PONE-D-24-01335Potential transferability and evolution of Tn6411 transposons carrying the blaIMP-1 gene in Pseudomonas aeruginosaPLOS ONE

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Reviewers' comments:

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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: Partly

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

Reviewer #1: N/A

Reviewer #2: N/A

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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

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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: Yes

Reviewer #2: 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: The manuscript by Wang and cols. analyzes the genetic detection of transposon Tn6411 flanking the blaIMP-1 carbapenem-resistant gene in two Pseudomonas aeruginosa of clinical oring. This manuscript is relevant because it reports the evolution and potential dissemination of Tn6411 in clones of P. aeruginosa not typically reported, considered not pandemic, which highlights its role in spreading last-resort antibiotics.

However, I have some recommendations that could improve the manuscript:

1. The quality of the two figures must be improved.

2. Line 81. Please include the version (year) of the LCSI guidelines.

3. Line 109-111. Why did the authors not include the OD600 as a normalization tool for the number of cells in the conjugation experiments? Why was a P. aeruginosa strain not used as a recipient instead of an E. coli? Please add more information about it in the discussion section.

4. Line 150-151. Why was strain 18083286 included in the conjugation experiments even though "no plasmid was detected"? Maybe the order of the experiments should be revised.

Reviewer #2: The paper “Potential transferability and evolution of Tn6411 transposons carrying the blaIMP-1 gene in Pseudomonas aeruginosa” is interesting. Describes a transposon that allows the mobility of resistance genes such as blaIMP-1. However, major corrections must be made to make it clear.

You must change Figure 1 because it cannot be seen.

Table 1 only describes the resistance profile of strain 18083286. If strain 18081308 has the same resistance profile, it should be said in the footer of the table or the title, and if not, the resistance profile of this strain should also be included.

The authors describe that this transposon carrying blaIMP-1 is found in one of the strains on the chromosome and in the other on a plasmid. However, they do not describe the plasmid carrying the transposon by sequencing, which would be important to see if it coincides with the results obteined in the assay of the conjugation.

In methods and results, the authors do not specify the strain used. The article, in general, describes one of the strains, and the question remains whether the other strain had the same thing or if there are changes, so it should include both strains.

The authors show that two methodologies were used for sequencing strain 18083286; however, they do not say if it made a hybrid assembly of the sequences.

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Reviewer #1: Yes: Gerardo Cortés-Cortés

Reviewer #2: No

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PONE-D-24-01335R1Potential transferability and evolution of Tn6411 transposons carrying the blaIMP-1 gene in Pseudomonas aeruginosaPLOS ONE

Dear Dr. Guo,

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.

Please submit your revised manuscript by Jul 11 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-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Mohamed O Ahmed

Academic Editor

PLOS ONE

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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: (No Response)

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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: Partly

**********

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

Reviewer #1: N/A

Reviewer #2: N/A

**********

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

**********

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

**********

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: Dear authors,

Thanks for addressing my comments.

I have some minor suggestions that I consider important to clarify:

1. I partially agree that in this case, "the clarity of the image is directly correlated with the reader's perception". However, once the figures meet the standards indicated in the guide for authors, it should be fine, and the production team will say something in case not.

2. Regarding the conjugation experiments, it is not clear to me why you wanted to validate the transferability of Tn6411-blaIMP-1 by conjugation in a plasmid-free strain. Why don't you evaluate this transferability by transformation instead? which could provide more comprehensive results.

Reviewer #2: The article “Potential transferability and evolution of Tn6411 a transposons carrying the blaIMP-1 gene in Pseudomonas aeruginosa” describes a transposon that allows the mobility of resistance genes such as blaIMP-1. However, it remains unclear on the following points:

1. The authors do not answer whether they carried out a hybrid assembly for strain 18083286 because they carried out two sequencing by two different methods. In that case, they could carry out a hybrid assembly that would provide them with more information. They could be surer of your information. I suggest that it be carried out or clarify why it was carried out by two sequencing methodologies, both in methods and results.

2. Line 222. The authors describe the Tn6411 transposon through bioinformatic analysis and compare it with others reported in GenBank. However, they cannot say that they determined the evolution of this transposon since, with this analysis, they can only say that there are changes. Other studies must be done to know evolution, so the word evolution must be eliminated from the title.

3. Line 198. The authors say that the observed modifications make Tn6411 and Tn6411-like stable, which must be demonstrated experimentally. Like in line 229, they should remove “forming a stable structure when you capture the type1 integron with blaIMP-1 because bioinformatic analysis cannot determine."

4. Line 116. It is unclear why a conjugation test should be performed on strain 18083286, given that the bioinformatic analysis showed that Tn6411 with blaIMP-1 is located on the chromosome. We know that it will only be transferred vertically.

5. The authors say that the plasmid cannot be detected because they have a low copy number; however, it is not a factor for not observing it in the type of sequencing they carry out because, in these cases, libraries are created that in some way would help with this problem. On the other hand, they detect it in strain 18081308. However, they do not describe it, at least what refers to its conjugation machinery, to relate it to what was found in the conjugation assay. So, they must change their conclusion (line 233).

6. I continue to insist that the figures are of very poor quality and do not look properly. These must be clear to any reader.

7. The authors give the title “Potential transferability and evolution”; however, the only thing they do is a description of the transposon found in their strains and a comparison with other transposons reported in GenBank, so they cannot talk about evolution or Potential transferability. Only with a bioinformatic analysis, in addition to this not being adequately documented in the article

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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: Yes: Gerardo Cortés-Cortés

Reviewer #2: No

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[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.

Comparative genomics of Tn6411 transposons carrying the blaIMP-1 gene in Pseudomonas aeruginosa

PONE-D-24-01335R2

Dear Guo,

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.

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PLOS ONE