PONE-D-19-27719

Comparison of sonication with chemical biofilm dislodgement methods using chelating and reducing agents: potential implications for the microbiological diagnosis of implant associated infection

PLOS ONE

Dear Prof. Trampuz,

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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Olivier Habimana

Academic Editor

PLOS ONE

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Additional Editor Comments (if provided):

The manuscript has now been reviewed by two experts in the field, with one recommended major revisions, and the other rejection. Major concerns pertained to the clinical relevance of the study that also is in need of more experimentation. The authors are, therefore, kindly requested to present a rebuttal addressing these concerns for further deliberation of the manuscript.

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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: 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 authors analyzed the activity (not efficacy as this is an in-vitro study) of chemical methods to be used for biofilm removal.

The manuscript is well written; the laboratory methods are suitable. The biofilm dispersion was determined by different methods. However, for this is a preliminary study. The purpose of removing biofilms from implants is medical microbiological diagnostics. For that, more microorganisms (incl. clinical strains) should be included. EDTA may interfere with viability of microorganisms. The supplementary use of chemicals to sonication should included in that study.

Minor remarks. Microorganisms associated with implant infections should be introduced.

How do the glass beads for biofilm formation correspond to clinically used material?

Reviewer #2: The authors performed an interesting in vitro study with the aim to evaluate the diagnostic performance of EDTA and DTT agents in comparison with the reference method (sonication) for bacterial dislodgement of S epidermidis and P aeruginosa biofilms.

Although the study has the following limitations:

- only 2 strains were tested (lacking information on S aureus, which is a well known biofilm producer),

- only reference strains were used,

- only glass beads as foreign body materials,

it undoubtely confirmed the higher bacterial detection rate of sonication method compared with other techniques. cThis finding has important consequences in terms of the clinical application of these new techniques, which might be considered as complentary, and not alternative, to sonication.

Detailed comment:

- Why 3 days of biofilm formation? Would the authors believe the results would be different with shorter or longer incubation time? Please comment on it

- The authors should also stress the potential use of sonication in implant infections other than orthopedics (ie intracardiac devices; neurosurgical shunts). Please add some references and a comment on it in the discussion.

- Insert a reference or an explanation on the use of porous glass beads as suitable model for biofilm formation in vitro in the method part.

- Please add a reference or an explanation for the timing of EDTA and DTT exposition for biofilm dislodgement in the method part, not only in the discussion.

- Furthermore, which is the rationale of choosing the EDTA and DTT concentrations? Please explain also in the method part.

- Insert the limit of detection in the method part (expressed as CFU/mL)

- Were the sonicated beads also submitted to vortexing process?

- The viability experiment should be better explained or addressed in an appropriate reference.

- When comparing all the three quantitative different groups, was the ANOVA test performed? In the case the ANOVA test was not used, please indicate the reason.

- Figure1 and Figure2; Figure5, 6. Please write per extenso EDTA and DTT. NS should be explained.

- Figure3 and figure4. Insert the different methods in the legend.

- Figure4. TTD should be explained. Which is the meaning of the arrow? Please explain.

- Figure3. 0,9% or 0.9%?. Please correct.

- Lines 151-153: the authors should describe that these CFUs obtained with these concentrations and timing derive from the above described experiments.

- Line 155: insert CFU/mL

- Lines 156-157: at which concentration and timing of EDTA and DTT for P. aeruginosa? Please describe.

- Lines 158-160: this sentence might be moved in a more appropriate place (ie, at the beginning of the paragraph).

- Lines 173-177: the results of P aeruginosa are similar to those observed for S epidermidis. Please re-phrase the sentence and remove the term “although”, which might be confounding for the reader.

- Lines 225-226. This sentence is not necessary since it has been already stated.

- It would be interesting to have a comment from the authors regarding other non-culture based methods of diagnosis of implant associated infection (ie, the metabolic assay BTA, BioTimer Assay, which has been recently investigated in intracardiac and central venous catheters infections as a complementary method to sonication)

- Please include a sentence highlighting the limitations of the study

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Reviewer #1: No

Reviewer #2: No

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PONE-D-19-27719R1

Comparison of sonication with chemical biofilm dislodgement methods using chelating and reducing agents: implications for the microbiological diagnosis of implant associated infection

PLOS ONE

Dear Prof. Trampuz,

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.

We would appreciate receiving your revised manuscript by May 01 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Olivier Habimana

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)

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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 #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: Thank you for letting me reviewing the revision of this manuscript. I’m impressed by the extensive and multimodal analysis of the activity of different biofilm-dislodging methods by the authors. The results are certainly of clinical relevance.

The former reviewers comments were addressed appropriately with a few exceptions:

Reviewer #2

2. only reference strains were used,

COMMENT: Explained in the manuscript.

“We did not use clinical strains as they typically show larger variability and are not suitable for

investigation of a new diagnostic method.”

3. only glass beads as foreign body materials,

COMMENT: We added the explanation and references in the method part.

“As a model to form the bacterial biofilm porous glass beads (diameter 4 mm, pore sizes 60 μm,

ROBU®, Hattert, Germany) were used. Due to the high volume-to-surface ratio, glass beads

were used for biofilm studies rather than smooth materials, as investigated in numerous

previous research works regarding biofilm formation and anti-biofilm activity [15-20].”

It undoubtedly confirmed the higher bacterial detection rate of sonication method compared with

other techniques. This finding has important consequences in terms of the clinical application of

these new techniques, which might be considered as complementary, and not alternative, to

sonication.

• These two limitations should be stated in the discussion. This in vitro analysis represents a fundament for further exploration in the clinical setting with clinical strains and real implants.

4. Why 3 days of biofilm formation? Would the authors believe the results would be different

with shorter or longer incubation time? Please comment on it

COMMENT: We incubated glass beads for 3 days for biofilm formation as described previously

by Drago et al. Clin Orthop Relat Res. 2012. The authors grew the biofilm for 3 days until a

visible biofilm was formed therefor further cultivation of mature biofilm is not needed.

• I agree with the reviewer. It remains unknown, whether the results would be different in more mature biofilms (as we are dealing with in the clinical setting). Comment this in the limitations setting.

9. Insert the limit of detection in the method part (expressed as CFU/mL)

COMMENT: We corrected in the manuscript as CFU/mL.

• Could not find the detection limit in the methods part. Please make sure to provide the limit of detection.

14. Figure3 and figure4 . Insert the different methods in the legend.

COMMENT: We corrected the information in the Figures.

• Please mention the different methods tested in the legend.

15. Figure4. TTD should be explained. Which is the meaning of the arrow? Please explain.

COMMENT: We corrected the information in the Figures.

• Please explain the meaning of the arrow in the legend

• Please make sure, that the figures are numbered correctly and mentioned accordingly in the manuscript text. Figure 2 is now Fig 5, Fig 3 is now Fig 6, etc.

18. Line 155: insert CFU/mL

COMMENT: We corrected in the manuscript as CFU/mL.

• Please add „CFU/ml“ in al the comparisons in the entire section (e.g. lines 170, 173, 178, 181)

Additional comments from my side:

• Make sure to use the terms dislodgement (vs. dislodgment) and NaCl (vs. saline) uniformally throughout the manuscript

• L 142: Please provide an introducing sentence before reporting the results. For example: „Figure 1 shows CFU counts of residual biofilms after exposure to chemical agents for variable durations.“

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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: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files 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.

Comparison of sonication with chemical biofilm dislodgement methods using chelating and reducing agents: implications for the microbiological diagnosis of implant associated infection

PONE-D-19-27719R2

Dear Dr. Trampuz,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Olivier Habimana

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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