Genetic characterization of Bacillus anthracis strains circulating in Italy from 1972 to 2018

Valeria Rondinone; Luigina Serrecchia; Antonio Parisi; Antonio Fasanella; Viviana Manzulli; Dora Cipolletta; Domenico Galante

doi:10.1371/journal.pone.0227875

Peer Review History

Original SubmissionAugust 20, 2019
21 Sep 2019 Decision Letter - Wendy C. Turner, Editor PONE-D-19-23489 Genetic characterization of Bacillus anthracis strains circulating in Italy from 1972 to 2018 PLOS ONE Dear Dr. Galante, 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. The reviewers provide important suggestions on clarifying both the methodology used and the interpretation of results regarding the ancestry of strains within Italy, and the occurrence/movements of these sub-groups regionally/globally. We would appreciate receiving your revised manuscript by Nov 05 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Wendy C. Turner Academic Editor PLOS ONE Journal Requirements: 1. When submitting your revision, we need you to address these additional requirements. 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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We ask that you please explain in more detail the human samples used in this study: i) Please clarify whether these are bacterial isolates only or some type of tissue or human fluid/excretion sample. ii) If they are human tissue/excretion samples, please explain how they were collected, who collected them, and whether they were anonymized when you received them. iii) Please also explain whether you obtained Institutional Review Board approval or ethics committee approval for the use of human tissue/material. In addition, we noticed you have some minor occurrence of overlapping text with previous publications: https://scielosp.org/article/aiss/2014.v50n2/192-195/en/ http://dx.doi.org/10.4236/ojvm.2012.22012 which needs to be addressed. In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed. [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 Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: 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: No 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: No ****** 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: Thank you for an interesting paper. The description of the genotypic distribution of B. anthracis isolates across Italy adds to the body of work which highlights the diversity of this pathogen internationally. That said, please be careful of your repetition of the word "results" in regards to canSNP lineages. I have made some editorial suggestions in comment boxes. Also, please check your references, especially with regards to the PCR's used. You referred to real time PCR and then referenced Fasanella et al. which describes conventional PCR. You must credit the designers of the primers used in your methodology references. You did not refer to Patra and Ramisse for the pag, lef, cap and BA813 primers. You also did not list which primers exactly were used. If indeed you did use qPCR in diagnostics this study, please state the chemistry employed; if not...adjust the method description accordingly. Would it be possible to add more detail surrounding the history of the isolates? Your references were only in relation to the epizootics involving tabanids/horseflies. More context would help flesh out your discussion and provide more evidence for solid conclusions. As it stands, you cannot conclude a common ancestor of strains using a limited panel of canSNP's and MLVA. If there is whole genome data to support this, you should refer to it. Your speculation around the introduction of the A.Br.005/006 also needs stronger evidence before you can call it a viable hypothesis. Otherwise, just call it speculation. Reviewer #2: The manuscript is sound, although they need to drop the term phylogenetic tree from their UPGMA analysis. Not much analysis in this paper overall, mostly descriptive in scope. Really needs some serious grammatical work. ****** 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 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 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. Attachments Attachment Submitted filename: PONE-D-19-23489_reviewer (2).pdf Attachment Submitted filename: PLOS_review_SB_Sep_18.docx https://doi.org/10.1371/journal.pone.0227875.r001
Revision 1
7 Nov 2019 Author Response Dear Editorial Office, we made and submitted the corrections required by the Journal and by the reviewers. The manuscript was revised by a professional English proofreading service as the revievers asked. Thank you for the precious suggestions and observations. We submitted the revised manuscript following PLOS ONE's style requirements and we replaced the overlapping text with previous publications as you asked. The human samples we used in this study are just 3 DNAs coming from people affected by cutaneous anthrax for genotyping analyses. We received them in anonymous form. We described the origin in the manuscript. Reviewer 1: We answered the questions directly in the pdf file he sent and in a separate word file. However we changed the reference he asked and we better described the primers used in the manuscript. We disecribed the Real Time PCR we used with Sybr Green technology. Most of the isolates we tested are all the isolates we collected all over the years during the Italian anthrax outbreaks in animals. The environmental samples were taken from soils and water following anthrax outbreaks. The references you are referring to are related to the description of the epidemic-like anthrax outbreaks occurred in Italy in Basilicata and Campania. They were not inserted for a description of all the samples we analyzed. As suggested we reworded the sentence with this hypothesis and deleted part of It. Reviewer 2: The term "phylogenetic tree" was replaced by the term "phylogram" and diversity index analysis was later performed in the manuscript. The manuscript was revised by a professional English proofreading service for grammar. Attachments Attachment Submitted filename: PONE-D-19-23489_reviewer (2) reply.pdf https://doi.org/10.1371/journal.pone.0227875.r002
4 Dec 2019 Decision Letter - Wendy C. Turner, Editor PONE-D-19-23489R1 Genetic characterization of Bacillus anthracis strains circulating in Italy from 1972 to 2018. PLOS ONE Dear Dr. Galante, 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. The reviewers note edits and minor corrections still necessary, but otherwise, a much improved paper. Please address these final comments. We would appreciate receiving your revised manuscript by Jan 18 2020 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Wendy C. Turner Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] 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: (No Response) Reviewer #2: 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: Partly Reviewer #2: Yes ****** 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: The document still requires a bit of editorial correction, although vastly improved from the previous round. I still have issue with some of the conclusions made in the discussion portion of the paper. While it is true that the trade history of Italy has been extensive over the centuries, conclusions regarding the genotypes of this study (limited to 1972-2018) cannot be compared to the middle ages unless you have solid data to support it. By the logic of your hypothesis, all isolates can be linked to trade with southern Africa. The time scales with which such genotypic distribution could have occured cannot be determined using MLVA (which inherently includes homoplasy/homoplasticity) and a limited panel of SNPs. Only whole genome sequencing and comparative genomics which includes diverse lineages can provide enough support for such a narrative. There is enough data to recommend this paper without the inclusion of such wild conclusions on the origins of these isolates. As to the inclusion of the Simpson's index: In my exprerience, the Shanon diversity better represents allelic diversity in VNTR loci. It will also make your work comparable to other papers. It reads much better overall and the methods are also much improved. Reviewer #2: Overall this MS has improved greatly from the previous version, although there is still extensive editing to be done in terms of grammar. I have tried to address all of these issues below but have likely still missed some. I would suggest the authors use the free program/web app Grammarly to specifically address the issue of the overuse of commas throughout. With the incorporation of the edits below I think this manuscript would be suitable for publication in PLOS One. Line 51: remove s at the end of “occupations” Line 53: add s at the end of “human”. Line 55: change “a fourth disease form” to “a fourth form of the disease” Line 57: change “for use as bacteriological” to “for use as a bacteriological” Line 66: remove comma Line 75: remove comma after “laboratory” Line 130: Remove comma after “analysis” and replace “we used” with “were used”. Line 139: Table 2: Make sure all rows are equally spaced (rows 6 through 9 are spaced farther apart then the rest) Lines 157-158: The authors should state what the reference numbers refer to (ncbi or ena, and what specific database) Line 159: Change “Phylogram”, to “A phylogram”. Line 171: Why does sentence end mid-page? Line 172: Remove comma Line 173: Remove “or” Line 174: Remove comma after Europe Line 175: Remove both commas surrounding “14th” Line 176: change “to” to “the” Line 183: remove comma after “VNTRs” Line 184: remove comma after “regions” Line 198: Table 4: Change “Simpson’s Index of Diversity” to “Simpson's Diversity Index” and throughout. Line 202: Remove extreme spacing between words Line 207: Change “Around the phylogram are shown, from the external part to the internal part” to “Circling the phylogram from the external to internal region are” Line 214: replace comma with “and” Line 215: change “purpose” to “purposes” Line 216-217: I’m not sure this sentence is true, and it is also grammatically incorrect. Does the high survivability of spores lead to genetic homogeneity? I don’t think so, I would argue that genetic homogeneity in this species is due to the fact that it has a single stranded chromosome and reproduces asexually. Persistence of spores in the environment complicate this but is not the defining feature that dictates homogeneity. Line 217-219: This sentence should be reworded to reduce the number of commas and read more coherently. Line 221: Remove comma Line 225: Remove comma after “GT-54” and “Veneto” Line 226: Remove comma after “observation” Line 228: Remove comma after “GT-55” Line 229: Remove comma after “Trentino” Line 228-229: This sentence is confusing because your allocating a genotype to a genotype. I would reword this to something like: “Furthermore, genotype GT-55; B.Br.CNEVA, isolated in Veneto and Trentino is highly differentiated from most other Italian strains examined here. Line 230: change “branch” to “genotype”, remove “mainly” and change “in particular” to “and found in” Line 236: Change “this group gave rise” to “this group is thought to have given rise” and cite the paper that originally presented this hypothesis. Line 238: Change “an effect of evolution of a common ancestral strain at territorial level” to “an effect of evolution on a common ancestral strain at the territorial level” Line 240, change “as well as” to “and” Line 241: change “seems” to “is postulated” Line 247: add the word “strains” after B.Br.CNEVA Line 248: remove the word “the” Line 254: remove comma after “regions” and change “can” to “may” Line 256: change “increasing the chances” to “which increases the chances” Line 257: Change “this” to “the” Line 263: Change “This” to “these” Line 264: change space in front of period ****** 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: Yes: Spencer A Bruce [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. Attachments Attachment Submitted filename: PONE-D-19-23489_Reviewer1_02122019.pdf https://doi.org/10.1371/journal.pone.0227875.r003
Revision 2
23 Dec 2019 Author Response Dear Editorial Office, we made and submitted the corrections required by the second revision of the Journal and of the reviewers. The corrections are reported in the attached files "response to reviewers" and "revised manuscript with track changes". Thank you for the further precious suggestions and observations. Attachments Attachment Submitted filename: RESPONSE TO REVIEWERS.docx https://doi.org/10.1371/journal.pone.0227875.r004
2 Jan 2020 Decision Letter - Wendy C. Turner, Editor Genetic characterization of Bacillus anthracis strains circulating in Italy from 1972 to 2018. PONE-D-19-23489R2 Dear Dr. Galante, 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, Wendy C. Turner Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0227875.r005
Formally Accepted
6 Jan 2020 Acceptance Letter - Wendy C. Turner, Editor PONE-D-19-23489R2 Genetic characterization of Bacillus anthracis strains circulating in Italy from 1972 to 2018. Dear Dr. Galante: 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 Dr. Wendy C. Turner Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0227875.r006

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