Bioactive triterpenoids from Solanum torvum fruits with antifungal, resistance modulatory and anti-biofilm formation activities against fluconazole-resistant candida albicans strains

Benjamin Kingsley Harley; David Neglo; Philip Tawiah; Mercy Adansi Pipim; Nana Ama Mireku-Gyimah; Clement Okraku Tettey; Cedric Dzidzor Amengor; Theophilus Christian Fleischer; Sayanika Devi Waikhom

doi:10.1371/journal.pone.0260956

Peer Review History

Original SubmissionJune 14, 2021
19 Aug 2021 Decision Letter - Horacio Bach, Editor PONE-D-21-19484 Bioactive triterpenoids from Solanum torvum fruits with Antifungal, Resistance Modulatory and Anti-Biofilm Formation Activities against Fluconazole-Resistant Candida albicans strains PLOS ONE Dear Dr. Waikhom, 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. As mentioned by Reviewer 1, your study needs a significant revision. Please provide a point-by-point letter addressing the concerns of the reviewer. In addition, please provide the following information: 1. The name of the botanist that identified the plant should be stated. 2. In Section 2.4, please add the percentage on the compounds following the weight. 3. Tables 1 and 2 expressed the results in mg/mL, whereas Table 3 in ug/mL. Please standarize the dimensions. 4. The word triplicate is not plural. 5. Please add a statistical analysis in Fig. 2. Please submit your revised manuscript by Oct 03 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. 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, Horacio Bach 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 you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. [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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: 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 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: It is an interesting study but requires major changes before being accepted for publication Vulnerable points: The whole (crude) is not a promising antifungal since the MIC were light, so the entire manuscript needs to be revised. Why has the study focused just on C. albicans isolates? It needs justification, as the non-Candida albicans species have increased in the last decades. To draw relevant conclusions from just four C. albicans isolates, in my opinion, is very bold. The English should be improved, maybe at the discretion of the editor. Specific points Abstract It is missing results details regarding the antifungal activity, What do the numbers in parentheses written after the compounds mean? Please delete this The obtained results did not support the conclusion “justifying its use as an antifungal agent in the treatment of vulvovaginal candidiasis….” Lines 21-22 Introduction Line 4: rephrase this sentence Line 27: delete the word “ opportunistic” Line 29: replace "The condition" by VVC Line 35: the reference [5] is unsuitable for this statement, please replace it. Line 62: Add the words of S. torvum after “The fruits” Objectives: Rewrite it focusing on the purified compounds (isolation and their antifungal activity) it is the most relevant in the present study. Methods Lines 98-101: The authors should supply more details on the purification of Fraction F5, originating the compounds, also cite a reference, and so on Lines 106-107: The growth in Sabouraud Dextrose Agar doesn't ensure the purity of the Candida isolates since this culture medium does not differentiate the colonial morphology between the species of this genus. Line 116: it is written "ml", while elsewhere, as in line 142 it is "mL", standardize it in the entire manuscript, please Line 126: the definition of the acronym STF .... ethanol extract (STF) is repeated other times, for instance, line 184 and others, please from the first definition (line 116) mention only STF Line 155: This experiment is confusing, if the crystal violet test was performed in microplates, why were the compounds diluted in test tube sets? Next, line 159 again "The tubes were then incubated at …..", please rewrite this paragraph, clarifying the biofilm inhibition assay. Results Line 171: Is it missing some letters in the first word? Fig 1: the caption is incomplete, it needs to be more informative, mentioning the compound names here instead of putting numbers every time compounds are cited in the text, as it makes reading confusing and tiring Line 191: add the information “ethanol extract” into the title of table 1 Lines 185-187: The results are confusing, are the two sentences contradictory? Or is the second one just an interpretation? If yes, please move the sentence “STF was considerably active against the fungal growth with MIC between 0.25 and 2 mg/mL” to the methods session. Line 229: Is the sentence “The isolated compounds, however, drastically reduced the activity of voriconazole” correct? I think it increased the activity of voriconazole Rephrase it, please Discussion The first paragraph should be deleted because it is confusing for instance (why was cited the reference 29?), and not relevant. Line 271: What does “ considerable antifungal activity” mean? Please mention the reference used to define the breaking points in the interpretation of your results Line 277: I disagree with the statement "low MIC values", as according to a recent systematic review by Alves et.al 2021 (https://doi.org/10.1155/2021/6653311), the values presented in Tables 1 and 2 (crude extract and fractions respectively) reveal only weak or no bioactivity. Please, clarify and discuss your data. Line 342: This statement is true, but it needs a reference Lines 360-361: The sentence “The reported activity justifies its use as an antifungal agent in the treatment of vulvovaginal candidiasis among the Ghanaian populace” must be deleted since it is not supported by data of the current study. Conclusion Lines 368-371: This subject is not a conclusion of the current study, maybe is possible to include it in the discussion session Reviewer #2: Dear authors, Solanum torvum is endemic in southern Mexico and South America, it is a widely studied plant. In their work they propose a possible therapeutic tool for antifungal treatment, making it a valuable proposal. 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: Yes:** Terezinha Inez Estivalet Svidzinski 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.0260956.r001
Revision 1
23 Oct 2021 Author Response The Editor-In-Chief PLOS ONE Cambridge, UK Dear Madam, RESPONSE TO REVIEWERS’ COMMENTS We hereby in this letter provide responses to the queries raised by the academic editor and reviewers of our manuscript titled “Bioactive triterpenoids from Solanum torvum fruits with Antifungal, Resistance Modulatory and Anti-Biofilm Formation Activities against Fluconazole-Resistant Candida albicans strains” submitted to your revered journal PLOS One. All datasets generated for this study are included in the manuscript and/or the supplementary files. Kindly find below our responses. We hope it will receive a favourable consideration. Thank you. Yours faithfully, Sayanika Devi Waikhom (Ph.D) (Corresponding Author) Academic Editor 1. The name of the botanist that identified the plant should be stated. We have included the name of the Botanist who identified the plant. Section 2.2, line 77 2. In Section 2.4, please add the percentage on the compounds following the weight. The percentage weight of the compounds per weight of the dried powdered material have been included. Section 2.4, lines 103-105 3. Tables 1 and 2 expressed the results in mg/mL, whereas Table 3 in ug/mL. Please standardize the dimensions. The units of the all the tables have been standardized to mg/mL Section 3.4 and Table 3. 4. The word triplicate is not plural. The spelling of triplicate has been corrected. Lines 136, 207, 220 and 232 5. Please add a statistical analysis in Fig. 2. The analysis we conducted on the figure 2 was to determine the percentage biofilm inhibition. Since we are not comparing the inhibition to a control. we cannot carry out any further statistical analysis other than comparing the percentage inhibitions of the compounds to each other in terms of which one was higher and lower. Reviewer 2 General comment 1. Why has the study focused just on C. albicans isolates? It needs justification, as the non-Candida albicans species have increased in the last decades. We agree to the fact that non-albicans candida species have increased in the recent years, however we decided to focus on C. albicans in this study because despite the increase in the other species of candida, still Most Candida infections in people are caused by Candida albicans [1]. The group however in other studies are investigating the effect of natural products on NAC such as C. glabrata and auris. We tried to justify the reason for our study on C. albicans in lines 28-29 and we provided an additional reference to support our justification. 2. To draw relevant conclusions from just four C. albicans isolates, in my opinion, is very bold. We selected the four C. albicans strains as they covered a range of diameters considered as fluconazole-resistant which are ≤ 14 mm. 3. The English should be improved, maybe at the discretion of the editor. We have improved the general English of the paper. ABSTRACT 1. It is missing results details regarding the antifungal activity We have included details regarding the antifungal activity in the abstract Line 13-16 2. What do the numbers in parentheses written after the compounds mean? Please delete this The number in parentheses represent the number assigned to the compounds in figure 1. This was done so readers could match the structure of the compound to the name. We have however deleted this as requested. 3. The obtained results did not support the conclusion “justifying its use as an antifungal agent in the treatment of vulvovaginal candidiasis….” Lines 21-22 The conclusion of the abstract has been re-written focusing on the isolated compounds. The statement “justifying its use as an antifungal agent in the treatment of vulvovaginal candidiasis….” has been deleted. Line 20-23 INTRODUCTION 1. Line 4: rephrase this sentence Unclear how the reviewer wants this sentenced rephrased. 2. Line 27: delete the word “ opportunistic” “Opportunistic” has been deleted Line 28 3. Line 35: the reference [5] is unsuitable for this statement, please replace it. Appropriate reference has inserted Reference [5] 4. Line 62: Add the words of S. torvum after “The fruits” Line 62: Add the words of S. torvum after “The fruits” Line 57 OBJECTIVES Rewrite it focusing on the purified compounds (isolation and their antifungal activity) it is the most relevant in the present study. The objectives have been re-written to focus on the isolation of the purified compounds and their subsequent testing. Line 67-69 METHODS 1. Lines 98-101: The authors should supply more details on the purification of Fraction F5, originating the compounds, also cite a reference, and so on Further details have been supplied on the purification of fraction F5. Line 99-106 2. Lines 106-107: The growth in Sabouraud Dextrose Agar doesn't ensure the purity of the Candida isolates since this culture medium does not differentiate the colonial morphology between the species of this genus. We have included how we identified and selected the C. albicans isolate Line 113-116 3. Line 116: it is written "ml", while elsewhere, as in line 142 it is "mL", standardize it in the entire manuscript, please The unit has been standardized and written as mL throughout the entire manuscript 4. Line 126: the definition of the acronym STF .... ethanol extract (STF) is repeated other times, for instance, line 184 and others, please from the first definition (line 116) mention only STF The repeated definition of the acronym STF in the text has been deleted. Line 132, 182, 195, 197, 277, 278, 281 5. Line 155: This experiment is confusing, if the crystal violet test was performed in microplates, why were the compounds diluted in test tube sets? Next, line 159 again "The tubes were then incubated at …..",please rewrite this paragraph, clarifying the biofilm inhibition assay. The paragraph and the experiment have been re-written with clarity. Section 2.5.5 lines 167-174 RESULTS 1. Line 171: Is it missing some letters in the first word? The missing letter has been included Line 182 2. Fig 1: the caption is incomplete, it needs to be more informative, mentioning the compound names here instead of putting numbers every time compounds are cited in the text, as it makes reading confusing and tiring As we stated earlier, the numbers before the names of the compounds are there to connect their structures to their names in fig 1. Using the numbers when the compounds are mentioned is a conventional practice of dealing with isolated compounds as seen in a number of journals including PLOS One. An example is this publication [2]. The caption of fig 1 has been modified. Figure 1 3. Line 191: add the information “ethanol extract” into the title of table 1 We have added the ethanol to the information relating to the title of table 1 Line 202 4. Lines 185-187: The results are confusing, are the two sentences contradictory? Or is the second one just an interpretation? If yes, please move the sentence “STF was considerably active against the fungal growth with MIC between 0.25 and 2 mg/mL” to the methods session. The first statement was a general summary on table 1 focusing on STF and its fractions. The second statement highlights the activity of STF alone before the 3rd statement finalizes on the most active fraction. Since it is a result, we do not think it will be appropriate to move it to the methods sections. 5. Line 229: Is the sentence “The isolated compounds, however, drastically reduced the activity of voriconazole” correct? I think it increased the activity of voriconazole Rephrase it, please The statement is correct as MIC values correlates indirectly with antimicrobial activity. The lower the MIC value the higher the activity. From the results, the MIC of voriconazole was lower than when it was in combination with the isolated compounds therefore voriconazole alone had a higher activity than when it was combined with the compounds. It is for this reason we indicated that isolated compounds reduced the activity of the voriconazole DISCUSSION 1. The first paragraph should be deleted because it is confusing for instance (why was cited the reference29?), and not relevant. The first paragraph served as an introduction to the discussion. We have however deleted 2. Line 271: What does “considerable antifungal activity” mean? Please mention the reference used to define the breaking points in the interpretation of your results We have re-written that aspect of the discussion and provided reference used to define the breaking points. We have also indicated that in the methods sections Line 278-279 3. Line 277: I disagree with the statement "low MIC values", as according to a recent systematic review byAlves et.al 2021 (https://doi.org/10.1155/2021/6653311), the values presented in Tables 1 and 2 (crude extract and fractions respectively) reveal only weak or no bioactivity. Please, clarify and discuss your data. We agree based on the break points and have deleted that statement. We have reviewed the publication you sent and have revised the discussion accordingly Line 281-286 4. Line 342: This statement is true, but it needs a reference A reference has been provided. Line 347, reference [55] 5. Lines 360-361: The sentence “The reported activity justifies its use as an antifungal agent in the treatment of vulvovaginal candidiasis among the Ghanaian populace” must be deleted since it is not supported by data of the current study. The statement has been deleted CONCLUSION 1. Lines 368-371: This subject is not a conclusion of the current study, maybe is possible to include it in the discussion session We have deleted that subject line Attachments Attachment Submitted filename: Response to reviewers .docx https://doi.org/10.1371/journal.pone.0260956.r002
22 Nov 2021 Decision Letter - Horacio Bach, Editor Bioactive triterpenoids from Solanum torvum fruits with Antifungal, Resistance Modulatory and Anti-Biofilm Formation Activities against Fluconazole-Resistant Candida albicans strains PONE-D-21-19484R1 Dear Dr. Waikhom, 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, Horacio Bach Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #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: N/A ****** 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: I carefully read the authors' responses and changes made to the new version of the manuscript. I realized that the vast majority of suggestions were accepted, improving the quality of the text. Therefore, I consider it suitable for publication in PLOS ONE. Morevoer, in my opinion the manuscript meets all requirements and recommendations mentioned above. ****** 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: Yes:** Terezinha Inez Estivalet Svidzinski https://doi.org/10.1371/journal.pone.0260956.r003
Formally Accepted
17 Dec 2021 Acceptance Letter - Horacio Bach, Editor PONE-D-21-19484R1 Bioactive triterpenoids from Solanum torvum fruits with Antifungal, Resistance Modulatory and Anti-Biofilm Formation Activities against Fluconazole-Resistant Candida albicans strains Dear Dr. Waikhom: 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. Horacio Bach Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0260956.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 .