PONE-D-23-32545Metagenomic Analysis of Sewage for Surveillance of Bacterial Pathogens: A Release Experiment to Determine SensitivityPLOS 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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: 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

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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

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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 aims to investigate the sensitivity of metagenomic sequencing for the detection of specific sewage bacteria in the context of wastewater-based epidemiology. This was conducted in situ by spiking known quantities of extraneous bacteria (Staphyloccus hyicus) into a sewer system before metagenomic sequencing of samples from the associated wastewater treatment plant. The authors highlight the potential for wastewater metagenomics to estimate the number of sick individuals with an increased sensitivity over previous non-metagenomic methods.

Overall, I found the manuscript to be well written, interesting, and novel as relatively few published studies have performed spiking/spot-release experiments in the context of wastewater-based epidemiology. However, I do believe clarification of some points, potential further analyses and additional discussion would enhance the manuscript.

I have following specific comments.

1. Line 137-140: Since these initial values are pivotal to the overall experiment and calculations of sensitivity, please provide more detailed justification on why a 200 g feces assumption was made (supporting reference?) and the same for CFU per gram - is based on observed amounts from Campylobacter spp. and Salmonella ssp infection stool samples? If so, please provide supporting reference

2. Line 220-222: The metagenomic sequencing depth is not clearly reported but can be partially inferred through the reported total fragments in Tables 1 and 2 (Line 299-311) or from the discussion (Line 328). This is of crucial importance and relevance to the manuscript analyses since sensitivity to detect specific targets in metagenomes is directly tied to metagenomic sequencing depth and sample complexity (https://doi.org/10.1186/s40793-019-0347-1). Please report the sequencing depth achieved per sample and discuss the impact of sequencing depth as a limitation (i.e. deeper sequencing would likely be required due to reduced/scattered real world release as compared to the large experimental spot-release)

3. Line 294-317: This section is very interesting and highlights potential sensitivity differences between results based on multiple samples and a single peak sample (as seen in other studies), but this is not currently mentioned in the discussion. Was there any factor which could explain the reduced sensitivity in Day 1-peak conc (e.g. weather, flow, taxonomic composition apart from S. hyicus). It would also be interesting to compare the sensitivities for all single samples and not just the sample with peak S. hyicus concentration

4. Line 320-343: The discussion section is very limited in scope and does not address several presented results which warrant discussion such as the similarities and differences between sensitivity of compound sample results (Table 1) and peak conc. (Table 2) – as above.

5. Line 329: The calculation of the value “1 in 260 to 2,250 people” is not clearly described. It can be inferred that it originates from from Tables 1 and 2 using Minimum amount required (n) and normalised by size of the WWTP through the conclusion only (Line 349-351). Please clarify at first mention.

Minor comments/typographical issues:

Line 32: Shotgun is more commonly accepted over “shot-gun” – please update

Line 139: Please update citation format to be consistent with numbering used throughout

Line 226: Please reference where the sequences were downloaded from to generate the reference database “genomic2_20191017”

Line 230: Please provides reference for Spades as done for KMA (Line 225-226)

Line 336: Typo “Cation” should be Caution

Reviewer #2: The manuscript, entitled "Metagenomic Analysis of Sewage for Surveillance of Bacterial Pathogens: A Release Experiment to Determine Sensitivity", provided new information about the implication of metagenomics for detecting pathogens from sewage. The manuscript is well-written and organized.

The results can be applied to monitoring bacterial pathogens.

For the methods, could you please explain the reason for using only the CFU of 1.66 and 1.91 but not different lower concentrations?

The second question is about the real sample from the community. The experiment revealed the sensitivity of the method for tracing the pathogens. However, is the method good for the real situation when the community has the patients caused by other pathogens? It would be more effective if the authors applied this method to a specific pathogen.

Thank you.

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Reviewer #1: No

Reviewer #2: No

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Metagenomic Analysis of Sewage for Surveillance of Bacterial Pathogens: A Release Experiment to Determine Sensitivity

PONE-D-23-32545R1

Dear Dr. Aarestrup,

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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Marwan Osman

Academic Editor

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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

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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: (No Response)

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

Reviewer #1: (No Response)

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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: (No Response)

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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: (No Response)

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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: (No Response)

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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 #1: Yes: Kevin K Chau

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