-->PONE-D-24-38694-->-->Molecular detection of super-antigenic Methicillin-Resistant Staphylococcus aureus from commercial cheese in Bangladesh-->-->PLOS ONE

Dear Dr. Hossain,

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.

==============================-->-->I have completed my evaluation of your manuscript. The reviewers recommend reconsideration of your manuscript following major revision. I invite you to resubmit your manuscript after addressing the comments below. Please resubmit your revised manuscript by Nov 04 2024 11:59PM.

When revising your manuscript, please consider all issues mentioned in the reviewers' comments carefully: please outline every change made in response to their comments and provide suitable rebuttals for any comments not addressed. Please note that your revised submission may need to be re-reviewed. -->-->

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

Please submit your revised manuscript by Nov 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,

Reham Mokhtar ELTarabili

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. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

3. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate ""supporting information"" files.

Additional Editor Comments :

The manuscript presents an investigation of MRSA isolated from commercial cheese. Reviewer #1 finds the manuscript generally good but recommends several major revisions, including formatting corrections, italic genes, and clarifications in the methodology and results sections. Reviewer #2 finds the manuscript covering important topic but raises significant methodological concerns, many missing data in many sections, the experimental design is simple, and test methods are routine.

The title does not comply with the subject of the study. Based on the reviewers' comments, the manuscript requires substantial revisions to address methodological concerns and improve clarity and formatting. Therefore, the editorial decision is to request a major revision. The authors should address the specific concerns raised by both reviewers.

[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: Yes

Reviewer #2: Partly

**********

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

Reviewer #1: Yes

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: No

**********

-->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: Title: Molecular detection of super-antigenic Methicillin-Resistant Staphylococcus aureus from commercial cheese in Bangladesh

Comments to authors:

The authors of this study characterize the Staphylococcus aureus strains isolated from cheese samples, according to their antibiotic susceptibility patterns, virulence factors, biofilm formation ability and their genetic similarity. The study includes interesting data. However, some part of the manuscript must be summarized (introduction, discussion and conclusions). I have the following comments which the authors should address before the paper is considered for publication.

Line 51: correct, enterotoxin gene sea was present in 16.7% of isolates, while sec and TSST-1 were identified in 8.33%

Line 53: correct, were producers of slime or were slime producers

Line 115: correct, affinity for the most of β-lactams molecules

Line 115: correct, mecA, in italic

Line 137: complete the sentence “(SEa, SEb, SEc, SEd, SEe etc), and toxic shock syndrome toxin (TSST1)”.

Line 169: why you have incubated the collected samples at 37°C? I think that you must maintained these samples at 4°C before their microbiological analysis.

Line 170: correct, isolation and identification of S. aureus

Line 172: I think that 1g is not sufficient for the isolation of S. aureus, you must take from each sample 10 or 25g

Line 176: correct, mannitol salt agar is not specific only for S. aureus but this media is used to isolate the staphylococci genus.

Line 177: you must precise how number of suspected colonies were chosen from each positive sample?

Line 182: for the evaluation of S. aureus load in each sample, you must use another media, Baird Parker, and you must count the number of characteristic and non-characteristic S. aureus colonies. I think also that 1g is not sufficient to estimate the S. aureus load.

Line 192: correct, nuc, mecA in italic

Line 201: correct, In vitro in italic

Line 204: delete, biofilm

Line 210: correct, all the identified isolates

Line 210: correct, for staphylococcal enterotoxin genes (sea, seb, sec, sed, see)

Line 211: correct, the genes in italic (eta, etb, tst, icaA, icaB, icaC, icaD)

Line 218: correct, antimicrobial resistance testing

Line 221: correct, CLSI, 2020

Line 222: correct, disc diffusion method

Line 265-272: summarize this paragraph

Line 294: correct, sea, seb, sec, sed, and see

Line 305: correct, antibiotic susceptibility profile

Line 306-307: reformulate the sentence and you must indicate the multi-drug resistance phenotypes, which are observed in this study.

Line 327: correct, can causes severe

Line 337: correct, Haque et al. (2018),

Line 344: correct, in which S. aureus was isolated or recovered

Line 350: correct, low quality of raw materials

Line 380: correct, that enterotoxin genes sea and sec

Line 408: correct, due to a number of virulence factors

Line 415: correct, carry one or more staphylococcal enterotoxin genes

Line 420: reformulate, our research reported the frequency of TSST-1 with a value of 8.33%, which

Line 458: correct, the zoonotic trait of MRSA or zoonotic properties of MRSA

Line 500: correct, S. aureus may contaminate cheese or cheese may be contaminated with S. aureus, which is …

Reviewer #2: This paper reported the prevalence of Methicillin Susceptible S. aureus (MSSA) and Methicillin Resistance S. aureus (MRSA) in commercial cheese in Bangladesh. Meanwhile, antimicrobial resistance, virulence profiles, biofilm-forming capabilities were revealed. Additionally, the phylogenetic relationships of the obtained only one isolate with those from other countries were shown. This study covers a very important topic, especially since microbial resistance to previously known substances is increasing day by day, which puts lots of lives in danger of foodborne diseases.

However, the experimental design is simple, and test methods are routine.

Title: Upon reviewing the methods employed in the study, it is evident that demonstrating only the presence of genes is insufficient to justify using the term 'super-antigenic. It is also necessary to show that the toxins are produced and active. The validation of super-antigenic properties typically requires both genetic analyses and experimental methods assessing the biological activity of the toxins.

Therefore, merely indicating the presence of genes is not enough to definitively confirm the presence of super-antigenic properties. It would be appropriate to update the title in this context. Also, they should rearrange their result and discussion in this context.

The abstract does not adequately reflect the content of the study. The abstract outlines methods for the isolation of S. aureus, determination of antibiotic resistance profiles, and characterization of toxin genes. However, the findings also focus on biofilm formation, biofilm-associated genes, and antibiotic-resistance genes. In this context, the objectives and methods should be rewritten for greater clarity.

The introduction and discussion sections of the manuscript are too long and contain many repetitions. Please review and reorganize them. Especially, Introduction sectin can be shorten.

In material and methods and results section,Table 1-3 are missing.

ısolation step already been covering in 2.2. section. However the section 2.3 is more clear and better. The authors should merge these two sections, or omitted section 2.2.

The results section unnecessarily includes material and methods statements rather than the actual findings. The obtained results should be written. In other words, the results are very complex and not understandable, more information should be provided on study results, and material and methods sentences should be removed.

Specific comments:

Gene names should be italicized throughout the article.

Line 48-49: i) should be added "classical" before staphylococcal enterotoxins

ii) should be added "genes" after (TSST-1)

L49-50:according to which legislation? ıs it local EU or FDA?, and what are the CFU counts the safety thresholds, please clarify

L51: The authors mentioned Stapylococcal enterotoxins. However, the authors did not mention the detection of SE with ELISA or other techniques in both the abstract and material method sections. Please rephrase accordingly.

L 52-54: The sentence should be rearranged according to the percentages obtained from the methods used and the amount that is meant by the significant percentage should be stated., For example, Notably, 66.67% and 58.33% of the isolates exhibited biofilm formation based on the Congo Red and microtiter plate techniques, respectively, with significant percentages (?) carrying the icaA and icaD genes.

L59. key words: should be added genes after Staphylococcal Enterotoxins

L 170: Isolation and Identification of Organism >>>> Isolation ........ of S. aureus

L171: ISO 6579:2002(E) standard is for horizantal method for the Detection of Slamonella spp. Please check and rewrite the S. aureus standard.

L172: desired organisim>>>>should be changed as S. aureus

L173:which serial dilution? two or ten fold?

L171-192:ısolation step already been covering in 2.2. section. However the section 2.3 is more clear and better. The authors should merge these two sections, or omitted section 2.2.

L177-180:The sentences is not clear. Please check it and rewrite

L194: Staphylococcus aureus>>>> S. aureus

L196-197: Table 1 and Table 4 is missing.I could not see the tables in the article

L202 and L204: slim layer>>>> slime layer

L208:Title should be shortened as "Multiplex PCR for detection of virulence factor genes

L213: Table 2 and Table 3 İs missing

L215: for 45 minutes (Molecular Cloning: A Laboratory Manual. Joseph Sambrook, David W. Russell, 2001): please recheck this reference according to guidelines of journal

L221: CST>>>(CLSI 2020, Adjei et al., 2022)

L234: according to CLSI guidelines of 2020>>>>should be removed

L236:according to (Weinstein & Clinical and Laboratory Standards Institute, n.d.)>>>>should be removed

L241:how did you chose mecA positive isolates? How much isolates subject to squence analyses? ad how did you perform the sequence analysis?

L244-255: these sentences are not necessary. ıt should be removed

L265: Out of 36 samples tested? your sample size seems to 72 in abstract and material method section. You mentioned 36 samples tested in here. Please clarified.

L267-272:These sentences is not necessary, it should be removed.

L274:should be added the range of CFU count from analysed samples (min, max and average)

L282-290:This paragraph is too long and contains unnecessary information. This pharagraf should be rearragened like this, based on the PCR result, 100% of the total sample showed the presence of S. aureus, from which 33.33% of them were mecA positive

In other words, the number of S. aureus isolates and how many of them are MRSA should be clarified.

L291-304:did you found any enterotoxin genes and/or exfoliative genes? results are confusing please simplify and focus on prevelance of these genes.

L306-307: More information should be provided AMR profile of isolates. Also Table 3 is missing.

L301-312: These sentences are not necessary. ıt should be removed

L352:EC regulations (COMMISSION REGULATION (EC)?? Please check it and correct

L352: MIM et al., 2019>>>> Mim et al.

L378 and L501: Methicillin-resistant S. aureus>>> SHOULD BE WRİTE ONLY MRSA. Since you have previously mentioned the full name of the bacterium, it is sufficient to use only its abbreviation

L501:25%??? İs this rate for MRSA? This rate seems to be 33% for MRSA in results. Please check and correct.

fİGURE 8: The colors in the first image should be described to indicate what they represent.(What do the purple and its shades in the first image represent?)

**********

-->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

**********

[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-24-38694R1-->-->Virulence Determinants and Toxin profile of Methicillin Resistant Staphylococcus aureus from Commercial Cheese in Bangladesh: A public Health Risk-->-->PLOS ONE

Dear Dr. Hossain,

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.

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

Two reviewers assessed your manuscript. They provided a number of valid comments and suggestions that must be carefully addressed by the authors upon revision.

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

Please submit your revised manuscript by Apr 24 2025 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,

Reham Mokhtar ELTarabili

Academic Editor

PLOS ONE

[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: All comments have been addressed

Reviewer #3: (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 #3: No

**********

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

Reviewer #1: Yes

Reviewer #3: 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

Reviewer #3: (No Response)

**********

-->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 #3: (No Response)

**********

-->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: Title: Molecular detection of super-antigenic Methicillin-Resistant Staphylococcus aureus from commercial cheese in Bangladesh

Comments to authors:

The authors of this paper characterize the Staphylococcus aureus isolates from cheese samples in Bangladesh, according to their antibiotic resistance patterns, enterotoxin gene profiles and their biofilm formation ability. The result of this study is interesting and should be of interest to the readership of your journal. I have the following observations which the authors should address before the paper is considered for publication.

Line 45: correct, comprehensive analysis

Line 48: correct, staphylococcal enterotoxin genes (sea, seb, sec, sed and see)

Line 51: correct, enterotoxin gene sea……………., while sec……………..

Line 59: correct, staphylococcal enterotoxin genes

Line 95: correct, during handling

Line 115: correct, for β-lactams

Line 115: correct, mecA in italic

Line 137-138: complete the sentence “(SEa, SEb, SEc, SEd, SEe etc), and toxic shock syndrome toxin (TSST”

Line 151: correct, by …..

Line 172: why you have sampled only 1g from each sample? . You must sample at least 10g

Line 176: correct, the mannitol salt agar is not specific for S. aureus species but specific for the Staphylococcus genus.

Line 177: you must precise the number of colonies chosen from each positive sample

Line 186: correct, Pétri plate

Line 201: correct, in vitro in italic

Line 210: correct, for staphylococcal enterotoxin genes (sea, seb,…….)

Line 210-211: you must put the targeted genes in italic

Line 223: why you have used the PBS for the preparation of the bacterial suspension?

Line 235: correct, and the strains were classified as susceptible, …..

Line 264: with the biochemical tests, you can not confirmed the S. aureus species, as you have many Staphylococcus species which are coagulase positive. For this, you must perform other tests and confirm the obtained isolates with molecular method (PCR).

Line 293: correct, All of the MRSA-positive isolates

Line 294: correct, such as sea, seb, sec, …..

Line 330: correct not for all but for the most β-lactam molecules

Line 340: correct, the results of this study do not agree with agree with those of Ifra …..

Line 413: correct, genes sea and sec

Line 421: correct, in S. aureus isolates from Minas ……

Line 424: you can compare your results with those obtained by TITOUCHE et al (2024). Investigation of Biofilm Formation Ability and Antibiotic Resistance of Staphylococcus aureus Isolates from Food Products.

Line 455: correct, however, the presence of these genes in S. aureus isolates from cheese samples

Line 500: correct, cheese may be contaminated with S. aureus…..

Line 504: correct, to decrease the contamination rate of

Reviewer #3: The authors performed this study to determine the virulence and toxin profiles of methicillin-resistant Staphylococcus aureus isolated from commercial cheese in Bangladesh. This type of study is really important for public health. Unfortunately, this study has many serious drawbacks. I found many discrepancies in the study design, especially in antibiotic selection. I believe the authors should check the CLSI guidelines properly before selecting any antibiotics. Also, I believe the authors need to perform the study again. However, please find my comments below.

Line 120: How much distilled water? Did you make the distilled water sterile before using it?

Line 145: Reference for boiling method?

Tables 2 and 5: I recommend using these tables as supplementary material.

Line 162: “2.6 Multiplex PCR for detection of virulence factor genes” – but you also mentioned resistance genes here. Please modify the name of the section.

Also, did you check resistance genes before determining their phenotypic resistance profiles? Please clarify this.

Line 176-195:

- So, you used oxacillin for S. aureus, right? How did you interpret the data based on CLSI 2020? You have to check their MIC; you can’t come to an outcome for oxacillin using the disk diffusion test. CLSI 2020 clearly mentions that oxacillin disk testing is not reliable for S. aureus. I believe this is a serious drawback of your study. You should go through your study again.

- Moreover, how did you interpret the resistance profiles of ampicillin and amoxicillin? As far as I know, you can only use penicillin (10 units) for S. aureus, which is another drawback.

- The same is true for streptomycin, cefotaxime, cefuroxime, and nalidixic acid. You should check the CLSI 2020 guidelines before using any antibiotics.

Line 189: “Gentamycin” should be “Gentamicin.” Please correct it where needed.

Line 200: “mecA” should be in italics. All the genes’ names should be in italics. Please correct it where needed.

Line 198-207: References for any software or methods you used are missing.

Line 209-214:

- I was just wondering why you used different versions of GraphPad Prism in the same study.

- “P” from “P-value” should be in italics.

- “study's findings and the authority” – what does that mean?

- You don’t need to provide the prevalence equation.

Line 246: You can’t determine MRSA by using only the mecA gene. There are several resistance genes that can also be responsible for MRSA, e.g., mecB, mecC, mecD, or others. This is another drawback of this study.

**********

-->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 #3: Yes: Md Saiful Islam

**********

[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-24-38694R2-->-->Virulence Determinants and Toxin profile of Methicillin Resistant Staphylococcus aureus from Commercial Cheese in Bangladesh: A public Health Risk-->-->PLOS ONE

Dear Dr. Hossain,

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 Oct 02 2025 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,

Abayeneh Girma

Academic Editor

PLOS ONE

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

[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 #4: All comments have been addressed

Reviewer #5: (No Response)

Reviewer #6: All comments have been addressed

Reviewer #7: (No Response)

Reviewer #8: 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 #4: Partly

Reviewer #5: No

Reviewer #6: Yes

Reviewer #7: Partly

Reviewer #8: Yes

**********

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

Reviewer #4: No

Reviewer #5: N/A

Reviewer #6: Yes

Reviewer #7: N/A

Reviewer #8: 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 #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: 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 #4: No

Reviewer #5: No

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: 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 #4: Virulence Determinants and Toxin profile of Methicillin Resistant Staphylococcus aureus

from Commercial Cheese in Bangladesh: A public Health Risk

PONE-D-24-38694_R2_reviewer

Comments on Revised version:

Abstract:

Line 28-30: In my opinion, the authors could represent this portion in another way. Suddenly started about MRSA in the very next sentence. The relationship of MRSA and cheese might come first and then objective of the research could be mentioned.

Line 35: Please rephrase this sentence.

Line 36: please write as……….. polymerase chain reaction (PCR)

Line 41-42: Please mention the safety threshold briefly.

Introduction:

Authors did not establish link between cheese and MRSA.

No research gap was mentioned.

Previous research on MRSA and cheese on global or national contexts was not mentioned.

Sources of contamination with MRSA should be discussed clearly.

Line 57-58: Please give reference.

Line 60-61: need reference(s).

Line 67-68: need reference(s)

Line 69-70: need reference(s).

Line 57-68: Need multiple references for each statement.

Materials and Methods:

Line `112-113: Only one raw milk cheese included? Why? Making comparison was not difficult?

Line 140-141: mention the rationale of using pour plate method.

Line 148-149: Why so many references for boiling method? Single reference was not enough? Why boiling method? Why did not any Kit?

Line 175: why CLSI, 2020? Why not recent?

Line 194-203: Use references.

Results:

As these findings were reviewed by previous reviewers, I don’t have queries.

Discussion:

Why 100%? Most of the authors found lower percentage of Staph. Aureus in their researches. The authors mentioned country and production procedure variations. But the commercial cheese production of renowned Brands follows the International standards, even in Bangladesh. In my opinion, authors should establish scientific basis of this finding.

As all samples exceeded the EU recommended standard, then what could be the authors decision about the commercial cheese in Bangladesh?

Conclusion:

Conclusion became too long. Should be more precise and shorter.

Line 427: After finishing the whole experiments, showing all findings, why did authors mention “may be’?

Reviewer #5: The manuscript entitled “Virulence Determinants and Toxin profile of Methicillin Resistant Staphylococcus aureus from Commercial Cheese in Bangladesh: A public Health Risk” presented the virulence and toxin factors of MRSA from commercial cheese in Bangladesh. The Authors also showed the AMR profile of MRSA by both phenotypic and genotypic approach. The manuscript has great importance in the field of AMR and food safety. However, the manuscript requires some corrections. There are areas of improvement. The knowledge gap should include why the study is different from other studies in Bangladesh. The discussion section has some repetitions. Please check the whole manuscript for English corrections. There is a serious issue in the methodology of bacterial load determination. All are given bellow-

Title: Why the word “public” starts with the small “p”.

Line no. 42: Please add “and” before “Food Safety and Standards Authority India (FSSAI)”.

Line no. 46: showed a moderate level of resistance. Check the sentence.

The gene name should be Italic. Check the uniformity of gene and species names throughout the manuscript.

Please check the whole manuscript for similar minor errors to improve the quality of the manuscript.

Line no. 110: Are all the brands from Bangladesh or imported as well?

Line 134-137: As per standard, the sample amount should be 10 grams. Thereafter, for ten-fold dilution, you must have to maintain 1:10 that is one part sample and 9 parts diluent. However, the Authors have not maintained this ratio. They used one gram sample and 900 microliter (0.9 ml) diluent that is 1: 0.9 ratio. your total volume is approximately 1.9mL. Your dilution factor is 1/1.9, which is approximately 10⁻⁰·²⁸, not 10⁻¹.

It is a serious mistake in the sample preparation and it will misinterpret the findings.

Line no. 150-151: Table 01, contains primers of nuc, mecA, and toxin genes.

Line no. 156: Table 01. Where is the supplementary table? After Table 1, where is Table 02?

Line no. 162-163: The sentence is not clear. Please mention the name of the test with the interpretation criteria.

Line no. 169-170: Approximate concentration of 1.5*10-8 CFU ml-1.

Line no. 247: total samples.

Line no. 380-382: Please mention the causes of differences in occurrences of MRSA in cheese particularly for Bangladesh.

Reviewer #6: Line 95: write the abbreviated form of International Institute for Diarrheal Disease Research within bracket

Line 132: change the heading 2.3 (Microbiological evaluation of Cheese sample). Aren’t the isolation, identification, molecular detection and antibiotic sensitivity test included in microbiological evaluation? Your writing resembles that only CFU counting represents the microbiological evaluation. You can change the heading as ‘’ total count of Staphylococcus genus’’.

Line 163: What are the modifications?

Line 213: Recheck this heading and make it shorter

Line 228: This study performed in Bangladesh. That’s why it is ok to compare the findings with the recommended limit of BFSA. But why the findings are compared to that of Standards Authority of India, and the European Union bypassing other countries like USA, Australia and others?

Line 228-229: Reference?

Line 286-287: 3.5 Toxin, Virulence, and antibiotic-resistant genes profile of Staphylococcus aureus isolated from cheese: What about SEb and SEd. Clarify their percentage.

Figure 6: it will be easier to understand if you represent the two pictures of PCR with two different figure names

Figure 8: same as figure 6

Figure 9: Try to increase the sharpness and clarity of the given diagram.

Line 341: Why is a matter of concern?

Discussion: line 388-394: Recheck the sequence and organization of the provided information. At first you wrote about the percentage of S. aureus and compared it with the existing data, then wrote about MRSA in the same manner. After that you repeated the S. aureus again. Try to write in more organized way.

Line 437: is it TEM or blaTEM?

Overall evaluation: Recheck the whole manuscript again for grammatical errors. The organism’s name and genes name must be in italic form. The headings under different section should be rechecked to make it simpler, smarter and shorter.

Reviewer #7: I have reviewed the manuscript "Virulence Determinants and Toxin Profile of Methicillin-Resistant Staphylococcus aureus from Commercial Cheese in Bangladesh: A Public Health Risk" for the first time. The authors have justified or corrected the observations made by reviewers in previous rounds. However, I believe there are still several errors or clarifications that need to be made, particularly in the methods and results.

Material and Methods

• Sections 2.4, 2.5, 2.6 and 2.7: Authors must include and mention the use of positive and negative controls.

• Table 1: The primers listed are not compatible with those used in the references indicated. Particularly in refs. 16 and 17, the primers are completely different. In the other cases (ref. 18), there are differences in some nucleotides. Authors should verify each primer in Tables 1, 3, and 4.

• Section 2.4/Line 148: It is excessive to use 5 references to cite the boiling method. Reduce to 1 or a maximum of 2.

• Section 2.6/Line 167: Authors must appropriately cite the CLSI manual used and not any other indirect reference.

• Sections 2.4 + 2.7: I recommend merging these two sections, as they are all PCR-based. Also, merge tables (1, 3, and 4)—note that Table 2 does not exist—and maintain the separation of primer groups by category as they were.

• These analyses are inappropriate and should be withdrawn. The fragment considered (233 bp) is very small and highly conserved in S. aureus (I blasted the OR096215 sequence and found nearly 2,000 sequences with 100% identity). The resolution of the phylogenetic tree does not allow for appropriate inferences between identical sequences. Any discussion or conclusion based on this analysis must be withdrawn.

Results:

• Since all PCR assays are from references and were not developed in the present study, I consider the gel figures (Figs. 3, 4, 6, 7, 8, 10B) unnecessary in the main body of the manuscript. I suggest transferring them as supplementary material.

• Figure 3: The authors indicate an expected amplification size of 280 bp for the nuc gene, but the figure shows bands above 300 bp. What is the reason for this?

• Figure 4: In the legend, the authors indicate that the samples in lanes 5 and 6 are negative. However, the same positive band can be seen in lanes 1 through 4.

• 3.4: The authors refer to Table 4, which currently lists primers against biofilm-producing genes. Verify.

• Figure 8: In panels A and B, the authors indicate other sizes such as "100 bp." Since the 100 band is not visible, I suggest only identifying the 500 band, which is the most intense. In panel B, something similar occurs as in Figure 3. The authors indicate that the detection band is 397 bp, however, in the gel photo, it appears to be below 300 bp. Is it possible that electrophoresis generated a curve? In any case, the use of positive controls is necessary to validate these results.

• Finally, I suggest a review of the language to avoid overly colloquial idioms and phrases.

Reviewer #8: The article is interesting and congratulations for your work. The authors did almost all required revisions.

**********

-->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 #4: No

Reviewer #5: Yes: Md. Abdus Sobur

Reviewer #6: No

Reviewer #7: No

Reviewer #8: Yes: Naglaa S. Elabd

**********

[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.

Attachments

Attachment

Submitted filename: plos one.docx

-->PONE-D-24-38694R3-->-->Virulence Determinants and Toxin profile of Methicillin Resistant Staphylococcus aureus from Commercial Cheese in Bangladesh: A public Health Risk-->-->PLOS One

Dear Dr. Hossain,

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 15 2026 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 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.

As the corresponding author, your ORCID iD is verified in the submission system and will appear in the published article. PLOS supports the use of ORCID, and we encourage all coauthors to register for an ORCID iD and use it as well. Please encourage your coauthors to verify their ORCID iD within the submission system before final acceptance, as unverified ORCID iDs will not appear in the published article. Only the individual author can complete the verification step; PLOS staff cannot verify ORCID iDs on behalf of authors.

We look forward to receiving your revised manuscript.

Kind regards,

Abayeneh Girma

Academic Editor

PLOS One

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

[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 #7: (No Response)

Reviewer #9: (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 #7: Partly

Reviewer #9: Partly

**********

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

Reviewer #7: Yes

Reviewer #9: 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 #7: Yes

Reviewer #9: 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 #7: Yes

Reviewer #9: No

**********

-->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 #7: The authors have appropriately corrected or justified almost all of the observations made in the preliminary round of my reviews.

However, they persist in maintaining the phylogenetic analysis and its discussions, which are inappropriate, as already argued in the previous round: "These analyses are inappropriate and should be withdrawn. The fragment considered (233 bp) is very small and highly conserved in S. aureus (I blasted the OR096215 sequence and found nearly 2,000 sequences with 100% identity). The resolution of the phylogenetic tree does not allow for appropriate inferences between identical sequences. Any discussion or conclusion based on this analysis must be withdrawn." Withdrawing this analysis does not detract from the merit of the study. On the contrary, persisting on this point undermines its scientific soundness. I insist on completely withdrawing this analysis (methods, results, and discussions) to avoid inappropriate or unjustified inferences.

Reviewer #9: This manuscript addresses a worthwhile public-health question and has potential value as a regional surveillance study on S. aureus/MRSA in commercial cheese.

However, in its current form, the paper has substantial methodological and reporting weaknesses that limit confidence in the quantitative conclusions. My comments as follows:

1. Unclear unit of analysis / isolate count

The manuscript does not clearly explain how many isolates were actually analyzed. It mentions 72 cheese samples, but many results use denominators that suggest only 12 isolates. It is unclear whether the study used one isolate per sample, per brand, or per positive culture, which makes prevalence and resistance/toxin frequencies hard to interpret.

2. Internal inconsistencies in results

The reported findings conflict across sections. Antibiotic resistance profiles differ between the abstract and Results, and the reported frequencies of biofilm and toxin genes are inconsistent. These are major result discrepancies, not minor wording issues.

3. Problems in microbiological methods

The described pre-enrichment in nutrient broth may be acceptable for detecting presence/absence, but not for quantitative estimation of bacterial load. Since the study also reports CFU/g and compares counts to safety thresholds, the workflow appears methodologically inconsistent. The dilution scheme described is also nonstandard and unclear.

4. Insufficiently robust identification strategy

The identification approach (MSA, Gram stain, catalase/coagulase, nuc PCR) is reasonable for confirming S. aureus, but the manuscript does not clearly explain isolate selection or controls. Given the unusually high claim of 100% S. aureus positivity in all cheese samples, stronger methodological detail is needed.

5. Weak justification for resistance-gene panel

Several screened resistance genes, especially ESBL/carbapenemase genes like CTX-M, CMY, SHV, and NDM-1, are more typical of Gram-negative bacteria and are not well justified in an S. aureus study. Their biological relevance and interpretation are not adequately discussed, and sequencing confirmation is lacking.

6. Overstated toxin conclusions

The study reports toxin genes by PCR, but the manuscript phrases this as if actual toxins were present in the cheese. Gene detection only shows the potential for toxin production, not confirmed toxin expression or toxin presence in the food.

7. Weak statistical analysis

The statistical section lacks a strong analytical framework. Comparing study findings directly to regulatory limits using p-values is not appropriate, and the paper does not provide confidence intervals, clear hypotheses, between-group comparisons, or detailed data presentation.

8. Inadequate phylogenetic analysis

The phylogenetic methods are not described in enough detail to support the claim of identifying a “new strain.” Key details are missing, including number of sequences, gene region, quality control, accession numbers, and reference selection.

**********

-->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 #7: No

Reviewer #9: 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.]

To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation.

NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

Virulence Determinants and Toxin profile of Methicillin Resistant Staphylococcus aureus from Commercial Cheese in Bangladesh: A public Health Risk

PONE-D-24-38694R4

Dear Dr. Hossain,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support.

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,

Abayeneh Girma

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments: