PONE-D-23-44006Phylogenetic diversity and virulence gene characteristics of Escherichia coli from pork and patients with urinary tract infections in ThailandPLOS ONE

Dear Dr. Suzuki,

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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Kwame Kumi Asare, Ph.D

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PLOS ONE

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“This work was supported, in part, by a grant from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, for the Joint Research Program of the Hokkaido University Research Center for Zoonosis Control toY.S., and, in part, by Japan Agency for Medical Research and Development (AMED) under Grant Numbers JP20jk0210005, JP20jm0110021, and JP20wm0125008 to Y.S.”

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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: Yes

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Partly

Reviewer #5: No

Reviewer #6: Partly

Reviewer #7: Yes

Reviewer #8: No

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: 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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: 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

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: 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 introduction should provide more context and background information on the significance of studying the transmission of virulence genes between pork and urinary pathogenic E. coli strains.

-In materials and methods, Were there any specific criteria used for selecting pork samples and UTI patient samples for inclusion in the study?

-The utilization of UPGMA clustering for analyzing the presence of virulence-associated genes and phylogroups is appropriate. However, the manuscript lacks clarity regarding the rationale for selecting this specific clustering method. Were alternative methods considered, and if so, why were they deemed less suitable? Additionally, the authors should provide more details regarding the interpretation of the dendrogram generated from the clustering analysis to aid readers in understanding the relationships between strains.

-We suggest that you refer to new research article doi: 10.3389/fcimb.2022.790184. and doi: 10.1007/s11033-023-09031-x in the discussion section.

Reviewer #2: Dear Authors,

I would like to say this study is very neat and has informative data that will be of interest for international readers of the journal.

Before publication, I recommend following my comment below:

Line 60: This paragraph provide some information about different E. coli pathotypes, I would suggest to use more references and not only one. I suggest to use this study: 10.30466/vrf.2022.550618.3416 as the second references for this paragraph.

Reviewer #3: Comments

*What were the aim and conclusion of your study, exactly? It is not clear to me the aim of the study on sharing virulence genes between PEC and UPEC. Did you aim to demonstrate that pork may serve as one possible source of UPEC? If so, how did you demonstrate that in the present work?

[Cluster analysis … demonstrated that pork contaminated with E. coli harboring UPEC virulence genes may serve as one possible source of UPEC in humans],[This finding may imply…. UPEC strains in this study were closely related to PEC]. [Our study highlights that PEC carrying UPEC virulence-associated genes may………further transmit to humans causing UTIs].

* Genotyping of isolates is needed to obtain accurate data and true prevalence. It is possible that your strains originated from a clone.

*[A total of 330 E. coli strains was obtained from the preserved stock culture collection]:

did you have exactly the same number of PEC and UPEC in your collection or you include 165 isolates (each group) from this collection in your study?

*What was your definition for UPEC?

*Grammatical errors and writing issues were seen in the manuscript.

*For abbreviations, provide their full term for the first mention.

*Information on the isolates is not provided. How were urines collected? What was the demographic data of patients?

* FimH is not only for UPEC. It is found in other E. coli types and in E. coli belonging to normal flora.

*Which tests were used to identify E. coli? Mention them.

*Did you test the antibiotic susceptibility of isolates?

*Some references such as reference 1 were referred to in the manuscript too much. Use other references instead of referring to 1.

*Figure titles need to be revised.

*Remove Table 1 and embed its data within the text

*Table 2: number of isolates is not enough to make a p-value, in some rows.

Best

Reviewer #4: Major concerns:

Please include the criteria used to characterize strains as UPEC, PEC and DEC in the Methods. A description of the approach is given in the Introduction (Lines 55 and 110), but the specific genomic markers used to make the determination should be listed. This impacts the finding on lines 184-185 that “165 isolates PEC and 165 ones UPEC” The collection criteria stated on lines 117-124 only select for an isolation source and make no distinction between UPEC, E.coli commensal or environmental contaminant. If the authors were able to subset the UPEC strains from this dataset, please provide the selection criteria. Otherwise, it would be more accurate to claim that the 330 isolates clustered with at least eight different groups of isolates of UPEC and PEC source. Further, were any tests performed to determine if isolates E1-165 were the causative agents of infection? If not, then additional justification is needed to categorize them as UPEC.

Reviewer #5: Firstly, I would like to thank you for the opportunity and trust in my work to review this scientific article. Below, I provide some comments:

Line 124:

I believe it would be interesting to describe the biochemical tests for E. coli characterization. Was there also molecular confirmation of the species?

The dendrogram image is of low quality, and it was not possible to analyze it.

Although there is a phylogenetic analysis in the paper (represented by the dendrogram that could not be analyzed), I believe that epidemiological data from the samples obtained would be necessary for the inferences made.

The work is interesting, but it should focus on the actual evidence obtained. The samples were obtained from a previous isolate bank. For the conclusions reached, it would be necessary to analyze epidemiological data that were not presented.

I suggest that epidemiological data be presented or that the conclusions and discussions obtained be reviewed.

I also suggest that a higher quality image of the dendrogram be provided.

Reviewer #6: Comment to the authors

Abstract

Have only the specific genes of UPEC been examined? The text raises this suspicion. If not, please change the text of the first lines in the abstract.

Line 36 – 40: The percentage of the genes in the UPEC is higher than in PEC, so it is better to present its name and its percentage first.

The conclusion of the abstract section is not based on the results and should be changed

Introduction

Line 56-59: First introduce EXPEC more. Mention and explain the virulence genes or pathogenic factors of this bacterium that cause urinary infections.

Line 67: Please write in full version for “STEC” for the first time.

Line 76: State the pathotypes of EXPEC here with more details along with its pathogenic genes.

Line 107-108: Please remove this sentence as a novelty, the authors have mentioned it several times in the text.

Material and method

More data should be provided on the sources of isolation of these bacteria, especially on human samples.

Does the study have a code of ethics, mention it.

The bootstrap value should be stated at the level of phylogenetic analysis.

How many times the same branch is observed when repeating the generation of a phylogenetic tree on a resampled set of data? The percentage of times each interior branch is given a value of 1 should be noted.

Reviewer #7: The paper entitled “Phylogenetic diversity and virulence gene characteristics of Escherichia coli from pork and patients with urinary tract infections in Thailand” has good and important subject, good writing, and interesting results. However some comments are as follow:

The phylogenetic method designed by Clermont et al has different protocol. Why did the authors change the PCR protocol?

In material and method, the authors described that the cluster analysis was performed according to phtlogenetic group and absence or presence of virulence genes, while in the fig 3. the strains with similarity profiles put in different clusters. I think the clustering is drawn according to phylogenetic groups only.

Page 23 Line 293. What is the reason for this difference?

Page 24 Lines 318 -319 are repeated.

Page 24 Lines 347-348 “Females have a higher risk of developing these infections due to the short distance between the rectum and the urethra” What is the connection between this sentence and the previous sentences?

Reviewer #8: The present manuscript evaluated the diversity of phylogenetic groups and virulence genes of E. coli isolates from pork (PEC) and human patients with urinary tract infection (UPEC) in Thailand. The objectives of the work were to compare the virulence genotype of UPEC and PEC. The authors reported that the PEC strains were grouped predominantly in groups A and B1 (commensal strains), while the UPEC strains, which carry a greater number of virulence genes, were classified in phylogroups B2 and C. In conclusion, they pointed to pork as a reservoir of genes associated with UTI, reporting a major public health concern.

The paper has some important limitations that prevent it from being accepted in this format. The main ones are:

1- There is no coherence between the results and the conclusions. How can we say that this is a major public health risk if the profile of the strains in the two groups was different? How can the risk be measured? It would be more appropriate to use more modern analysis tools such as sequencing types or dendrograms generated by comparing WGS data. The frequency of genes between the two groups is important information, but on its own it doesn't serve to support the hypothesis or measure the risk.

2- The Clermont classification used is outdated. Currently, 9 phylogroups are considered. See Clermont et al. (2019) - Group G.

3- There is no hybrid of DAEC and UPEC, as the fimH gene is not a UPEC marker. On the contrary, it is a highly prevalent fimbria in enterobacteria that can be detected in non-pathogenic strains of the microbiota. This information needs to be reviewed and is considered a lack of definition of the concept of hybrid strains.

4- The statistics between the frequency of virulence genes and phylogroups is completely unnecessary information, given that the concept of phylogroups is based on the evolutionary process of acquiring virulence genes in the B2 group. So this result is to be expected and has no significance whatsoever in light of the proposal.

5- The results of group C are of local epidemiological data, bute the importance should be understood in more in-depth studies in Thailand.

So, I think this paper needs to be modified prior the publication.

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Reviewer #1: Yes: Mehrdad Halaji

Reviewer #2: Yes: Mahdi Askari Badouei

Reviewer #3: No

Reviewer #4: Yes: Hami Kaboosi

Reviewer #5: No

Reviewer #6: No

Reviewer #7: No

Reviewer #8: Yes: Terezinha Knöbl

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PONE-D-23-44006R1Phylogenetic diversity and virulence gene characteristics of Escherichia coli from pork and patients with urinary tract infections in ThailandPLOS ONE

Dear Dr. Suzuki,

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 Jun 24 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,

Kwame Kumi Asare, Ph.D

Academic Editor

PLOS ONE

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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 #2: All comments have been addressed

Reviewer #3: (No Response)

Reviewer #4: All comments have been addressed

Reviewer #6: (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 #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Yes

Reviewer #6: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: N/A

Reviewer #6: 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 #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #6: 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 #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #6: 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: This article is accepted in this form

This article is accepted in this form

This article is accepted in this form

Reviewer #2: The authors addressed all the questions properly, and I would like to recommend for acceptance of this article.

Reviewer #3: The authors made an extensive effort to improve the manuscript. There are still some ambiguities.

*Sharing virulence genes between E. coli groups is important data, but it can’t support your hypothesis. The dendrogram and cluster analysis of virulence genes also can’t be used to support the aim of the present study. Therefore, please change and revise the corresponding lines.

[Comment 1: What were the aim and conclusion of your study, exactly? ….…. Response 1 : Yes, this study aimed to demonstrate that pork may serve as one possible source of UPEC in humans. The presence of virulence genes in pork strains in this study may imply that these strains may serve as a possible source of UPEC in humans. Moreover, the results from cluster analysis showed that some strains of pork and urine samples were clustered in the same branch of the tree. However, a molecular epidemiology study of these strains will be confirmed in further study.]

*Some simpler and cheaper methods than WGS could be used to analyze the relationships between the isolates. Furthermore, the genotyping may help to support the hypothesis that :pork may serve as one possible source of UPEC in humans

*Please add more references to the first paragraph in Introduction: lines 51-59.[Comment 11: Some references were referred to in the manuscript too much. Use other references instead of referring to 1. Response 11: The other references were used instead of using only reference 1].

Reviewer #4: The authors have responded to the reviewer queries adequately and implemented the changes that have improved the accuracy and readability of the manuscript. I am satisfied with the changes implemented by the authors in this revised manuscript. However there are some more needs to revise the manuscript. First, Fig. 2 should be transferred to supplementary file. Second, in dendrogram it is unknown that what is the tolerance and optimization rate in analysis. There are some strains whose patterns are the same with 100 % similarity. But the dendrogram clustered them into different patterns. Like strains U20 and U19, also strains U21 and U22. Or some bands have been missed between isolates like strains U16 and U35 between P39 and P30 (U30). It seems that the authors should analyze the bands again with new tolerance and less sensitivity. So that after analysis these strains will got into the same pattern. After re-analysis the dendrogram all the text should be revised based on the results.

Reviewer #6: At first, the authors did not adequately answer some questions. For example, one of the questions was why the researchers used the method of UPGMA clustering for cluster analysis and why the other clustering methods were not suitable. They suggested that due to the limited facilities, it was not possible to perform other tests such as a complete genome examination, which was a wrong answer to this question. In fact, they should have explained other clustering methods, and why they did not use those methods. Another example is my question “Have only the specific genes of UPEC been examined?” they didn’t answer it correctly. They stated that the purpose of their study was to examine virulence genes related to UPEC isolates, but they have other types of E. coli isolates. In this section, writing should have been done in such a way that the importance of all isolate types was also shown.

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Reviewer #1: No

Reviewer #2: Yes: Mahdi Askari Badouei

Reviewer #3: Yes: Safoura Derakhshan

Reviewer #4: Yes: Hami Kaboosi

Reviewer #6: No

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Phylogenetic diversity and virulence gene characteristics of Escherichia coli from pork and patients with urinary tract infections in Thailand

PONE-D-23-44006R2

Dear Dr. Suzuki,

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. If you have any questions relating to publication charges, 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,

Kwame Kumi Asare, Ph.D

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 #2: All comments have been addressed

Reviewer #4: 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 #2: Yes

Reviewer #4: Yes

**********

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

Reviewer #2: Yes

Reviewer #4: 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 #2: Yes

Reviewer #4: 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 #2: Yes

Reviewer #4: 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 #2: (No Response)

Reviewer #4: The authors have responded appropriately to comments and incorporated feedback into the revised, and improved version of the manuscript.

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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 #4: Yes: Hami Kaboosi

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