PONE-D-22-27001Molecular epidemiology and antimicrobial susceptibility of diarrheagenic Escherichia coli isolated from children under-five with diarrhea and healthy controls in Central EthiopiaPLOS ONE

Dear Dr. Zelelie,

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Reviewers' comments:

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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: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

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Reviewer #1: TITLE:

The title is misleading as it indicates that healthy controls were used; however, the methods imply that children without diarrhea were enrolled, without referring to their health status. Having no diarrhea is not indicative of sound health, as all the children enrolled into the study were seeking care. So, the title needs to be revised to depict exactly what the study was about in context of children’s health.

ABSTRACT:

The abstract indicates;

- That bacterial identification, antibiotic sensitivity testing, and pathotype determination were determined with PCR however, this is not correct especially for bacterial identification and antibiotic sensitivity testing that were phenotypically determined. It’s only DEC pathotypes that were molecularly determined. Revise.

- That 476 specimens were analyzed; were this one specimen per child? How many specimens from each child were analyzed? Revise.

- Results for all the children are grouped together, yet as per the title and/or methods, the study seems to have subgroups i.e., healthy children / children without diarrhea vs. sick children / children with diarrhea. It would be proper to first report findings per subgroup.

- Percentage figures with and without corresponding fractions; it would be better to consistently use percentages with their corresponding fractions.

- Change ‘The overall prevalence rate -------’ to ‘The overall prevalence -------’

INTRODUCTION:

In the second paragraph, revise,

- ‘Diarrheagenic E. coli (DEC) is the major causes of diarrhea -----’ to ‘Diarrheagenic E. coli (DEC) is the major cause of diarrhea -----’

- ‘EPEC is an attaching and effacing (A/E) pathogens -----’ to ‘EPEC is an attaching and effacing (A/E) pathogen -----’

- The acronyms ‘tEPEC’ and ‘aEPEC’ are not defined at their first use

MATERIALS AND METHODS:

- In the section ‘Study area and participants’ nowhere is it mentioned that healthy control children were enrolled as depicted in the title; revise.

- If healthy control children were indeed recruited, the specimen collection procedure described is for only sick children; could you describe the specimen collection procedure for healthy children?

- ESBLs, and so are several acronyms in this manuscript, are introduced without definition at first use.

- As well, ESBLs and carbapenemases are introduced without context and/or rationale, and the procedure for their detection is inadequate.

RESULTS:

- Review presentation for the results of the ESBL- and carbapenemase producing isolates; it would be less confusing if the data for ESBL- and carbapenemase- producing isolates are presented first before showing comparisons with DEC and antibiotic sensitivity data.

- Nothing is mentioned about carbapenemase producing isolates yet the methods are described. Revised.

Reviewer #2: Reviewer’s report

Molecular epidemiology and antimicrobial susceptibility of diarrheagenic Escherichia coli isolated from children under-five with diarrhea and healthy controls in Central Ethiopia by Zenebe et al.

The research article of Zenebe et al. described “Molecular epidemiology and antimicrobial susceptibility of diarrheagenic Escherichia coli isolated from children under-five with diarrhea and healthy controls in Central Ethiopia”. Moderately, the research work was well executed and the manuscript was well written. However, there are some fundamental observations that I have on the way data were presented and interpreted in the manuscript. There are lots of errors, misinterpretations, and misinformation of data in the article. In addition, there are lots of grammatical/typographical errors in the manuscript that require serious overhauling. My comments and observations are as listed below:

Major Comments:-

1. Under the Methodology section, sub-section: “Detection of DEC virulence genes” on page 9: The author gave only one PCR condition for the three different multiplex PCR done. Is the same PCR condition used for the three multiplex reactions? Is the annealing temperature of 60 oC used for the PCR processes all through the experiments? The author needs to clarify this and probably give additional information on the process.

2. Under the Result section, sub-section: “Epidemiology of DEC and associated factors” on page 12:

i. The statement – “Under-five children who visited a health-care facility during winter had a less likelihood…” seems to be ambiguous. Is the author saying that visitation of clinics during the summer periods brings about higher infection with the DEC compared to the winter, as if it is hospital-acquired? Or, is the author trying to say that there is a higher infection rate in the summer than in the winter thereby resulting in many children visiting the clinic due to DEC infections?

ii. The statement – “Children who lived in a family with a monthly income of below $36

were more likely to acquire DEC (AOR=2.364, CI=1.354, 4.126) compared…” seems to be misrepresenting the data presented in Table 2. Family income range of $37-115(middle) actually has a higher number of children positive for DEC (74) compared to 61 recorded for family income of <$36 (low).

iii. The statement – “Children who were cared for by their mother had a lower risk of DEC (AOR=0.423, CI=0.211, 0.846) compared…” Like above, the statement seems not tally with the value presented in Table 2 where a higher number of children taken care of by their mother (98/183, 53.6%) actually came down with DEC infections.

iv. The statement – “Under-five children who began their supplementary food before 6 months old were more likely to acquire DEC (AOR=3.660, CI=1.322, 10.128) than those….” Same as above, the misinterpretation of actual data as expressed in Table 2. Children who began supplementary food at 6-12months have a higher incidence of DEC infections of 150 (82.0%).

v. The statement – “Children that lived in a compound with domestic animals had 2 times the likelihood (AOR=1.555, 1.016, 2.381) of being positive for DEC…” Same as above, misinterpretation of actual data expressed in Table 2. Children who were not having contact with domestic animals showed higher cases of DEC infections, precisely 114 (62.3%) compared to 69 (37.7%) observed for children that lived in a compound with domestic animals.

It could be observed that the author wrote this last statement correctly based on the data expressed in Table 2: “Children whose family obtained water in shift had a greater change of acquiring DEC (AOR=1.735, OR=1.046, 2.879) compared to those with a continuous daily supply of water.” I wondered why all other ones highlighted above were done differently as if the author interpreted the data with premeditated notions.

It could also be noted that the author used these wrongly expressed results to discuss findings from this research work under the Discussion section.

3. Under the Result section, sub-section: “Antimicrobial resistance (AMR) profile” on page 15 and Table 4 on page 16:

It could be observed that data for STEC and DEAE are conspicuously missing in Table 4, whereas, their values were included in the n=183 that was used to arrive at the values presented in the table. Likewise, the correctness of the values presented in the table should be properly checked. For example, why is the author not specific about the number of DEC isolates that were resistant to ampicillin by writing ≥85% on Line 1 under the sub-section?

Also in Table 4 EAEC = 76, whereas, if values for EAEC are summed up in Table 3, it is equal to 86. It seems there are lots of errors in the data presented in the manuscript.

Other Comments:

ABSTRACT SECTION

Under methods, last line, page 2: Consider revising the statement “Bacterial identification, antimicrobial susceptibility, and pathotype determination using polymerase chain reaction (PCR) were evaluated” to “Bacterial isolation, identification, antimicrobial susceptibility, and pathotype determination using polymerase chain reaction (PCR) were done”.

Under results, line 7, page 2: delete “of” before “non-diarrheic”

INTRODUCTION SECTION

Paragraph 2, Last sentence, page 4: The statement: “Perhaps state that recently mixed pathotypes have been identified that have virulence factors associated with more than one pathotype.” It seems something is missing in the statement or it needs to be revised to make better meaning. Also, I think cited reference “(7)” is missing there too.

Paragraph 3, Line 5-7, page 4: Same as above, the statement: “DEC pathotypes cause different outbreaks include in Germany in 2011(15) and Japan in 2016 (16), a haemolytic uremic syndrome caused by STEC, and in Nottingham, UK in 2014 due to EIEC (17)” needs revision.

MATERIALS AND METHODS SECTION

Sub-section: Specimen collection- third to the last line on page 7, add “isolation” after bacterial”

Sub-section: Bacterial Identifications on page 7 should be corrected to Bacterial Isolation and Identifications

Sub-section: Detection of DEC virulence genes, paragraph 2, line 2, page 8: the statement “Briefly, detection of DEC virulence genes were performed…” Should be corrected as: "Briefly, the detection of DEC virulence genes was performed..."

Sub-section: Detection of DEC virulence genes, paragraph 2, line 3, page 8: “…using the primer oligonucleotide DNA sequences…” should be corrected as “…using the primer sequences…”

Sub-section: Detection of DEC virulence genes, paragraph 2, last statement, page 9: the statement: “Known and whole genome sequenced positive DEC strains were used during optimization of the PCR assay.” is not clear.

DISCUSSION SECTION

Paragraph 1, Line 1, page 17: The statement: In Ethiopia, molecular epidemiology of DEC and its resistance profile is unknown” sounds to be outrageous. What about works done by Getaneh et al., 2021; Belete et al., 2022 and so on? Even the author in his systematic review article (Zenebe et al., 2020) gave some insights into the occurrence of DEC in children in Ethiopia. The authors have even failed to give credit to any of the previous works done on E. coli/DEC in Ethiopia, maybe so as to prove the assertion made here. If so, it is grossly unacceptable/unscientific.

Paragraph 5, Line 6, page 19:

The statement: “EPEC classified in to typical…” should be revised as “EPEC are classified into typical...”

Paragraph 7, Line 3-5, page 20: The statement: “In the present study children who visited a

health-care facility during winter were less likely to be positive for DEC (table 2) compared to

children who visited during summer.” Should be revised to make an intended meaning.

Paragraph 7, Line 6, page 20: “Mexco” should be corrected

Paragraph 7, Line 7/8, page 20: The statement “It has been reported that bacterial caused diarrhea….” Could be written as “It has been reported that bacterial-caused diarrhea…” Same thing with “…viral caused diarrhea…” in Line 8, could be written as “…viral-caused diarrhea…”

Paragraph 7, Line 11, page 20: “protective” should be corrected as “protected”

Paragraph 8, Line 12-14, page 21: What is the significance of the statement “DEC pathotypes isolated from diarrheic children showed significantly higher resistance to ceftazidime and cefotaxime compared to those from non-diarrheic children (p=0.010).”? It should be stated.

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Reviewer #1: Yes: David Patrick Kateete

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Reviewers report.docx

PONE-D-22-27001R1Molecular epidemiology and antimicrobial susceptibility of diarrheagenic Escherichia coli isolated from children under-five with and without diarrhea in Central EthiopiaPLOS ONE

Dear Dr. Zelelie,

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.

==============================

A thorough revision of the provided methodology, results presentation and interpretation is desired. The points raised by the esteemed reviewer are indicative. Authors would like to check the manuscript thoroughly and amend it accordingly.

==============================

Please submit your revised manuscript by Feb 16 2023 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Samer Singh, Ph.D.

Academic Editor

PLOS ONE

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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 :

The authors should pay close attention to the provided methodology details, results and interpretation in addition to that indicated by the reviewer. A thoroughly corrected version is desired.

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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: (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

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

Reviewer #1: Yes

Reviewer #2: 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

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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

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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 #1: The authors have addressed all the concerns raised in the original submission. I recommend publication of the current version.

Reviewer #2: Reviewer’s report on the Revised Manuscript: PONE-D-22-27001R1

Molecular epidemiology and antimicrobial susceptibility of diarrheagenic Escherichia coli isolated from children under-five with diarrhea and healthy controls in Central Ethiopia by Zenebe et al.

There is no doubt that the quality of the manuscript has increased considerably following the revisions made by the authors. However, I have some minor observations, comments, and inputs.

1. I would like to use one of my initial comments and the authors’ response to point out my observation below:

“Comments: v. The statement – “Children that lived in a compound with domestic animals had 2 times the likelihood (AOR=1.555, 1.016, 2.381) of being positive for DEC…” Same as above, misinterpretation of actual data expressed in Table 2. Children who were not having contact with domestic animals showed higher cases of DEC infections, precisely 114 (62.3%) compared to 69 (37.7%) observed for children that lived in a compound with domestic animals.

Responses:

• The odd for DEC positive with animal is (69/156=0.442); the odd for DEC positive with no animals is (114/137=0.832). The lower the odd is more likely to occur. The odd ratio was computed with logistic regression.”

My observation/question is, what is the relevance of Table 3 if the data presented in it does not give direct support to the information expressed in the manuscript except an additional calculation is done by the reader of the manuscript in order to understand claims by the authors? For example, in the above comment and response, the authors have to manually calculate another set of odd values (69/156=0.442 and 114/137=0.832) to explain their point that “Children that lived in a compound with domestic animals had 2 times the likelihood (AOR=1.555, 1.016, 2.381) of being positive for DEC…” I think the authors should be able to use the data expressed in the table to write their results and discuss them in a manner that ordinary readers could understand without having to pick calculator to start doing additional calculations.

2. Under the Introduction Section:

i. On Page 4, the revised last statement in paragraph 2, “Recently mixed pathotypes have been identified that have virulence factors associated with more than one pathotype (7).” I am surprised the citation (7) is dated 2012 whereas the authors made the statement “Recently…|”

ii. On Page 5, paragraph 4, line 4, I guessed the word “systemic” is supposed to be “systematic”. The authors should check it up. Also, the word: “presences” should be corrected to “presence” in the last statement of the same paragraph

3. Under the Methodology Section:

i. Sub-section: Phenotypic detection of ESBLs and Carbapenemases production, line 7, page 8. The statement “Briefly, 1μL loopful colony of test isolate from overnight blood agar plate suspended…” should be revised. I can’t figure out how a 1μL loopful colony of test isolate could be taken from an agar medium.

ii. Sub-section: Detection of DEC virulence genes, second to the last statement on page 10. The statement: “Previously used positive known DEC strains (whole genome sequenced) were used for the optimization of the PCR assay.” should be revised for better meaning. Previously used positive strains by who?

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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: David Patrick Kateete

Reviewer #2: Yes: Kayode Olayinka Afolabi

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Attachments

Attachment

Submitted filename: Reviewers Report 2.docx

PONE-D-22-27001R2Molecular epidemiology and antimicrobial susceptibility of diarrheagenic Escherichia coli isolated from children under-five with and without diarrhea in Central EthiopiaPLOS ONE

Dear Dr. Zelelie,

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 12 2023 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,

Samer Singh, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments:

The manuscript is improved from the last version but still rife with deficiencies, as indicated by a reviewer. The decision of major revision is just to indicate gravity of the minor issues that still needs to be fixed. The points raised by the learned reviewer are indicative; a thorough revision of the manuscript is desired. The authors would like to pay attention to the precise presentation of facts and methodology. Catalog no. of chemicals and required details/ permissions as expected for the study be indicated or provided. The inclusion of relevant details with clarifying statements would tremendously improve its quality.

For language improvement, which could be partially contributing to the indicated issues, the help of native speakers may be sought by the authors.

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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 #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: (No Response)

Reviewer #3: Partly

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

Reviewer #2: (No Response)

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: (No Response)

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 #2: (No Response)

Reviewer #3: No

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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: (No Response)

Reviewer #3: Major Comments:

The present study was carried out in Ethiopia and showed the prevalence of DEC pathotypes in children under the age of five years. The authors were trying to find out and reported the prevalence of different strains of DEC pathotypes such as EAEC, ETEC, EIEC, and some hybrid strains also. The authors reported the prevalence of MDR bacteria and ESBL pathogens.

1. Diarrheagenic Escherichia coli (DEC) Pl. italicized the Escherichia coli.

2. Diarrheagenic Escherichia coli (DEC) is the major cause of diarrhea in under-five children. Pl. revised the sentences carefully.

3. The present study aims to determine molecular epidemiology and antimicrobial resistance profiles of DEC and identify contributing factors for acquisition among under-five children in Central Ethiopia. What is the mean of under-five children? An author should write it like children under the age of five.

4. Ethiopia is one of the 15 pneumonia and diarrhea are high-burden countries. The author should Pl. revised the sentence.

5. A systemic review and meta-analysis study done in Ethiopia reported a 22% pooled prevalence of diarrhea among under-five children. What do you mean? The author should kindly revise the sentence.

6. A systemic review and meta-analysis study done in Ethiopia reported a 22% pooled prevalence of diarrhea among under-five children. What are under five children? The author should rephrase it.

7. Specific combinations of virulence traits group DEC into six common pathotypes, including enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shiga-toxin producing E. coli (STEC)/enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and diffusely adherent E. coli (DAEC). The following sentence is not clear. Pl. revised the sentence.

8. EPEC is an attaching and effacing (A/E) pathogen known by the A/E lesion formation. Pl. revised the sentence.

9. Based on the E. coli adherence factor (EAF) plasmid (pEAF) encodes bundle-forming pili (BFP). Pl. revise the sentence properly.

10. EPEC are classified into typical EPEC (tEPEC) (presence of BFP) and atypical EPEC (aEPEC). What do you mean? Pl. revised the manuscript.

11. STEC causes mild to bloody diarrhea, foodborne outbreaks, and severe illness, including hemorrhagic colitis and hemolytic uremic syndrome, and is known by its potent toxin production, Shiga toxin 1 or 2 genes (stx1, stx2). The author should rephrase the sentence and make it clear and comprehensible.

12. Transcription activator (virF). What is the role of virF?

13. Hybrid DEC pathotype strains are increasingly reported elsewhere in the world. What does it mean? Is it elsewhere???

14. These include EPEC/ETEC in India [10], STEC/EAEC in Germany [11] and Indonesia [12], EPEC/STEC [13] and EPEC/EAEC [14] in Brazil, and STEC/ETEC in Sweden [15]. What does it mean? Is EPEC strain also contain ETEC virulence factor?

15. Outbreaks of diarrheal diseases due to different DEC pathotypes had been reported in different countries including Germany in 2011[16], Japan in 2016 [17], and Nottingham, UK in 2014 [18]. Are any recent outbreaks of diarrhea due to DEC pathotypes like in 2018-2022?

16. Among MDR strains extended spectrum ß-lactamase (ESBLs) and carbapenemase-producing DEC pathotypes are emerging globally (20). Typological error. Pl. correct it.

17. The occurrence of hybrid DEC [10-13], outbreak-causing DEC [16-18], and resistant DEC [20, 21] reported so far showed the possible emergence of hyper-virulent DEC strains anywhere to cause severe diseases or outbreaks. The author should Pl. revised the sentence and make it easy to read and understand.

18. Epidemiological data of DEC will assist public health personnel in identifying and tracking bacterial outbreaks and severe diarrheal diseases. Thus, understanding the distribution pattern of enteric pathogens within the community will help control or prevent fatal outbreaks. Authors suddenly jumped from different strains of DAE to epidemiological factors.

19. The author should submit the structured questionnaire as the supplementary file which was used for the study.

20. How Author has prevented the contamination of urine during stool sample collection?

21. Which type of sample container was used for the stool sample collection? Pl. describe.

22. The author should briefly describe how aseptic conditions were maintained during stool sample collection.

23. A large number of typological errors were present throughout the manuscript.

24. How stool samples were inoculated on MacConkey agar?

25. Stool samples were diluted or directly streaked on MacConkey agar?

26. The author should briefly describe the inoculation of stool samples on MacConkey agar.

27. 16% glycerol stock was used?

28. Why author should store the E. coli isolates in Brain Heart Infusion agar? Generally, this agar is used for the cultivation of streptococci, meningococci, etc.

29. Ampicillin concentration was 10mcg?

30. Imipenem concentration was 30 mcg?

31. MDR was defined when the isolate was nonsusceptible to at least one agent among three or more antimicrobial categories. Is this the definition of MDR bacteria? The author should refer to the following paper for reference.

a. Singh AK, Das S, Kumar S, Gajamer VR, Najar IN, Lepcha YD, Tiwari HK, Singh S. Distribution of antibiotic-resistant Enterobacteriaceae pathogens in potable spring water of eastern Indian Himalayas: emphasis on virulence gene and antibiotic resistance genes in Escherichia coli. Frontiers in Microbiology. 2020 Nov 5;11:581072.

b. Singh AK, Das S, Singh S, Gajamer VR, Pradhan N, Lepcha YD, Tiwari HK. Prevalence of antibiotic resistance in commensal Escherichia coli among the children in rural hill communities of Northeast India. PloS one. 2018 Jun 18;13(6):e0199179.

32. What is the positive and negative control used for the Antibiotic Susceptibility test/

33. What are the inclusion and exclusion criteria used for the present study?

34. What are the basic criteria used for the selection of antibiotics?

35. by using a 1μL size loop? What is the standard size of the inoculation loop? Is it 1 μL?

36. A confirmatory test for ESBLs activity was conducted for isolates resistant to cefotaxime (30μg) and ceftazidime (30μg) by the combination disk method. The author should briefly describe the screening test method and antibiotics used in the confirmatory test.

37. E. coli resistance to ceftazidime and cefotaxime were considered ESBL producers.

38. What is the role of clavulanic acid during ESBL detection?

39. Without using clavulanic acid author confirmed the ESBL pathogens.

40. Subsequently, the meropenem disks were removed from the broth using an inoculaton loop. Inoculaton????

41. DEC strains positive for the relevant virulence gene (s) stored in the Armauer Hansen Research Institute from previous works and CCUD (Culture Collection University of Gothenburg) 24T E. coli were used as the positive and negative control, respectively. What is 24T E. coli? The author should describe what is the positive and negative control.

42. An author should briefly and separately describe the number of samples collected both diarrheal as well as non-diarrheal during the dry season and rainy season.

43. All 428 E. coli isolates were tested by PCR, of which 183 (42.8%, 183/428) were positive for DEC pathotype. The overall prevalence of DEC in the present study was 38.4% (183/476), and 58.5% (107/183) of the DEC types were isolated from Addis Ababa and the remaining from Debre Berhan. The line shows contradictory results to each other. The overall prevalence of DEC in the present study was 42.8% but again in the second line author mentioned that the overall prevalence of DEC type is 38.4%. The author should briefly revise the sentence.

44. The overall prevalence of DEC in the present study was 38.4% (183/476), and 58.5% (107/183) of the DEC types were isolated from Addis Ababa and the remaining from Debre Berhan (Table 3, Fig1 a & b). The current result and table 3 data were not clear.

45. In Table 3 what are the total cases meaning? And what is total control?

46. Dry season? What is the meaning of the dry season? The author did not mention any data on the dry season in table 2.

47. Under-five children were more likely to acquire DEC during the rainy season compared to the dry season (AOR=0.529, CI= 0.335; 0.835) (Table 2). In table 2 author mentioned the data of summer, Winter, and Spring whereas in the manuscript it was written as dry season and rainy season as well as some places' rainy season. The author should write it properly in the either rainy and dry seasons and spring, summer, and winter in tabl2 as well as the manuscript write-up.

48. The data of table 2 shows that the most prevalent season for DEC was winter (45.9%) followed by summer (32.2%) and spring (21.9%). Kindly check it.

49. Resistance to ceftazidime and cefotaxime was higher in DEC isolates obtained from diarrheic compared to non-diarrheic children (p=0.010). All the STEC and DAEC, 15% ETEC (6/39), and 22% EAEC (17/76) were resistant to ciprofloxacin. Kindly rephrase the sentence properly.

50. ETEC (5%, 2/39) and EAEC (3%, 2/76) were resistant to meropenem and ertapenem. The author should separately describe what percentage of ETEC as well as EAEC were resistant to meropenem as well as entrapenem.

51. This finding agrees with reports from Nigeria [42], and Iran [40]. The author should add the percentage DEC in diarrheic and non-diarrheic in Nigeria and Iran.

52. Only LT-positive ETEC was found in the non-diarrheic children in the present study. ST-positive ETEC strains are commonly associated with diarrhea compared to LT-positive. LT toxin is not responsible for diarrhea?

53. EAEC (41.5%) was the predominant DEC pathotype identified in the present study. What could be the plausible region?

54. The second most prevalent DEC pathotype was ETEC (21.3%) which was consistent with studies done in Nigeria [42], China [21], Sudan [38], and Iran [40]. The author should kindly add the data of these countries.

55. In table 2, the acquisition of DEC in the rainy season (32.2%) looks less than in the dry season (45.9%). It is quite confusing some tables show data for winter, spring, and summer whereas the author discusses the dry season and rainy season. The author should kindly revise it thoroughly and use either winter, summer as well as spring or dry and rainy seasons. What could be the possible reason for the prevalence of DEC pathotypes in winter as compared to the rainy season? The author should discuss it with suitable reason.

56. In table 2, the acquisition of DEC in the rainy season (32.2%) looks less than in the dry season (45.9%). However, it was statistically significant that under-five children were more likely to acquire DEC during the rainy season compared to the dry season in the present study, resulting in children visiting the health facility in the summer due to DEC infections. If children have visited the health facility center in the rainy season then why is the prevalence of DEC pathotypes shown higher in the winter season?

57. Children who were cared for by their mothers (37.5%) had statistically significant protection than those who were cared for by others (grandmothers, close family members, and daycare) (14.0%). The author should recheck the data properly. It is 37.5% or 53.6%??

58. This may be due to that the mother is more likely careful than other caregivers, potentially contributing to the incidence of DEC in the children. What could be the possible reason for the prevalence of DEC pathotypes in more who cares by mother as compared to other caregivers?

59. Any significant effect of the number of children in a particular family on the prevalence of DEC pathotype in that particular family?

60. What is the meaning of total control which showed in table 3?

61. All 428 E. coli isolates were tested by PCR, of which 183 (42.8%, 183/428) were positive for DEC pathotype. The overall prevalence of DEC in the present study was 38.4% (183/476), and 58.5% (107/183) of the DEC types were isolated from Addis Ababa and the remaining from Debre Berhan (Table 3, Fig1 a & b). The author should kindly revise it properly. It creates confusion like 42.8%% were DEC type and the next line overall prevalence was 38.4%.

62. In table 3 the DAEC prevalence was showing 14.5% whereas in the manuscript it was written as 41.5%. The author should revise the tables and write either in percentages or numbers.

63. The author should revise the manuscript thoroughly and correct the data in the table as well as the manuscript.

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Reviewer #2: Yes: Kayode Olayinka Afolabi

Reviewer #3: Yes: Dr. Ashish Kumar Singh

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Attachments

Attachment

Submitted filename: Plosone_Major_Comments.docx

PONE-D-22-27001R3Molecular epidemiology and antimicrobial susceptibility of diarrheagenic Escherichia coli isolated from children under age five with and without diarrhea in Central EthiopiaPLOS ONE

Dear Dr. Zelelie,

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 Jul 01 2023 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Samer Singh, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

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:

There are some issue with the language at few places as indicated by the reviewer. The changes suggested are indicative. Authors would like to check the manuscript thoroughly for typographical and grammatical errors and suitably correct them as needed. 

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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 #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: Yes

********** 

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

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

Reviewer #3: Yes

********** 

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: Q.1 Then, the disks were placed on Mueller-Hinton agar plates (Oxoid, UK) freshly inoculated with a 0.5 McFarland suspension of Escherichia coli ATCC 25922. The results from the ESBL confirmation and carbapenemase production were interpreted according to CLSI guidelines.

Kindly italicized the coli also.

Q.2 DEC pathotypes (whole genome sequenced strains) confirmed positive for the target virulence gene(s) from previous works used as a positive control. CCUD (Culture Collection University of Gothenburg) 24T E. coli strain confirmed negative to the target gene (s) was used as a negative control. Kindly correct the typological errors.

Q.3 A clinical presentation of children under age five positive for DEC is presented in S2Table. Pl. correct the typological errors.

Q.4 Of the total DEC pathotypes, 58.5% (107/183) were from Addis Ababa, and the remaining 41.5% (76/183) were from Debre Berhan (Table 3, Fig1 a & b). Table 3 or Table 2?? Pl. correct it carefully.

Q.4 Placed in a freezer at −20°C for 10 minutes. Kindly correct the typological errors.

Q.5 Then, 100μL of the supernatant was transferred into a nuclease-free Eppendorf tube and stored at −20°C until use. Kindly correct the typological errors.

Q.6 PCR reaction 3 contained primer mix 3 (M-3) for the detection of EAEC and DAEC targeting virulence genes aggR, astA, and daaF. Why italicized the 3?

Q.7 The PCR thermal conditions were set with an initial denaturation of 94°C for 2 min, followed by 35 cycles of 92°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 30 sec, and a final extension at 72°C for 5 min in a PCR machine (Biometra TRIO Thermal Cycler, Analytik Jena). Kindly correct the typological errors.

Q.8 Table 3 and Table 4 were showing the number of isolates as well as the percentage also. It creates confusion for the readers. Kindly show the data only in percentages is better to understand.

Q.9 Author is showing two types of data one is the proportion of DEC pathotypes and the second one is the prevalence of DEC pathotypes. It will create confusion for the readers.

Q.10 What could be the plausible reason to collect the diarrheal stool sample from the children who did not receive the antibiotics in the past three weeks?

Q.11 Table 4. Antimicrobial resistant profile of DEC pathotypes (n=183) isolated from diarrheic and non-diarrheic under -five children, Addis Ababa and Debre Berhan, Ethiopia. Kindly correct it properly.

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Reviewer #3: Yes

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Attachments

Attachment

Submitted filename: Minor_Comments (1).docx

Molecular epidemiology and antimicrobial susceptibility of diarrheagenic Escherichia coli isolated from children under age five with and without diarrhea in Central Ethiopia

PONE-D-22-27001R4

Dear Dr. Zelelie,

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,

Samer Singh, 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 #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 #3: Yes

**********

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

Reviewer #3: Yes

**********

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

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

Reviewer #3: Yes

**********

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

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

Reviewer #3: Yes

**********

6. Review Comments to the Author

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

Reviewer #3: (No Response)

**********

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: Yes: Dr. Ashish Kumar Singh, Ph.D.

**********