Transcriptomic analysis of biofilm formation in strains of Clostridioides difficile associated with recurrent and non-recurrent infection reveals potential candidate markers for recurrence

Daira Rubio-Mendoza; Carlos Córdova-Fletes; Adrián Martínez-Meléndez; Rayo Morfín-Otero; Héctor Jesús Maldonado-Garza; Elvira Garza-González

doi:10.1371/journal.pone.0289593

Peer Review History

Original SubmissionFebruary 6, 2023
9 May 2023 Decision Letter - Rajesh P. Shastry, Editor PONE-D-23-03435Transcriptomic analysis of biofilm formation in strains of Clostridioides difficile associated with recurrent and nonrecurrent infection reveals potential candidate markers for recurrence.PLOS ONE Dear Dr. Garza-González, 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 Jun 23 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, Rajesh P. Shastry 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. Please make sure that all information entered in the 'Ethics Statement' section regarding ethics approval is also included in the Methods section of the manuscript. 3. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information. If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”). For additional information about PLOS ONE ethical requirements for human subjects research, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research. 4. Thank you for stating the following financial disclosure: [copy in funding statement]. 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. 5. 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. 6. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service. Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://aje.com/go/plos) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services. If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free. Upon resubmission, please provide the following: ● The name of the colleague or the details of the professional service that edited your manuscript ● A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a supporting information file) ● A clean copy of the edited manuscript (uploaded as the new manuscript file) 7. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. [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: Partly Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: I Don't Know ****** 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: 1. This is good attempt to investigate the role of differentially regulated genes during biofilm formation. 2. Objective, materials and methods and results have been described very clearly. Data analysis has been done in details and representation of data is concise. But the paper lacks any conclusive evidence about the role of genes in biofilm formation or R-CDI. There is more of speculation or information-oriented paper rather than pinning down the mechanisms for R-CDI. 3. RT-qPCR validation of few genes might give a hint towards the mechanism involved in biofilm formation, but orthogonal experiments with some gene modifications- like gene deletion, gene knockdown or over expression etc. are essential. 4. It is surprising that other proteins involved like bile acid receptor (CspC), germination receptor (like GerK etc.), which have been previously shown are not affected in the transcriptomic analysis. Reviewer #2: Dear authors, Many thanks for this interesting article which highlights the impact of biofilm formation on recurrence of C. difficile. However, I would have some points that would be nice if they could be addressed. I am not sure if a pool of probes is really a good way to evaluate this research question. Recurrence of C. difficile is complex but one common denominator are patients' characteristics as well. Because a strain did not cause clinically a recurrent disease does not automatically imply that biofilm formation is lower. By the way in ~50% of rCDI the patient has been infected with another strain he acquired from the environment. In the other 50% the strain persists. The genetic diversity is rather low, since only RT001 and RT027 were included. I would suggest the testing on a broader variety of strains (including the CD630 global RT012 reference strain) and each strain for itself. If I have seen it correctly, there were no strains with low bioflim formation included? Minor: Please harmonize the Material and methods part: sometimes the manufacturer is given with city, then with the state etc. Abstract: Please shorten the Material and method part, e.g. you may not give manufacturers name in this section. "nonrecurrent" or non-recurrent? (also in the title), please check. Some small English language editing can be considered. I would insert a "space" before the references. With the best regards. ****** 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.0289593.r001
Revision 1
4 Jul 2023 Author Response Rajesh P. Shastry Academic Editor PLOS ONE Dear Editor: Please find below the response for comments kindly provided by reviewers to our Manuscript PONE-D-23-03435 Transcriptomic analysis of biofilm formation in strains of Clostridioides difficile associated with recurrent and non-recurrent infection reveals potential candidate markers for recurrence. The professional service Scribbr edited our manuscript (https://order.scribbr.es/, order 2417109, revised by Debra, https://www.scribbr.com/about-us/editors/?_ga=2.73435346.1109912598.1685462982-2142197220.1685462982) This research was funded by Mexico’s National Council for Science and Technology, CONACYT, grant 284042. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Regarding figures, we are sending the original un crop image. We hope that this revision will answer the concerns and suggestions. Yours sincerely, The Authors Reviewer #1: 1. 2. Objective, materials and methods and results have been described very clearly. Data analysis has been done in details and representation of data is concise. But the paper lacks any conclusive evidence about the role of genes in biofilm formation or R-CDI. There is more of speculation or information-oriented paper rather than pinning down the mechanisms for R-CDI. Response: In our study, we detected that CAJ70148, CAJ68100, CAJ69725 and CAJ68151 genes were differentially expressed in biofilm and strains associated with R-CDI. A significant limitation in the results is that some of these genes have not yet been studied, and the proteins they encode and the pathways in which they participate are unknown. To elucidate the exact mechanisms, multiple investigations are required to determine the function of the unknown genes. Our results are the basis for further experiments, including some gene modifications- like gene deletion, gene knockdown or over-expression- to identify the role recurrence. This comment was added in the new manuscript, Discussion section. These comments were added in the new manuscript. 3. RT-qPCR validation of a few genes might give a hint towards the mechanism involved in biofilm formation, but orthogonal experiments with some gene modifications- like gene deletion, gene knockdown or over-expression etc. are essential. Response: In our study, RT-qPCR experiments were performed to validate differential expressions in the microarrays. Genes were selected based on their expression and presence in recurrence. Our results are the basis for further experiments, including some gene modifications- like gene deletion, gene knockdown or over-expression- to identify the role recurrence. This comment was added in the new manuscript, Discussion section. 4. It is surprising that other proteins involved like bile acid receptor (CspC), germination receptor (like GerK etc.), which have been previously shown are not affected in the transcriptomic analysis. Response: Germination begins when a germinating molecule interacts with the germinative (Ger) receptor. C. difficile spores do not share homologs of the gerA, gerB, and gerK spreout receptors commonly recognized in Bacillus spp. and other clostridia (Clin Microbiol Infect. 2018;24(5):476–82). In C. difficile, the first step of germination is the binding of germinants to subtilin-like serine protease (Csp)-like proteins encoded at the cspBAC locus, where the cspBA and cspC genes that code for the CspBA and CspC which are key regulators of germination signal transduction (Trends Microbiol. 2020;28(9):744–52). CspC functions as a receptor for bile salts. In addition to activating and regulating, it transmits the signal through protein-protein interactions to CspB. Overexpression of cspC and cspAB has been found in a biofilm model of C.difficile 630Δerm grown at 37°C in continuous-flow glass micro-fermentor in Tryptone Yeast Extract supplemented with 0.1% sodium thioglycolate incubating for 72 h (Front Microbiol. 2018 Sep 12;9:2084). The cspC gene was not overexpressed in the present work, probably because our methodology, the medium used (BHIS medium), had no thioglycolate. These comments were added to the new manuscript, Discussion section. Reviewer #2: I am not sure if a pool of probes is really a good way to evaluate this research question. Recurrence of C. difficile is complex but one common denominator are patients' characteristics as well. Because a strain did not cause clinically a recurrent disease does not automatically imply that biofilm formation is lower. Response: The use of pool of samples was included to minimize the effects of individual patients, and different strains. We included the same patients characteristics, as this is the common denominator for preparation of the pools. We agree with the reviewer that the association of a strain with recurrent disease does not imply that biofilm formation is lower. Indeed, we observed variation in the level of biofilm production among strains associated with recurrent infection. The use of pools is intended to minimize this effect. This comment was added to the new manuscript. By the way in ~50% of rCDI the patient has been infected with another strain acquired from the environment. In the other 50% the strain persists. Response: It has been reported that in CDI-R, 70 to 81% is due to the same strain and 15 to 30% is due to a different strain (J Infect Dis. 2014;209(9):1446–51., J Hosp Infect. 2015;90(2):108–16). Unfortunately, we have no strains collected in the initial CDI and the recurrent-CDI to define is the strain is the same or not. The use of pools is intended to minimize this effect. This comment was added to the new manuscript. The genetic diversity is rather low, since only RT001 and RT027 were included. I would suggest the testing on a broader variety of strains (including the CD630 global RT012 reference strain) and each strain for itself. Response: Some ribotypes are frequently associated with recurrence, including RT012 (Eur J Clin Microbiol Infect Dis. 2017;36(11):2251–8.), RT078, RT244 (S D Med. 2017;70(9):422–3), and RT027 (PLoS One. 2014;9(6)., J Clin Microbiol. 2012;50(12):4078–82., mSphere. 2018;3(3):1–8.). In contrast, other ribotypes have no clear association with recurrence, including RT001, RT106, RT017 and 018 (APMIS. 2013;121(2):153-7.,J Hosp Infect. 2017;95(4):394-399. PLoS One. 2016;11(11):e01661595–17). We selected a common ribotype clearly associated with recurrence and a second ribotype not clearly associated whit. Our results provide an overview of the transcriptomic differences among strains associated with CDI-R and CDR-NR in a 3-day biofilm, depending on the ribotype. The inclusion of other ribotypes will be of interest to clearly define the importance of ribotypes in the recurrence of infection. This comment was added in the new manuscript, Discussion section. If I have seen it correctly, there were no strains with low bioflim formation included? Response: Biofilm production was quantified after 3 days of incubation. Of all the strains included in the study, all the strains included RT027 associated with CDI-NR and CDI-R were strong biofilm producers. In the case of RT001, of the strains associated with CDI-NR, 1 was a strong producer, 2 were moderate producers, and 2 were non-producers, and for the strains associated with CDI-R, 5 were strong producers. (S1 Table. Classification of biofilm production in C. difficile). Minor: Please harmonize the Material and methods part: sometimes the manufacturer is given with city, then with the state etc. Response: Manufacturers were harmonized in the Material and methods section. The country is included in manufacturer details. Abstract: Please shorten the Material and method part, e.g. you may not give manufacturers name in this section. Response: Abstract was shortened in the method section "nonrecurrent" or non-recurrent? (also in the title), please check. Response: The term non-recurrent is now used I all the manuscript Some small English language editing can be considered. Response: English language was performed I would insert a "space" before the references. Response: A space was added before the references Attachments Attachment Submitted filename: respose260523.docx https://doi.org/10.1371/journal.pone.0289593.r002
24 Jul 2023 Decision Letter - Rajesh P. Shastry, Editor Transcriptomic analysis of biofilm formation in strains of Clostridioides difficile associated with recurrent and nonrecurrent infection reveals potential candidate markers for recurrence. PONE-D-23-03435R1 Dear Dr. Garza-González, 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, Rajesh P. Shastry Academic Editor PLOS ONE Additional Editor Comments (optional): I have included the some minor changes as suggested by the reviewer. Please address the changes in the manuscript before finalization. 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: 1. All the comments during the first round were carefully addressed and the manuscript is ready for acceptance. 2. For further studies, it would be interesting to see how the phenotypes of R-CDI and NR-CDI strains looks like in different media. BHIS is a very rich medium and C. difficile might not always encounter rich environmental conditions in the gut. Suggestions for few minor changes are below: a) Line 63: Change ‘nonmutant’ to wild type (WT). b) Line 99: “After, 200 uL from each suspension……”. Delete the word “After”. c) Line 102: “… the biofilm was fixed with 200 uL with methanol…..” Edit the sentence as “…. Was fixed with 200 uL of methanol…” ****** 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 ******** https://doi.org/10.1371/journal.pone.0289593.r003
Formally Accepted
27 Jul 2023 Acceptance Letter - Rajesh P. Shastry, Editor PONE-D-23-03435R1 Transcriptomic analysis of biofilm formation in strains of Clostridioides difficile associated with recurrent and non-recurrent infection reveals potential candidate markers for recurrence. Dear Dr. Garza-González: 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. Rajesh P. Shastry Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0289593.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 .