The first comparative metagenomic analysis of diarrheal and non-diarrheal gut microbiome leading to the identification of prospective prognostic markers and probiotics to protect from diarrhea: A Brief Report
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Introduction: Diarrhea is a leading contributor of mortality. Its disease burden can be assuaged using enhanced prognostics and therapeutics. A cross-sectional gut microbiome analysis of non-diarrheal and diarrheal fecal samples was conducted to meet the goals of the Global Action Plan for Pneumonia and Diarrhea. Hypothesis: Next-generation Sequencing as a tool for epidemiological surveillance helps in the comparison of structural diversity of the gut microbiome of diarrheal and non-diarrheal samples and can identify prognostic and therapeutic candidates. Aim: The pilot study was designed to identify taxa that are enriched in non-diarrheal samples and to predict gut microbial interactions. Methodology: 16SrRNA amplicon sequencing and analysis were deployed for taxonomic profiling and abundance interpretation of OTUs (Operational Taxonomic Units). Results: The findings suggested significant differences in structural composition between the two groups. Firmicutes was the most abundant phylum in most samples. B/F ratio was consistently <1 in all diarrheal samples. Proteobacteria was more abundant in diarrheal samples. Prevotellaceae was the most abundant family in non-diarrheal samples. Streptococcaceae was the most abundant family in 60% diarrheal samples.Where Streptococcaceae was suppressed, Bacteroideaceae and Nocardeaceae were the most abundant. In non-diarrheal samples where Bifidobacteriaceae was the most abundant other families were significantly low. A negative correlation was observed between Prevotellaceae and Bacteroideaceae in non-diarrheal samples. Prevotella copri was the most abundant species in 70% non-diarrheal samples and was significantly suppressed in diarrheal samples. Proteus mirabilis was identified in all the non-diarrheal samples and were absent in diarrheal samples. Conclusion: The OTUs associated with diarrheal dysbiosis can serve as prognostic markers. This is the first report on the comparative analysis of diarrheal and non-diarrheal microbiome. The study addressed gut microbiome dysbiosis from the context that can lead to the development of prognostic markers and probiotics for protecting the endemic population from diarrhea and help in reaching Sustainable Development Goals 2 and 3.
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Thank you for submitting your revised manuscript to Access Microbiology. It has now been reviewed by the same reviewers that provided feedback in the previous round of review, whose comments can be found at the bottom of the email. Although corrections have been performed to some extent, the reviewers, and in particular Reviewer 1, still have concerns about the manuscript and point out that many of the suggested corrections have not been applied (for example, table formats), which have been specially difficult to verify as neither tracked-changes Word document of the manuscript nor point-by-point responses to the reviewer comments were provided. Please provide a revised version of the manuscript paying special attention to the previous suggestions given by the reviewers to fulfil their queries, along with the aforementioned …
Thank you for submitting your revised manuscript to Access Microbiology. It has now been reviewed by the same reviewers that provided feedback in the previous round of review, whose comments can be found at the bottom of the email. Although corrections have been performed to some extent, the reviewers, and in particular Reviewer 1, still have concerns about the manuscript and point out that many of the suggested corrections have not been applied (for example, table formats), which have been specially difficult to verify as neither tracked-changes Word document of the manuscript nor point-by-point responses to the reviewer comments were provided. Please provide a revised version of the manuscript paying special attention to the previous suggestions given by the reviewers to fulfil their queries, along with the aforementioned tracked-changes version of the manuscript and the point-by-point response to their comments within one month.
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Comments to Author
The responses are not point wise, and I can't follow the changes made in the revised manuscript (track changes can't be seen in the PDF version). Furthermore, I feel that all the questions I raised were not addressed in the revised MS/response letter. For eg. None of the minor comments I made are addressed in the revised version. Also, the tables are still in the same format although I have recommended the revision. In its current format, I cannot review this revised MS.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of …
Comments to Author
The responses are not point wise, and I can't follow the changes made in the revised manuscript (track changes can't be seen in the PDF version). Furthermore, I feel that all the questions I raised were not addressed in the revised MS/response letter. For eg. None of the minor comments I made are addressed in the revised version. Also, the tables are still in the same format although I have recommended the revision. In its current format, I cannot review this revised MS.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
concerns addressed
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Thank you for submitting your manuscript to Access Microbiology. It has now been reviewed by two experts in the field whose comments are attached at the bottom of this email. They both have important concerns on the structure, scope, literature references and interpretation of the results. Please address them carefully in a revised version of the manuscript. Please submit a revised version of the manuscript (including a tracked-changes document) along with a point-by-point response to the reviewer comments within 2 months.
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Comments to Author
In the current manuscript, the authors compared the gut microbiome between diarrheal and non-diarrheal populations using metagenomic analysis. They found significant differences in structural composition between the two groups. However, the study populations were too small, especially for the diarrheal group (only 5). Moreover, the causative agent for diarrhea may be viruses (such as norovirus and rotavirus), parasites (such as Giardia lamblia and Entamoeba histolytica), or bacteria (such as Salmonella and Shigella). The authors did not distinguish viral diarrheal group from bacterial diarrheal group. The findings may be different under the circumstance of different pathogens.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the …
Comments to Author
In the current manuscript, the authors compared the gut microbiome between diarrheal and non-diarrheal populations using metagenomic analysis. They found significant differences in structural composition between the two groups. However, the study populations were too small, especially for the diarrheal group (only 5). Moreover, the causative agent for diarrhea may be viruses (such as norovirus and rotavirus), parasites (such as Giardia lamblia and Entamoeba histolytica), or bacteria (such as Salmonella and Shigella). The authors did not distinguish viral diarrheal group from bacterial diarrheal group. The findings may be different under the circumstance of different pathogens.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Not at all
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-
Comments to Author
The MS by the authors described the microbiome analysis in diarrheal and non-diarrheal individuals using comparative 16S based metagenomic analysis. The study will be of importance to understand the dysbiosis and possible interventions. I append my comments as follows: General comment: 1. The MS has careless drafting errors and lack proofreading. Use of jargons is there. 2. Introduction lack enough literature survey. Not identifying the gaps in the literature. 3. There are literatures that need to be included in the background. Some eg. but not limited to are as follows. doi.org/10.1016/j.mib.2022.01.006 doi.org/10.1186/gb-2014-15-6-r76 doi.org/10.1080/19490976.2017.1361093 doi.org/10.1126/science.aad3503 4. Diarrhea is the disease of the low-income countries as described in the MS. How will the cost …
Comments to Author
The MS by the authors described the microbiome analysis in diarrheal and non-diarrheal individuals using comparative 16S based metagenomic analysis. The study will be of importance to understand the dysbiosis and possible interventions. I append my comments as follows: General comment: 1. The MS has careless drafting errors and lack proofreading. Use of jargons is there. 2. Introduction lack enough literature survey. Not identifying the gaps in the literature. 3. There are literatures that need to be included in the background. Some eg. but not limited to are as follows. doi.org/10.1016/j.mib.2022.01.006 doi.org/10.1186/gb-2014-15-6-r76 doi.org/10.1080/19490976.2017.1361093 doi.org/10.1126/science.aad3503 4. Diarrhea is the disease of the low-income countries as described in the MS. How will the cost intensive metagenomics can be used for such resource poor settings? Methodology: 1. Sample size between diarrhea and non-diarrhea vary significantly. The results might be affected by the outliers particularly in diarrhea groups. 2. Ages of the individuals in diarrhea and non-diarrhea groups? 3. During the sample collection, did a comprehensive Q&A was carried out? This reviewer is interested in knowing if the individuals with diarrhea were on antibiotics before sample collection. Similarly, for non-diarrheal cases, did by any chance they have taken the antibiotics for non-related illness. If the information is available, this will aid the MS discussion. 4. How many days post onset of diarrhea, samples were collected? 5. What were the possible etiological agents in the 5 diarrheal cases? 6. Why 16S rRNA based metagenomic analysis was carried out when shotgun metagenomics can demonstrate higher level of phylotype resolution. Presentation of Results: 1. Although the submission of the MS is format neutral, still the authors should have considered preparing the tables in the basic publication format. 2. Same is for the figures. No need to separately describe the x-axis and y-axis. Rather, they should be in the axis itself. 3. In tables, decimal places are not unified and significant figures are not considered. 4. Core microbiome/conservation of OTUs in diarrhea cases should have been discussed with another study from the authors (Ref. 4) Referencing: Usage of full stops/periods is improper at multiple places. References are not unified. Other comments: Ln29: Add space after 'samples'. Ln56, 59: Add apace between numbers of the references. Ln63: Remove period after 'methods' Ln124: Add a space before 'correlation' Ln127: p=0.057914; p
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
Yes: I have known one of the co-author through my meetings with them at conferences. Though I don't have any colloborative works, projects or publications with them.
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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