Antibiotic resistant bacteria and commensal fungi are common and conserved in the mosquito microbiome

Josephine Hyde; Courtney Gorham; Doug E. Brackney; Blaire Steven

doi:10.1371/journal.pone.0218907

Peer Review History

Original SubmissionJune 10, 2019
16 Jul 2019 Decision Letter - Patrick Butaye, Editor PONE-D-19-16438 Antibiotic resistant bacteria and commensal fungi are common and conserved in the mosquito microbiome PLOS ONE Dear Dr Steven, 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. Both reviewers are positive about your manuscript. I leave it p to you if you want to have the fungi included yes or no. Please reply to the other comments. We would appreciate receiving your revised manuscript by Aug 30 2019 11:59PM. When you are 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. 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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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A 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: This reviewer enjoyed very much in reading this excellent manuscript. This work combines the fields of the vector-borne diseases, antimicrobial resistance and microbiology. The manuscript is well written, and the conclusion is well supported by the data. Main comments: 1) I strongly feel like that the portion of commensal fungi can be left out for another independent publication. Please focus on the beauty of this work, antimicrobial resistance and mosquitoes. Do not get distracted by the addition of fungi, in which you did not get any isolates, and cannot characterize the nature of their antimicrobial resistance; 2) For the field-caught mosquitoes, did you try to wash the mosquitoes before performing bacterial isolation etc? There are constant contact between mosquitoes and animal hosts/environmental surfaces etc. The identified microbiome could be on the surface of the mosquitoes, or their inside? Please provide more information, and make meaningful discussion; 3) As indicated in Fig 2 (Schematic diagram of bacterial culturing), you used LB initially, followed by the utilization of different selective media. Please justify this approach. What would you get if you would use the selective media directly without the use of LB media? Even LB is not a type of selective media, the process of LB culture may still provide some preference of certain bacteria which overgrow over others; 4) It is great that the authors used the bioinformatics to identify bacterial OUTs in this work. In the meantime, it would be more clinically more important and meaningful if you could identify the genus level and even species level of the isolated bacterial from the antibiotics-resistant plates. You are very close to provide this valuable information since these isolates are available to you, and you can even ID them by the use of the whole-length 16S rRNA sequencing. Reviewer #2: Well done paper. On line 95 I think you are missing the word resistant - it states antibiotic populations and I think you meant to say antibiotic resistant populations. That is the only change that I have. ****** 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: Chengming Wang Reviewer #2: Yes: James F Lowe [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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0218907.r001
Revision 1
18 Jul 2019 Author Response Reviewer #1: This reviewer enjoyed very much in reading this excellent manuscript. This work combines the fields of the vector-borne diseases, antimicrobial resistance and microbiology. The manuscript is well written, and the conclusion is well supported by the data. Response: We would like to thank the reviewer for their kind comments. Main comments: 1) I strongly feel like that the portion of commensal fungi can be left out for another independent publication. Please focus on the beauty of this work, antimicrobial resistance and mosquitoes. Do not get distracted by the addition of fungi, in which you did not get any isolates, and cannot characterize the nature of their antimicrobial resistance; Response: We feel that the fungal data should remain in the current manuscript. Our reasoning is as follows: These data are based on isolates. As we state in line 356 “through our efforts to culture antibiotic resistant bacteria we opened up a niche to culture fungal isolates from the mosquitoes”. We hypothesize that we recovered these fungal isolated by reducing the bacterial load on the antibiotic plates. We did not actually set out to culture fungi. In this regard our base media (LB) was not optimized for fungal recovery, as such the identification of fungi was a spurious finding in this study, not an objective. This data would not likely be sufficient for an independent manuscript. 2) For the field-caught mosquitoes, did you try to wash the mosquitoes before performing bacterial isolation etc? There are constant contact between mosquitoes and animal hosts/environmental surfaces etc. The identified microbiome could be on the surface of the mosquitoes, or their inside? Please provide more information, and make meaningful discussion; Response: This is something that we took care to control for but failed to mention in the methods. We have revised the methods to read “Prior to culturing individuals were washed in a solution of 90% ethanol to remove external adhering bacteria.” line 148 3) As indicated in Fig 2 (Schematic diagram of bacterial culturing), you used LB initially, followed by the utilization of different selective media. Please justify this approach. What would you get if you would use the selective media directly without the use of LB media? Even LB is not a type of selective media, the process of LB culture may still provide some preference of certain bacteria which overgrow over others; Response: The selective plates were LB plates supplemented with antibiotics. The purpose of this was to determine the proportion of antibiotic resistant cells in the “total” population. To avoid confusion we have added “LB media supplemented with antibiotics” to the figure legend of Figure 2 to make it clear that the media formulation is the same between all of the plates. We agree with the reviewer that only using LB media likely influenced the bacteria that we recovered. We have added the following “However, as all culturing was performed on LB media these data likely only represent a proportion of the total bacterial diversity and favor fast growing bacterial species” to the results. 4) It is great that the authors used the bioinformatics to identify bacterial OUTs in this work. In the meantime, it would be more clinically more important and meaningful if you could identify the genus level and even species level of the isolated bacterial from the antibiotics-resistant plates. You are very close to provide this valuable information since these isolates are available to you, and you can even ID them by the use of the whole-length 16S rRNA sequencing. Response: While we agree with the reviewer that full-length sequencing could improve some of the ability to discern OTU’s, we argue that performing full length sequencing is not warranted for the following reasons. 1. Even full-length 16S rRNA gene sequencing is not generally considered to be sufficient to differentiate species or strains. This level of discrimination is generally performed by multi-locus sequencing, DNA-DNA reassociation kinetics, or whole genome sequencing, which is beyond the scope of this study. 2. Even with the short fragment that we sequenced ~75% of the isolates were classified to genus with >80% confidence (line 232). In this respect, there is likely to be little improvement in our data with full-length gene sequencing. 3. We have dedicated a section of our discussion (line 356) to discuss the potential limitations of using a short sequence fragment, so have already addressed this concern in the manuscript. Reviewer #2: Well done paper. On line 95 I think you are missing the word resistant - it states antibiotic populations and I think you meant to say antibiotic resistant populations. That is the only change that I have. Response: The suggested change has been made. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0218907.r002
1 Aug 2019 Decision Letter - Patrick Butaye, Editor Antibiotic resistant bacteria and commensal fungi are common and conserved in the mosquito microbiome PONE-D-19-16438R1 Dear Dr. Steven, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Patrick Butaye, DVM, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0218907.r003
Formally Accepted
5 Aug 2019 Acceptance Letter - Patrick Butaye, Editor PONE-D-19-16438R1 Antibiotic resistant bacteria and commensal fungi are common and conserved in the mosquito microbiome Dear Dr. Steven: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Patrick Butaye Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0218907.r004

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