Combination treatment to improve mucociliary transport of Pseudomonas aeruginosa biofilms

Kaitlyn R. Rouillard; Christopher P. Esther; William J. Kissner; Lucas M. Plott; Dean W. Bowman; Matthew R. Markovetz; David B. Hill

doi:10.1371/journal.pone.0294120

Peer Review History

Original SubmissionSeptember 11, 2023
10 Oct 2023 Decision Letter - Abdelwahab Omri, Editor Transfer Alert This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present. PONE-D-23-29054Combination Treatment to Improve Mucociliary Transport of Pseudomonas aeruginosa BiofilmsPLOS ONE Dear Dr. Hill, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Nov 24 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Abdelwahab Omri, Pharm B, Ph.D, Laurentian University Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 3. Thank you for stating the following financial disclosure: "KRR (Cystic Fibrosis Foundation ROUILL22F0) DBH (Cystic Fibrosis Foundation HILL19G0, HILL20y2-OUT) This work also benfited from Cores funded by the Cystic Fibrosis Foundation (BOUCHE19R0) and NIH (1P01HL164320)". Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 4. Thank you for stating the following in the Acknowledgments Section of your manuscript: "Funding for this work was provided by the Cystic Fibrosis Foundation (ROUILL22F0, HILL19G0), HILL20Y2-OUT, BOUCHE19R0), and the NIH (P30DK065988 and 1P01HL164320)". We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "KRR (Cystic Fibrosis Foundation ROUILL22F0) DBH (Cystic Fibrosis Foundation HILL19G0, HILL20y2-OUT) This work also benfited from Cores funded by the Cystic Fibrosis Foundation (BOUCHE19R0) and NIH (1P01HL164320)" Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 5. Thank you for stating the following in your Competing Interests section: "No authors have competing interest". Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state "The authors have declared that no competing interests exist.", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now This information should be included in your cover letter; we will change the online submission form on your behalf. 6. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. In your revised cover letter, please address the following prompts: a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. We will update your Data Availability statement on your behalf to reflect the information you provide. 7. Please note that in order to use the direct billing option the corresponding author must be affiliated with the chosen institute. Please either amend your manuscript to change the affiliation or corresponding author, or email us at plosone@plos.org with a request to remove this option. 8. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well. 9. Please upload a copy of Supporting Information Figure/Table/etc. Supplemental Figure 1, 2, 3, 4, 5 and 6 which you refer to in your text on pages 21, 22, 23 and 24. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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 ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The manuscript reports a study on combinations of antibiotic with biofilm matrix or/and mucus modulating actives to improve chronic bacterial infections therapy in patients with muco-obstructive diseases. The aim of the drug combinations is to simultaneously kill the bacterial pathogen and increase the mucociliary clearance. The effect of mono and dual and triple drug combinations was investigated i) on rheology of mucus biofilm ii) ciliary transport rates using an in vitro cell system and iii) bacteriocidal efficacy against P. aeruginosa (PAO1). The study is well planned, performed and described. A model to investigate the mucociliary clearance in controlled in vitro setting without ethical concerns of animal experiments is an interesting set-up which could help in development of mucolytic formulation of anti-infectives. The selected drug combinations are based on authors experience and literatur and are well chosen. Few minor adjustments are recommended before acceptance in PLOS ONE for publishing. • Please spell out the full name, or even further specification of the surfactant NP40 (e.g., avoiding possible confusion with Nonidet-P40). • NP40 can be used for cell lysis. Some comment on the used concentration of this surfactant would be welcome • In MCT rates determination, the procedure is described to filter out stuck beads (i.e. MC<01. µm/s). Figure 2 reports CBFs and MCTs are sufficiently reproducible over cultures and quadrants. Is the removal of stuck beads essential to achieve the reproducibility or only facilitating the calculation? Is the fraction of stuck beads approx. equal for quadrants and culture samples? • Supplemental Figure 4: Please clarify in the caption if the 4 Videos show 4 technical replicates or different regions form the same sample (treatment 10mM TCEP+ 1mg/mL Tob). Reviewer #2: The data presented here make a compelling case for combining therapies targeting mucus clearance with antibiotics in order to more effectively eradicate chronic Pseudomonas aeruginosa infections. The triple combination approach is interesting, and the data showing that viscoelastic behavior of the biofilm switching from a viscoelastic solid to a viscoelastic liquid is particularly compelling. However, there are some weaknesses that the authors should consider to enhance the impact of the data. 1. The experiments are done only on non-diseased human bronchial cells. Adding in cells from a donor with muco-obstructive pulmonary disease would be important to understand if this approach will be efficacious in disease. 2. Table 1 is helpful for the reader but not entirely clear. Why was the listed combination chosen? In particular, why was DNAse not included in a triple combination approach given that it is a currently approved therapeutic? 3. In figure 4B, it is not clear which data set indicates biofilm vs planktonic forms of the bacteria. 4. In Figure 4D, the change in CFUs is important, but it may be more impactful to compare the CFUs to tobramycin, since this is the current standard of care. 5. Table 2, and the biological impact of this data, is not clear. 6. In Figure 5, it is not clear why the MCT rates go down with the addition of tobramycin for all the groups except for DNAse. Are there CFU data to match this experiment? 7. It is understandable why the authors chose to start the experiments with PAO1. It will be much more impactful if these results are confirmed in the presence of a clinical isolate or mucoid strain. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0294120.r001
Revision 1
20 Oct 2023 Author Response We thank the reviewers for their kind and constructive review of our manuscript. We have addressed each concern with tracked changes in the submitted document. Below are our point-by-point replies: Reviewer 1: 1. NP40 is nonyl phenoxypolyethoxylethanol. We have updated the manuscript to include this full name. 2. NP40 was used at 0.1% which is below the critical micelle concentration. We wanted to avoid any complications from micelle formations in the rheological impact of treatment. Additionally, the 0.1% dose was the lowest dose necessary to induce significant decreases in macroscopic and microscopic complex viscosity values in both 2% and 4% mucus. These data are included in a different manuscript submission specific to mucus rheology. We have referenced the previous study that included the dosages used for the other components. 3. The removal of the stuck beads is critical for an accurate quantification of MCT rates. A large portion of the beads have effectively transport rates of zero, which decreases the ensemble rate greatly. The number of stuck beads across videos ranges from 0-25, depending on the properties of the transporting sample. More heterogeneous and viscous samples are associated with more stuck beads, such as untreated biofilms and mucus + planktonic bacteria. The treated biofilms that have a compromised matrix, particularly those treated with the triple combinations, have far fewer stuck beads. For transparency, we have included the number of stuck beads removed from each sample into a table in the supporting information. 4. Each video is acquired in a different region of the culture. We have updated the caption to specify this. Reviewer 2: 1. Transport in diseased HBE cells can be especially challenging, particularly with decreased MCT rates in the diseased cultures. Also, with the decreasing availability of CF lungs, CF cells are being used for more precious experiments. We are currently investigating the use of conditionally reprogrammed cells (CRC) to expand CF cells to compare mucus biophysical properties. When these studies are complete, we will be able to utilize CF HBE mucus and cells for transport experiments. For the purpose of this study, HBE mucus was prepared at 3% solids to represent mild pulmonary disease. We believe that the present work lays the foundation necessary for more complex studies with diseased HBE cells, like CF HBE CRC cultures, that can then benefit from a dose-response study with the triple combination treatments screened in this study. 2. Treatments that maximized biofilm disruption (increase or decrease in biofilm complex viscosity) and reductions in bacterial viability were combined with tobramycin for the triple combination treatment. DNase was excluded from triple combination treatments due to minimal impact on biofilm rheology in high mucus concentrations, which we have published on previously. TCEP, NP40, and HA have demonstrated more promising biofilm disruption capabilities in this study and our previous work. This study laid the groundwork necessary to evaluate a single treatment (TCEP/NP40/HA) in an infected murine model, which is upcoming. 3. Throughout the document, PAO1 3% is used to indicate biofilms grown in 3% mucus while 3% HBE + PAO1 is used to indicate mucus with planktonic bacteria. The caption includes this information as well. We have also included a clarifying statement into the text to highlight the nomenclature and remove any confusion. 4. The individual agents do not significantly decrease bacterial viability, and ultimately, the goal is to optimize reductions in biofilm complex viscosity and viability. Thus, we must ensure that there are no antagonistic interactions between the biofilm disruption agent (e.g., TCEP) and the antibiotic. 5. We have previously published on the non-Gaussian parameter (κ) for PAO1 biofilms grown in mucus. Greater κ values indicate greater heterogeneity of the biofilm biophysical properties, which may be used as a measure of biofilm disruption. The combinations that induced the greatest changes in κ values were TCEP, HA, and NP40, each with tobramycin. The PTMR data for each of these conditions is included in Supplemental Figure 2 where we qualitatively observe changes in biofilm biophysical behavior (i.e., complex viscosity distribution). These values suggest that combinations of these agents may result in superior biofilm disruption that may be associated with increased MCT rates. Indeed, the triple combinations of these agents significantly increased MCT rates. We have added a statement to this effect into the discussion section. 6. The CFU data for the combinations are included in Figure 4, but treatment with the single agent had little effect on viability. We hypothesize that biofilm disruption with the single agent (e.g., TCEP, HA) was sufficient to increase MCT rates but not eradicate bacteria. However, the addition of tobramycin resulted in cell death that contributed to slightly increased complex viscosity values (4C) and decreased MCT rates (Fig 5). We have included a statement to this effect in the results section. 7. We have a reproducible and robust biofilm growth model in HBE mucus with PAO1 for in vitro studies. The benefit to PAO1 for these experiments is its broad availability to other labs as well. The goal of this work was to screen and evaluate combination treatments in order to select an optimal in vitro candidate for in vivo studies. Because the in vitro susceptibility of even clinical isolates is often different from in vivo behavior, we wanted to optimize biophysical changes with PAO1 before moving to an isolate that’s been optimized for an infected murine model. These studies are ongoing. Attachments Attachment Submitted filename: MCT response to reviewers.docx https://doi.org/10.1371/journal.pone.0294120.r002
26 Oct 2023 Decision Letter - Abdelwahab Omri, Editor Combination Treatment to Improve Mucociliary Transport of Pseudomonas aeruginosa Biofilms PONE-D-23-29054R1 Dear Dr. David B Hill, 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, Abdelwahab Omri, Pharm B, Ph.D, Laurentian University Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0294120.r003
Formally Accepted
10 Nov 2023 Acceptance Letter - Abdelwahab Omri, Editor PONE-D-23-29054R1 Combination Treatment to Improve Mucociliary Transport of Pseudomonas aeruginosa Biofilms Dear Dr. Hill: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Abdelwahab Omri Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0294120.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .