The effects of antibiotics and illness on gut microbial composition in the fawn-footed mosaic-tailed rat (Melomys cervinipes)

Tasmin L. Rymer; Neville Pillay

doi:10.1371/journal.pone.0281533

Peer Review History

Original SubmissionOctober 16, 2022
14 Nov 2022 Decision Letter - Mohammad Tauqeer Alam, Editor PONE-D-22-28589The effects of antibiotics and illness on gut microbial composition in the fawn-footed mosaic-tailed rat (Melomys cervinipes)PLOS ONE Dear Dr. Rymer, 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 go through the reviewers comments and address their concerns specially about the animal health and obvious microbial changes due to antibiotic treatment. Please submit your revised manuscript by Dec 29 2022 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, Mohammad Tauqeer Alam, PhD Academic Editor PLOS ONE Journal requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. 3. As part of your revision, please complete and submit a copy of the Full ARRIVE 2.0 Guidelines checklist, a document that aims to improve experimental reporting and reproducibility of animal studies for purposes of post-publication data analysis and reproducibility: https://arriveguidelines.org/sites/arrive/files/documents/Author%20Checklist%20-%20Full.pdf Please include your completed checklist as a Supporting Information file. Note that if your paper is accepted for publication, this checklist will be published as part of your article. 4. Thank you for stating the following financial disclosure: “The author(s) received no specific funding for this work. TR received general funding from James Cook University, and several donors contributed to a crowd funding campaign. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” At this time, please address the following queries: a) Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution. b) State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” c) If any authors received a salary from any of your funders, please state which authors and which funders. d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 5. Thank you for stating the following in the Acknowledgments Section of your manuscript: “We would like to thank James Cook University College of Science and Engineering for funding this project and to all the contributors in a Pozible.com crowd-funding campaign. We are grateful to numerous volunteers for their assistance in maintaining the mosaic-tailed rat colony. Special thanks to Dr Misha Rowell for her care of the colony during the illness, and for the collection of faecal samples.” We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: “The author(s) received no specific funding for this work. TR received general funding from James Cook University, and several donors contributed to a crowd funding campaign. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 6. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Additional Editor Comments: Dear Dr. Rymer, The review process of your manuscript is now complete. The reviewers have found your manuscript interesting and it has several good results. Also, the experimental design of the study and follow-up analysis approaches were appropriate. They did however raise several important issues. Additionally, both reviewers have suggested modifying the results and conclusions sections and briefly mentioning the animal health, and discussing the obvious shift in microbial structure due to antibiotic treatments. Given the concerns and suggestions from both reviewers, unfortunately, we can not accept your manuscript in its current form. However, if you can address the reviewer's comment then we will be happy to consider the revised manuscript. With best regards [Note: HTML markup is below. Please do not edit.] 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: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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 ****** 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 ****** 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 paper described and quantified gut bacterial community composition changes in response to illness and treatment with antibiotics in a native Australian rodent, the fawn-footed mosaic-tailed rat (Melomys cervinipes), mainly by high throughput genome sequencing. This study seems to be an extension of a previously published study on a mosaic-tailed rat and builds on additional data on the dynamics of gut bacterial community composition in response to illness and treatment with antibiotics, and it is adequately planned, executed, and well-presented. NGS data could have been explored more with much deeper insight into the reason for changes in the gut bacterial community. In addition, it would have been interesting to include antibiotic-resistant bacteria (ARB) and genes (ARG), including functional analysis of bacterial community. Reviewer #2: The authors summarized the effects of antibiotic treatment and 1-year post-treatment on gut microbial diversity in mosaic-tailed wild and captive rats. Overall, the manuscript is well-organized and well-written. Although the study was opportunistic having no controls and no significant rationale, my suggestion is to provide more detailed information on the overall health of the animal because of changes in gut microbial diversity. It would be worth adding some conclusions and suggestions for future studies on the basis of your observation otherwise this data is just information about different microbial species decreasing and increasing with antibiotics treatment. This is very obvious that gut microbial species change with antibiotics treatment and regained new homeostasis after some time with normal feed. It must be taken into account in the results section of the study to show the most important results that the research reached without going into excessive detail. In terms of the well-being of animals kept under captive conditions (it is one of the points highlighted in the article), authors must suggest how to manage the health of animals after antibiotic treatment according to their research results. ****** 6. 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: MUNAWWAR ALI KHAN Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0281533.r001
Revision 1
4 Jan 2023 Author Response We have carefully considered the comments and respond to each comment below in bold. Where line numbers are provided, these refer to the line numbers in the unmarked manuscript (labelled “Manuscript”). Responses to journal requirements: 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. We have followed the style requirements as requested. 2. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. We originally included an ethical note in the methods sections (L108-123). We now include a statement that no animals were sacrificed for this experiment (L121-122), and that all deaths were natural a consequence of the undiagnosed illness (L122-123). In the original manuscript, we stated that all animals received antibiotics (L137-139). No anaesthesia or analgesia was used/administered. We have added that sick individuals were transported to a veterinarian once symptoms started, but four died prior to receiving treatment (L118-120). As stated, all animals received antibiotics, which alleviated suffering (we stated on L120-121 that they all recovered completely). 3. As part of your revision, please complete and submit a copy of the Full ARRIVE 2.0 Guidelines checklist, a document that aims to improve experimental reporting and reproducibility of animal studies for purposes of post-publication data analysis and reproducibility: https://arriveguidelines.org/sites/arrive/files/documents/Author%20Checklist%20-%20Full.pdf Please include your completed checklist as a Supporting Information file. Note that if your paper is accepted for publication, this checklist will be published as part of your article. We have completed the checklist as requested. 4. a) Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution. James Cook University provides researchers with facilities such as a laboratory, access to printing, access to the internet and research databases (as similar). This is what is meant by “general funding”. No grant or organization supported this work per se. This funding helped support the maintenance of the colony and provided some funds towards several projects. b) State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” We stated this originally on submission. We state again: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. c) If any authors received a salary from any of your funders, please state which authors and which funders. No authors received a salary from any funders. d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.” We stated this originally on submission. We state again: “The authors received no specific funding for this work.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Included in the above queries as requested. 5. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. We have removed this information from the Acknowledgements section. Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Please see below amended statement: “The author(s) received no specific funding for this work. TR received general funding (e.g. access to a research laboratory, printing, research databases, etc.) from James Cook University, and several donors contributed to a crowd funding campaign that generated general research funding (e.g. not specific to this project, but allowed for the colony to be maintained). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No authors received a salary from any funders.” 6. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Included as requested and provided in-text citations to match as provided in the guidelines. Additional Editor Comments: The review process of your manuscript is now complete. The reviewers have found your manuscript interesting and it has several good results. Also, the experimental design of the study and follow-up analysis approaches were appropriate. They did however raise several important issues. Additionally, both reviewers have suggested modifying the results and conclusions sections and briefly mentioning the animal health, and discussing the obvious shift in microbial structure due to antibiotic treatments. Thank you for the comments. We have responded to each reviewers’ comments below in bold. Reviewers' comments: 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: No We have addressed each of the reviewers’ comments below each in bold. 5. Review Comments to the Author Reviewer #1: The paper described and quantified gut bacterial community composition changes in response to illness and treatment with antibiotics in a native Australian rodent, the fawn-footed mosaic-tailed rat (Melomys cervinipes), mainly by high throughput genome sequencing. This study seems to be an extension of a previously published study on a mosaic-tailed rat and builds on additional data on the dynamics of gut bacterial community composition in response to illness and treatment with antibiotics, and it is adequately planned, executed, and well-presented. Thank you for the comments. There have been several other studies on mosaic-tailed rats in this colony. However, this is the first study to explore the gut bacterial community, so we have no prior knowledge or data as suggested. NGS data could have been explored more with much deeper insight into the reason for changes in the gut bacterial community. This study was opportunistic. As we have no specific controls, we are unable to state with certainty that the responses are seeing are definitively a consequence of the antibiotic treatment, the illness itself or a combination of both. Given the broad approach taken to this study, we do not feel that we can provide more insight into the reason for the changes. Rather, we document the changes at the taxonomic level, and suggest that future studies could explore these changes with more controlled experiments. We have included a bit more information on the overall variation in biodiversity indices, as well as some specific bacterial species variations at each time period (L244-251, L486-514, L630-666). In addition, it would have been interesting to include antibiotic-resistant bacteria (ARB) and genes (ARG), including functional analysis of bacterial community. 25 bacterial species were identified through the ACE pipeline analysis. Of these, only 6 occurred in both time periods. We discuss one of these, Clostridioides difficile (L657-666). Furthermore, 2 bacterial species were identified to only occur during the period of treatment, namely Pseudomonas aeruginosa and Stenotrophomonas maltophilia (L633-649). We provide a discussion on these two species. During the period following treatment, while we identified some potentially interesting pathogenic, antibiotic resistant bacteria, their low occurrence and presence in only a few individuals suggests no relationship to this particular incidence (L650-656). Given the nature of the study, and the fact that we cannot state definitively the cause of changes in the gut microbiota, we conducted a simple analysis using phyloseq to explore whether there were differences in biodiversity indices of species between the two time periods, and then explored the aforementioned species in more detail, looking very briefly at functional pathways and genes (L244-251; L630-666). Reviewer #2: The authors summarized the effects of antibiotic treatment and 1-year post-treatment on gut microbial diversity in mosaic-tailed wild and captive rats. Overall, the manuscript is well-organized and well-written. Although the study was opportunistic having no controls and no significant rationale, my suggestion is to provide more detailed information on the overall health of the animal because of changes in gut microbial diversity. Thank you for the comments. We have included that sick animals showed weight loss (associated with inappetence and diarrhoea as already stated) but no other symptoms were observed (L93-94). We have provided some additional information in the methods on healthy animals (L97-99). We have also provided information on what occurred once symptoms were detected in the first four individuals (L119-119). It would be worth adding some conclusions and suggestions for future studies on the basis of your observation otherwise this data is just information about different microbial species decreasing and increasing with antibiotics treatment. In the original manuscript, we stated the following (L685-689): “Future studies on the gut microbial composition of wild fawn-footed mosaic-tailed rats will be useful for understanding how captivity affects the microbiome independently of illness and antibiotic treatment, providing new insights into the effective management of this species in captivity.” We have now added “and related species” to this sentence. We also add (L689-691): “Furthermore, studies with carefully considered controls and deliberate manipulation of antibiotics could equally provide insights into the specific reasons for why we observed changes in the gut microbial communities of mosaic-tailed rats.” This is very obvious that gut microbial species change with antibiotics treatment and regained new homeostasis after some time with normal feed. It must be taken into account in the results section of the study to show the most important results that the research reached without going into excessive detail. Unfortunately, because this was an opportunistic study, we could not provide controls to definitively state whether the changes in community composition were a consequence of the illness, the antibiotics or both. Therefore, we chose to be cautious in our presentation and discussion of the results. We feel that we have dealt appropriately with the data at each taxonomic level at a level that is sufficient for this opportunistic study. However, we have added in some information on changes in abundance and more information on some specific species (L486-514, L630-666). If the reviewer can provide more specific recommendations on what they consider should be included and how, we will gladly consider this. In terms of the well-being of animals kept under captive conditions (it is one of the points highlighted in the article), authors must suggest how to manage the health of animals after antibiotic treatment according to their research results. We have provided some additional information as requested (L686-680). Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0281533.r002
26 Jan 2023 Decision Letter - Mohammad Tauqeer Alam, Editor The effects of antibiotics and illness on gut microbial composition in the fawn-footed mosaic-tailed rat (Melomys cervinipes) PONE-D-22-28589R1 Dear Dr. Rymer, 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. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Mohammad Tauqeer Alam, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Dear Dr Tasmin, The review process of your manuscript is now complete. I am pleased to see that the reviewers are happy with the revised version of the manuscript. We would like to thank you for addressing the comments in the review process! Kind regards 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) ******** 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: Partly ****** 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? 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 ****** 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 ****** 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: Thank you for revising the manuscript and addressing a few minor concerns in the previously submitted manuscript. I hope this study will lay the foundation for future in-depth research on similar or closely related study. Reviewer #2: (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 #1: Yes: MUNAWWAR ALI KHAN Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0281533.r003
Formally Accepted
14 Feb 2023 Acceptance Letter - Mohammad Tauqeer Alam, Editor PONE-D-22-28589R1 The effects of antibiotics and illness on gut microbial composition in the fawn-footed mosaic-tailed rat (Melomys cervinipes) Dear Dr. Rymer: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Mohammad Tauqeer Alam Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0281533.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .