Antibacterial, antibiofilm and anti-quorum sensing activities of 1,2,3,5-tetrazine derivatives linked to a benzothiazole moiety

Jean Paul Dzoyem; Joseph Tsemeugne; Boniface Pone Kamdem; Rostand Foyou Meupiap; Boris Arnaud Kuate; Pierre Mkounga; Fabrice Fekam Boyom; Lyndy Joy McGaw

doi:10.1371/journal.pone.0318135

Peer Review History

Original SubmissionJanuary 11, 2025
11 Jan 2025 Author Response https://doi.org/10.1371/journal.pone.0318135.r001
28 Mar 2025 Decision Letter - M. Alejandro Dinamarca, Editor PONE-D-25-01821Antibacterial, antibiofilm and anti-quorum sensing activities of 1,2,3,5-tetrazine derivatives linked to a benzothiazole moietyPLOS ONE Dear Dr. Pone Kamdem, 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. The submitted manuscript has scientific merit and it is positively valued to work with chemically synthesized compounds by the authors' own team in the context of bacterial quorum sensing inhibition. However, in light of the review, the manuscript must correct and clarify aspects related to the methodology, specifically the relationship between MICs, MIBCs, and the biomass necessary to perform QS and form biofilms. Additionally, the authors should delve into the chemical and molecular mechanisms that explain the observed results. This should be done by proposing chemical or molecular biology assays that can elucidate the pathways through which the derived triazines exert their action. Please submit your revised manuscript by april 25th. 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. 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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, M. Alejandro Dinamarca, Dr. 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 Additional Editor Comments: The article titled Antibacterial, antibiofilm and anti-quorum sensing activities of 1,2,3,5-tetrazine derivatives linked to a benzothiazole moiety, is a work that evaluates a series of organic compounds derived from chemical synthesis conducted by the group of authors. Specifically, the authors evaluate their synthesis products in two related bacterial behavior systems, which are quorum sensing communication and biofilm formation. The evaluations are conducted on a series of bacterial pathogens or pathogenicity models such as: Klebsiella aerogenes ATCC 130148, Acinetobacter baumannii ATCC BAA-1605, Enterococcus faecium ATCC700221, Staphylococcus epidermidis. Additionally, the ability to inhibit the quorum sensing mechanism is evaluated using the production of violacein in Chromobacterium violaceum ATCC12472 as a model. To methodologically relate their work, the authors propose an experimental design based on determining the respective MICs and MIBCs of each compound in concentrations of µg/ml. The results of the work are expressed in percentage values through known formulas. The results are consistent with the experimental design; however, they present a methodological question that must be clarified by the authors. Specifically, if bacteria need a specific concentration to perform QS communication, how does this relate to the concentrations related to MICs and MIBCs used for each evaluated triazine? The same is considered for the formation or eradication of biofilms. This must be adequately clarified in the methodology section and in the results, with a special mention in the discussion section. Finally, although the article is coherent and it is positively valued to work with compounds derived from synthesis, it is required to delve deeper methodologically or in the discussion regarding the molecular and chemical mechanisms that may be involved in the observed results. The manuscript must be presented in the format required by PLOS. [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A ****** 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 ****** 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 ****** 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 addresses a relevant topic and evaluates new potential antimicrobial and antivirulence compounds. While the evaluated compounds show potential against relevant pathogens, there are some aspects of the microbiological assays that are not included and without which a solid conclusion can not be generated. Major comments There are some major methodological issues regarding the evaluation of biofilm inhibition and violacein production. While authors test different concentrations of the selected compounds below MIC and MBC, they do not take into consideration the potential effect of subinhibitory concentrations of these on bacterial growth. Therefore, it is not possible to determine if the lower biofilm formation and violacein production is due to the selective effect of the compounds on these cellular processes or to a reduction of bacterial cells. Normalization by total cell number (CFU or Optical density at 600 nm) or growth curves at the selected concentrations of the compounds should be included to support the conclusions. Minor comments: Introduction, L5: change "The main mechanisms of ..... includes" for "include" (plural) Introduction, L7: "degradation" may be replace bt a more accurate concept as "structural modification" Methods, 2.2.1: Please indicate why C. violaceum was cultured at 30 ºC instead of 26 ºC (its optimal growth temperature) Methods 2.2.3: Delete ")" after gentamicin. Please specify gentamicin concentrations used as control condition. "The MBC was determined as the ...that completely inhibited the growth of bacteria". Because authors are evaluating bactericidal effect, the more accurate concept should be "that inhibited bacterial viability". Methods, 2.3.1. Please specify vanillin concentration Results, 3.1.2: Authors should include ATCC numbers for all strains (or none). Results, 3.4.1, Figure 2. According to the figure and its Y-axis, lower concentrations of the selected compouds produced higher ihnibition of violacein production. Please check this information and plot´s axis names. Discussion "By contrast, compound 4c bears only two nitro groups, whereas compound 4c do not contain any nitro moiety". Authors mention compound 4c twice, please check. Conclusion. Authors do not expose the conclusion of their research. Instead, in this section, they summarize the relevance and of their results. Please, include the conclusions. ****** 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 ******** [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.0318135.r002
Revision 1
25 Apr 2025 Author Response Manuscript number: [PONE-D-25-01821] - [EMID:c96f77a474010caa] Title: Antibacterial, antibiofilm and anti-quorum sensing activities of 1,2,3,5-tetrazine derivatives linked to a benzothiazole moiety Answers to Reviewers and Editor Comments Additional Editor Comments: The article titled Antibacterial, antibiofilm and anti-quorum sensing activities of 1,2,3,5-tetrazine derivatives linked to a benzothiazole moiety, is a work that evaluates a series of organic compounds derived from chemical synthesis conducted by the group of authors. Specifically, the authors evaluate their synthesis products in two related bacterial behavior systems, which are quorum sensing communication and biofilm formation. The evaluations are conducted on a series of bacterial pathogens or pathogenicity models such as: Klebsiella aerogenes ATCC 130148, Acinetobacter baumannii ATCC BAA-1605, Enterococcus faecium ATCC700221, Staphylococcus epidermidis. Additionally, the ability to inhibit the quorum sensing mechanism is evaluated using the production of violacein in Chromobacterium violaceum ATCC12472 as a model. To methodologically relate their work, the authors propose an experimental design based on determining the respective MICs and MIBCs of each compound in concentrations of µg/ml. The results of the work are expressed in percentage values through known formulas. The results are consistent with the experimental design; however, they present a methodological question that must be clarified by the authors. Specifically, if bacteria need a specific concentration to perform QS communication, how does this relate to the concentrations related to MICs and MIBCs used for each evaluated triazine? The same is considered for the formation or eradication of biofilms. This must be adequately clarified in the methodology section and in the results, with a special mention in the discussion section. Finally, although the article is coherent and it is positively valued to work with compounds derived from synthesis, it is required to delve deeper methodologically or in the discussion regarding the molecular and chemical mechanisms that may be involved in the observed results. Response: We sincerely appreciate the Editor’s thoughtful comments and suggestions for improving our manuscript. We have now clarified in the methodology section that the sub-inhibitory concentrations tested (1/2x, 1/4x, 1/8x, 1/16x, and 1/32x MIC) were specifically selected to evaluate bacterial growth and biofilm formation while avoiding growth effects. We have also expanded the discussion to include potential molecular targets of the tested compounds. The manuscript must be presented in the format required by PLOS. Response: We have now presented the manuscript in the format required by PLOS. The changes have been colored red with yellow stripes across the manuscript. Reviewers' comments: Comments to the Author Reviewer #1: Major comments There are some major methodological issues regarding the evaluation of biofilm inhibition and violacein production. While authors test different concentrations of the selected compounds below MIC and MBC, they do not take into consideration the potential effect of subinhibitory concentrations of these on bacterial growth. Therefore, it is not possible to determine if the lower biofilm formation and violacein production is due to the selective effect of the compounds on these cellular processes or to a reduction of bacterial cells. Normalization by total cell number (CFU or Optical density at 600 nm) or growth curves at the selected concentrations of the compounds should be included to support the conclusions. Response: We appreciate the reviewer’s insightful comment regarding the potential effect of subinhibitory concentrations of the tested compounds on bacterial growth. To clarify this point, we provided data of the kinetics of bacterial growth and biofilm formation in response to subinhibitory concentrations of the tested compounds, alongside our biofilm and violacein production experiments. We have now explicitly discussed this point in the revised manuscript to ensure clarity for readers. The changes have been colored red with yellow stripes in the main text. Thank you for raising this important consideration. Minor comments: Introduction, L5: change "The main mechanisms of ..... includes" for "include" (plural) Introduction, L7: "degradation" may be replace bt a more accurate concept as "structural modification" Response: The appropriate correction has been made. Methods, 2.2.1: Please indicate why C. violaceum was cultured at 30 ºC instead of 26 ºC (its optimal growth temperature). Response: The mistake has been corrected. Methods 2.2.3: Delete ")" after gentamicin. Please specify gentamicin concentrations used as control condition. Response: The gentamicin range of concentrations used has been provided. "The MBC was determined as the ...that completely inhibited the growth of bacteria". Because authors are evaluating bactericidal effect, the more accurate concept should be "that inhibited bacterial viability". Response: The appropriate correction has been made. Methods, 2.3.1. Please specify vanillin concentration Response: The appropriate correction has been made. Results, 3.1.2: Authors should include ATCC numbers for all strains (or none). Response: ATCC numbers are now provided only once, in the methodology section. Results, 3.4.1, Figure 2. According to the figure and its Y-axis, lower concentrations of the selected compouds produced higher ihnibition of violacein production. Please check this information and plot´s axis names. Response: We appreciate the reviewer’s comments and suggestions for improving our manuscript. The mistake has been corrected. Y-axis of Figure 2 is the % of violacein production, where higher concentrations of the selected compounds produced lower violacein production. Discussion "By contrast, compound 4c bears only two nitro groups, whereas compound 4c do not contain any nitro moiety". Authors mention compound 4c twice, please check. Response: We agree with the reviewer’s inquisitiveness. The change has now been amended as suggested. Conclusion. Authors do not expose the conclusion of their research. Instead, in this section, they summarize the relevance and of their results. Please, include the conclusions. Response: The conclusion section has now been re-written as suggested. We request the Editorial office for re-evaluation and kind consideration. Attachments Attachment Submitted filename: Response to the reviewers comments of 24th April.docx https://doi.org/10.1371/journal.pone.0318135.r003
6 May 2025 Decision Letter - M. Alejandro Dinamarca, Editor Antibacterial, antibiofilm and anti-quorum sensing activities of 1,2,3,5-tetrazine derivatives linked to a benzothiazole moiety PONE-D-25-01821R1 Dear Dr. Boniface Pone Kamdem, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, M. Alejandro Dinamarca, Dr. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0318135.r004
Formally Accepted
Acceptance Letter - M. Alejandro Dinamarca, Editor PONE-D-25-01821R1 PLOS ONE Dear Dr. Pone Kamdem, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, 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 customercare@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 Mr M. Alejandro Dinamarca Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0318135.r005

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