PWAT-D-23-00061

Microbial community function and pathogen composition in pit latrines in peri-urban Malawi

PLOS Water

Dear Dr. de los Reyes III,

Thank you for submitting your manuscript to PLOS Water. After careful consideration, we feel that it has merit but does not fully meet PLOS Water'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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Kind regards,

Silvia Monteiro

Academic Editor

PLOS Water

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Additional Editor Comments (if provided):

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does this manuscript meet PLOS Water’s publication criteria? Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe methodologically and ethically rigorous research with conclusions that are appropriately drawn 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 (please refer to the Data Availability Statement at the start of the manuscript PDF file)?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception. 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 Water 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: This study characterized the microbial communities and inferred functions within 55 lined pit latrines. This study was well designed and the results appear to be reliable. The results of this study would be useful to PLOS Water readers.

The benefits of implementing WBE in pit latrines are not clear. An advantage of WBE is the accessibility of population-based data and anonymity when applied to a centralized sanitation system. WBE for a decentralized system carries the risk of violating the privacy of the pit owner if it is not a shared facility. This issue may not be relevant to the main topic of this study, but authors should be careful about this point and clearly describe the advantages or motivation of WBE over this disadvantage.

The authors investigated the species richness of microbial pathogens (Figure 5). In practice, richness of quantity is important information to know the prevalence of infectious diseases. I believe that many readers will be interested in the degradation of genetic material of pathogens in a pit. Therefore, it must be valuable for authors to discuss both species and quantity richness in the surface, middle and bottom areas of a pit. Please discuss the appropriate depth of sampling based on quantity and diversity richness.

Specific comments follow.

"Pathogen" in the title should be changed to "bacterial pathogen".

It was not clear what "water source" means. Please provide a definition and the types of water sources identified in this study.

Line 130: The addition of a process control is necessary in wastewater monitoring to evaluate the recovery of pathogens through the processes of concentration, extraction, and quantification (Haramoto et al., 2018). Please explain how the authors evaluated the recovery of pathogens from sludge.

Haramoto, E., Kitajima, M., Hata, A., Torrey, J. R., Masago, Y., Sano, D., Katayama, H., 2018. A review on recent progress in the detection methods and prevalence of human enteric viruses in water. Water Research 135, 168–186.

Line 197: "Richness" is used for both quantity and diversity. Please clearly define that the authors assessed species richness.

Line 234: Detailed information on pit latrines is not provided. Many readers expect the environment of a pit latrine to affect the microbiological and physico-chemical characteristics within it. I recommend that the authors provide the following for each pit: population served, moisture content, years since last emptying, depth. I expect that the population will affect the microbial diversity in the pit sludge.

Line 280: Microbiome data in activated sludge, anaerobic digestion and human gut are not available in this manuscript. Please explain how the authors obtained the data. Please add appropriate references if necessary.

Figure 4: Is this the average of 55 pit latrines?

Line 327: This result should not be generalised as sampling was only done once in this study. The level of pathogens in a pit may vary seasonally, affecting the species diversity of pathogens in a pit.

Table 1: Please give the raw data and the distribution of each factor. Readers will suspect that there are biases in the factors.

Result: Please give the limit of quantification (LoQ) and limit of detection (LoD) of the pathogens.

Line 381: This section is well written. Although I am not familiar with the microbiome and functional analysis, I think this section is useful and all the discussion is insightful. However, it is a bit difficult to grasp the main points found in this study. I would appreciate it if the authors could provide a summary for the following questions: What was a common function of the microbes in all the pit latrines that was not observed in other microbiological treatments? What was a unique function observed in each pit latrine?

Many readers expect that metabolism takes place in anaerobic environment in deeper area and in aerobic environment in shallower area. I understand that the authors confirmed this. The novelty or unexpected findings related to bacterial function in this study were not clear to me.

Reviewer #2: Overall:

The manuscript presents interesting in relevant information regarding the microbial (bacteria and archaea) and pathogenic (bacteria) communities in pit latrines and compares results with mcrobiomes of anaerobic digestion of municipal sewage sludge and human gut. The manuscript is well-written and well-structured. Below some minor comments.

Specific comments:

ABSTRACT

Line 40. Remove word ‘only’.

INTRODUCTION

Line 47. I suggest ‘to peri-urban and urban settings’.

Lines 53-55. Sentence deserves reference.

Lines 63-65. Sentence deserves reference.

Lines 67-68. I suggest adding location information regarding the studies performed by Torondel et al (2016) and Ijaz et al (2022).

MATERIAL AND METHODS

lines 140-141. By mixing and then centrifugation? I suggest clarifying this.

Lines 172-175. Why comparing the communities present in pit latrines (predominantly anaerobic) to the ones in activated sludge (aerobic?)

RESULTS

* Microbial community analyses and comparisons to similar biological systems.

I suggest presenting graphical results of both bacteria (Figures S2 and S3) and archaea (Figure 1) populations in the manuscript (instead of presenting bacteria results in the Supporting Information).

Lines 280-288. It was not observed shared genera between activated sludge and pit latrines (please see comment for lines 172-174). In addition, I suggest adding Figure S5 to the manuscript (instead presenting it in the Supporting Information).

Figure 4. Indicate the groups with statistical differences in the chart?

* Pathogens and public health implications

Lines 303-314. As relative abundances were quite low for pathogens, I’m not sure if presenting Figure 5 here is ideal. I would tend to mention in the text the pathogens who appeared the most and/or were common between sites and levels and/or with higher abundance. Another option would be to change the y-axis scale (e.g,. 0.000-.005) and add notes to those with high cumulative abundance (9, 10, 14 in Blantyre; 19, 21, 24, 25 in Mzuzu). Finally, in line 314, it’s ‘(Figure S7)’, and not ‘(Figure S7Figure S710)’.

* Guidance for sampling pit latrines

It’s clear that sampling from 8 pit latrines only would be representative regardless of depth (Figure 6) and location (Figure 7). However, from a visual analysis of both Figures, sampling from only 5 pit latrines (perhaps even 4) would also be representative, and obviously easier. I suggest further exploring this.

DISCUSSION

Line 385. Again, not sure about activated sludge.

Line 388. Which Figure?

Lines 404-405, 415-416, 430-431. Sentences deserve references.

Lines 494-495. See comment about the topic ‘Guidance for sampling pit latrines’

Final questions:

Could the findings of this research be used for the improvement of pit latrines? For example, changes that could be made in the pits to increase degradation of organic matter, reduction of pathogens and production of methane (energy recovery)? I understand this was not the aim of the manuscript, but mentioning such topics could highlight its relevance.

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Reviewer #1: Yes: Wakana Oishi

Reviewer #2: No

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Attachments

Attachment

Submitted filename: PWAT-D-23-00061.docx

Microbial community function and bacterial pathogen composition in pit latrines in peri-urban Malawi

PWAT-D-23-00061R1

Dear Dr. de los Reyes III,

We are pleased to inform you that your manuscript 'Microbial community function and bacterial pathogen composition in pit latrines in peri-urban Malawi' has been provisionally accepted for publication in PLOS Water.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow-up email from a member of our team. 

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Water.

Best regards,

Silvia Monteiro

Academic Editor

PLOS Water

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Reviewer Comments (if any, and for reference):

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

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2. Does this manuscript meet PLOS Water’s publication criteria? Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe methodologically and ethically rigorous research with conclusions that are appropriately drawn based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available (please refer to the Data Availability Statement at the start of the manuscript PDF file)?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception. 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

**********

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

PLOS Water 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

**********

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 #2: (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.

Do you want your identity to be public for this peer review? If you choose “no”, your identity will remain anonymous but your review may still be made public.

For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

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