PONE-D-23-27779Whole-genome comparison of two same-genotype macrolide-resistant Bordetella pertussis isolates collected in JapanPLOS ONE

Dear Dr. Koide,

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 Jan 12 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,

Daniela Flavia Hozbor

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.  

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

3. 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.

[Note: HTML markup is below. Please do not edit.]

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: Partly

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: N/A

Reviewer #2: N/A

Reviewer #3: Yes

**********

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

**********

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

Reviewer #3: Yes

**********

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 submitted for publication in Plos One by Kentaro Koide et al entitled “Whole-genome comparison of two same-genotype macrolide-resistant Bordetella pertussis isolates collected in Japan” presents the genomic characterization of two macrolide-resistant Bordetella pertussis isolates collected in Tokyo and Osaka between June and July 2018.

One isolate was already reported in a previous publication (reference 5). This study provides 2 additional whole genome sequences of isolates resistant to macrolides recently collected in Japan and compares them to resistant isolates from China.

Abstract L32-34 should be removed: this is not the key message of this work.

L48- authors should mention that 3 copies of 23sRNA are present in the genome of B. pertussis and that resistant isolates mostly present the same mutation in the 3 copies of 23sRNA. Bartkus et al., 2003 publication (doi: 10.1128/JCM.41.3.1167-1172.2003) that first describes the mutation associated to erythromycin resistance in Bordetella should be added

L75-76: “However, despite their simultaneous occurrence, a distinguishable epidemiological link between these isolates could not be established => based on what?

L90-91: why where information acquired in April 2022 for isolates collected in 2018?

L91-93: where the 3 copies of 23sRNA mutated?

L105: why was PCR-based genotyping done since whole genome sequencing is performed?

L112-113: please give the accession Number of data from Genbank database for determination of alleles.

L124-L127. Information about reads quality should also be added.

L215: Figure 1: was the tree rooted? Which isolate correspond to the longer branch? It seems there is only 1 isolate ptxP3 and resistant in the dataset? Are MRBP from other country than China also included in the tree? Resolution of Figures 1&2 is not good on my pdf.

Authors used a SNP cut off determined in another study but were phylogenetic analysis the same as in the present study?

Authors should discuss mutations that differentiate the 2 MRBP isolates.

L293-304: this paragraph has to be shortened.

L470-475: Supporting information titles are not provided on the pdf.

Reviewer #2: Koide et al. investigated whole genome sequences of two macrolide-resistant Bordetella pertussis isolates in Japan. They show that 11 SNPs exist between two isolates, indicating these isolates independently transmitted from PRC to Japan. These information would contribute to understandings for Drug-resistant Bordetella pertussis.

Specific comments:

Line 186; Please correct 3256.

Line number should not add to Tables and foot note of Tables : Line 103-104, Line 191-202, Line 243-245.

Line 291: The authors described “They were likely introduced from other cities in China.” However, it is not shown the reason why Beijing, where the most similar strain was isolated, could be excluded from the origin.

Line 295: “Typically, short-read sequencing …”, This paragraph should discuss on what NGS technique is the best for analyzing MRBP genome in regard with transmission and subcluster. Descriptions in the manuscript is a bit too general.

Reviewer #3: Abstract:

- There is no previous sentence about the close relationship of your MRBP strains with Chinese strains. It is suggested to revise the sentence to clearly show that the phylogeny analysis suggested this close relationship.

- It is better to include a sentence in the abstract explaining why MRBP strains are important.

Introduction:

- Please rewrite lines 43-46 as a more accurate history of MRBP strains. The current sentence implies that MRBP strains were first found in China, which is not true. You also mentioned sporadic strains in Asian countries, but there are reports from other continents as well.

- Please rewrite lines 62-72 to make the paragraph more cohesive and clearer.

- The sentence in line 72 is ambiguous. Is WGS used widely in diagnostic labs or research labs? Please clarify this sentence.

- In line 131 why B616, one of the MRBP clinical strains, was chosen as a reference. Have you aligned your samples against TohamaI as well? The reviewer requested that you explain.

- What strain was used as an outgroup for phylogenetic analysis? Since most of the isolates were ptxP1, why did you not use the global reference genome Tohama I for SNP detection and phylogeny analysis?

- It is suggested to investigate the divergence time for MRBP isolates if possible.

- The authors did not explain and discuss the mechanisms of resistance to macrolides due to the mutation in 23srRNA either in the introduction or discussion.

-Since the authors emphasize the ptxP1 and ptxP3 strains in their manuscript it is suggested to explain their importance and genomic profiles either in the introduction or discussion section. For the majority of readers who do not have pertussis knowledge but are interested in antimicrobial resistance microorganisms, it is not clearly explained what ptxP1 and PtxP3 are and what is their role in BP and its virulence.

- It is suggested to the authors to discuss the importance of MRBP isolates. Since the majority of isolates in Japan are PtxP3 and rare ptxP1 isolates are isolated in current years why these strains are important.

- It is suggested to discuss why most of the MRBP isolates are ptxP1 and not ptxP3. Is there any ptxP profile information for MRBP strains of other countries?

**********

6. 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: No

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.

Whole-genome comparison of two same-genotype macrolide-resistant Bordetella pertussis isolates collected in Japan

PONE-D-23-27779R1

Dear Dr. Kentaro Koide,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Daniela Flavia Hozbor

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: