-->PONE-D-25-53029-->-->Identification, Antibiogram assessment and Molecular Detection of Methicillin Resistant Staphylococcus aureus in Beef Line in two Municipal Abattoirs, Northwest Ethiopia-->-->PLOS ONE

Dear Dr. Abey,

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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We look forward to receiving your revised manuscript.

Kind regards,

Md. Tanvir Rahman, DVM, MSc (Canada), PhD (UK), FBAS

Academic Editor

PLOS ONE

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Please see the comments of the reviewers amd address them, do experiment where applied.

[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: No

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

-->3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: No

Reviewer #2: Yes

**********

-->4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

Reviewer #2: Yes

**********

-->5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: Overall Assessment:

The study “Identification, Antibiogram Assessment and Molecular Detection of Methicillin Resistant Staphylococcus aureus in Beef Line in Two Municipal Abattoirs, Northwest Ethiopia“addresses a relevant One Health issue concerning MRSA in abattoirs, which is important for food safety and public health. However, the manuscript has limited novelty and requires improvement in methodological justification, data analysis, and interpretation to meet publication standards. Below are the comments on the article that needs to be addressed before consideration for publication.

Novelty:

The topic is not new; similar studies have been conducted in Ethiopia. The authors should better highlight what new information this study contributes, such as unique molecular or phylogenetic findings.

Methodology:

Sampling strategy and sample size lack statistical justification.

Only 15 isolates were tested for mecA gene—this needs justification.

Phenotypic identification without molecular confirmation (e.g., nuc gene) limits accuracy.

PCR controls and validation details are missing.

Results and Analysis:

Statistical tests (e.g., Chi-square) should be applied to strengthen findings.

The discrepancy between phenotypic and genotypic MRSA detection (100% vs 33.3%) is not well explained.

Discussion:

Too descriptive—should focus more on interpretation and comparison with recent studies.

The phylogenetic results could be better discussed in terms of evolutionary or epidemiological significance.

Conclusion:

Valid but too general. Include clearer recommendations for abattoir hygiene, AMR monitoring, and One Health interventions.

Recommendation:

Major Revision — The manuscript provides useful local data but requires stronger methodological rigor, analytical depth, and clearer discussion of its novel contributions before publication.

Reviewer #2: The authors describe the prevalence of MRSA amongst two Municipal abattoirs in Northwest Ethiopia. While previous studies have described MRSA prevalence amongst abattoirs with variable outcomes, this study evaluates its prevalence in an unexplored region. However, the study is deeply flawed in its overall execution. For instance, it is unclear how these MRSA are identified. Why is methicillin used for screening when it is not the most ideal method to screen for MRSA. It is also not clear why the authors have sequenced the mecA gene, which is quite conserved amongst MRSA strains. I would request the authors to comment on the utility for mecA gene sequence for source attribution or transmission analysis in introduction. In any case the premise for the study and methodology adapted is weak. Perhaps using MLST, spa, SCCmec typing, PFGE or WGS would have been much more appropriate techniques for assessing MRSA transmission patterns amongst workers, environment and the carcasses.

Other comments:

Title: Title is too generalized. Please modify the title to reflect basic outcome of the study

Line 55: kindly rephrase this sentence; It produces preformed enterotoxins in food, which cause food poisoning and gastrointestinal illness. What is meant by pre-formed in this context?

What is the reason for focusing on the mecA gene for sequence analysis?

Line 131-133: Why the authors chose to screen using methicillin rather than cefoxitin which is better recommended for MRSA detection? Methicillin has limited utility to detect MRSA due to heterogeneous expression, high sensitivity to variations in laboratory conditions (inoculum size, incubation time, temperature, and salt concentration in the culture medium), leading to inconsistent or false-negative results. Furthermore, hyperproduction of beta-lactamase can also lead to borderline resistance that is difficult to distinguish with routine methicillin tests. As one of the aims of the study was to screen for mecA gene, this would greatly mis represent the number of MRSA amongst isolates. It would also be useful to include vancomycin in testing.

Line 199: Change Subculture to subcultured

Line 160: Italicize mecA

Remove last two sentences in Data Management and Analysis.

Line 208: Change Antibiogram assessment to antibiotic susceptibility profiling.

Table 3 is very confusing. What is the difference between number of resistant vs total number of resistant isolates? I assume first column describes the combination of resistance against different class combination of antibiotics. Kindly merge last column with first. For example; Three classes of antibiotics (n=16).

Line 219: How were the 15 isolates selected for mecA gene screening? I assume these were on the basis of phenotypic resistance against methicillin/ oxacillin/ cefoxitin?

Lines 229-232: Why the same isolate (MRSA-BahirDar-01-2021) has different accession numbers?

Line 243-250: These are more suited to the methods description and does not mention the results obtained from the analysis.

Line 273-276: The authors discuss how workers hands compared to clothes have a higher S. aureus carriage rate and thus indicate contamination due to carcass handling. Could the higher prevalence also be indicative that it is part of normal flora of the human skin? To infer that this is entirely due to carcass handling may therefore not be accurate.

Line 292-294: Please discuss some data related to long-term, indiscriminate use of β- lactams in both veterinary and human medicine in Ethiopia.

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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.

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Reviewer #1: Yes: Prof. Dr. Tahir Usman

Reviewer #2: No

**********

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

-->PONE-D-25-53029R1-->-->Methicillin-Resistant Staphylococcus aureus Along the Beef Production Line: Phenotypic Resistance and mecA Phylogeny in Two Ethiopian Municipal Abattoirs-->-->PLOS One

Dear Dr. Abey,

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 31 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,

Md. Tanvir Rahman, DVM, MSc (Canada), PhD (UK), FBAS

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.

Additional Editor Comments :

Please revised the manuscript as suggested.

Comments:

The manuscript addresses an important public health issue by investigating Methicillin-resistant Staphylococcus aureus along the beef production chain. The integration of phenotypic resistance profiling with mecA phylogenetic analysis is valuable and relevant. However, the manuscript requires careful revision to improve clarity, consistency, and scientific accuracy. Several sections contain repetition, inconsistent formatting (particularly for scientific names, gene names, and antibiotic nomenclature), and methodological ambiguities. Addressing these issues will significantly enhance the overall quality and readability of the manuscript.

Lines 166–167: Reference 32 (Mehrotra et al., 2000) does not correspond to the same primers authors used for mecA amplification. The primers described in the manuscript differ from those in the cited reference. Please provide an appropriate and accurate reference.

Additionally, Please provide a clearer, high-resolution PCR gel image and verify the reported product size.

Lines 104–109: This section repeats information presented in the following paragraph. Please remove the redundant text to improve clarity and avoid duplication.

Line 136: The term “Gram” should be written in lowercase (i.e., “gram”).

Lines 144–146: Please group antibiotics according to their respective classes and clearly mention the class names. Additionally, ensure consistency in formatting—antibiotic names should follow a uniform style throughout the manuscript (e.g., consistent capitalization).

Line 152: Scientific names should be written in italics.

Line 160: The sentence is unclear. It is recommended to revise it as: “Genomic DNA was extracted from Staphylococcus aureus.”

Line 183: The DNA extraction method has already been described earlier. Repetition is unnecessary—please remove this section.

Line 250: Scientific names should be written in italics.

Line 251: The gene name mecA is inconsistently formatted (italicized in some places and not in others). Please adopt a consistent style throughout the manuscript.

[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: Yes

**********

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

Reviewer #1: I Don't Know

Reviewer #3: I Don't Know

**********

-->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: 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 #3: 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 #1: The grammar needs a bit improvement. The S. aureus should be uniformly italic across the article. The rest, I have no more comments.

Reviewer #3: The manuscript addresses an important public health issue by investigating Methicillin-resistant Staphylococcus aureus along the beef production chain. The integration of phenotypic resistance profiling with mecA phylogenetic analysis is valuable and relevant. However, the manuscript requires careful revision to improve clarity, consistency, and scientific accuracy. Several sections contain repetition, inconsistent formatting (particularly for scientific names, gene names, and antibiotic nomenclature), and methodological ambiguities. Addressing these issues will significantly enhance the overall quality and readability of the manuscript.

Lines 166–167: Reference 32 (Mehrotra et al., 2000) does not correspond to the same primers authors used for mecA amplification. The primers described in the manuscript differ from those in the cited reference. Please provide an appropriate and accurate reference.

Additionally, Please provide a clearer, high-resolution PCR gel image and verify the reported product size.

Lines 104–109: This section repeats information presented in the following paragraph. Please remove the redundant text to improve clarity and avoid duplication.

Line 136: The term “Gram” should be written in lowercase (i.e., “gram”).

Lines 144–146: Please group antibiotics according to their respective classes and clearly mention the class names. Additionally, ensure consistency in formatting—antibiotic names should follow a uniform style throughout the manuscript (e.g., consistent capitalization).

Line 152: Scientific names should be written in italics.

Line 160: The sentence is unclear. It is recommended to revise it as: “Genomic DNA was extracted from Staphylococcus aureus.”

Line 183: The DNA extraction method has already been described earlier. Repetition is unnecessary—please remove this section.

Line 250: Scientific names should be written in italics.

Line 251: The gene name mecA is inconsistently formatted (italicized in some places and not in others). Please adopt a consistent style throughout the manuscript.

**********

-->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: Yes: Tahir Usman

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.]

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.

Methicillin-Resistant Staphylococcus aureus Along the Beef Production Line: Phenotypic Resistance and mecA Phylogeny in Two Ethiopian Municipal Abattoirs

PONE-D-25-53029R2

Dear Dr. Abey,

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,

Md. Tanvir Rahman, DVM, MSc (Canada), PhD (UK), FBAS

Academic Editor

PLOS One

Additional Editor Comments (optional):

Thanks for the revision.

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

**********

-->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

**********

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

Reviewer #3: I Don't Know

**********

-->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

**********

-->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: The authors have adequately addressed all comments and made the required revisions. The manuscript has improved and is now suitable for publication.

**********

-->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

**********