PONE-D-23-35234

Antibacterial activities of Miang extracts against selected pathogens and the potential of the tannin-free extracts in the growth inhibition of Streptococcus mutans

PLOS ONE

Dear Dr. Khanongnuch,

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.

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Academic Editor

PLOS ONE

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Additional Editor Comments:

Dear Dr. Khanongnuch, I have made a decision about your manuscript, please review each reviewer's comment point by point, changes should be highlighted so we can track. 

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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: Yes

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Partly

Reviewer #5: Yes

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: 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

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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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

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: 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 PONE-D-23-35234 entitled “Antibacterial activities of Miang extracts against selected pathogens and the potential of the tannin-free extracts in the growth inhibition of Streptococcus mutans” has described solvent extraction of Miang (fermented tea leaves) and tea leaves, raw material for Miang production and determination of antibacterial activities of the obtained extracts, and those treated with tannin-free against selected bacterial pathogens, examination of compounds. The results have found that tannin-free Miang extracts showed more potential to inhibit growth of pathogens, specifically Streptococcus mutans than tannin-free tea leaf extracts. In addition, the manuscript has described analysis of the extracts by HPLC-MS in order to examine the chemical compounds that are relevant to growth inhibition of Streptococcus mutans. The identified compounds were used to determine interactions between each identified compound and sortase A enzyme, the key enzyme associated with biofilm formation of S. mutans. The authors conclude that the tannin-free Miang extracts could be used as a natural active pharmaceutical ingredient for applications in oral hygiene products.

After the evaluation, this manuscript presents sufficient experimental designs and statistical analysis. However, there is unclear information and errors. To meet the standard manuscript for publication, the current form of the manuscript must be substantially revised. The following comments and suggestions would help increase the completement of the manuscript.

General comments and suggestions

Please check the writing format of the scientific name of all microorganisms. There are “Streptococcus” and “Salmonella” which can be abbreviated as “S.”. This would make readers confused. It is suggested to present “S.” for Streptococcus mutans and “Sm”. for Salmonella.

To describe Salmonella enterica serovar Typhimurium, it is recommended to state it as “Salmonella var. Typhimurium” or “Salmonella Typhimurium” or “Sm. Typhimurium”. Please correct it in the whole manuscript.

Please indicate strain to all microorganisms used in this study.

Full name of a microorganism must be present for the first time of the main section and then it should be abbreviated. However, when the authors state it as a subject of the sentence, it is recommended to state its full name.

It is confusing when the authors state NFP and FFP as Miang because either NFP or FFP is a process of Miang fermentation. It is recommended to state it as “NFP Miang” or “FFP Miang”. Please also correct these words throughout the manuscript.

Comma symbol (,) must be added after a specific word such as respectively, especially, etc.,

Considering the type of extracts used in this study and their consistency, please reconsider these words: non-neutral, neutral, acidic, and pH 7. If tea leaf extracts (YTL and MTL) and NFP and FFP Miang extracts are generally acidic, it could state them as “non-neutralized extracts”. After the authors have neutralized the acidic extracts, it could state them as “neutralized extracts”.

Due to Miang is made from tea leaves, it is relevant to express only young tea leaves or mature tea leaves rather than unfermented young tea leaves and unfermented mature tea leaves, respectively (Line 228 and others in discussion section).

Please italicize p of p-value and t of t-value in whole manuscript.

It is more relevant to replace “PVPP-treated extract’ supernatant” by “PVPP-treated extract”. (lines 148, 398, 408, …)

Major and minor comments

Introduction section, add more information about role of sortase A enzyme in dental plaque formation. This information would at least let readers primarily understand why the authors chose molecular docking to investigate interactions between active compounds in Miang extracts and the sortase A enzyme.

Line 109-107 how did the authors express the unit of all extract? Is it mg/mL total polyphenol content or mg/mL total tannin content, or mg extract/mL, etc., Please add more information.

Line 127, is it non-neutralized Miang extracts?

Line 139, is it neutralized Miang extracts? Please also consider as above mentioned.

Line 133, indicate diameter of borer.

Line 134, indicate temperature.

Line 163, remove “be”

Line 172, edit to “Streptococcus mutans” when it was first used as the subject.

Line 173, edit to “an optical density at 600 nm (OD600)”

Liness 223, 247, and 253 add the strain of S. mutans.

Lines 230 and 239 how did the authors calculate percentages of inhibition? Please clarify.

Table 1, the authors can categorize the extracts into two groups including non-neutralized (acidic) and neutralized extracts. Each group consists of YTL, MTL, NFP, and FFP.

Line 236-237, different alphabets represent significant differences vs control. The control was just water. If the authors compared the experimental values to the control, extract samples exhibited the clear zone will have significant differences. From the point of view, multiple comparisons were made after ANOVA results showed significant differences (p < 0.05). It is supposed that all pairwise comparisons were used to analyze the data within the same row, not just compared with the control. It is noted that data with positive results are always significant when they are compared with the control. Please consider this comment for Fig 3., Table 2, Fig 5, and Fig 6.

Lines 245 and 251, it is recommended to revise the figure legend as follows:

Fig 1. Antibacterial activities of non-neutralized tea leaf extracts and non-neutralized Miang extracts against Bacillus cereus TISTR 747 (A), Escherichia coli ATCC 22595 (B), Salmonella Typhimurium TISTR 292 (C), Streptococcus mutans DMST 18777 (D). Sterile water was used as the control.

Fig 2. Antibacterial activities of neutralized tea leaf extracts and neutralized Miang extracts against Bacillus cereus TISTR 747 (A), Escherichia coli ATCC 22595 (B), Salmonella Typhimurium TISTR 292 (C), Streptococcus mutans DMST 18777 (D). Sterile water was used as the control.

Lines 256-277, it is recommended to initially describe line 257-260 (Antibacterial quantitative assays showed …….at acidic and neutral extracts’ pH respectively). However, the phrase “at acidic and neutral extracts’ pH respectively” should be revised to proper format as previously suggested. For example, Antibacterial quantitative assays showed an overall better MIC and MBC for NFP and FFP Miang extracts compared to the YTL and MTL when they were tested against all the pathogens, respectively.

Line 278, it is recommended to revise the figure legend as follows:

Fig 3. Antibacterial activities of non-neutralized and neutralized extracts of Miang and tea leaves against Bacillus cereus TISTR 747 (A), Escherichia coli ATCC 22595 (B), Salmonella Typhimurium TISTR 292 (C), Streptococcus mutans DMST 18777 (D).

Line 282, Different asterisk (*) represent significant differences vs control. What was the control of this experiment? Is it the initial cell concentration or just sterile water? This phrase could be modified to “Different asterisk symbols * and ** represent significant differences of MIC and MBC, respectively (p < 0.05)”.

Line 289, please consider modifying description of table 2 as follows:

Table 2. The inhibition zone of tannin-free extracts of Miang and tea leaves against Streptococcus mutans DMST 18777. Avoid extension of figure description.

Line 293 the comment is similar to that is given for Line 236-237

Line 296, it is unnecessary to add Fig 4 because the authors have already displayed the results in Table 2.

Lines 309-314 it is necessary to compare effect of tannin-free extracts and their original extract (in the presence of tannin)

Lines 384-386 Streptococcus mutans has been reported to recruit sucrose-dependent mechanisms where glucosyltransferase (GTF) reacts with sucrose and convert into dextran polymer or glucan that can be further bound with glucan-binding proteins (GBP) which leads to the formation of dental plaque. Please consider modifying the sentence.

Lines 411-425 please shorten principal discussion about role of sortase A in plaque formation but discuss more about finding of this study. How could active compounds from PVPP-treated Miang extracts inhibit sortase A? Are there any literatures reported active compounds that have potential to inhibit the enzyme?

Line 438-440 the sentence in the conclusion is not relevant to what the author proposes in introduction section (Lines 51-53, and 90-93).

Reviewer #2: It was advantageous to discover a plant extract that inhibited pathogen growth. Several concerns, however, needed to be elucidated in order to have a better knowledge of Miang extracts' antibacterial activity.

It was unclear how the authors obtained the quantities of Miang extracts utilized in Table 1 to establish zones of inhibition against bacteria.

Please double-check whether it was Turkey's or Tukey's test (line 237).

Only triplicate samples used to run a t-test comparing tannin rich extract to tannin-free extract were sufficient to accept the results (Table 2). Please specify the concentration of Miang extracts that were used.

Please modify the titles of all figures to improve comprehension.

In the conclusion section, the authors should summarize the optimal concentration of each Miang extract that can be employed to inhibit bacterial growth based on the MIC:MBC ratio of Miang extracts.

Reviewer #3: Materials and Methods

1. Line 126 to 136 talks about ‘Determination of antibacterial activities of neutralized (pH 7) and non-neutral (pH 4.0 – 4.6) Miang extracts’. The description, however, misses out on how these extracts were obtained before using them for the microbial tests. The ‘Preparation of Miang extracts’ is also silent on how the neutral and non-neutral extracts were obtained. Authors should kindly take a look at that.

2. What was the rationale for testing neutralized and non-neutralized extracts in the study? Could the justification be clearly demonstrated in the write-up?

3. Line 127 – I guess ‘non-neural’ should be ‘non-neutral’.

4. Kindly take a second look at the format for writing your units. At one point, I see CFU/mL, then at other places, I see CFU mL-1.

5. Line 163-164 : authors should kindly the sentence ‘The separation of the chemical compounds was be conducted……..’

Results

1. From Table 2, the zone of inhibition for tannin-rich and tannin-free extracts against Streptococcus mutans were significantly different for each type of sample. How do you authors then arrive at the observation that ‘tannin-free NFP and FFP extracts had significantly (p> 0.05) comparable zones of inhibition with the tannin-rich extracts’ and refer to figure 4, which I suppose is the primary source of data for Table 2?

2. Based on the observations in Table 2, it would be appreciated if authors could reconsider the point on tannin-free producing similar effects as tannin-rich extracts. That obviously also affect the reasoning that

Reviewer #4: This article explain on the antibacterial activity of Miang extract againts selected foodborne pathogens and the finding showed that the extract had the ability to inhibit the growth of pathogens. However, there is no problem statement regarding foodborne pathogens eventhough at the the author mention about foodborne problem at the beginning. Author is suggested to do proof read on their article content. This research give new info on the antibacteria activity of miang extract

Reviewer #5: I extend my appreciation to the authors for their dedication and commendable efforts in completing this excellent piece of research. This work is of considerable importance within the context of oral health, addressing a notable gap in the current literature and searching for novel antimicrobial agents is of great interest. I recommend its publication in PLOS for the following reasons:

• The title effectively reflects the study, and the abstract is clear and concise for readers.

• The comprehensive literature review demonstrates a good understanding of existing research, particularly in the quest for alternative antimicrobial agents.

• The methodology is well-structured, offering a clear and systematic approach to the research, which can serve as a valuable guide for future researchers. I just suggest standardizing the writing style concerning bacteria.

• The data analysis is well-performed, and the presentation is clear.

• The discussion is thoughtful, providing significant interpretations of the results.

• The author displays a high level of proficiency in English.

• The use of references is appropriate.

Overall, this work makes a noteworthy contribution to the field in the search for a new antimicrobial agent and aligns well with the standards of PLOS. I strongly recommend its publication.

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6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

**********

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Attachments

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Submitted filename: PONE-D-23-35234_Comments.docx

Attachment

Submitted filename: Comments PLos one.pdf

PONE-D-23-35234R1Antibacterial activities of Miang extracts against selected pathogens and the potential of the tannin-free extracts in the growth inhibition of Streptococcus mutansPLOS ONE

Dear Dr. Khanongnuch,

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.

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

ACADEMIC EDITOR:

Dear author, I received the opinion on your manuscript. Review reviewers' recommendations point by point

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

Please submit your revised manuscript by May 04 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Mozaniel Santana de Oliveira, Ph.D

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.

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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: All comments have been addressed

Reviewer #2: (No Response)

Reviewer #3: 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

Reviewer #2: Partly

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: N/A

Reviewer #3: 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: No

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: 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: Dear Authors,

Thank you for your revised manuscript. All of my comments and suggestions have been almost completed. It would be great if the authors could include response 12 in the manuscript.

Reviewer #2: Although the authors responded to the comments, some points needed to be elucidated.

The authors should determine which active ingredients in Miang extract prevent antibacterial growth.

To describe the study's findings, the authors should include the concentration of Miang extract that was observed to limit bacterial growth.

Reviewer #3: All the comments that I raised have been addressed adequately. I comend the authors for the efforts put into addressing all the comments posed by the reviewers.

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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

Reviewer #3: 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.

Antibacterial activities of Miang extracts against selected pathogens and the potential of the tannin-free extracts in the growth inhibition of Streptococcus mutans

PONE-D-23-35234R2

Dear Dr. Khanongnuch,

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.

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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,

Mozaniel Santana de Oliveira, Ph.D

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 #2: 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 #2: Yes

**********

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

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 #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 #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 #2: The authors had responded to all comments. Please remove value 3.76 from Table 3, as it did not reflect any parameter.

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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 #2: No

**********