Identification of pathogenic Leptospira species and serovars in New Zealand using metabarcoding

David A. Wilkinson; Matthew Edwards; Jackie Benschop; Shahista Nisa

doi:10.1371/journal.pone.0257971

Peer Review History

Original SubmissionMarch 28, 2021
7 Jun 2021 Decision Letter - Kalimuthusamy Natarajaseenivasan, Editor PONE-D-21-10197 Identification of pathogenic Leptospira species and serovars in New Zealand using metabarcoding. PLOS ONE Dear Dr. Wilkinson, 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. Appropriate negative control is emphasized to validate the applied technology for the species level identification of Leptospira. Quality of the figures to be improvised. Please submit your revised manuscript by 3rd SEP 2021. 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: http://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, Kalimuthusamy Natarajaseenivasan 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. In the interests of reproducibility, in your Methods, please provide the name and location of the farm from which samples were obtained from your study. If you have been advised by an institutional data protection officer or equivalent authority that the identity of the farm should remain anonymous, please update your Data Availability Statement to mention this. 3.Thank you for stating the following in the Funding Section of your manuscript: "This project was funded by the Hawke’s Bay Medical Research Foundation and the Palmerston 386 North Medical Research Foundation. Our research into leptospirosis in New Zealand is 387 additionally supported by the Health Research Council." 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." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 4. 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. [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: Partly Reviewer #3: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: N/A Reviewer #3: N/A ****** 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 Reviewer #3: No ****** 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 Reviewer #3: 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: Overall, the manuscript is well-written. However, the manuscript could be further strengthened in describing the gap of knowledge or research problem in a clear manner and stating the challenges faced during trials prior to obtaining the results. Reviewer #2: This Research Article by Wilkinson et al, described the introduction of a new methodology to identify pathogenic Leptospira species in enriched environmental cultures. The authors identified a partial region of the glmU gene as a suitable locus for the discrimation of Leptospira species and endemic serovars of New Zealand and explored the use of this locus using the MinION sequencing platform. Since current molecular methods used for Leptospira detection are unable to discriminate species and serovars, the introduction of this technology in the field of leptospirosis will be a valuable tool for epidemiological surveys and will also increase the understanding of Leptospira diversity in New Zealand allowing the implementation of control strategies of this important zoonotic disease. Major comments: 1. The Nanopore Sequencing Technology is used for identification of bacterial communities in the field and provides real-time data enabling immediate access to results such as species identification and abundance (Urban, Holzer, et al. eLife 2021;10:e61504. DOI: https://doi.org/10.7554/eLife.61504). My major concern for this study is about the enrichment of the environmental samples by culturing in EMJH broth for 13 weeks. One important information is to know which species of Leptospira are present in the environment and at which levels. The direct sequencing of DNA from environmental samples overcomes enrichment and biases common to culturing (i.e.: some Leptospira species growth faster than others). Did the authors try to sequence glmU by using the DNA extracted from the environmental water samples (without culture enrichment)? 2. This study should include appropriate negative controls such as bacterial strains that are not expected to be detected with the designed primers (i.e. Leptospira subclade S1 and/or other environmental bacterial species) and create mixed mock communities including DNA preparations from negative controls. 3. A shorter incubation time than 13 weeks to enrich cultures will be practical from the clinical point of view if this method is going to be use for diagnosis. What is the rationale for incubating EMJH cultures 13 weeks? Detectable leptospiral DNA might be present in the enriched culture before leptospires are visible by dark field microscopy. Did the author test DNA extracted from younger cultures? 4. The taxonomy of the genus Leptospira was revisited by Vincent et al. (2019) and the genus was divided into 2 clades and 4 subclades (P1, P2, S1 and S2). Subclade P1 can further split into “virulent” and “low-virulence” pathogens defined by distinct accessory gene patterns and ability to cause or not severe infections in animals and humans (Thibeaux et al, https://doi.org/10.1099/mgen.0.000144). Virulent pathogens species (L. interrogans, L. borgpetersenii, L. kershneri, among others) are the main agents of leptospirosis, while the virulence in humans for some “low-virulence” pathogens species is still controversial. Thus, I suggest to update the classification of the genus Leptospira in Introduction (lines 50-51), and describe the Results with the proper Discussion considering the concept of “low-virulence”. Minor comments: 1. Abstract, line 38: correct “environmental samples” to “enriched environmental cultures”. The detection of Leptospira species from environmental water samples have been described (Gamage et al., doi: 10.1371/journal.pntd.0008437; Urban, Holzer, et al. eLife 2021;10:e61504. DOI: https://doi.org/10.7554/eLife.61504); thus, the proper terminology throughout the MS is recommended. 2. Abstract, line 30-31: what is the meaning of “reported cases that have not been attributed to the infecting serovar or genomospecies”? These are clinically-diagnosed cases but not laboratory-confirmed cases? Please explain. 3. Introduction, line 80: “pathogenic species”. Please specify which ones or described to which subclade they belong to. 4. Introduction, line 96. Please give a reference for “different reservoir distributions for different serovars”. 5. Introduction, line 101: correct the spelling for “Leptospira”. 6. Discussion (lines 334-344). Discuss Leptospira species and serovars by explaining the virulence of each Leptospira species in humans. 7. Discussion (lines 352-355). Vincent et al. (Reference No. 36 of the current MS) reported that the ppk gene can be used to discriminate Leptospira genomospecies. Did the authors consider this gene? Please discuss about this locus. 8. Discussion lines 368-369: “in the context of New Zealand where low genomospecies and serovar diversity”... ? Since one of the conclusion of this study is that several species are present in environmental water samples of New Zealand , and Leptospira species diversity seems to be higher than expected, the phrase “low genomospecies diversity” is contradictory. Please re-phase. Reviewer #3: The authors tried to detect pathogenic Leptospira DNA from environmental samples of New Zealand. They focused on long read DNA sequencing technology using Oxford Nanopore MinION to distinguish intra-species variations of pathogenic Leptospira. I think the aim of this study is very nice and preferable. The prior identification of a suitable marker gene glmU based on genome sequence analysis of Leptospira species is very valuable. In my opinion, however, unfortunately, the Figures of the present manuscript seems to be low quality. (1) Figure 1: OTU names is invisible and unreadable. (2) Figure 2: the correspondence between bar plot and the caption is very obscure. The green bar and the caption "Tarassovi" (?) seem to be already misaligned. The correct caption of the blue and magenta bars on the right side is not identifiable. (3) Figure 3: OTU names, names of the "Isolates", and values in the "Mock Communities Composition Ratios" are unreadable. (4) Figure 5: OTU names and captions below the heatmap panel are unreadable. The legends of the figures does not cover these insufficiencies of the figures above. Accordingly, in my view, the readers are not able to evaluate the validity of the results and discussion based on these figures. I think the manuscript is better to be rejected once, and recommended to resubmit after correction of the figures and the related things. Minor points: Line 173: the version of the MEGA X used should be described. Line 221: "amplified" would be a possible typo of "amplify." Line 222: "LipL32" may be expressed as italicized "lipL32." Line 296: "of24" seems to miss the space. Line 321: I think the s of the "16s rRNA" is usually described in uppercase. (16S rRNA; Sedimentation coefficient) Line 323: "LipL32" may be expressed as italicized "lipL32." Line 483: "... controls, results are ..." may be a typo of "... controls. Results are ...". ****** 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:** Lesley Maurice Bilung Reviewer #2: No Reviewer #3: 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.0257971.r001
Revision 1
22 Jul 2021 Author Response Dear PLOS Editor, We would like to thank you and the three reviewers of our manuscript for taking the time to appraise our work. We have been through the comments carefully and edited our manuscript accordingly. We believe that the modifications have significantly improved the clarity of the article, and we hope that the revised version of our paper can now be considered for publication in PLOS ONE. We provide point-by-point responses below. Reviewers’ comments are in red italic, our responses are in black. Yours Sincerely, Dr David A Wilkinson EDITORIAL COMMENTS: 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 We have been through the article and modified the document formatting in accordance with the PLOS ONE template. Many thanks for these guidelines. 2. In the interests of reproducibility, in your Methods, please provide the name and location of the farm from which samples were obtained from your study. If you have been advised by an institutional data protection officer or equivalent authority that the identity of the farm should remain anonymous, please update your Data Availability Statement to mention this. The owners of the farm would like its exact location to remain anonymous. We have added a statement to the Data Availability section to this effect. 3.Thank you for stating the following in the Funding Section of your manuscript: "This project was funded by the Hawke’s Bay Medical Research Foundation and the Palmerston 386 North Medical Research Foundation. Our research into leptospirosis in New Zealand is 387 additionally supported by the Health Research Council." 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." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. The funding statement has been added to the revision’s cover letter and removed from the body of the manuscript. 4. 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. Done Review Comments to the Author Reviewer #1: Overall, the manuscript is well-written. However, the manuscript could be further strengthened in describing the gap of knowledge or research problem in a clear manner and stating the challenges faced during trials prior to obtaining the results. Many thanks for this comment. We have added text to the introduction and discussion sections that make the aim of the study, its context and the issues faced in leptospirosis diagnostics in New Zealand more explicit. We hope that this has clarified those aspects that the reviewer found to be lacking. Reviewer #2: This Research Article by Wilkinson et al, described the introduction of a new methodology to identify pathogenic Leptospira species in enriched environmental cultures. The authors identified a partial region of the glmU gene as a suitable locus for the discrimation of Leptospira species and endemic serovars of New Zealand and explored the use of this locus using the MinION sequencing platform. Since current molecular methods used for Leptospira detection are unable to discriminate species and serovars, the introduction of this technology in the field of leptospirosis will be a valuable tool for epidemiological surveys and will also increase the understanding of Leptospira diversity in New Zealand allowing the implementation of control strategies of this important zoonotic disease. Many thanks for this positive assessment. We also believe that these sorts of barcoding approaches will be of great use in leptospirosis diagnostics (as highlighted recently in a paper by Guernier et al.). Major comments: 1. The Nanopore Sequencing Technology is used for identification of bacterial communities in the field and provides real-time data enabling immediate access to results such as species identification and abundance (Urban, Holzer, et al. eLife 2021;10:e61504. DOI: https://doi.org/10.7554/eLife.61504). My major concern for this study is about the enrichment of the environmental samples by culturing in EMJH broth for 13 weeks. One important information is to know which species of Leptospira are present in the environment and at which levels. The direct sequencing of DNA from environmental samples overcomes enrichment and biases common to culturing (i.e.: some Leptospira species growth faster than others). Did the authors try to sequence glmU by using the DNA extracted from the environmental water samples (without culture enrichment)? We agree with the reviewer that it is not ideal to rely on 13-week enrichment cultures for Leptospira sequencing. This is, indeed, one of the main reasons for using barcoding techniques – to bypass the need for culture-dependent methods. However, the specific samples that we tested in this study are the perfect example of why barcoding techniques can be useful when culture-dependent techniques fail. We had attempted to isolate clonal cultures from these samples for a long period of time, specifically Pacifica, without success. Sanger sequencing of PCR products gave mixed signals, and there was high cellular heterogeneity in dark field observations from these cultures. It was only by developing this barcoding technique that we were able to identify what was in these mixed cultures (without resorting to cloning PCR products). We agree that the enrichment culture is likely to have introduced biases in the distributions of different lineages in each sample, however, determining the distributions of species of Leptospira in the environment was not the primary purpose of this body of work. For this study, we were exploring the use of thie glmU locus as a molecular barcoding tool via the Oxford Nanopore Technology (ONT) sequencing platform MinION i.e. method validation rather than researching the environment. The technique itself will work on any sample from which a PCR product can be obtained. Our estimates of the limit of detection for our PCR primers suggest that this would even be true from very dilute cultures – and many environmental samples prior to enrichment. 2. This study should include appropriate negative controls such as bacterial strains that are not expected to be detected with the designed primers (i.e. Leptospira subclade S1 and/or other environmental bacterial species) and create mixed mock communities including DNA preparations from negative controls. As stated in the manuscript, negative controls were included in all limit-of-detection and mixed mock community PCR amplifications. In addition, we have additional data from animal urine and kidneys that suggests there is little or no “false positive” amplification from samples that have tested negative using alternative PCR systems such as the lipL32 system. It was not (and is not) possible for us to include Leptospira controls from other subclades, as we have no access to these strains and the rules preventing import of potential zoonotic pathogens to New Zealand are very strict. 3. A shorter incubation time than 13 weeks to enrich cultures will be practical from the clinical point of view if this method is going to be use for diagnosis. What is the rationale for incubating EMJH cultures 13 weeks? Detectable leptospiral DNA might be present in the enriched culture before leptospires are visible by dark field microscopy. Did the author test DNA extracted from younger cultures? As stated in major comment 1 above, the described method will work for any sample from which a PCR product can be obtained. The limit of detection of the PCR system was calculated, meaning we know that PCR products of sequencing quality can be obtained from samples that have concentrations as low as 10 leptospires per ml. 4. The taxonomy of the genus Leptospira was revisited by Vincent et al. (2019) and the genus was divided into 2 clades and 4 subclades (P1, P2, S1 and S2). Subclade P1 can further split into “virulent” and “low-virulence” pathogens defined by distinct accessory gene patterns and ability to cause or not severe infections in animals and humans (Thibeaux et al, https://doi.org/10.1099/mgen.0.000144). Virulent pathogens species (L. interrogans, L. borgpetersenii, L. kershneri, among others) are the main agents of leptospirosis, while the virulence in humans for some “low-virulence” pathogens species is still controversial. Thus, I suggest to update the classification of the genus Leptospira in Introduction (lines 50-51), and describe the Results with the proper Discussion considering the concept of “low-virulence”. This has been updated as requested, and we now refer to relevant subclades P1, P2, S1 and S2 throughout the manuscript. Specifically, lines 39-45 in the introduction and lines 355-356 in the discussion. Minor comments: 1. Abstract, line 38: correct “environmental samples” to “enriched environmental cultures”. The detection of Leptospira species from environmental water samples have been described (Gamage et al., doi: 10.1371/journal.pntd.0008437; Urban, Holzer, et al. eLife 2021;10:e61504. DOI: https://doi.org/10.7554/eLife.61504); thus, the proper terminology throughout the MS is recommended. We have corrected the way in which we refer to these samples and used “enriched environmental cultures” throughout. 2. Abstract, line 30-31: what is the meaning of “reported cases that have not been attributed to the infecting serovar or genomospecies”? These are clinically-diagnosed cases but not laboratory-confirmed cases? Please explain. This refers to individual cases that have been diagnosed as positive for leptospirosis through PCR – only positive/negative data is available, but no typing information is available… for example, in contrast to what would be obtained when using MAT which would give serological results against one or several specific serovars. We have slightly altered the wording of this sentence to clarify the message. 3. Introduction, line 80: “pathogenic species”. Please specify which ones or described to which subclade they belong to. We have edited this and are referring to this as the P1 species throughout the paper. 4. Introduction, line 96. Please give a reference for “different reservoir distributions for different serovars”. A suitable reference has been added to the text. 5. Introduction, line 101: correct the spelling for “Leptospira”. Done. 6. Discussion (lines 334-344). Discuss Leptospira species and serovars by explaining the virulence of each Leptospira species in humans. Thank you for raising this. Additional discussion has been added to lines 333-344. 7. Discussion (lines 352-355). Vincent et al. (Reference No. 36 of the current MS) reported that the ppk gene can be used to discriminate Leptospira genomospecies. Did the authors consider this gene? Please discuss about this locus. This is a very good point. The ppk locus is indeed a good choice for the discrimination of different Leptospira. Vincent et al. report that it is a core gene that allows the accurate reconstruction of the genome-level phylogeny. Indeed, when we investigated the discriminatory power of this locus for New Zealand strains, it does allow the identification of all known endemic human pathogenic Leptospira. However, we did not consider this locus because no conserved primer scheme has been proposed for its amplification to date (to our knowledge). Following the reviewer’s comment, we examined the locus in more detail. Upon alignment of ppk sequences from all available Leptospira genomes, we were unable to identify conserved flanking sequences that would allow for its effective use. It is possible that the use of degenerate primers may allow its amplification however, we specifically avoided the use of degenerate primers in our study – as there may exist differences in sensitivity for the different primer sequences. We have added a sentence to the discussion of the article specifically mentioning ppk as a potential locus for further investigation of metabarcoding protocols. 8. Discussion lines 368-369: “in the context of New Zealand where low genomospecies and serovar diversity”... ? Since one of the conclusion of this study is that several species are present in environmental water samples of New Zealand , and Leptospira species diversity seems to be higher than expected, the phrase “low genomospecies diversity” is contradictory. Please re-phase. This is a good point. When we refer to low Leptospira diversity, specifically we are talking about the diversity of known species associated with human disease. The phrase has been altered accordingly. Reviewer #3: The authors tried to detect pathogenic Leptospira DNA from environmental samples of New Zealand. They focused on long read DNA sequencing technology using Oxford Nanopore MinION to distinguish intra-species variations of pathogenic Leptospira. I think the aim of this study is very nice and preferable. The prior identification of a suitable marker gene glmU based on genome sequence analysis of Leptospira species is very valuable. Many thanks for these positive comments. In my opinion, however, unfortunately, the Figures of the present manuscript seems to be low quality. (1) Figure 1: OTU names is invisible and unreadable. (2) Figure 2: the correspondence between bar plot and the caption is very obscure. The green bar and the caption "Tarassovi" (?) seem to be already misaligned. The correct caption of the blue and magenta bars on the right side is not identifiable. (3) Figure 3: OTU names, names of the "Isolates", and values in the "Mock Communities Composition Ratios" are unreadable. (4) Figure 5: OTU names and captions below the heatmap panel are unreadable. The legends of the figures does not cover these insufficiencies of the figures above. Accordingly, in my view, the readers are not able to evaluate the validity of the results and discussion based on these figures. I think the manuscript is better to be rejected once, and recommended to resubmit after correction of the figures and the related things. I am sorry that you were unable to interpret the figures for the manuscript. A major reason for this may be due to the revision system, which can often generate low quality figures due to the pdf conversion process. Given that other reviewers were able to read and interpret the figures, I believe this to be a conversion issue and not an actual issue with the figures. However, in an attempt to make the figures more legible, we have increased font sizes where possible and corrected any errors that have been pointed out. We have also submitted vector format .eps files for each figure, which should allow PLOS to provide a high-resolution figure when in print. Hopefully this will make the figures easier to interpret. Minor points: Line 173: the version of the MEGA X used should be described. The version number has been added to the text. Line 221: "amplified" would be a possible typo of "amplify." Well spotted. This has been changed. Line 222: "LipL32" may be expressed as italicized "lipL32." We have italicized lipL32 whenever it has been used to refer to a specific genetic locus in the text. Line 296: "of24" seems to miss the space. Changed. Line 321: I think the s of the "16s rRNA" is usually described in uppercase. (16S rRNA; Sedimentation coefficient) Absolutely correct. We have changed the text. Line 323: "LipL32" may be expressed as italicized "lipL32." We have italicized lipL32 whenever it has been used to refer to a specific genetic locus in the text. Line 483: "... controls, results are ..." may be a typo of "... controls. Results are ...". Changed. Attachments Attachment Submitted filename: rebuttal_FINAL.docx https://doi.org/10.1371/journal.pone.0257971.r002
15 Sep 2021 Decision Letter - Kalimuthusamy Natarajaseenivasan, Editor Identification of pathogenic Leptospira species and serovars in New Zealand using metabarcoding. PONE-D-21-10197R1 Dear Dr. Wilkinson, 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, Kalimuthusamy Natarajaseenivasan 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 Reviewer #2: All comments have been addressed Reviewer #3: (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: (No Response) Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: (No Response) Reviewer #3: N/A ****** 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: (No Response) Reviewer #3: 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: (No Response) Reviewer #3: 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) Reviewer #3: I think that the authors adequately addressed the concerns from the reviewers regarding previous version of the manuscript. It seems that the revised version of the manuscript is now acceptable. Only the following minor points, I hope that authors will confirm. I think this potential correction could be done after acceptance of the paper, at the stage of proof production: (1) The digit expression, please re-check and unify: Line 120: at 8 000 x ... Line 134: from 200 000 to ... Line 191: 1500 fmol Line 237: of 1:10240 while ... Line 238: of 1:1280 ... Line 357: the 2138 bp ppk ... ****** 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 Reviewer #2: No Reviewer #3: No https://doi.org/10.1371/journal.pone.0257971.r003
Formally Accepted
20 Sep 2021 Acceptance Letter - Kalimuthusamy Natarajaseenivasan, Editor PONE-D-21-10197R1 Identification of pathogenic Leptospira species and serovars in New Zealand using metabarcoding. Dear Dr. Wilkinson: 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. Kalimuthusamy Natarajaseenivasan Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0257971.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 .