PONE-D-21-12554

Rapid resistance development to three antistaphylococcal therapies in antibiotic-tolerant Staphylococcus aureus bacteremia

PLOS ONE

Dear Dr. Berti,

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 all the points raised by the reviewers during the review process.

Please submit your revised manuscript by Jul 17 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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Herminia de Lencastre, Ph.D.

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: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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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 from Miller et al. documents a clinical case report of rapid resistance development to three MRSA therapies in a patient with methicillin-resistant Staphylococcus aureus bacteremia. Genetic, phenotypic and metabolic studies have been done using blood samples recovered over a six-week clinical course. The study technically sounds and this is an area of increasing interest. Authors attributed the rapid emergence of resistance to the daptomycin tolerant phenotype of the index isolate and ruled out the hypermutator phenotype through the determination of spontaneous mutation rate to rifampin in vitro. They were able to justify the appearance of mutations as a result of the antibiotic therapy that was used. It is missing from the manuscript some important information, namely: if the nostrils of the patient have been screened for staphylococcal colonization during the clinical course; the number of colonies picked at each time point and the number of colonies sequenced at each time point.

Instead of having a table in Figure 1, authors may consider to create an infographic timeline combining the information of that table with the antibiotic therapy used and including in the timeline the intermediate blood surveillance cultures negative for MRSA.

Minor points of criticism:

- In the abstract section, line 28 please substitute “the isolates demonstrated” by “the index isolate demonstrated”.

- Please substitute in line 108 “Patients isolates are” by “BSN14S1 is”.

- In results section, line 114 please substitute “All patient isolates” by “All comparator isolates”

- Please state the name to which the acronym BL in line 143 refers to.

- What do the asterisks in Table 2 mean?

- It is missing from materials and methods the conditions of the DNA extraction for WGS.

- Did the authors used default parameters for SNP calling? If yes please state it.

Reviewer #2: This manuscript reports the case of a patient with methicillin-resistant Staphylococcus aureus (MRSA) bacteremia which developed resistance to several antibiotics classically used for MRSA infections (ceftaroline and daptomycin) under the course of treatment (over a period of 44 days). Isolates also showed high minimal inhibitory concentrations (MICs) for vancomycin. Five different isolates were obtained on days 1, 3, 28, 31 and 44. These isolates were sequenced using both Nanopore MinION and Illumina. All isolates were confirmed to be isogenic. Several mutations were present in recurrent isolates compared to the initial one, in genes already described in literature as associated with resistance to daptomycin or heteroresistance to glycopeptides. Then the authors explored several hypotheses to explain the ability of this S. aureus strain to develop these mutations and to understand the potential role of the mutations detected by whole-genome sequencing (WGS). Interestingly they showed that this strain was antibiotic tolerant.

WGS sequences have been deposited to Genbank.

Major comments

- The results of this study are difficult to extrapolate since they only concerned one patient case but they provide insights on the potential link between antibiotic tolerance and development of antibiotic resistance. Nevertheless, data presented here did not allow to affirm that antibiotic tolerance facilitates rapid resistance development.

- In my opinion, the manuscript is not easy to read, probably due to the fact that authors have chosen not to separate results and discussion, and it is difficult to understand what is really new in these results from that is already described in the literature. For instance, the authors described mutations in mprf or in vraT but they did not precise if the exact mutations present in this patient’s isolates have already been described and associated with antibiotic resistance or not.

- Data already published about the link between antibiotic tolerance and development of antibiotic resistance should be more developed by the authors in the introduction and in the discussion. The authors should highlight in the manuscript which of their data provide new insights in the field.

- The authors should provide the complete antimicrobial susceptibility testing profile of the isolates (and not only oxacillin, vancomycin, ceftaroline, daptomycin, linezolid).

- They report the isolates as resistant to vancomycin but isolates have a MIC equal to 2 mg/L, which is susceptible according to CLSI recommendations. If the authors considered the isolates as heteroGISA, they should have performed population analysis profile to confirm this. So the title of the manuscript seems not adapted if the authors included vancomycin in the three antibiotics concerned by development of resistance. Moreover, authors described the appearance of a vraT mutation but the vancomycin MIC of the isolates did not increase over time so it is difficult to assign a role in antibiotic resistance to this mutation.

- In the “Patient case” section, the authors first described the antimicrobial treatments received by the patient and then the results of bacteriological cultures. As the choice of antibiotic therapy depends on the bacteriological results, these latter should be presented before or with antibiotic therapies. Instead of figure 1 presented as a table, a chronological timeline including both bacteriological results and changes in antibiotic therapies would better illustrate the patient case.

- Isolate BSN14RB showed a decreased ceftaroline MIC compared to the previous isolates: did the authors have a hypothesis about this? The authors do not discuss what would be the mechanism of resistance to ceftaroline in isolates R1 and R2.

- The method used to study the binding of cytochrome C is not described in the section “Materials and Methods”.

- The authors described the selection of mutations potentially involved in the development of resistance. Did they test several colonies on the initial S. aureus population or the following isolates to check for the absence of a heterogeneous population?

Minor comments

- Line 70: precise that bacteria were MRSA

- Line 121: results of doubling time are presented in Figure 2 and not Table 2

- Lines 213, 217, 244: please italicize S. aureus

- In the text, define the abbreviations TBA and MDK

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Reviewer #1: No

Reviewer #2: No

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PONE-D-21-12554R1Rapid resistance development to three antistaphylococcal therapies in antibiotic-tolerant Staphylococcus aureus bacteremiaPLOS ONE

Dear Dr. Berti,

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 additional points raised during the review process by the two reviewers. Please submit your revised manuscript by Oct 22 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,

Herminia de Lencastre, Ph.D.

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: (No Response)

Reviewer #2: 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

**********

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

Reviewer #1: Yes

Reviewer #2: 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: No

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: Authors have addressed reviewers' comments and they still have added additional in vitro experiments to support their arguments, namely, that antimicrobial tolerance may promote the development of antimicrobial resistance. There are still some changes authors can made in this version of the manuscript for its improvement.

1. Lines 78-79 - Please clarify. Based on Figure 1 after the 11th day and for two weeks there was no weekly surveillance cultures.

2. Line 84 - "Blood cultures cleared day 31", shouldn't be day 33?

3. Authors refer that they perform PAP analysis but these results are not shown.

4. Please avoid referring to the isolates by the month of their isolation. Please use instead the day of the isolation or the name of the isolate to be in accordance with Figure 1.

5. Lines 289-291 - Only volumes used were provided, no reference for the concentration of the solutions used is provided.

6. Line 332 - "0.25 mg/L or 0.5 mg/L" of what?

7. Figure 1 - Please define antibiotics' abbreviations in the legend.

8. Figure 4 - In material and methods is referred that three biological replicates of each strain have been used. As so, why are represented 6 replicates (n=6) for BSN14S1?

Reviewer #2: The authors have addressed all the points raised during the first review. Thus the clarity and robustness of the manuscript’s conclusions have been strongly improved.

I have some minor comments.

- Line 19: index isolate is not susceptible to trimethoprim/sulfamethoxazole so change “all (non-beta-lactam) antibiotics” for “almost all” or “all… except trimethoprim/sulfamethoxazole”

- Line 23: isolate BSN14RB is not susceptible to ceftaroline (table S1, MIC=2, as previous isolates R1 and R2)

- Line 93: genetic variations are not indicated in Figure 1

- Line 111: please indicate that data are presented In Figure 2. Results in the text are not consistent with those in Figure 2 (23-38 vs 23-28 for BSN14S1)

- Line 216: data are shown in Table S2

- Line 332: precise that tubes contained daptomycin

- Figure 1: what does ⴕ mean in the table?

- Table S1: if the table is published, it would be useful to add the date of collection of each isolate as in Figure 1.

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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-21-12554R2Rapid resistance development to three antistaphylococcal therapies in antibiotic-tolerant Staphylococcus aureus bacteremiaPLOS ONE

Dear Dr. Berti,

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 additional points raised by reviewer #1.

Please submit your revised manuscript by Nov 07 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,

Herminia de Lencastre, Ph.D.

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

**********

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

**********

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

Reviewer #1: 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

**********

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

**********

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 addressed all the comments raised during the previous round of review. However, I still have some minor comments:

- There is a typo in the beginning of the title of the manuscript. Please remove the letter "z".

- Please clarify the susceptibility profile of the blood cultures mentioned in lines 22-23. In accordance to figure 1 and table S1, the blood culture remained with intermediate resistance to vancomycin and resistant to daptomycin and ceftaroline susceptibility was not available for this blood culture in the patient chart.

- Line 160 - Please remove the word "in".

- The interpretation criteria used to define vancomycin phenotype in table S1 (VSSA, hVISA or VISA) should be provided, as well as the PAP graphics. In table S1 why was isolate BSN14S2 considered as VSSA and isolates BSN14R1 and BSN14RB as hVISA?

- Please provide the name of the software used for the determination of AUC.

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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

[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.

Rapid resistance development to three antistaphylococcal therapies in antibiotic-tolerant Staphylococcus aureus bacteremia

PONE-D-21-12554R3

Dear Dr. Berti,

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,

Herminia de Lencastre, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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

**********

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)

**********

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

Reviewer #1: (No Response)

**********

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)

**********

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)

**********

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: Authors have changed the order of the AUCs data presented in table S1, however they did not change the values of "PAP/AUC ratio" in the second line of the table accordingly. Please revise that line too.

**********

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