PONE-D-21-06872

Hedera rhombea inhibits the biofilm formation of Candida, thereby increases the susceptibility of antifungal agent, and reduces virulence.

PLOS ONE

Dear Dr. kim,

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 May 13 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.

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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.

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We look forward to receiving your revised manuscript.

Kind regards,

Roy Aziz Khalaf

Academic Editor

PLOS ONE

Additional Editor Comments:

Dear Dr. Kiyoung Kim

Greetings. Two expert reviewers have commented on your manuscript. As you can see there are many issues with the paper before it is suitable for publication, notably in the materials and methods section an how the extract was prepared and with regards to English language. Please make sure to have the manuscript proofread by a native speaker. Please make sure to address all there comments below. In addition, as far as plant extract if a hot water extract from leaves of this plant was performed then this most likely will be acidic. You then concentrated it down and dried it resuspending in DMSO for use. The control is described their control as being no extract. It probably should be DMSO and something should be done about pH change such as buffering (there is no MOPS RPMI), or use an equivalent acid in DMSO as their control.

Thank you

Roy

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3. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0167470

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

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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: No

**********

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: No

**********

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: Language and Format

A native English speaker should be consulted to address language issues. The errors are generally minor, but they are found throughout the paper. For instance, the first sentence of the introduction should read "...with impaired response is able to persist...and often leads to..."

In Candida, gene names are italicized

Title

As noted below, virulence is not tested in this paper.

Abstract

Reference is made to biofilm cellular density, but there are no results regarding this in the paper.

There is reference to bacterial growth.

Introduction

What else is known about extracts from this plant?

Why is there no reference to the following paper, in particular the impact on growth: Choi, H.A.; Cheong, D.E.; Lim, H.D.; Kim, W.H.; Ham, M.H.; Oh, M.H.; Wu, Y.; Shin, H.J.; Kim, G.J. Antimicrobial and anti-biofilm activities of the methanol extracts of medicinal plants against dental pathogens Streptococcus mutans and Candida albicans. J. Microbiol. Biotechnol. 2017, 27, 1242–1248, doi:10.4014/jmb.1701.01026.

Materials and Methods

There are a number of non-standard methods and incomplete descriptions.

In biofilm formation, the inoculum concentration is half standard an no reference is made to inoculum volume. Hypha inducing medium (YPD+serum) is used, but is incubated at yeast temperature (30C). Was the destaining solution transferred to a fresh well for reading the OD?

In the qPCR description, what was the time of the culture with the compound? Was the incubation with the compound done shaking or was this static? How was the cDNA synthesis primed? How were the results normalized?

In the time kill assay, there is a typo (fatal for fetal). Growth is described as being in the YPD and YPD+serum, but the graph has only one base medium.

Results.

It would be helpful if the species name was included at the top of each graph so that the reader didn’t need to keep consulting the figure legend.

In figure 3, it’s not clear if the graphed results are from one replicate or the sum total of all results. There is no n value for the number of cells examined. Why is the cell density so much lower in panel subpanel D of panels B and D than in the other panels? In spite of figure 7, this makes it look like the compound is affecting viability, not just hypha formation. Was the “0” sample without any additive, or with same solvent as the extract?

In figure 4, how many cells were counted?

The times in the graph for Figure 7 do not match those in the methods.

Discussion.

There is a statement that various plant extracts “may not always do their work”. What is that supposed to mean? Figures 3 and 4 do not assess virulence of C. albicans although they examine filamentation and adhesion which are important virulence trait. There are a number of examples of C. albicans strains with adhesion defects which retain high virulence, so this statement needs to be modified.

Reviewer #2: This paper describes the influence of plant extract from Hederea rhombea on Candida albicans. The paper suggest a wide ranging influence of the plant extract making this a paper with potentially high impact.

Their are issues throughout the paper with the English which start with the title. Virulence should not be used as it is not assessed. It should probably read something like "Hedera rhombea inhibits the biofilm formation of Candida and increases the susceptibility of the antifungal agent Miconazole". The issues continue throughout the paper and this really should be improved before further review.

The paper also doesn't address competing data from (https://doi.org/10.4014/jmb.1701.01026 J. Microbiol. Biotechnol. (2017), 27(7), 1242–1248) which showed less influence from Hederea rhombea. This should be referenced and discussed.

The are issues with methods where they are non-standard and not fully described. The controls are either inappropriate or not fully described and this should be remedied. The biofilm data needs error bars and statistical analysis where significant difference is inferred. The protocols for biofilm formation are missing details...like how was the culture prepared prior to preparing cells for biofilm formation. How were the cells prepared for biofilm formation post initial growth. Non-standard film growth conditions are specified but not really justified/explained why 30 not 37 celsius? Why the prolonged incubation with crystal violet. Are they really scoring the actual biofilm and not the destain?. For the dimorphic assay... which RPMI was used how was it made/supplemented. How was it prepared to induce filamentation. The images also need to be better. Without this it is hard to evaluate these experiments. A better label could also be used for graph axis labels than H. rhombea. Perhaps use an acronym for your extract or just put extract.

Below are line by line suggestions that should improve the document. I would also suggest having some very fluent in English review the document.

Abstract suggestions:

change the first line to " Candida is an opportunistic pathogen and common cause of fungal infections worldwide. Anti-fungal use against Candida infections has resulted in the appearance of resistant strains. The limited choice of anti-fungal therapy means alternative strategies are needed to control fungal infectious diseases."

The inhibition of biofilm.....(from line 6) should read something like "Biofilm formation was assessed using the crystal violet assay which showed a dose dependent reduction in the presence of extract"

Genes names should be in italics.

Introduction.

line 1 should read "....pathogen that is responsible for systemic infections in immunocompromised patients....."

line 10 should read "....most antifungal agents, ....."

Line 12. Replace "Thereby" with Therefore.

Line 14 either delete Increasing or change to Increasingly

Line 16 change to "Medicinal plants are used for diverse traditional methods....". end the sentence with "many other diseases"

Line 23 change to "....several fungi..."

Line 24 change to "Particularly, anti-biofilm activity....."

The methods need more detail. Please see above.

Discussion.

Line 3. Change to "Antifungal drugs can cause side effects and resistance and there have been multiple recent reports of resistance including in the emerging problematic organism Candida auris.

At the bottom of the first paragraph. rephrase "those may not always do their work" to be more explicit about what their problem is.

In the third paragraph (and later) ".....further studies must be conducted..." not followed!

Line 18 "... biofilm formation is related to the susceptibility..."

Line 20 "....cell adhesion was significantly reduced by treatment with H. rhombea extract"

on the following/last page

line 3 ".... and in vivo testing must be carried out."

Line 7 is adjuvant really the right word. Perhaps simply say treatment option.

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-21-06872R1

Hedera rhombea inhibits the biofilm formation of Candida, thereby increases the susceptibility of antifungal agent, and reduces virulence.

PLOS ONE

Dear Dr. kim,

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.

Dear Sir

The reviewers have assessed your manuscript and both agree that even though it is vastly improved it still needs some work. Please modify according to the reviewer comments, especially those of reviewer two.

Thank you

Please submit your revised manuscript by Jul 29 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,

Roy Aziz Khalaf

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

Dear Sir

The reviewers have assessed your manuscript and both agree that even though it is vastly improved it still needs some work. Please modify according to the reviewer comments, especially those of reviewer two.

Thank you

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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: (No Response)

Reviewer #2: (No Response)

**********

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: Partly

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

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

Reviewer #2: 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: No

Reviewer #2: 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: Methods and Results

Please make sure that the temperatures in the methods match the temperatures in the figure legends.

As a formatting issue, I’m not sure why the figure legends are incorporated into the results section.

I have concerns with the results reported in figure 3 (inhibition of filamentation). The following description is difficult to understand “This method, which was necessary because the hyphae-specific identification cells could not be quantified, also resulted in an under expression of the number of mycelial cells in the population. The reported percentage of hyphae is normalized to the percentage of probability hyphae when a numerator is not added. Since the number of cells observed in D is small, it seems to be the part where the number of observed cells is small.”

What does “the percentage of probability hyphae” mean?

The table in the supplementatal information indicates that the percentage graphed is the average number from a particular treatment as a percentage of the number in the untreated control. Presumably these numbers are the number of hyphal cells. I don’t see the rationale for expressing this information as a percentage of the control. Surely graphing the percentage of hyphal cells out of the total number of cells counted in a particular treatment would be clearer.

Numerous language issues remain.

Throughout it should be susceptibility to an antifungal, not susceptibility of an antifungal.

Line 52 should have “pathogenesis” instead of pathogenic.

Line 57 The following does not make sense, although I have an idea of what the second part is trying to say “The biofilm matrix acts as an intestinal for sessile cells, avert the inlet of the majority used antifungal agents, therefore give drug resistance.”

Line 65 should read “Traditional medicine is proposed to have lower adverse reactions compare with typical medicines.”

Line 92 What does “Controls without test compounds were served” mean?

Line 95 something like “for 24h, non-adherent cells were removed by washing with PBS and then 100ul of 1% aqueous crystal violet was applied for 30 minutes” might be better

Line 112 (and others) should be fetal, not fatal.

Line 116: the term bud would be best, not “sprout cell” and this whole description could be simplified.

Line 133 should read “non-adherent” for “none adhere”

Line 158. This might be better described as a growth inhibition assay, as that is what is being assessed, not killing.

Line 187 should read “Extract was applied”

Line 189 should read “increased the susceptibility to miconazole”

Line 190 I think this line should read “The extract also increased the susceptibility to plant-derived antifungal candidates including”

Fig 2 Legend should read increased the susceptibility of C. albicans to miconazole etc.

Line 205: what is meant by “serious infection effect”?

Line 286: perhaps “further studies must be conducted.”

Line 293: gene names must be italicized

Line 294: perhaps “that influence adhesion” in place of “and is also participated to the adherence”

Line 295 “could” would be better than “should”

Line 296 should read “Further studies, including…”

In figure 5 Planktonic has been misspelled

Reviewer #2: This revised manuscript is much improved, I thank the authors for their hard work.

I still have some remaining concerns and a few language corrections.

My major concern relates to their plant leaf extract and the use of RPMI. Many plant leaf extracts are acidic.

RPMI as used in the candida field is typically heavily buffered with MOPS. It is not clear from the description of the methods that RPMI was buffered. Candida filamentation and biofilm formation are heavily influenced by pH. Please clarify the situation with the RPMI.

My second concern with the revision is that the discussion still does not address competing (and conflicting data) (https://doi.org/10.4014/jmb.1701.01026 J. Microbiol. Biotechnol. (2017), 27(7), 1242–1248) which showed less influence from Hederea rhombea. This should be referenced and discussed.

The controls still aren't clear. Presumably these are just DMSO. But this needs to be clearly stated when it is used. There is a comment that "controls without test compounds were served" but it should clearly state somewhere is the 0/control used is DMSO

The title is misleading and should really read something like "Hedera rhombea inhibits the biofilm formation of Candida and increases the susceptibility to antifungal agents"

Below are minor language changes to improve the manuscript.

Abstract/Page 8

line33 should read "Treatment with 6.25....." and "...increased susceptibility to miconazole..."

line39 should read "...analysis was done and the extract was found to dose dependently reduce the expression....

Page 9

line 44 should read " biofilm formation is good screen for developing.....

Introduction Page 10

line 50 should read "....persist inside the host and can be aided by drug resistance traits which often lead to failure of....."

line 52 should read "...species pathogenicity is their..."

line 56 should read "...is a finely controlled process that involves attachment to surface and embedment in the exopolymer...."

line 57 should read "the biofilm matrix acts as a haven for sessile cells..."

line 58 should read "....averting the inlet of the majority of anitfungal agents, thereby resulting in resistance"

line 59 I think the authors mean to say "adherent C. albicans cells without specific drug resistant gene expression..."

line 67 should read "...provenance of replacement remedies...."

line 73 should read "Interestingly, the activity of the extract also increased susceptibility to antibiotics."

Methods

line 92 should probably read "DMSO controls without test compounds were used"

???line 93 ...was this RPMI MOPS buffered????

line 112... I think autocorrect likely altered this as fatal bovine serum is back/still there. It should be fetal or foetal. This error recurs...see line 130

Results

line 185 should read "H. rhombea extract increased the efficacy of antifungal agents...."

If you want to use susceptibility then the extract would increase the susceptibility of C. albicans to antifungals.

Discussion

line 285-286. should read "....but further studies should be undertaken."

**********

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

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.

PONE-D-21-06872R2

Hedera rhombea inhibits the biofilm formation of Candida, thereby increases the susceptibility to antifungal agent, and reduces infection.

PLOS ONE

Dear Dr. kim,

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.

==============================

Dear Dr. Kim.

Greetings. The paper is much improved. However minor revisions are still required especially the issue of the RPMI media highlighted by reviewer 2 before the manuscript can be officially accepted. Please address the reviewer comments below.

Thank you

==============================

Please submit your revised manuscript by Oct 02 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,

Roy Aziz Khalaf

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

Dear Dr. Kim.

Greetings. The paper is much improved. However minor revisions are still required especially the issue of the RPMI media highlighted by reviewer 2 before the manuscript can be officially accepted. Please address the reviewer comments below.

Thank you

[Note: HTML markup is below. Please do not edit.]

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: (No Response)

Reviewer #2: (No Response)

**********

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

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: 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

Reviewer #2: 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

Reviewer #2: 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 appreciate the changes made by the authors and I have only a handful of minor edits remaining.

Line 57 should read “The biofilm matrix acts to structure microbial communities and includes sessile cells that are frequently much more resistant to antifungal agents.”

Line 74 I think “and reducing fungal infection” needs to be removed since virulence wasn’t assessed in the manuscript.

Line 125 should read “nonadherent”

Line 179 remove “was applied”

Line 196 should just read “virulence” not “virulence-mediated”

Line 290 I think this should read ”Candida albicans biofilm formation is determined by various transcription factors including BCR1, EFG1, TEC1, and NDT80 that function as components in several pathways and also influence adherence of Candida, suggesting that even though biofilm formation was initially tested, adherence or infection factors could be also regulated by treatment of H. rhombea extract.”

Figure 1 Legend has one last “fatal” instead of “fetal”

Figure 5 be sure that in the figure the label is “planktonic”.

Reviewer #2: This revised manuscript is much improved, I thank the authors for their

hard work. My main remaining concern and is with regards to the RPMI based experiments. The authors provided references to respond to this question. The frontiers paper that was referenced clearly describes the RPMI they used as supplemented with L-glutamine and buffered with MOPS to pH 7. (see extract 1 below..copied from the article).

The other two papers reference the CLSI standard and then they describe RPMI without sodium barcarbonate at pH7. The CLSI standard recommends the use of RPMI without sodium bicarbonate and with glutamine...but it also says to use a buffer and recommends mops (see second extract below).

If this is accurate for the experiments described here then a similar line should be added to methods. If not perhaps the authors could include a line about the pH of their extract. Or comment on the color of the media as the phenol red is in RPMI as a pH indicator.

Alternatively RPMI can use a sodium bicarbonate/Co2 buffer system....but that is usually for cell culture.

[extract 1] taken from Front. Microbiol., 15 November 2019 | https://doi.org/10.3389/fmicb.2019.02642 "A round-bottom 96-well microplate (Corning Inc., Corning, NY, United States) was filled with RPMI medium supplemented with L-glutamine (Cellgro; Corning, United States) and buffered with 165 mM morpholinepropanesulfonic acid (MOPS) (Thermo Fisher Scientific Inc., United States) at pH 7.0 "

[extract 2] taken from CLSI M27 standard for antifungal testing: "Media should be buffered to a pH of 7.0 ± 0.1 at 25 °C. A buffer should be selected that does not antagonize antifungal agents. Tris buffer is unsatisfactory, because it antagonizes the activity of flucytosine. Zwitterion buffers are preferable to buffers that readily traverse the cell membrane, such as phosphate buffers, because, theoretically, the latter can produce unexpected interactions with antifungal agents. One buffer that has been found to be satisfactory for antifungal testing is MOPS [3-(N-morpholino) propanesulfonic acid] (final concentration 0.165 mol/L for pH 7.0). The pH of each batch of medium is to be checked with a pH meter immediately after the medium is prepared; the pH should be between 6.9 and 7.1 at room temperature (25 °C). MIC performance characteristics of each batch of broth are evaluated using a standard set of quality control organisms."

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Hedera rhombea inhibits the biofilm formation of Candida, thereby increases the susceptibility to antifungal agent, and reduces infection.

PONE-D-21-06872R3

Dear Dr. kim,

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