for pointing this out. It has been replaced by 16S RNA |
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Comments 12: When characterizing the resistome, it is intriguing to reveal the source of resistance genes. To this end, the reviewer recommends searching for mobile genetic elements and revealing the associations with resistome elements.
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Response 12: There are no mobile elements, we used Mob-recon for the construction of the plasmids but it doesn't have any, it's all chromosomal. |
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Comments 13: The genome comparisons lack depth, hiding potential insights. Revealing the resistome components in other Aeromonas genomes would certainly provide a better understanding of the distribution of antibiotic resistance capacity within the species in general and the isolated strain as well.
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Response 13: Thank you for pointing this out. This is added at the end of the discussion. Lines 350-369 |
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Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have improved the manuscript according to the previously provided comments. Below are my minor suggestions:
- Line 225: Please write the entire sentence in English, as the word "y" appears in Spanish. Additionally, it is unclear whether two OXA-type enzymes were identified or if it is the same enzyme detected by two different programs. Please clarify this point further.
- Line 232: The authors state that no plasmids were found. Were insertion sequences or other elements indicative of mobilization within the genome also investigated?
- Line 271: Please italicize bla.
Author Response
For research article
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Response to Reviewer 1 Comments
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1. Summary |
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Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted in the re-submitted files.
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2. Point-by-point response to Comments and Suggestions for Authors |
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Comments 1: Line 225: Please write the entire sentence in English, as the word "y" appears in Spanish. Additionally, it is unclear whether two OXA-type enzymes were identified or if it is the same enzyme detected by two different programs. Please clarify this point further.
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Response 1: Thank you for pointing this out. I've clarified all this information. It's on lines 225 and 230. |
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Comments 2: The authors state that no plasmids were found. Were insertion sequences or other elements indicative of mobilization within the genome also investigated? |
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Response 2: Thank you for pointing this out. I've clarified all this information. It's on lines 234 and 239. |
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Comments 3: Line 271: Please italicize bla. |
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Response 3: Thank you for pointing this out. This modification has been made.
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Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsI have carefully checked the revisions made and confirm that the authors have addressed all the issues mentioned, improved the structure, discussion, and added the necessary data. I could now recommend the article for publication.
Author Response
Thank you for the recommendation
