-->PONE-D-26-07244-->-->Prevalence, virulence profiles and antibiotic susceptibility patterns of Escherichia coli O157:H7 (STEC) among children 6-59 months in Longido, Arusha-Tanzania.-->-->PLOS One

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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: No

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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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

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Reviewer #2: No

Reviewer #3: No

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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 submitted by Michael Martin et al. evaluates the prevalence of Escherichia coli producing Shiga toxin O157:H7 (STEC) in children with diarrhea in Longido District, Arusha, Tanzania. The detection of virulence genes confirms the pathogenic potential of the isolates, and the 20% prevalence suggests a relevant role in childhood diarrheal disease. In addition, resistance to some commonly used antibiotics was observed. The study provides important data for epidemiological surveillance and for preventing infections and complications such as Hemolytic Uremic Syndrome. The study addresses an important public health issue; however, several aspects of the manuscript require clarification and improvement before publication.

The samples were collected between July and August 2025. Considering the potential seasonal variation of enteric pathogens, could the authors discuss whether the reported prevalence may vary across other seasons?

The discussion attributes the high prevalence to factors such as human–animal interaction, sanitation practices and food habits; however, these variables were not directly measured in the study. Could the authors clarify whether these interpretations are supported by collected data or moderate this conclusion?

The definition of Shiga toxin–producing Escherichia coli O157:H7 relies on the presence of stx1 and/or stx2 genes. However, several isolates (N014, N069, N045, N050, N079, N115, N032, N010B, N006B, N014B) reported in Table “ANTIBITIC_SUSCEPTITY_DATA_PLOS_ONE_SUBMISSION” appear to be positive for the rfbE gene but negative for both stx1 and stx2. Could the authors clarify whether these isolates were still classified as STEC and how this was considered in the prevalence calculation?

The table “ANTIBITIC_SUSCEPTITY_DATA_PLOS_ONE_SUBMISSION” includes both virulence gene detection and antimicrobial susceptibility data. For clarity, the authors may consider either revising the table title to reflect both datasets or separating the information into two tables. In addition, this dataset is currently provided in the supplementary material. It would improve readability if a summarized table of virulence gene prevalence and antimicrobial susceptibility were included in the main manuscript.

In Figure 2, the correspondence between gel lanes and the Sample IDs listed in Table “ANTIBITIC_SUSCEPTITY_DATA_PLOS_ONE_SUBMISSION” is not indicated. The authors should clarify which isolates correspond to each lane. In addition, the expected band sizes for each virulence gene should be annotated in the gel, and the image quality (resolution/contrast) could be improved to facilitate interpretation of the PCR products.

Title: Remove “(STEC)”

Line 68 = change “Shiga toxin-producing E. coli STEC” by only “STEC”

Line 239 = change “aea” to “eae”

Line 323 = change “Shiga toxin producing E.coli” to “STEC”

Line 334 = change “aea” to “eae”

Line 334 = “…hemolysin gene EHEC hly was identified…” this gene is the hlyA? describe the full name of the hemolysin gene

Reviewer #2: The manuscript presents a study detecting STEC in children under 5 years of age in 4 locations in Longido, Arusha, Tanzania, during July and August 2025. 150 samples were analyzed, from which 30 STEC isolates were identified. The presence of some virulence genes and antibiotic susceptibility were determined in these STEC strains. Additionally, some sociodemographic and clinical variables were collected during sample collection.

While the study is interesting and the frequency of STEC O157:H7 (20%) is striking, the article requires a more in-depth analysis of the collected data. A more thorough analysis of the sociodemographic and clinical information in relation to the isolates obtained is lacking. In all cases, only percentage values are mentioned, without any correlation analysis. It is noteworthy that, given the high frequency of STEC, these patients were not followed up. Another weakness of the manuscript is the presentation of the results, which presents raw data without adequate statistical analysis.

The manuscript provides too much information, not necessarily related, especially in the introduction. The discussion is weak, and given the lack of statistical analysis, it is not possible to draw clear conclusions.

Reviewer #3: General comment

This manuscript addresses an important public health issue by investigating the prevalence, virulence gene profiles, and antimicrobial susceptibility of Escherichia coli O157:H7 (STEC) among children in a semi-pastoral community in Tanzania. The study is relevant within a One Health framework, particularly given the close interaction between humans, livestock, and environmental exposures in the study setting. The reported prevalence of STEC O157:H7 (20%) is striking and, if robust, represents a potentially significant contribution to the epidemiology of diarrhoeal diseases in underrepresented populations.

However, there are several methodological, conceptual, and structural limitations that need to be addressed. These include issues related to study design, microbiological characterization, and data presentation. In addition, several aspects of the manuscript would benefit from improved clarity, updated references, and better organization. In its current form, the manuscript requires major revision before it can be considered for publication.

Introduction

The introduction provides a general overview of STEC as a zoonotic pathogen and highlights its relevance in diarrhoeal disease. However, the logical flow of ideas needs to be improved, as the current structure is somewhat fragmented and occasionally repetitive. Some statements require careful revision for accuracy. For example, the assertion that “the majority of diarrhoeal disease outbreaks, sporadic cases, and severe clinical diseases such as hemorrhagic colitis and hemolytic uremic syndrome are attributed to STEC infection” (Lines 46 – 48) is an overstatement and not fully supported by current epidemiological evidence. While STEC is indeed an important pathogen, it is not responsible for the majority of diarrhoeal disease cases globally.

The introduction would also benefit from the inclusion of more recent references. Epidemiological data on STEC prior to 2020 are now considered outdated in many contexts, and more recent studies should be incorporated to reflect current trends. That said, it is understood that in the African context, data on STEC are limited, and the use of older studies may sometimes be unavoidable. This limitation should be acknowledged explicitly.

Additionally, the objective of the study should be reformulated to avoid implying causality. The current phrasing suggests that the study aims to “reveal the roles” of STEC in diarrhoeal disease, which is not appropriate for a cross-sectional design. A more accurate formulation would focus on determining prevalence and molecular characteristics.

Methodology

The methodology section describes a hospital-based cross-sectional study, which is suitable for estimating prevalence but not for establishing causation.

An important omission in this section is the lack of explicit mention of ethical approval and informed consent within the Methods section itself. Although an ethics statement appears elsewhere in the manuscript, it should be clearly integrated into the methodology.

The microbiological and molecular methods are generally appropriate, including the use of selective culture media, serological confirmation, and multiplex PCR targeting key virulence genes.

The antimicrobial susceptibility testing follows CLSI guidelines, which is appropriate, but the limited panel of antibiotics restricts the interpretation of multidrug resistance. Moreover, the criteria used to define multidrug resistance are not specified. That is, more than 2 antibiotics or?

The statistical analysis is basic and limited to descriptive statistics and chi-square tests. No multivariate analysis is performed, and potential confounding factors are not addressed.

Results

The results section would benefit from improved organization. The subsections titled “Participant’s characteristics,” “Participant’s socio-demographic characteristics,” and “Participant’s clinical signs” should be merged into a single, cohesive section to improve readability and avoid redundancy.

There are also issues with table organization and consistency. The numbering of tables does not always follow the order in which they are cited in the text, and this should be corrected. Additionally, Table 3 presents data only for the children, despite being described as including parents or guardians. Table 6 appears to repeat data already presented in Table 3, and this redundancy should be addressed.

The reported prevalence of STEC O157:H7 is high and noteworthy, but its interpretation is limited by the absence of a control group and the inability to distinguish between infection and asymptomatic carriage. The manuscript would benefit from a clearer discussion of this distinction.

The virulence gene data raise some concerns. While it is known that some STEC O157:H7 strains may be eae-negative, such strains are relatively rare. The finding that only 40% of isolates carried the eae gene is unusual and warrants further clarification. The presentation of these results should be revised to improve clarity and facilitate interpretation.

Discussion

The discussion section addresses several relevant points but would benefit from improved structure and focus. It is recommended to remove the current subheadings to allow for a more fluid and integrated narrative.

The manuscript should emphasize the importance of studying antimicrobial resistance in STEC within the appropriate clinical context. Notably, STEC infections are generally not treated with antibiotics due to the potential risk of hemolytic uremic syndrome, and this important consideration is not discussed.

The consumption of unpasteurized milk in the study population is a highly relevant finding and represents a distinctive epidemiological feature of this community. The discussion would benefit from incorporating studies that have investigated STEC contamination in raw milk or similar settings, as well as comparisons with populations that have comparable dietary practices.

Another important aspect that is not addressed is the possibility of asymptomatic carriage of STEC in the study population. This is particularly relevant in endemic settings and should be discussed as a potential explanation for the observed prevalence.

Finally, the manuscript does not adequately address its limitations, including sampling bias and the constraints of the cross-sectional design. A dedicated limitations paragraph is strongly recommended.

Minor comments

There are several minor issues related to formatting and terminology that should be corrected. For example, references to tables should follow a consistent format, such as “Table 1” and “Table 2,” rather than “table one” or “table two.” This issue appears multiple times throughout the manuscript, including in lines 168, 169, and 219. Additionally, the term WAZ should be defined when first introduced, as it may not be familiar to all readers.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Attachments

Attachment

Submitted filename: comments.docx

-->PONE-D-26-07244R1-->-->Prevalence, virulence profiles and antibiotic susceptibility patterns of Shiga toxin producing Escherichia coli O157:H7 among children 6-59 months in Longido, Arusha-Tanzania.-->-->PLOS One

Dear Dr. Martin,

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 31 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:-->

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• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

-->

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If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

As the corresponding author, your ORCID iD is verified in the submission system and will appear in the published article. PLOS supports the use of ORCID, and we encourage all coauthors to register for an ORCID iD and use it as well. Please encourage your coauthors to verify their ORCID iD within the submission system before final acceptance, as unverified ORCID iDs will not appear in the published article. Only  the individual author can complete the verification step; PLOS staff cannot  verify ORCID iDs on behalf of authors.

We look forward to receiving your revised manuscript.

Kind regards,

Fernando Navarro-Garcia

Academic Editor

PLOS One

Journal Requirements:

1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

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

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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 #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 #1: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

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

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

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

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

Reviewer #1: Yes

Reviewer #3: No

**********

-->6. Review Comments to the Author

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

Reviewer #1:  (No Response)

Reviewer #3:  General Comment

My primary concern is that the revised manuscript does not correspond to the tracked-changes version provided by the authors. The two documents differ substantially, making the review process unnecessarily difficult, as it essentially required reviewing two different manuscripts. The authors should ensure that the correct tracked-changes file is submitted so that reviewers can accurately assess the revisions made in response to previous comments.

Introduction

The statement "It is estimated to cause millions of infections annually, with a proportion progressing to severe disease requiring hospitalization" lacks quantitative support. How many millions of infections are being referred to? In the original version, the authors cited data from 2014, whereas the tracked-changes version included data from 2020. In the revised manuscript, these data have been removed. The authors should provide up-to-date epidemiological estimates and appropriate references to support this statement.

Regarding the tracked-changes version, the authors improved the Introduction by incorporating more recent references and providing a clearer epidemiological context for STEC in Africa. However, several paragraphs still require language editing, and some grammatical and stylistic issues remain. For example, in the paragraph spanning lines 79–86, the term “Tanzania” is repeated three times unnecessarily.

Additionally, in line 137, there is a repeated phrase “The results of this study provided The findings of this study”

The study objective has been reformulated and now adequately reflects the scope of the results presented.

Methodology

The authors have still not clarified the criteria used to define multidrug resistance (MDR). Specifically, it remains unclear whether MDR was defined as resistance to more than two antimicrobial agents, resistance to three or more antimicrobial classes, or according to another established criterion. A clear definition should be provided.

The tracked-changes version also contains several typographical and grammatical errors. For example:

• Line 187: “Table one”

• Line 188: “Table two”

These should be revised according to the journal style.

In line 202, a punctuation error is present: "Bauer disk diffusion technique (Sangeda et al., 2021) According to the guidelines..." A period is missing after the citation. I recommend that the authors carefully proofread the entire manuscript to identify and correct similar issues.

I also note that the revised manuscript includes an Ethical Clearance section, whereas this information is absent from the tracked-changes version. This discrepancy further highlights the inconsistency between the two submitted documents.

Results

The Results section is presented quite differently in the revised manuscript and in the tracked-changes version. The revised manuscript is more consistent with the recommendations provided during the first round of review and appears to better address the concerns previously raised.

In line 218, the authors refer to “STEC status.” What do they mean by STEC Status?

Overall Assessment

Overall, the authors have addressed several of the comments raised during the previous review round. However, it remains difficult to accurately assess the revisions because the tracked-changes document does not correspond to the revised manuscript currently under review.

The remaining minor comments on the revised version should be addressed. A final round of revision appears necessary, and I strongly recommend that the authors submit the correct tracked-changes version so that reviewers can properly evaluate the modifications made.

**********

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

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

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

Reviewer #1: No

Reviewer #3: No

**********

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Prevalence, virulence profiles and antibiotic susceptibility patterns of Shiga toxin producing Escherichia coli O157:H7 among children 6-59 months in Longido, Arusha-Tanzania.

PONE-D-26-07244R2

Dear Dr. Martin,

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.

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PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

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-->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)

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

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Reviewer #3: Yes

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

Reviewer #3: Yes

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Reviewer #3: Yes

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Reviewer #3: Yes

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-->6. Review Comments to the Author

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Reviewer #3: The authors have addressed all comments raised by this reviewer, except for clarifying what they mean by "STEC status."

I have no further comments.

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Reviewer #3: No

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