Dear Dr. Gebeyehu,

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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?

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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Reviewer #1: The research is interesting and raises serious concerns about the potential public health risks associated with the consumption of raw beef in Ethiopia. I support its publication after appropriate modifications, as outlined below.

The names of bacterial species should be written in italics. There are some omissions, so please check carefully and correct them.

In microbiological food analysis, standardized methods require the use of control strains to ensure the validity of the results. Hence, it is necessary to specify which bacterial strains were used as controls (positive control and negative control, respectively).

Most antibiotics tested to assess the susceptibility of isolated strains, except ciprofloxacin, are not currently used to treat infections with E. coli, Salmonella, or Shigella due to recognized antimicrobial resistance, toxicity, and the availability of superior alternatives. Therefore, the authors should explain the criteria used to select the antimicrobials for this study.

The presence of E. coli strains in raw beef is not unusual, and not all strains of E. coli are potentially pathogenic (with the exception of E. coli O157:H7 or other STEC). In fact, E. coli is an indicator of hygiene in food industry, whose presence is allowed in a limited count, depending on the legislation or reference standard. Therefore, the authors should reconsider these statements in the discussion section.

In Conclusions section the authors need to insert a complex sentence highlighting the study limitation and further perspectives in the approached research area.

Reviewer #2: This manuscript by Gebeyehu et al. addresses an important and timely public health issue: antimicrobial-resistant foodborne pathogens associated with raw beef consumption in Ethiopia, framed within a One Health perspective. The topic is relevant, and the dataset appears substantial. However, In its present form, the manuscript does not yet meet the standards for publication in a high-impact, international journal. I invite the authors to address the following concerns, specifically:

The manuscript claims to conduct a “health risk assessment,” yet the approach used is largely descriptive and semi-quantitative, without a clearly defined or validated risk assessment model. The risk scoring system (likelihood × severity) is insufficiently justified, not formally validated, and relies heavily on subjective scoring of hygiene practices. The manuscript does not clearly articulate what is novel relative to prior Ethiopian studies on raw meat contamination and AMR. Several similar prevalence studies are cited, but the incremental contribution of this work remains unclear. Therefore, the authors must clearly define whether this study is a microbiological prevalence study, an AMR surveillance study, or a formal risk assessment. If it is a risk assessment, the authors must justify the framework, explain assumptions, and clarify how it differs from existing Codex/WHO quantitative or semi-quantitative approaches. In addition, they must explicitly state the novel contribution in the Introduction and Discussion sections.

Identification of Salmonella, Shigella, and E. coli O157:H7 relies entirely on culture and biochemical methods, without molecular confirmation.Given the high reported prevalence of Shigella and E. coli O157:H7, the absence of PCR or serological confirmation raises concerns about misclassification. Shigella is rarely isolated from meat compared to Salmonella and E. coli; the unusually high prevalence requires stronger validation. Therefore, the authors must acknowledge the limitations of culture-based identification explicitly and clarify whether any serotyping or molecular confirmation was attempted. Likewise, temper claims related to E. coli O157:H7 and Shigella prevalence, or justify them with stronger methodological support.

Within the antimicrobial susceptibility testing section, the antibiotic panel includes drugs of questionable relevance for Gram-negative enteric pathogens (e.g., erythromycin). Chloramphenicol is reported as 100% effective, yet this drug is largely obsolete or restricted in many settings due to toxicity. CLSI breakpoints for some antibiotics used (especially erythromycin) are not clearly applicable to the organisms tested. The authors must justify the choice of antibiotics based on national treatment guidelines or surveillance relevance and clarify CLSI breakpoint applicability for each organism–antibiotic combination. What about chloramphenicol results and their interpretation? Avoid presenting it as a practical treatment option.

The hygiene scoring system (binary compliant/non-compliant) lacks validation and weighting. The reviewer wonder, why? The calculation of “cumulative gaps” (e.g., 52%, 76%, 100%) is arbitrary, with no sensitivity analysis. Risk categorization (low/medium/high) is asserted rather than statistically or probabilistically demonstrated. Please clarify these concerns, by providing a clear mathematical description of how risk scores were derived and justify thresholds for “high risk” using published frameworks.

Statistical methods are largely descriptive. Associations between hygiene practices and contamination are mentioned, but results of statistical tests are not presented (e.g., test statistics, p-values). Please clarify this issues!

The manuscript frequently uses strong causal language (“poses a serious health threat,” “urgent need”) without sufficient causal evidence. The One Health framing is conceptually invoked but not operationalized through data from animal health, environmental sampling, or policy analysis.

Last, but not least, language and grammar require substantial professional editing.

Reviewer #3: The manuscript discusses the public health risks of consuming raw beef in Ethiopia, especially in relation to bacterial contamination and antimicrobial resistance (AMR). The topic is highly relevant within the One Health concept, linking food safety, antimicrobial resistance, and consumer health. The manuscript provides useful primary microbiological data, hygiene observations, and risk assessment, making it potentially valuable for policymakers and public health researchers. Overall, this manuscript is well-written but needs some revisions and explanation. I recommend this manuscript for a major revision. I explain my concerns in more detail below. I ask that the authors specifically address each of my comments in their responses.

Comments:

1. Introduction:

• Why in this manuscript E. coli, Salmonella and Shigella were prioritized? Please add deep explanation or references.

• Research gap could be sharpened

• The introduction would benefit if the Authors can give clearer explanation about how impact the AMR to raw beef meat consumers.

2. Material and methods:

• Please add how to select 50 restaurant and 250 swab samples.

• Study design: The study is use cross-sectional, yet sampling occurred over a six-month period (January–June 2023). Clarification is needed on whether samples were collected once per site or repeatedly, and how temporal variability was addressed.

• 2.5.1 and 2.5.2 The authors should add molecular data like PCR, etc to confirm the bacteria and MDR.

• Could the Authors explain, why for antibiotic resistance testing use 6 antibiotics? Please give the additional explanation.

• Why the Authors use Erythromycin and Amoxicillin? Because there are not relevant to the Gram-negative bacteria.

• Data analysis: Criteria for significance are not mentioned and the rationale for using Chi square & Fisher’s test should be briefly stated.

3. Results:

• Explanation in 3.3 more appropriate for the discussion

• The criteria used to define multidrug resistance (MDR) should be explicitly stated in the Results section before MDR percentages are reported.

• The Results section would be strengthened by presenting confidence intervals or measures of uncertainty for key prevalence estimates.

• Although the Methods section mentions chi-square and Fisher’s exact tests, the Results does not report corresponding test statistics, degrees of freedom, or p-values. Where associations are implied (e.g., between hygiene practices and contamination), statistical outcomes should be reported.

4. Discussion:

• Deeper analytical interpretation rather than descriptive comparison

• Insufficient acknowledgment of study limitations

• Please be careful with the statement of treatment failure or severe clinical outcomes because this study does not provide direct clinical evidence of infection, therapeutic failure, or adverse health outcomes. The data only from meat and restaurant environments.

5. Conclusion:

• Please make it simply, the conclusion is too long. Conclude the result from your manuscript.

• Add future research in this section.

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes:Dhandy Koesoemo WardhanaDhandy Koesoemo WardhanaDhandy Koesoemo WardhanaDhandy Koesoemo Wardhana

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Attachments

Attachment

Submitted filename: Manuscript Number PONE-D-25-56353_review.pdf

Dear Dr. Gebeyehu,

Please submit your revised manuscript by Apr 16 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.. 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.. 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.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If 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 . 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 . 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 . 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,

Guadalupe Virginia Nevárez-Moorillón, Ph.D.

Academic Editor

PLOS One

Journal Requirements:

1. 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.

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

Additional Editor Comments:

Please address the comments of Reviewer 3

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Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

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.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 Response)

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

Reviewer #1: (No Response)

Reviewer #2: The authors correctly acknowledged all of my raised concenrs in the previous review round. Congratulation!

Reviewer #3: I would like to thank the authors for their efforts in revising the manuscript and for addressing many of the previous comments. However, there remain some important issues that need further clarification and refinement.

1. The inclusion of erythromycin and amoxicillin in the antimicrobial susceptibility testing requires further clarification. Although the authors justify their selection based on local antimicrobial usage and surveillance value, previous studies consistently demonstrate that these antibiotics are generally ineffective against Gram-negative bacteria such as E. coli, Salmonella, and Shigella due to intrinsic resistance. Therefore, the scientific rationale for testing these agents should be more clearly articulated. If resistance is expected to approach 100% based on well-established intrinsic mechanisms, the purpose and added value of reporting these resistance rates need to be explicitly explained.

The authors should also clearly state whether antimicrobial susceptibility testing was performed and interpreted according to internationally recognized standards, such as the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST), and specify the breakpoints applied for each organism–antibiotic combination.

If the inclusion of these antibiotics was intended to reflect local antimicrobial usage patterns (e.g., in veterinary practice), the manuscript should provide supporting data or appropriate references documenting such usage. Furthermore, it should be clarified whether intrinsically resistant agents were excluded from multidrug resistance (MDR) calculations to avoid potential overestimation of resistance levels.

Without these clarifications, the interpretation, scientific validity, and epidemiological relevance of the susceptibility data remain uncertain.

2. Discussion part

The Discussion section is currently largely descriptive and comparative in nature. Many of the interpretation follows the pattern of reporting prevalence values and comparing them with findings from other countries, followed by generalized explanations such as differences in hygiene practices. While such comparisons are useful, the section lacks sufficient analytical depth expected for publication in an international peer-reviewed journal.

The authors can give deep explanation about the results, for example:

- What biological or ecological factors could explain the unusually high prevalence?

- Could supply chain conditions contribute to the contamination?

- Does this suggest potential failure in cold chain management during transportation or storage?

- Whether the reported resistance (particularly to amoxicillin and erythromycin) reflects intrinsic resistance rather than acquired resistance.

- Whether there is evidence of antibiotic selection pressure in livestock production systems.

- The possible role of unregulated or excessive antimicrobial use in veterinary practice.

and many additional aspects that could be discussed based on your findings.

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). 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 For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..-->

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

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Antimicrobial-resistant pathogens on the plate: a semi-quantitative hygiene risk evaluation of raw beef consumption in Ethiopia within a One Health context

PONE-D-25-56353R2

Dear Dr. Gebeyehu,

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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Kind regards,

Guadalupe Virginia Nevárez-Moorillón, Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments: