Examining the bacterial diversity including extracellular vesicles in air and soil: implications for human health

Hyunjun Yun; Ji Hoon Seo; Yoon-Keun Kim; Jinho Yang

doi:10.1371/journal.pone.0320916

Peer Review History

Original SubmissionOctober 29, 2024
29 Oct 2024 Author Response https://doi.org/10.1371/journal.pone.0320916.r001
27 Jan 2025 Decision Letter - Jian Xu, Editor PONE-D-24-49243Examining the Bacterial Diversity including Extracellular Vesicles in Air and Soil: Implications for Human HealthPLOS ONE Dear Dr. Yang, 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 submit your revised manuscript by Mar 13 2025 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, Jian Xu, Ph.D. 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. Thank you for stating the following financial disclosure: “This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (RS-2023-00244833).” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. [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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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: Dear editor, I have reviewed the paper titled “Examining the Bacterial Diversity including Extracellular Vesicles in Air and Soil: Implications for Human Health”. This study aims to examine the bacteria and BEV microbiota and predict functional pathways in aerosol based on 16S rRNA gene sequencing in outdoor air and mountain soil through a field study. The results indicate that the biodiversity of bacteria and BEVs differ between air and soil. This study provides a reference point for the impact of microorganisms on human health. However, while the study provides insights, there is a need to be revised to improve overall quality. The detailed comments are in the following. 1. Page 4, Line 78, “impairment in in vivo tests”, please check that. 2. Please give the novelty or hypothesis in the end of Introduction. 3. Page 5, Line 112, why emphasize that it's a sunny summer day? 4. Page 7, Line 143-144, the sequencing length in this manuscript ranges from 350bp to 550bp. Is this data based on other references or is it filtered through your own sequences? 5. Page 7, Line 160-162, the meaning of this statement is that the data needs to be rarefied before performing alpha diversity analysis? Please rewrite this description to make it clearer. 6. Page 7, Line 166, the “between” should be “among”. 7. The “Materials and Methods” only has a small section on macrogenomes and some description could be added. 8. Page 10, Line 232, significance here is not reflected in the data. Is there an additional significance test? 9. Discussions about specific bacteria in air and soil are similar to the description of the results. Discussion is not a conclusion. In addition, “environmental factors strongly influence the microbiome” is based on a summary of other literatures, and there are no experiments in this manuscript. Is it possible to add some. 10. Similarly, the discussion of human health is not effectively integrated with the conclusions of this manuscript. It is not enough to summarize that “it is important to analyze the BEV microbiome for human health”. Please resolve this discrepancy. 11. Page 13-14, Line 308-329, the discussion of aerosol in this place is a bit abrupt and some introduction could be added to the “Introduction” section. 12. Figure can be optimized a bit more, such as colors. 13. The author should embellish some expressions. Please check the format and grammar of writing carefully. ****** 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: 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.0320916.r002
Revision 1
14 Feb 2025 Author Response We sincerely appreciate your thorough review of our manuscript, “Examining the Bacterial Diversity including Extracellular Vesicles in Air and Soil: Implications for Human Health”. Your insightful comments and suggestions have been invaluable in refining our study. Based on your feedback, we have carefully revised the manuscript to enhance its clarity, structure, and scientific rigor. Below, we provide a detailed response to your comments, highlighting the specific modifications made in the revised version. comment1 Page 4, Line 78, “impairment in in vivo tests”, please check that. response1 Thank you for pointing this out. We acknowledge the redundancy in the phrase "impairment in in vivo tests". We have revised this sentence for clarity and conciseness. The updated sentence now reads: Revised sentence (Page 4, Lines 78): "impairment observed in in vivo tests." comment2 Please give the novelty or hypothesis in the end of Introduction. response2 Thank you for your suggestion. We have revised the final paragraph of the Introduction section (Page 4, Lines 102-106) to explicitly highlight the novelty of our study and clarify the hypothesis. Revised sentence (Page 5, Lines 103-111): "While previous studies have examined airborne and soil microbiomes separately, there has been no in-depth study comparing bacterial communities and bacterial extracellular vesicles (BEVs) between these two environments. Furthermore, little is known about the functional roles of BEVs in environmental microbiomes and their potential implications for human health. Therefore, this study aims to provide a comprehensive analysis of bacterial and BEV microbiota in air and soil using 16S rRNA sequencing, with a specific focus on their biodiversity and predicted functional pathways. We hypothesize that the bacterial community composition and functional pathways of BEVs differ significantly between air and soil, which may influence human health in distinct ways." comment3 Page 5, Line 112, why emphasize that it's a sunny summer day? response3 Thank you for your comment. We acknowledge that specifying “sunny summer day” may not be necessary unless weather conditions significantly impact microbial composition. The intent was to indicate that sampling was conducted in the absence of extreme weather conditions, such as rain or strong winds, which could influence bioaerosol concentrations. To improve clarity, we have revised the sentence as follows: Revised sentence (Page 6, Line 116-118): "Ambient air sampling was conducted on the rooftop of a building during a period without precipitation or strong winds, after obtaining access permission." comment4 Page 7, Line 143-144, the sequencing length in this manuscript ranges from 350bp to 550bp. Is this data based on other references or is it filtered through your own sequences? response4 Thank you for your insightful question. The sequencing length in this manuscript (ranging from 350 bp to 550 bp) was determined based on both established guidelines and our own sequence filtering criteria. As noted in [36], taxonomic identification of bacteria via 16S rRNA sequencing generally requires at least 300 bp of high-quality sequence data. Our sequencing approach ensures that we capture at least 350 bp, which includes two phylogenetically informative variable regions of the 16S rRNA gene. This strategy is in line with the '16S center primer method' described in [36], which enhances sequencing accuracy and phylogenetic resolution. Illumina recommends targeting regions that result in an amplicon that when sequenced with paired‐end reads has at least ~50 bp of overlapping sequence in the middle. For example, if running 2x300 bp paired‐end reads Illumina recommends having an insert size of 550 bp or smaller so that the bases sequenced at the end of each read overlap [16S Metagenomic Sequencing Library Preparation Guide, Illumina]. comment5 Page 7, Line 160-162, the meaning of this statement is that the data needs to be rarefied before performing alpha diversity analysis? Please rewrite this description to make it clearer. response5 Thank you for your question. To improve clarity, we have revised the statement as follows: Revised sentence (Page8, Lines 167-169) "To adjust for uneven sequencing depth among the samples, the samples were rarefied to even depths of 10,000 before diversity analysis." comment6 Page 7, Line 166, the “between” should be “among”. response6 Thank you for pointing this out. We have revised it as follows: Revised sentence (Page 8, Line 172): "Correlations among microbiome, functional pathways, and orthologous groups were analyzed using Pearson correlation coefficients. All statistical analyses were performed using R version 4.3.2." comment7 The “Materials and Methods” only has a small section on macrogenomes and some description could be added. response7 Thank you for your valuable comment. In this study, we focused on 16S rRNA-based metagenomic analysis, rather than whole macrogenome sequencing. Our primary objective was to characterize the bacterial community structure and functional potential in airborne and soil environments using taxonomic profiling and predictive functional analysis (Tax4Fun). We acknowledge that macrogenomic analysis could provide additional insights into the genetic potential of microbial communities. However, given the scope and objectives of this study, we opted for a 16S rRNA approach to efficiently compare bacterial and extracellular vesicle (BEV) compositions. Future studies may incorporate whole-genome sequencing to further explore microbial functional capacities in greater detail. We have clarified this point in the Materials and Methods section to avoid potential misunderstandings. Revised sentence (Page 7, Line 136-141): “In this study, we employed 16S rRNA-based metagenomic analysis to characterize bacterial communities rather than whole macrogenome sequencing. This approach was chosen to efficiently compare bacterial and BEV compositions in airborne and soil environments. Bacterial genomic DNA targeting the V3–V4 hypervariable regions of the 16S rRNA gene was amplified with the 16s_V3_F and 16s_V4_R primers [34,35].” comment8 Page 10, Line 232, significance here is not reflected in the data. Is there an additional significance test? response8 Thank you for your valuable comment. We have reflected the significance in Figure 4. comment9 Discussions about specific bacteria in air and soil are similar to the description of the results. Discussion is not a conclusion. In addition, “environmental factors strongly influence the microbiome” is based on a summary of other literatures, and there are no experiments in this manuscript. Is it possible to add some. response9 Thank you for your valuable feedback. We have revised the Discussion to focus more on interpretation, comparisons with previous studies, and the significance of our findings in the context of environmental microbiology and human health. The revised sections are highlighted in red. • Line 256-265 • Line 269-272 • Line 282-296 • Line 325-330 • Line 353-357 comment10 Similarly, the discussion of human health is not effectively integrated with the conclusions of this manuscript. It is not enough to summarize that “it is important to analyze the BEV microbiome for human health”. Please resolve this discrepancy. response10 Thank you for your insightful comment. To better integrate the discussion on human health with the conclusions, we have revised sections where we discuss the implications of BEVs and bacterial communities in air and soil on health. • Line 282-296 • Line 379-388 comment11 Page 13-14, Line 308-329, the discussion of aerosol in this place is a bit abrupt and some introduction could be added to the “Introduction” section. response11 Thank you for your insightful comment. To improve the logical flow of the manuscript, we have revised the Introduction to provide better context for aerosol exposure. Revised sentence (Page 3, Lines 52-59): "Recently, interest in the microbiome has shifted from composition to functional roles in human health. Furthermore, public concern for ultrafine particles, including bioaerosols, has increased due to their ability to remain airborne for extended periods and penetrate deep into the respiratory tract. Among these bioaerosols, bacterial extracellular vesicles (BEVs) are emerging as key factors in airborne microbial exposure, potentially influencing immune responses and respiratory health [7, 8]. Understanding the differences between airborne and soil microbiomes, including BEVs, is critical for assessing their environmental and health implications." comment12 Figure can be optimized a bit more, such as colors. response12 Thank you for your valuable comment. We have optimized the figures by adjusting the colors for better visual clarity and enhancing the readability of significance levels through appropriate annotations. comment13 The author should embellish some expressions. Please check the format and grammar of writing carefully. response13 Thank you for your valuable feedback. We have carefully reviewed the manuscript for grammatical accuracy, formatting consistency, and overall clarity. In response to your suggestion, we conducted a thorough revision of the entire manuscript, making necessary corrections to improve readability and ensure precise scientific communication. Attachments Attachment Submitted filename: Response to comments.docx https://doi.org/10.1371/journal.pone.0320916.r003
27 Feb 2025 Decision Letter - Jian Xu, Editor Examining the Bacterial Diversity including Extracellular Vesicles in Air and Soil: Implications for Human Health PONE-D-24-49243R1 Dear Dr. Yang, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Jian Xu, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): 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 ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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) ****** 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: Dear editor, Thank you for inviting me to review the revision “Examining the Bacterial Diversity including Extracellular Vesicles in Air and Soil: Implications for Human Health”. I have carefully read the revised manuscript submitted by the authors and found that significant improvements have been made in response to the previous review comments. ****** 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: No ******** https://doi.org/10.1371/journal.pone.0320916.r004
Formally Accepted
Acceptance Letter - Jian Xu, Editor PONE-D-24-49243R1 PLOS ONE Dear Dr. Yang, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Jian Xu Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0320916.r005

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 .