PONE-D-21-18023

Genomic analysis of shiga toxin-containing Escherichia coli O157:H7 isolated from Argentinean cattle

PLOS ONE

Dear Dr. Cataldi,

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 by both reviewers.

Quality of the figures was borderline when sent out for initial review and I would appreciate if you could include higher quality figures and label them accordingly. Please also address the concerns raised by reviewer 2 in regards to data/software availability. Considering the limited number of isolates analyzed conclusions should not be overstated. As suggested by reviewer 2 "Results/Discussion" could be shortened to avoid redundancies without hurting the overall information content of this manuscript.

Please submit your revised manuscript by Aug 16 2021 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Mark Eppinger

Academic Editor

PLOS ONE

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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

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

Reviewer #1: Yes

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: No

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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: This study reports a comparative genomic analysis of eight EHEC strains isolated from cattle in Argentina, that were previously classified into clades based on SNPs. These strains were sequenced using PacBio technology and used adequate bioinformatic software. They made comparative analysis founding virulence genes related to LEE genomic island, toxin effectors and stress resistance. Among novelty information, the authors found prophage distribution between strains which is relatively different to previous reports and were capable to group into families. Its findings are very interesting and give new insights into diversity of this important pathotype.

I only have a few observations and suggestions to make your manuscript better understood:

1. “Pathogenicity island” is a no longer used term, is preferred “genomic Island” even if it harbors virulence genes, my suggestion is to change the terms.

2. When you mentioned “geneious” you are referring to the software so, you should specify it in order to avoid confusion particularly to novel readers into bioinformatics

3. Shiga-toxin detection and cytotoxicity in Vero cells is a well performed assay and according to previous bibliography and provides important information but it seems to be not related with the title of the manuscript, later in text, the importance of this assay arises so I suggest to justify it when you introduce the results of the experiment

4. All the figures are blurry even if you download it as .tif format, I´m not sure if they are formatted in the correct specification. It makes difficult to follow the text referring to the figure.

5. Lines 229-230. You are talking about cytotoxicity referring to Figure 1, but it is an Artemis map not a toxicity graph

6. Lines 370-375. You are indicating panel (A) and (B) but they are not indicated in the figure, please correct it

Reviewer #2: This manuscript presents a genomic comparison of eight E. coli O157:H7 strains, isolated from the same region of Argentina. Comparative genomic tools were applied to screen for genomic features well-described by this field, including clade typing, prophage analysis, stx2 genotyping, and virulence gene identification. The authors identify characteristics reported before in similar studies, including diverse stx2a prophage but less diversity in stx2c prophage, polymorphisms in stx2-associated genes, and genomic rearrangements. While most of their findings were expected, the experiments were performed properly and for the most part (see below), their interpretations were supported by the data.

Major comments;

1. With hundreds of O157:H7 sequenced genomes available, this seems like a missed opportunity not to perform a larger comparison with other clade 8's. This would add much more value to the paper.

2. In the absence of a deeper genomic analysis, the phrase “sheds light about the genetic basis of hypervirulence” must be removed. This study does not report any experiments that provide new insights into in vivo virulence potential.

3. It appears a decent amount of prophage analysis was done with a “custom Perl script”. Without access to the script, the reader cannot repeat any this portion of the study. This should be made available on GitHub or similar site.

4. What statistical analysis was done to make the “larger prophages” conclusion in lines 261-262?

5. Line 363: Did you test whether this strain makes Shiga toxin using your ELISA? If not, explain in the paper why.

6. The Discussion is a reiteration of the Results. These two sections should be combined.

7. Many of the Figures, even when downloaded via the link provided, are really blurry.

Minor comments:

1. Lines 34-35: sentence fragment

2. Line 66: stx2h and 2i have been reported as well

3. Line 224: temper this language, as there may be additional prophage that weren’t identified by Phaster.

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Reviewer #1: Yes: Edwin Barrios-Villa

Reviewer #2: No

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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-21-18023R1

Genomic analysis of shiga toxin-containing Escherichia coli O157:H7 isolated from Argentinean cattle

PLOS ONE

Dear Dr. Cataldi,

Thank you for submitting your manuscript to PLOS ONE. 

Please address the comment raised by one of the reviewers in regards to the discussion of stx2 suballeles and references.

"The authors added "stx2h" and "stx2i" as previously suggested, but use an incorrect 2012 reference (these variants were first reported around 2018-2019).  Please see doi:10.1038/s41598-018-25233-x and doi: https:doi.org/10.4315/ 0362-028X.JFP-18-291."

Also, a more recent article reports on stx2k (Yang, X. et (2020). Escherichia coli strains producing a novel Shiga toxin 2 subtype circulate in China. Int J Med Microbiol 310, 151377.).

Please submit your revised manuscript by Nov 01 2021 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,

Mark Eppinger

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 (if provided):

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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: Yes

********** 

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

Reviewer #1: Yes

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: As mentioned in past review, this study worth to be communicated. All recommendations have been addressed

Reviewer #2: The authors added "stx2h" and "stx2i" as previously suggested, but use an incorrect 2012 reference (these variants were first reported around 2018-2019). Please see doi:10.1038/s41598-018-25233-x and doi: https:doi.org/10.4315/ 0362-028X.JFP-18-291.

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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: 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.

Genomic analysis of shiga toxin-containing Escherichia coli O157:H7 isolated from Argentinean cattle

PONE-D-21-18023R2

Dear Dr. Cataldi,

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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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,

Mark Eppinger

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: