PONE-D-22-23426Prevalence of Salmonella spp. and Escherichia coli in the feces of free-roaming wildlife throughout South KoreaPLOS ONE

Dear Dr. Yung Chul Park,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

ACADEMIC EDITOR:Please address all of the reviewers' comments.Take particular attention to  describing and explaining the association between wildlife, agricultural produce, and food poisoning of humans in South Korea. Describe also the risk factors that might impacted the contamination of produce by wildlife. 

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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: Partly

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

Reviewer #1: No

Reviewer #2: Yes

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.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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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: No

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: The manuscript did not thoroughly explain the association between wildlife, agricultural produce, and food poisoning in South Korea. The authors did not fully present the logic or reference whether the food poisoning in South Korea was caused by the consumption of agricultural produce which might be contaminated by wildlife feces. Statistical analysis was not explained comprehensively. This manuscript did not present in an intelligent fashion: more than ten sentences in the Introduction and Discussion sections require adding or changing references. The naming of the collection points was not consistent and a lot of typos and punctuation errors. It is recommended to use a professional copyediting service.

Line 50: References are not sufficient. The authors talked about wild animals but references 1-4 were about chickens, food animals, and humans.

Line 51: Add reference for humans.

Lines 51-53: The sentence is confusing. Is fecal-oral transmission the only transmission route? Salmonella can spread from animals to people and from people to people.

Lines 57-58: Is this for humans or is this include animals? Bai et al. (2016) reported non-O157 STEC.

Lines 59-60: Mogren et al (2018) discussed the hurdle approach to control pathogenic bacterial contamination of leafy green vegetables.

Line 62: High prevalence is not clear. It is suggested to elaborate on the prevalence and host ubiquity written by Kim et al. (2020)

Line 67: Add areas.

Lines 76-79: Do you have stats or references in South Korea?

Line 88: You also collected samples from Seoul and Busan cities.

Line 94: What kind of methods did you use for the sample size calculation? Is this for the detection of disease? What was the confidence level and the detection level?

Line 98: ‘More samples’ are not clear. Do you have comparison ratios among the seasons?

Line 101: The sentence is not clear. Jang et al. (2020) collected samples from Seoraksan National Park, not the whole area of your study.

Line 111: How did you check the size? Did you take pictures with a ruler on the sampling date?

Line 115: The study area from Lim et al. (2015) was the Odaesan National Park, not the whole area of your study.

Table 1: Seul city: it should be Seoul city; Gyong-buk-Bonghwa: This should be Bonghwa, Gyeongsangbuk-do

Line 125: The year should be 2015-2017.

Line 131: Hanm: this should be Hanam.

Line 132: Gyong-buk- Bonghwa: this should be Bonghwa, Gyeongsangbuk-do.

Line 201: FigS2 does not have A-B

Line 203: S2A-B Fig: this is FigS3 A-B

Line 240: S3Fig: this is FigS2.

Line 317: This sentence is not clear. Did you build two models for E.coli and STEC? Why did you conduct regression analysis? You did not explain the regression analysis in the Results or Discussion sections.

Line 335: Table 2: what is TSI?

Line 392: Table 3: you didn’t explain what Cov and Ident mean.

Line 394: Gyong-buk- Bonghwa: this should be Bonghwa, Gyeongsangbuk-do.

Line 406-407: You didn’t explain what NJ and ML mean.

Line 417: Fig 3 is about stx1, not stx1 + stx2

Line 472-474: Table 3 talks about BHG, BUS, CCG, HMS, SKG, and TBG.

Line 486-488: Is this about table 1? Did you talk about the statistical test in the Materials & Methods section: What kind of test did you use? There’s no explanation of this test in MM (lines 317-324).

Lines 489-490: This sentence is confusing. You can write that the percentage of positive samples was the highest in spring, followed by summer and fall.

Line 504: This is the S3 Table, not S2 Table. This sentence is confusing. Oct and Jul had the highest percentage and number of stx1 gene, but spring has the highest prevalence of stx1. You have to explain it.

Line 510: Fig 6. It is recommended to delete winter because you didn’t collect during winter. You need the number of samples per bar.

Line 520: Add references.

Line532: It is not clear why you add Ju et al. (2011).

Line 535: It will be better to add that Jay et al. (2007) collected samples from California.

Line 539-544: It is not clear what the authors try to say. Why did you add this sentence? Are you trying to say that deer feces is not important because of the low level of E.coli O157 from deer feces with no cross-contamination from infected livestock?

Line 560: Add references.

Lines 561-563: Why do you jump to GAP? Do Korean GAPs include control of wildlife? Do you want to discuss how Korean GAPs contribute to the improvement of food safety?

Lines 586-594: What is Fig FB? What does the high genetic diversity among Korean Salmonella strains mean?

Lines 604-606: You found STEC and Salmonella from wildlife feces, BUT you didn’t prove that wildlife samples are a source of fecal-borne pathogen dissemination. Is there any chance that the irrigation water contaminated with livestock feces transmits pathogenic bacteria?

Reviewer #2: S2A-B Fig. Please also use the italics style for bacterial designation and genes

line 237: β-lactum typos need to be corrected

272: serovars need to be given with an initial capital letter and not in italics

Please also check the references section for the use of the italics style for bacterial and gene designation

Nice work

Reviewer #3: Park et al. present a very large survey of foodborne bacteria presence in wildlife feces throughout S. Korea. This is a valuable sample set with the potential for impactful conclusions to be made regarding produce safety and sampling. Unfortunately, the rich ecological data collected is not presented in a meaningful way. Rather than simply giving prevalence for different cities, I'd like to see if patterns were detected regarding proximity of the positive specimens to the produce - were they at the edges of the field? Or interspersed with the plantings? Near water sources? Also, what is more problematic are the inferences about diversity based on partial gene Sanger sequencing. This is totally antiquated and not scientifically justified. Phylogeography and source tracking of bacteria should be based on whole genome sequencing. This is widely commercially available now for not much more than the cost of PCR.

Data availability: The indicated NCBI numbers are not showing up when I search for them.

Line 117: Were all samples confirmed with the cameras? If not, what proportion?

Line 93: On what assumptions was the sample size calculation based on?

Table 1: Clarify what numbers are given in the "No. of Salmonella detected sample" column

Line 147: Colony morphology is not a confirmatory test

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Prevalence of Salmonella spp. and Escherichia coli in the feces of free-roaming wildlife throughout South Korea

PONE-D-22-23426R1

Dear Dr. Park,

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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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,

Csaba Varga, DVM MSc PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for addressing all of the comments!

I have carefully reviewed the manuscript and the authors addressed all of the peer-reviewers' suggestions. Reviewer 3 rejected the manuscript, however the  whole-genome sequencing analysis that she suggested should be a future research direction and not a requirement for publication of this current study.

The article as currently stands qualifies for publication based on PLOS ONE publication criteria.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

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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 #2: Yes

Reviewer #3: No

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

Reviewer #2: N/A

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

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

Reviewer #2: Yes

Reviewer #3: No

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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 #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

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

Reviewer #2: The revision have been carefully addressed. There are no further comments of this reviewer to improve the quality of the manuscript.

Reviewer #3: I am not satisfied with the revisions, in particular for my comment on use of partial gene sequence for Salmonella typing, which was essentially ignored. The paper is still quite disorganized.

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

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

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

Reviewer #2: No

Reviewer #3: No

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