PONE-D-23-38194Antibiotic Resistance and Multilocus Sequence Typing of Aeromonas Isolated from Freshwater Fish in Hebei ProvincePLOS ONE

Dear Dr. Lee,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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We look forward to receiving your revised manuscript.

Kind regards,

Mahmoud Abdel Aziz Mabrok, PhD

Academic Editor

PLOS ONE

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4. Thank you for stating the following financial disclosure: "This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-RS-2023-00275307) and Basic Science Research Capacity Enhancement Project through Korea Basic Science Institute (National research Facilities and Equipment Center) grant funded by the Ministry of Education (Grant No. 2019R1A6C1010033)."

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments to authors:

-The current study is interesting; however, the authors should address the following comments to improve the quality of the manuscript:

-The manuscript should be revised for English editing and grammar mistakes.

- Please write the scientific names of bacterial pathogens and genes in the correct form all over the manuscript and the references section.

Title:

I think the work would benefit from the title that contains the main conclusion of the study (should be derived from the conclusion). Please modify the title.

Abstract:

- The abstract must illustrate the used methods and the most prevalent results (give more hints about methods and results). Besides, rephrase the aim of the work and the main conclusion of your findings.

- Add the full expression before the abbreviations.

-Introduction: (it needs to be more informative):

-Give a hint about the virulence factors and the mechanism of disease occurrence, and infecions caused by Aeromonas spp.

- The authors should illustrate the public health importance concerning the emergence of multidrug-resistant (MDR) bacterial pathogens that reflect the necessity of new potent and safe antimicrobial agents. Several studies proved the widespread MDR- bacterial pathogens;

Authors could add the following paragraph:

Multidrug resistance has been increased all over the world that is considered a public health threat. Several recent investigations reported the emergence of multidrug-resistant bacterial pathogens from different origins that increase the necessity of the proper use of antibiotics. Besides, the routine application of the antimicrobial susceptibility testing to detect the antibiotic of choice as well as the screening of the emerging MDR strains. You are advised to cite the following valuable studies:

1. PMID: 36819057

2.PMID: 36365013

3. PMID: 32235800

4. PMID: 36439817

5. PMID: 35971557

5. PMID: 35532863

6. PMID: 31170450

- Illustrate the mechanism of action different virulence factors of Aeromonads.

-Rephrase the aim of the work to be clear and better sound.

Material and methods:

- Support all methods with updated specific references.

• Add the company, city, and country of the used chemicals and reagents.

- Bacterial Strains:

Please illustrate the methods used for isolation and identification of Aeromonas spp. :

Besides, specific references should be added.

• Add the company, city, and country of the used bacterial media and reagents that were used in the biochemical identification of isolates. Also, enumerate all used biochemical reactions.

- Antimicrobial susceptibility testing:

-Please, explain in detail

•Add the names of the antimicrobial classes.

•The authors are advised to classify the tested isolates to MDR , XDR, and PDR as described by Magiorakos et al. Also, the MAR index was determined according to Krumperma.

Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012; 18:268–81. doi:10.1111/j.1469-0691.2011.03570.x.

Krumperman PH. Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of fecal contamination of foods. Applied and Environmental Microbiology. 1983;46(1):165-70.

- The detection of virulence and antimicrobial resistance genes in the recovered isolates should be performed. Afterwards, the correlation between phenotypic and genotypic multidrug resistance should be performed.

Besides, the correlation coefficient (r) should be determined among the identified resistance genes and the tested antimicrobial agents.

-Where are the Statistical analyses? Add more details about the used software.

-Results:

- Please add a starting paragraph to the results section to briefly introduce the topic, your goals and

hypothesis and a short summary of what you did in this work.

-Add this subtitle: Phenotypic characteristics of the recovered isolates:

• Illustrate in detail the phenotypic characteristics of the recovered A. hydrophila and A. veronii isolates.

-Antimicrobial susceptibility testing

• -Illustrate in a new table the occurrence of MDR (Multidrug resistance) among the recovered isolates as the following (illustrate the names of the antimicrobial classes and different antibiotics):

No. of strains % Type of resistance

R, MDR, and XDR Phenotypic multidrug resistance

(Antimicrobial classes and different antibiotics). The antibiotic-resistance genes

-The correlation (Correlation coefficient) between phenotypic and genotypic multidrug resistance should be performed.

-Increase the resolution of figures (must be 600 dpi).

-Discussion:

- Please illustrate different mechanisms of antimicrobial resistance in Aeromonas spp.

-Conclusion

- Should be rephrased to be sounded. A real conclusion should focus on the question or claim you articulated in your study, which resolution has been the main objective of your paper?

Reviewer #2: Dear Author

Thank you for your manuscript submission. The present study is well-done and well-designed. However, a Minor Revision is needed as below:

1. The Introduction section is too long. Please do shorten it.

2. Please do add all the related references in association with the used protocols within the manuscript.

3. Please do add all the related references associated with the used primers in the present study.

4. It is recommended to add a flow chart to Materials and Methods section to show all the procedures done within this study in brief.

5. The Methodology of the manuscript is bisectional. A section is experimental and the other section is bioinformatic. Thus, it is recommended to add "Wet Lab" term for the experimental section and "Dry Lab" term for bioinformatic section.

In this regard, please do read and add the following paper to References section of the manuscript to have a fruitful Methodology section:

DNA microarray technology and bioinformatic web services. Acta Microbiol Immunol Hung. 2019 Mar 1;66(1):19-30. doi: 10.1556/030.65.2018.028. Epub 2018 Jul 16. PMID: 30010394.

6. As the Results section is interesting, it is recommended to add a schematic figure to Results section to show the most effective Results in the present study.

Reviewer #3: This study proposed that the identification selective power of Aeromonas spp. might be increased by employing the Multilocus Sequence Typing technique. However, this is an interesting work. But it has some challenges that need to be addressed before it is ready for publication.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Payam BEHZADI

Reviewer #3: No

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Attachments

Attachment

Submitted filename: Reviewer comments.docx

PONE-D-23-38194R1Multilocus sequence typing and antibiotic resistance of Aeromonas isolated from freshwater fish in Hebei ProvincePLOS ONE

Dear Dr. Lee,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

In the results section, we focus exclusively on presenting our own findings without including results from other studies or any references, thus address the following comments;

Results:

Line 402: remove the reference (61), Page 15.

Line 413: remove the reference (32), Page 16.

Line 572: remove the reference (62), Page 21.

==============================

Please submit your revised manuscript by Mar 07 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-emailutm_source=authorlettersutm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Mahmoud Abdel Aziz Mabrok, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have carried out significant changes to the manuscript. They have addressed most of the suggested corrections and comments. Really, it's an interesting study that has a significant impact. Now, the manuscript could be accepted.

Only one minor comment should be addressed in the final version: The journals names should be added in the references section.

Reviewer #2: (No Response)

Reviewer #3: Dear authors, in the Results section, we focus exclusively on presenting our own findings without including results from other studies or any references, thus address the following comments;

Results:

Line 402: remove the reference (61), Page 15.

Line 413: remove the reference (32), Page 16.

Line 572: remove the reference (62), Page 21.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Payam BEHZADI

Reviewer #3: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: Reviewer comments - R2.docx

Multilocus sequence typing and antibiotic resistance of Aeromonas isolated from freshwater fish in Hebei Province

PONE-D-23-38194R2

Dear Dr. Ho-Seong Lee

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Mahmoud Abdel Aziz Mabrok, PhD

Academic Editor

PLOS ONE