Rosemary essential oil and its components 1,8-cineole and α-pinene induce ROS-dependent lethality and ROS-independent virulence inhibition in Candida albicans

Zinnat Shahina; Raymond Al Homsi; Jared D. W. Price; Malcolm Whiteway; Taranum Sultana; Tanya E. S. Dahms

doi:10.1371/journal.pone.0277097

Peer Review History

Original SubmissionJune 15, 2022
29 Jul 2022 Decision Letter - Roy Aziz Khalaf, 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-22-17200Rosemary essential oil and its components 1,8-cineole and α-pinene induce ROS-dependent cell death and inhibit Candida albicans virulence in a ROS-independent mannerPLOS ONE Dear Dr. Dahms, 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. ============================== Your manuscript has been reviewed by 2 specialists in the field and as you can see from the revisions the manuscript requires minor non experimental revisions before it is fit for publication. 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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 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 study by Shahina et al. describes the potential mechanisms of action and antifungal effects of two major components of rosemary essential oil (alpha-pinene and 1,8-cineole) against Candida albicans. The authors make a detailed description of the potential mechanisms of action, in addition to a very careful and accurate choice of the proposed experimental design. It explores numerous universally accepted and recognized methodologies and presents the results clearly and accurately. All experiments are strictly controlled and the results are presented in tables and images of high technical and graphic quality. The theme is of interest to the biomedical community and relevant within medical mycology. The results expand the prerogative of using rosemary in pharmaceutical preparations with antifungal potential. I suggest publishing the study after the authors consider minor revisions: Candida albicans ATCC10231 is resistant to several antifungals (such as fluconazole, anidulafungin, miconazole). In addition, the susceptibility profile of the clinical isolates used should be indicated in the supplementary material section. This information is important as it may perhaps help to explain the difference in susceptibility to alpha-pinene and 1,8-cinelol of isolates used in the study. The authors report a weak synergistic interaction of alpha-pinene and 1,8-cinelol. In assays of genes linked to resistance to these two agents, very distinct patterns were observed between the compounds. This is a strong indication that each one acts on a different target, thus inhibiting different biochemical processes in the fungal cell. Inhibition of distinct metabolic pathways is one of the cornerstones of synergism between molecules and these correlations should be further explored in the Discussion section. Reviewer #2: The manuscript provides promising outcomes on the efficacy of essential oil from rosemary on C. albicans. Although the manuscript shows excellent presentation, some comments still need to be addressed including 1. Title: need to be modified and concise. I would suggest "Rosemary essential oil and its major components 1,8-1 cineole and α-pinene induce ROS-dependent and ROS-independent inhibition of Candida albicans" 2. MIC need to be identified in the abstract, for example is it MIC100? also include SE 3. Also in the abstract, include fold difference compared to control when talking about mechanisms. 4. Please follow binomial system when talking about organism names. when plant name alone should be lower case. for example Rosmarinus officinalis, while rosmeray should be used. please also include plant family name and taxonomic name at first use in the abstract and materials. 5. Please include CAT# and company, country of all purchases and equipment 6. Add more details regrading the use of Software employed 7. Please give details about all negative controls and if they contain all components except the compound or drug. In other words DMSO and TWEEN can cause antifungal effect if not in suitable concentration. 8. Explain and mention in the manuscript the reason for using different CFU from experiment to another? 9. Please rewrite the text under the first subheading in the results. Also some metabolites should be considered major and the authors identified as minor. However they can identify that cineole and Pinene are the highest. 10. A section in the methods about the fungal strains in terms of source and way of isolation and maintenance should be included, in addition to any ethical approval should be used. 11. The concentration of the oil employed in the study is very high, which may suggest cytotoxicity. Can the author elaborate on this 12. Please revise the labels on Figure 8, I believe MIC should be instead of 1/2 MIC under pinene. also revise all labels within all figures through out the manuscript . Further, please make all letters in the figure legend bold to differentiate from text. 13. Although the English is fairly used, so many mistakes, spelling errors, incomplete sentences and sentences construction are not satisfactorily used and need to be carefully revised. ****** 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: William Gustavo Lima Reviewer #2: Yes: Sameh Soliman ******** [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.0277097.r001
Revision 1
14 Oct 2022 Author Response We thank the reviewers for their careful review which ultimately improve our submission. All changes in the manuscript appear in blue font. ________________________________________ 5. Review Comments to the Author Reviewer #1: The study by Shahina et al. describes the potential mechanisms of action and antifungal effects of two major components of rosemary essential oil (alpha-pinene and 1,8-cineole) against Candida albicans. The authors make a detailed description of the potential mechanisms of action, in addition to a very careful and accurate choice of the proposed experimental design. It explores numerous universally accepted and recognized methodologies and presents the results clearly and accurately. All experiments are strictly controlled and the results are presented in tables and images of high technical and graphic quality. The theme is of interest to the biomedical community and relevant within medical mycology. The results expand the prerogative of using rosemary in pharmaceutical preparations with antifungal potential. I suggest publishing the study after the authors consider minor revisions: Candida albicans ATCC10231 is resistant to several antifungals (such as fluconazole, anidulafungin, miconazole). In addition, the susceptibility profile of the clinical isolates used should be indicated in the supplementary material section. This information is important as it may perhaps help to explain the difference in susceptibility to alpha-pinene and 1,8-cinelol of isolates used in the study. We have included the MIC information for each antifungal and each strain, including clinical isolates in the supplementary material, S2 Fig entitled “MIC of various C. albicans strains and EOC synergy.”. In comparison with the clinical reference strain (ATCC10231), only the genital and blood strains show resistance to �-pinene. The authors report a weak synergistic interaction of alpha-pinene and 1,8-cinelol. In assays of genes linked to resistance to these two agents, very distinct patterns were observed between the compounds. This is a strong indication that each one acts on a different target, thus inhibiting different biochemical processes in the fungal cell. Inhibition of distinct metabolic pathways is one of the cornerstones of synergism between molecules and these correlations should be further explored in the Discussion section. Thank you for pointing out this oversight. Indeed there could be inhibition of distinct metabolic pathways which leads to synergism, and we have added this idea to the discussion (lines 801-803). Reviewer #2: The manuscript provides promising outcomes on the efficacy of essential oil from rosemary on C. albicans. Although the manuscript shows excellent presentation, some comments still need to be addressed including 1. Title: need to be modified and concise. I would suggest "Rosemary essential oil and its major components 1,8-1 cineole and α-pinene induce ROS-dependent and ROS-independent inhibition of Candida albicans" We thank you for this suggestion to improve the cumbersome title. We have accordingly changed the title to: Rosemary essential oil and its major components 1,8-cineole and α-pinene induce ROS-dependent lethality and ROS-independent virulence inhibition of Candida albicans" We think it is important to distinguish between lethality and inhibition of virulence. 2. MIC need to be identified in the abstract, for example is it MIC100? also include SE Yes, this is MIC100, which we have now indicated in the abstract (line 33). The MICs determined from three biological replicates were identical, which would represent a SEM of 0. We have modified S2 Fig legend to indicate the number of biological replicates, and typical error (~0.01) associated with 100% inhibition corresponding to a null OD600 reading (lines 1347-1348). 3. Also in the abstract, include fold difference compared to control when talking about mechanisms. The fold change is dependent on oil type and strain, so in the abstract we have included the maximal fold changes for mycelial and biofilm formation (line 42). 4. Please follow binomial system when talking about organism names. when plant name alone should be lower case. for example Rosmarinus officinalis, while rosmeray should be used. please also include plant family name and taxonomic name at first use in the abstract and materials. Thank you for pointing out this error. We have corrected the abstract (line 27) and methods (line 105) accordingly, and consistently use lower case for “rosemary” (lines 378). 5. Please include CAT# and company, country of all purchases and equipment We have corrected this oversight by including the CAT# and all suppliers (lines 107-115, 176-177, 202, 211-212, 268, 306, 362-363). 6. Add more details regrading the use of Software employed We have added software for chemogenomic data processing (lines 185-187), AFM (215 and 220), fluorescence microscopy (lines 243, 258), ImageJ (lines 280-281) and statistical analyses (367-368). 7. Please give details about all negative controls and if they contain all components except the compound or drug. In other words DMSO and TWEEN can cause antifungal effect if not in suitable concentration. Thank you for pointing out this oversight. We have included a description of negative controls (lines 114-116). 8. Explain and mention in the manuscript the reason for using different CFU from experiment to another? Upon further review, we realize that the way in which the methods were written was confusing, so we have edited this section for clarity (lines 130-140). Included are the guidelines for yeast determined the CFU used for the MIC assays. The standard most commonly used in the clinical microbiology laboratory is the 0.5 McFarland Standard (1.2 x 105 CFU/mL). We now state that all other experiments started with a higher CFU/mL (107), at mid log phase of the growth curve where cells are more metabolically. Microscopic experiments required a higher cell density, and so were not further diluted prior to sample preparation, whereas each well for biochemical assays were normalized to 1 x 105 cells/mL, a cell density that prevents signal saturation. 9. Please rewrite the text under the first subheading in the results. Also some metabolites should be considered major and the authors identified as minor. However they can identify that cineole and Pinene are the highest. Thank you for pointing out the lack of clarity in this paragraph, which we have rewritten (lines 379- 385). 10. A section in the methods about the fungal strains in terms of source and way of isolation and maintenance should be included, in addition to any ethical approval should be used. C. albicans strains were not collected by our laboratory, rather the Regina Qu’appelle Health Region microbiology laboratory, with all patient identifying information associated with clinical isolates removed prior to transfer to our laboratory. Therefore the study did not require ethical approval. We now indicate the source of each strain, which are described in the supplementary S1 Table, and we have clarified this at the beginning of our methods section (lines 91 -97). In that same paragraph, under Strains, media and growth conditions, we describe how the strains were maintained. 11. The concentration of the oil employed in the study is very high, which may suggest cytotoxicity. Can the author elaborate on this? We agree, which is why we do not propose their use as therapeutics, rather we suggest them as possible prophylactics: “a promising avenue for developing novel sanitizing formulations and medical coatings aimed at preventing infection”. It is possible that the chemical structures of these compounds could serve as a starting point for future therapeutics. 12. Please revise the labels on Figure 8, I believe MIC should be instead of 1/2 MIC under pinene. also revise all labels within all figures through out the manuscript . Further, please make all letters in the figure legend bold to differentiate from text. We assume that the reviewer is referring to the close proximity of the legend to the labels in Figure 8, where we have now added a space. All Figure labels now appear in bold font, along with letters in the figure legends. 13. Although the English is fairly used, so many mistakes, spelling errors, incomplete sentences and sentences construction are not satisfactorily used and need to be carefully revised. Agreed. We have tried to eliminate all typos, incomplete sentences and grammatical errors. Attachments Attachment Submitted filename: Response to Reviewers3.docx https://doi.org/10.1371/journal.pone.0277097.r002
19 Oct 2022 Decision Letter - Roy Aziz Khalaf, Editor Rosemary essential oil and its components 1,8-cineole and α-pinene induce ROS-dependent lethality and ROS-independent virulence inhibition in Candida albicans PONE-D-22-17200R1 Dear Dr. Dahms, 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, Roy Aziz Khalaf Academic Editor PLOS ONE Additional Editor Comments (optional): all reviewer required corrections were performed Reviewers' comments: https://doi.org/10.1371/journal.pone.0277097.r003
Formally Accepted
7 Nov 2022 Acceptance Letter - Roy Aziz Khalaf, Editor PONE-D-22-17200R1 Rosemary essential oil and its components 1,8-cineole and α-pinene induce ROS-dependent lethality and ROS-independent virulence inhibition in Candida albicans Dear Dr. Dahms: 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. Roy Aziz Khalaf Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0277097.r004

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