PONE-D-23-27461Global population structure of Lacticaseibacillus (formerly Lactobacillus) rhamnosusPLOS ONE

Dear Dr. Ozer,

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 Nov 19 2023 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,

António Machado

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. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. 

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

3. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well. 

4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. 

Additional Editor Comments:

Dear authors,

Several major concerns were posed by both reviewers, since the title, abstract, introduction, and methodologies procedures. I strongly recommend a careful revision of the manuscript wit properly answering all comments and/or suggestions of the reviewers.

Best regards,

António

[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: Yes

Reviewer #2: No

**********

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

Reviewer #1: Yes

Reviewer #2: No

**********

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: No

**********

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

**********

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 and Suggestions for Authors

The manuscript examine the population structure of Lacticaseibacillus rhamnosus and investigate the utility of using bacterial genomics to identify the source of invasive Lacticaseibacillus infections. Although the paper is well written and structured and the experiments are well planned, some questions need to be explained or clarified, as indicated as follows:

INTRODUCTION

1. " formerly Lactobacillus" appears in the Line 4, which should be supported by references.

2. The introduction has no logic, please rearrange it.

1. Why should we study the relationship between Lacticaseibacillus rhamnosus and endocarditis, or what is the necessity, please revise and add.

2 in the introduction. Author thought WGS is not accurate enough, why should this method be used for your research?

3. At the end of the introduction, the author believes that WGS cannot identify the real source of bacteria. What is the purpose of the research after that?

RESULT

1. Please explain whether the different branches of Lactobacillus rhamnosus are related to the source.

2. The result has no logic, please rearrange it. For example, to supplement the relevant information of the genome, the relationship between different branches and isolates.

DISCUSSION

1. Please analyze the discussion section in conjunction with other references and do not continue to describe the content of the results.

2. Lacticaseibacillus rhamnosus was detected in the blood of the patient. The possible cause is not discussed, please add.

3. The relationship between Lacticaseibacillus rhamnosus and geography and origin is not clearly discussed in this paper. In addition, Lacticaseibacillus rhamnosus in blood may come from food, which is not discussed in detail in this paper. The authors may refer to other relevant studies on the genome of L. rhamnosus, such as A large-scale comparative genomics study reveals niche-driven and within-sample intra-species functional diversification in Lacticaseibacillus rhamnosus(doi.org/10.1016/j.foodres.2023.113446)

References

1. Please unify the format

Reviewer #2: TITLE: It is suggested that the authors revise the title and make it more concise and appealing.

ABSTRACT

In background, Correct to formally knowns as

Some rephrasing is needed.

What do you mean by population structure?

LAY SUMMARY

What is meant by the population structure

INTRODUCTION

In first line correct to ‘and to prevent…’

Insert reference for the statement ‘a common human and animal commensal species…’

specify which type of commercial products.

What do the authors mean by saying ‘For this reason…’?

What is meant by What do you mean by "escape from GIT" Clarify.

cite reference for the statements where ingestion of lactobacillus containing foods poses a risk for patients and sick individuals.

The use of the term strain ought to be used instead of isolates

The third paragraph of the introduction section carries statements which are more suited for results and conclusion sections, for example ‘We demonstrate that food, probiotic, GI commensal, and

infection isolates are intermixed across the phylogenetic tree and frequently show surprisingly

little strain-to-strain sequence variability. These findings suggest that WGS may not be useful in

determining the source of L. rhamnosus infections….’ Similarly, the last 4 lines of the same para are also repetitive and belong to the conclusion section and not the introduction part of the article.

Rephrase statement given against cited reference 10

RESULTS

The authors must give a clear conceptual explanation of the term's unique whole genome assemblies and Illumina read sets in the context of their rationale of doing this work and experimental design

can we conclude from the sequencing data if there is significant variability in the data (supplementary table 1) such as # of read pairs/bases, alignment coverage and genome size? or even missing for many strains... explain why this is not the case here!

Again, great variation in sequencing data provided in supplementary table 1. So, is it justifiable to interpret only 10 or 11 SNVs difference? The total number of bases were provided only for DSM. Why is it so?

The result section ends with a summary which is the main conclusion of your study? Was this not obvious already?

DISCUSSION

Explain the rationale ‘of combining WGS and assemblies…’

MATERIAL & METHODS

In the section of public sequences, the manner of excluding assembly sequences appears arbitrary, contig counts or total genome size limits are vague, must be clarified, and restated.

Clarify how ‘read sets were further filtered to randomly select one set of reads from multiples…’?

How do you compare the quality of the 419 read sets with WGS? What are the benchmarks? It would be giving this comparison in a tabulated form

no detail of illumina read sequences has been provided. What is the quality of reads? is it comparable? for example, we cannot compare 60% quality reads with 95-100%

In the section Single nucleotide variant identification, ‘What is meant by SNV quality of scores of less than 200, selection of read consensuses, read depths?

In the section Phylogenetic analysis, what are separate complete sequence alignments. What is the basis of this consensus building?

In the section Patient Consent Statement, the authors make some conflicting statements regarding patient consent, they state that patient consent is not possible because of death of the patient but in the same paragraph, they also claim that it is not a requirement.

Kindly provide reference or describe the exact rule which supports that the ‘the study is not considered human subject's research

**********

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

**********

[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.

PONE-D-23-27461R1The global population structure of Lacticaseibacillus rhamnosus and its application to an investigation of a rare case of infective endocarditis.PLOS ONE

Dear Dr. Ozer,

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 Mar 15 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,

António Machado

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.

Additional Editor Comments:

Dear authors,

I am pleased to inform you that one reviewer already endorsed the revised manuscript and the second reviewer (Reviewer 3) requested major revisions for future publication endorsement. Please carefully answer the reviewer 3's concerns and rectify the manuscript following the reviewer's comments.

Thank you and best regards,

António Machado

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

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: (No Response)

Reviewer #3: (No Response)

**********

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: (No Response)

Reviewer #3: Partly

**********

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

Reviewer #1: (No Response)

Reviewer #3: 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: (No Response)

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: (No Response)

Reviewer #3: No

**********

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: (No Response)

Reviewer #3: The article written is important for the area of probiotics and the conclusions obtained may generate controversy, mainly because the text establishes a direct relationship between the presence of the Lactobacillus species in the patient's bacteremia and in yogurts that are of mass public consumption. Therefore, it is important to carry out an exhaustive review of the text and of the statements that are being used. In addition to this, it is important to make the following changes:

- Replace the terminology "flora" (in disuse) by microbiota.

- Include more bibliographic references in lines 56, 86-90, 92, 274-275 (original manuscript) and in all the sentences that are convenient to better support the statements that are made

- Restructure the results to make them more understandable, including subtitles to separate the various analyses performed.

- Indicate whether the yogurt brands analyzed include the one that the patient consumed (based on the relatives' statement) and justify why these brands were chosen (were they randomly selected? How can one justify the selection of 4 yogurt brands as a significant sample of all the yogurt brands offered on the market?)

- In the abstract the "core genome" is mentioned, but nothing else is indicated in the rest of the paper. Justify why the analysis was done only on the basis of the core genome, how it was defined and what the core genome comprises.

- In the discussion, cite other studies that have performed similar analyses or indicate that this is the first one.

- And finally, clarify that the condition of the patient from whom the strain was obtained was very critical and his decompensation is due to several factors. Also clarify that more analyses like this one (and even with greater statistical support) are needed to affirm that there is a relationship between probiotic consumption and the generation of serious infections.

**********

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 #3: Yes: Sandra Pamela Cangui Panchi

**********

[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.

The global population structure of Lacticaseibacillus rhamnosus and its application to an investigation of a rare case of infective endocarditis.

PONE-D-23-27461R2

Dear authors,

I am pleased to inform you that the revised manuscript was accepted for publication by both reviewers.

Thank you for submitting your work to the PLOS ONE journal and best regards,

António Machado

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 #3: All comments have been addressed

**********

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 #3: Yes

**********

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

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 #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 #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 #3: (No Response)

**********

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 #3: No

**********