PONE-D-20-04063

Cell death and biomass reduction in biofilms of multidrug resistant extended spectrum β-lactamase-producing uropathogenic Escherichia coli isolates by 1,8-cineole

PLOS ONE

Dear Dr Galvan,

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.

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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: Partly

Reviewer #2: Partly

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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: No

**********

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: Vazquez and colleagues present a manuscript that highlights an important issue - antibiotic resistance. With growing resistance to currently available therapeutics - natural compounds are of growing interest as potential biofilm inhibitors.

The manuscript is well written - however I have some concerns about the methodology used and the associated explanation for the chosen methods.

- Why did the authors choose a 3 day biofilm assay? Response to antimicrobial strategies in bacterial populations can be observed at the transcript level (RNA) after ~30 minutes treatment, by growing biofilms for 3 days - my concern at this small scale is that cells within the biofilm are under stress from depleting nutrients and oxygen, and therefore will impact any live/dead assessment being made for the inhibitor.

A time course with treatment would be a good way to test how the inhibitor's efficacy is impacted by depleting biofilm health.

-Were flow experiments only performed on dispersed cells? These are likely to be live as they are active - flow should be performed on biofilm cells also to calculate quantity of live/dead. This was not clear in the manuscript.

- 1,8 - cineole may be acting as a dispersal agent - what are the potential health consequences of this in a patient? This should be discussed by the authors. Also additional dispersal assays would strengthen the manuscript. Is motility involved?

Reviewer #2: This paper's major issue is that the English is not of sufficient quality to review. I believe that the experiments conducted and the methods used are likely sufficient to address the question but at this time the paper must be largely re-written. Due to this I have been unable to fully review the paper but this was my review of the parts I could review.

I dislike the term essential oil in a scientific paper. It does not convey a clear description of what the product is. Clearer terms such as plant oil. It can also be misappropriated by alternative medicine proponents. The authors are I assume not working under the guise that the aroma of the oil is in anyway contributing to the antibacterial nature of the oil. If that hold true then the use of ‘essential oil’ should be avoided.

In terms of scientific questions, the authors layout the following approach. Using the plant extracted oil a substantial reduction in viable cells was observed (a 3-fold log reduction). This was result was obtained in both antibiotic resistant and sensitive cells. This is then expanded to say that there is a reduction in biomass. The remaining biomass was then subjected to confocal analysis of live/dead staining and showed part of the remaining adhered biomass was actually dead. Cell no longer adhered we found to be mostly dead by flow cytometry analysis.

The use of statistics seems reudementary but without access to the raw data (as required by PLOS one) it is hard to determine. I would argue that given your hypothesis and previous work a two tailed test is not correct, you are working under the hypothesis that the plant extract will reduce the biofilm and or cell viability.

Regards

James Gurney

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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.

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Reviewer #1: No

Reviewer #2: Yes: James Gurney

[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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-20-04063R1

Cell death and biomass reduction in biofilms of multidrug resistant extended spectrum β-lactamase-producing uropathogenic Escherichia coli isolates by 1,8-cineole

PLOS ONE

Dear Dr. Galvan,

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 24 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Monica Cartelle Gestal, PhD

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 #2: All comments have been addressed

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 #2: Partly

Reviewer #3: Yes

**********

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

Reviewer #2: Yes

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 #2: Yes

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 #2: No

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 #2: Second review of the proposed plos one article

Cell death and biomass reduction in biofilms of multidrug resistant extended spectrum β-lactamase-producing uropathogenic Escherichia coli isolates by 1,8-cineole

The authors have made several key improvements to the English as evident by the tracked changes version, however there are a number of flaws still remaining that will need addressing. Plos One does not copy edit so they must be fixed before acceptance.

The improved English has allowed me to complete a full review. I have 4 major concerns and a number of minor concerns; I will include the errors in English under the minor concerns where I have spotted them, I will not correct them only point out where I believe there is a flaw.

Major concerns

How much of the reduction is due to the presence of tween 80? Previous work using the same compound did not make use of the surfactant. Why was it used and why was it not controlled for? On line 219 the authors make mention of reduced bacterial growth. They should present this data. Did they do a tween negative control? If not, they should. Line 389-392: has to be expanded significantly, currently other published work shows a much higher level for both MIC and bactericidal of the oil. What rational do the authors have for this discrepancy?

Given that the majority of clinical isolates did not make biofilms the authors must address the rational for using a biofilm disrupting treatment. I see three possibilities, and all should be discussed. Either the medium (M9) is not sufficient to recapitulate the clinical environment, reducing the enthusiasm for this work. Or the presence of biofilm forming strains in actual clinical samples is limited raising question about the usefulness of this treatment. Third, perhaps samples collected from clinical are bias for not biofilm forming as they would presumably be easier to collect. I would ask the author to at least address point 1 and 2 and remove all work related to the strain that did not produce biofilms.

The authors use 3 different methods of approximating biofilms. Crystal violet, CFUs, and confocal live dead staining. All 3 methods gave wildly different levels of reduction. For example, why did the CFU have 3-4 log reduction while the confocal had around a 1-2 log fold? Why don’t these methods agree? This I find further concerning as the MIC does not agree with the biofilm data. I would revise the section from line 364-379 to address this problem.

Section starting on line 309. Are the cells actually released from the biofilm or are they just growing planktonically? If they are released than according to fig 5A the ethanol treatment is better at releasing cells. If they are just growing planktonically this section needs to be revised.

Minor

Line 35: English

Line 38: English

Line 63: Needs a citation

Line 68: needs a citation

Line 74: why differentiate between fungi and yeast?

Line 83-84 I would remove the QS section. It adds nothing.

Line 116: English in two instances. Freshly streaked doesn’t mean anything and you do not streak *in* agar but *on* it.

Line 117: what is the volume used for the overnights?

Line 136: Why did you only use duplicates here? This data is not robust for statistically analysis. Please state all replicate numbers in figure legends

Line 146: space between tween 80 is missing.

Line 149: state what the vehicle is and how much was used.

Line 152: was only 1 assay done as the sentence implies? Or should it be biofilm biomass and cell viability?

Line 155: English

Line 182: English

Line 216: table say equal or greater to, but text suggests it is sensitive to 2%

Line 236: English

Line 248: and other parts, the authors say at least 3 replicates, are there uneven tests? What did the authors do to reduce the P-hacking of sampling at different rates per treatment? Or is this a case of an English mistake?

Line 254-255: Why does biomass (Fig 1) not correlate with figure 3?

Line 260: English

Line 282: What is the replicate number for data in table 3

Line 288 English

Line 316 & 318: give exact numbers.

Line 366: English

Line 411: English

Reviewer #3: General comments. Vazquez and colleagues present a manuscript evaluating the antibiofilm activity of 1,8-cineole against pre-formed mature biofilms of uropathogenic multidrug-resistant E. coli clinical strains.

The manuscript is well written and experiments are presented on a rational base.

However I have the following concerns:

Specific comment #1. The authors evaluate biofilm formation of ten E. coli isolates. Only one of them presented a substantial biofilm formation and two a mild formation. Raw data presented supports these conclusions. However data dispersion of biofilm formation in biofilm-forming strains is unusual. Authors should discuss these anomalies.

Specific comment #2. Authors should analyze the low frequency of biofilm-forming isolates in the context of evaluating a possible biofilm inhibition treatment.

Specific comment #3. Fig2A. The authors show cell viability on biofilms when they increased 1,8 cineole concentration. In raw data authors present five assays for treatment experiment but only one for control. Although differences between treatment and control with concentrations > 0.5% are important, authors must demonstrated this with a test. I don´t think the curve representation is the best suitable for these result.

Authors can consider present results in two parts: ethanol in extracts is not detrimental for survival and on the other side, dependence of viability to 1,8 cineole concentration compared to non 1,8 cineole (first column on raw data).

Specific comment #4. Table 3. The dispersion values between assays are noteworthy. Although differences are significant authors should address why they have such differences between assays. Dispersion was not observed in raw data for fig 1. I assume that some disruption of biofilm was produced during washes during treatment.

Specific comment #5. Authors described viability of detached cells arguing “It has been postulated that a good antibiofilm agent should not only attack bacteria into the biofilm but also display an action against biofilm-released cells”. The results is in concordance with high rate of dead cells on biofilm. It would be more interesting and will significantly improve the impact of the work to investigate if a post-treatment biofilm is able to growth.

Specific comment #6. Authors did not demonstrate that high biofilm biomasses yielded by strains are consequence of thicker extracellular matrix. Statement in L388-L393 should be modified.

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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 #2: Yes: James Gurney

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.

PONE-D-20-04063R2

Cell death and biomass reduction in biofilms of multidrug resistant extended spectrum β-lactamase-producing uropathogenic Escherichia coli isolates by 1,8-cineole

PLOS ONE

Dear Dr. Galvan,

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 Sep 26 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Monica Cartelle Gestal, PhD

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 #2: All comments have been addressed

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 #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #2: Yes

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 #2: Yes

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 #2: Yes

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 #2: (No Response)

Reviewer #3: Although most of the comments have been addressed, there are still a number of flaws that need to be addressed.

# one

The authors cite an article by Kragh et al to justify the stochastic variation in the biofilm results for the 72-hour biofilm and discourage the use of the CV staining method to draw conclusions. However, variations in biomass quantification were observed by microscopy (CV results were not shown) and were not as large as presented in this manuscript.

Authors must discuss the comment more extensively than citing the article by Kragh et al.

# two

Specific comment # 5. I disagree with the authors. I think the suggested experiment would improve significantly

impact of work. The authors present a new drug capable of altering and killing the cells presented in the biofilm life form. It is important to determine if the cells can grow.

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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 #2: Yes: James Gurney

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.

Cell death and biomass reduction in biofilms of multidrug resistant extended spectrum β-lactamase-producing uropathogenic Escherichia coli isolates by 1,8-cineole

PONE-D-20-04063R3

Dear Dr. Galvan,

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,

Monica Cartelle Gestal, PhD

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 #2: All comments have been addressed

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 #2: Yes

Reviewer #3: (No Response)

**********

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

Reviewer #2: Yes

Reviewer #3: (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 #2: Yes

Reviewer #3: (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 #2: Yes

Reviewer #3: (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 #2: While I signed off on the last version of this manuscript, I agree with reviewer 3 that the requested additions have strengthened the paper. My old comment is that I think the regrowth control should not have been a culture that had reach carrying capacity. Instead, I would have preferred to see the authors disrupt and dilute the biofilm so that the CFU was close to the treated level and watch for regrowth. However given the clear results of lower regrowth, I think the current assay is sufficient.

James Gurney

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 #2: Yes: James Gurney

Reviewer #3: No