PONE-D-24-09897Antimicrobial proteins from oyster hemolymph improve the efficacy of conventional antibioticsPLOS ONE

Dear Dr. Summer,

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Additional Editor Comments:

The manuscript and work carried out by authors it is of interest since it addresses a critical issue in antimicrobial therapy and presents promising data on the use of oyster hemolymph proteins.

However, there are some points that should be clarified and improved, and additional information should be added. Please follow reviewer indications.

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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: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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: No

Reviewer #2: Yes

Reviewer #3: 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

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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: Overall, this manuscript addresses a critical issue in antimicrobial therapy and presents promising data on the use of oyster hemolymph proteins. With detailed methodologies and a balanced discussion, it has the potential to make a significant impact in the field of antimicrobial research. This manuscript provides a comprehensive and detailed description of covering the preparation and treatment of hemolymph protein extracts (HPE), proteomic analyses, antibacterial and antibiofilm assays, and cytotoxicity assays. However, there are some areas where clarity and conciseness could be improved, and additional information could enhance reproducibility.

Specific Comments

2.1 HPE Preparation

Hemolymph Collection from SRO (Section 2.1.1):

Ensure that the referenced method [36] is accessible and sufficiently detailed for replication.

The statement "Hemolymph was withdrawn from multiple oysters, combined to obtain 5 mL pools" would benefit from specifying how many oysters were used on average per pool, as this could impact the consistency and yield of hemolymph.

Hemolymph Fractionation (Section 2.1.2):

The freeze-drying process is well-explained, but adding details about the reconstitution process (e.g., the volume of Milli-Q water used) would improve clarity.

HPE Heat Treatments (Section 2.1.3):

The heat treatment conditions are well-documented. It would be beneficial to include a brief rationale for the chosen temperatures and durations to understand their relevance to the study's objectives.

2.2 Proteomics

Protein Quantification and Visualization (Section 2.2.1):

Ensure that the sources of all reagents and materials are consistently specified (e.g., "Bio-Rad, Australia" for the protein dye reagent).

Protein Identification by HPLC-MS/MS (Section 2.2.2):

Mentioning the specific version of the database used for searching spectra (e.g., the release date or version number of the Uniprot Mollusca database) would enhance reproducibility.

The protein extraction and digestion process is well-described, but clarifying the final volume in which peptides are redissolved before analysis could prevent confusion.

Protein Data Analysis (Section 2.2.3):

Including an explanation for choosing NSAF and the specific clustering methods (PCA and hierarchical clustering) would provide context for readers unfamiliar with these techniques.

2.3 Antibacterial and Antibiofilm Assays

Media and Reagents (Section 2.3.1):

Ensure that any deviations from standard protocols (if any) are mentioned.

Bacteria Preparation (Section 2.3.2):

The preparation of bacterial cultures is detailed and aligns with standard procedures. Specifying the rationale for selecting the particular strains and clinical isolates would add context.

Antibacterial-biofilm Inhibition Coupled Assays (Section 2.3.3):

The assay setup and conditions are well-documented. The choice of concentrations for HPE and antibiotics could be briefly justified to highlight their relevance to the study.

The statistical methods used for data analysis are appropriate. Consider providing additional details on the criteria for determining significant differences.

Biofilm Treatment Assays (Section 2.3.4):

It would be helpful to explain why specific controls were chosen (e.g., EDTA and ciprofloxacin) to contextualize their relevance as benchmarks.

2.4 Cytotoxicity Assays

Cell Viability Using the MTS Assay (Section 2.4.3):

Justifying the range of HPE concentrations tested would add value, as would a brief discussion on the selection of statistical methods for data analysis.

The discussion is comprehensive and aligns well with the study's findings and objectives. Enhancing the structure, providing more detailed comparisons with existing research, and elaborating on future research directions will strengthen the manuscript.

Antibacterial Efficacy: Provide detailed comparisons with other AMPs.

Synergistic Effects: Expand on mechanisms and cite relevant studies.

Clinical Relevance: Discuss potential integration into clinical protocols.

Challenges: Elaborate on production costs and efficacy barriers.

Future Research: Specify next steps and potential collaborative efforts.

Reviewer #2: Authors did interesting research on Antimicrobial proteins from oyster hemolymph improve the

3 efficacy of conventional antibiotics and they found that HPE is a semi-purified extract of SRO hemolymph proteins showing strong antibacterial activity specific to Streptococcus sp. which could be used as potent agent to develop drug against streptococcus infections. I highly recommended the manuscript and have a future scope. Some grammar and spelling mistakes should be removed in revised thesis.

Reviewer #3: I have reviewed the manuscript entitled “Antimicrobial proteins from oyster hemolymph improve the efficacy of conventional antibiotics” submitted for possible publication in the journal “PLOS ONE”. The authors have done good efforts in compiling the results and all of the other relevant information. The work is thorough and the authors have described it very well. The manuscript could be beneficial for the reader and scientific community specially in terms of antibiotic resistance. After critically reviewing the manuscript, I did not noticed any flaws of major problems. However, few grammatical corrections have to be considered before proceeding it further towards publication process. My specific comments are:

1. Line 154: Correct the sign “°C”.

2. Line 155: correct “up to 30 50ms”

3. Figure 1 should be improved. The authors can use simply PPT to make the labelling in the figure.

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Reviewer #1: No

Reviewer #2: Yes: Saiqa Andleeb

Reviewer #3: Yes: Naveed Ahmed

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Antimicrobial proteins from oyster hemolymph improve the efficacy of conventional antibiotics

PONE-D-24-09897R1

Dear Dr. Summer,

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,

Maria del Mar Ortega-Villaizan

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 #3: All comments have been addressed

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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 #3: Yes

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

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 #3: Yes

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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 #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 #3: (No Response)

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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 #3: No

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