DNA microarray analysis of Staphylococcus aureus from Nigeria and South Africa

Adebayo Osagie Shittu; Tomiwa Adesoji; Edet Ekpenyong Udo

doi:10.1371/journal.pone.0237124

Peer Review History

Original SubmissionJuly 18, 2020
26 Oct 2020 Decision Letter - Monica Cartelle Gestal, Editor PONE-D-20-22325 DNA microarray analysis of Staphylococcus aureus from Nigeria and South Africa PLOS ONE Dear Dr. Shittu, 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 revise the manuscript taking into account the suggestions and comments of the reivewers. These cahnges will substantially improve the manuscript. Please send the revised version as soon as your convenience. Please submit your revised manuscript by Dec 10 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Monica Cartelle Gestal, PhD Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: No ****** 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: 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 ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This manuscript describes the typing of isolates of Staphylococcus aureus from South Africa and Nigeria. The manuscript is a survey of historic strains from these two countries and compares spa typing to DNA microarray. Overall, the manuscript is well written. As this manuscript is mainly descriptive the analysis is limited but satisfactorily covers the results. More detail of the methods is required to allow for interpretation of the results and conclusions. Major issues: 1. The methods section lacks the details required to understand what was done in the experiments. How were the strains grown? How was DNA obtained? Did you carry out any statistical analysis? How many biological and technical repeats were carried for each experiment? 2. You have mentioned 2 outliers. With SA32 did you confirm that ermC gene was intact by sequencing? This would tell you if it is a technical problem or not. This approach could also be used for SA17 and blaZ but a negative result would be inconclusive. Minor: Line 216: The abbreviation for mupirocin resistance should be MuR throughout. Line 218: insert full stop after references. Reviewer #2: Staphylococcus aureus is an important human pathogen with an arsenal of virulence factors and a propensity to acquire antibiotic resistance genes. The understanding of the global epidemiology of S. aureus through the use of various typing methods is important in the detection and tracking of novel and epidemic clones in countries and regions. However, detailed information on antibiotic resistance and virulence genes of S. aureus, and its population structure is still limited in Africa. The current manuscript by Shittu et al. screens S. aureus isolates collected in South Africa (n=38) and Nigeria (n=2) from 2001-2004 for a variety of virulence factors and antimicrobial elements using DNA microarray analysis. This paper basically summarizes the findings of the screen and the concludes that “DNA microarray assay provides information on antibiotic resistance and virulence gene determinants and can be a useful tool to identify gene markers of specific S. aureus clones in Africa”. So while the data reported could be useful, more information and additional analyses are needed for this the report to be of value to the scientific community. Specific comments and suggestions are listed below for consideration: 1) The title is misleading. The current study only includes 40 isolates from Nigeria and only 2 from South Africa. How much value is placed on an “N” of two? To remedy this situation, the number of isolates evaluated for each location should be included in the title. For example “Microarray analysis of 40 Staphylococcus aureus isolates from Nigeria and 2 Staphylococcus aureus isolates South Africa” as one possibility for improvement. 2) Table 1 is confusing. The percentage of selected genes from microarray is not really useful information. Either instead of or in addition to the data provided, the authors should screen the isolates for the presence of a reference nucleotide sequence for each gene. Then report the percent nucleotide identity of each gene in the isolate to the reference nucleotide sequence. This will provide much more useful data! 3) The full nucleotide sequence of each gene in the microarray needs to be provided. Currently no sequence or length of the sequence is provided. Length of the sequence is important because in some instances the length for a microarray target might be only 75 nucleotides. Any search or table for a study of this nature, needs to include the full nucleotide sequence as part of the minimum requirements for publishing! ****** 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.] 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.0237124.r001
Revision 1
31 Dec 2020 Author Response Re: Revised manuscript: PONE-D-20-22325 – DNA microarray analysis of Staphylococcus aureus from Nigeria and South Africa On behalf of other co-authors, I forward herewith the revised manuscript for your kind consideration. It has been formatted to meet the PLOS ONE’s publication criteria. Furthermore, the comments of the reviewers have been examined and we provide a point-by-point response as indicated below. 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 Response: The revised manuscript has been formatted according to the requirements with PLOS ONE. 2. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files. Response: Tables 1 and 2 have been included in the revised manuscript. The previous Table 1 is now Supplementary 2. 3. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please also include your statement in the online submission form via 'Edit submission'. Response: The ethics statement has been transferred to the Methods section of the manuscript. Reviewers' comments: Reviewer #1: This manuscript describes the typing of isolates of Staphylococcus aureus from South Africa and Nigeria. The manuscript is a survey of historic strains from these two countries and compares spa typing to DNA microarray. Overall, the manuscript is well written. As this manuscript is mainly descriptive the analysis is limited but satisfactorily covers the results. More detail of the methods is required to allow for interpretation of the results and conclusions. Major issues: 1. The methods section lacks the details required to understand what was done in the experiments. How were the strains grown? How was DNA obtained? Did you carry out any statistical analysis? How many biological and technical repeats were carried for each experiment? Response: We appreciate the comments of the reviewer. Additional information on the strains including DNA isolation is provided in the revised manuscript. The tests were generally not repeated due to their high level of reproducibility. Spa typing and microarray were only repeated in a few instances where the results were ambiguous. We have provided information on some constraints in the conclusion of the manuscript, nevertheless, they do not affect the overall objective of the study. Statistical analysis: The percentage agreement between the phenotypic (antibiotic susceptibility testing) and genotypic (microarray) methods in the identification of isolates as ‘susceptible’ and ‘resistant’ was calculated from the 2 X 2 tables. Moreover, the Cohen Kappa (κ) test was employed to determine the level of agreement of the two methods. The data is described in the revised manuscript. 2. You have mentioned 2 outliers. With SA32 did you confirm that ermC gene was intact by sequencing? This would tell you if it is a technical problem or not. This approach could also be used for SA17 and blaZ but a negative result would be inconclusive. Response: We did not conduct a further investigation including sequencing of the blaZ and ermC genes on the isolates that were resistant to penicillin and susceptible to erythromycin, respectively. This is indicated as a limitation of the study in the conclusion. Our focus was to determine the percentage and level of agreement between antibiotic susceptibility testing and DNA microarray. We have provided data (in the revised manuscript) which showed a high level of agreement with the two methods. Minor: Line 216: The abbreviation for mupirocin resistance should be MuR throughout. Response: We are grateful for the comment of the reviewer. However, we prefer the use of mupR (which is widely used) than MuR as suggested. Line 218: insert full stop after references. Response: The observation is noted and the amendment is in the revised manuscript. Reviewer #2: Staphylococcus aureus is an important human pathogen with an arsenal of virulence factors and a propensity to acquire antibiotic resistance genes. The understanding of the global epidemiology of S. aureus through the use of various typing methods is important in the detection and tracking of novel and epidemic clones in countries and regions. However, detailed information on antibiotic resistance and virulence genes of S. aureus, and its population structure is still limited in Africa. The current manuscript by Shittu et al. screens S. aureus isolates collected in South Africa (n=38) and Nigeria (n=2) from 2001-2004 for a variety of virulence factors and antimicrobial elements using DNA microarray analysis. This paper basically summarizes the findings of the screen and the concludes that “DNA microarray assay provides information on antibiotic resistance and virulence gene determinants and can be a useful tool to identify gene markers of specific S. aureus clones in Africa”. So while the data reported could be useful, more information and additional analyses are needed for this the report to be of value to the scientific community. Specific comments and suggestions are listed below for consideration: 1) The title is misleading. The current study only includes 40 isolates from Nigeria and only 2 from South Africa. How much value is placed on an “N” of two? To remedy this situation, the number of isolates evaluated for each location should be included in the title. For example “Microarray analysis of 40 Staphylococcus aureus isolates from Nigeria and 2 Staphylococcus aureus isolates South Africa” as one possibility for improvement. Response: We appreciate the comments of the reviewer. The small and disproportionate number of S. aureus isolates from the two African countries is a limitation to the study, and this is highlighted in the manuscript. Nevertheless, the origin and number of the isolates are indicated in the abstract, hence we believe it is not necessary to change the title of the manuscript. Moreover, we reiterate that the main objective of the study was to provide detailed information on antibiotic resistance and virulence-related genes, and the population structure of S. aureus isolates (from Africa) based on the microarray assay. We strongly believe that despite the limitation indicated above, the investigation has fulfilled this objective. 2) Table 1 is confusing. The percentage of selected genes from microarray is not really useful information. Either instead of or in addition to the data provided, the authors should screen the isolates for the presence of a reference nucleotide sequence for each gene. Then report the percent nucleotide identity of each gene in the isolate to the reference nucleotide sequence. This will provide much more useful data! Response: We are grateful for the comment of the reviewer. The microarray assay (Identibac S. aureus genotyping Kit 2.0) is a widely acknowledged tool specifically developed (Alere Technology, Jena, Germany [now Abbott Rapid Diagnostics GmbH, Jena, Germany]) for genotyping of S. aureus isolates. Furthermore, the list of primer/probe sequences has been published previously (Monecke et al., 2011), and this reference is provided in the revised manuscript. Monecke S, Coombs G, Shore AC, Coleman DC, Akpaka P, Borg M, Chow H, Ip M, Jatzwauk L, Jonas D, Kadlec K, Kearns A, Laurent F, O'Brien FG, Pearson J, Ruppelt A, Schwarz S, Scicluna E, Slickers P, Tan HL, Weber S, Ehricht R. A field guide to pandemic, epidemic and sporadic clones of methicillin-resistant Staphylococcus aureus. PLoS One. 2011 Apr 6;6(4):e17936. doi: 10.1371/journal.pone.0017936. Information on the proportion of isolates positive for the antibiotic resistance and virulence genes of S. aureus isolates based on the microarray assay has been described in some recent publications including: 1. Peterson JC, Durkee H, Miller D, Maestre-Mesa J, Arboleda A, Aguilar MC, Relhan N, Flynn HW Jr, Amescua G, Parel JM, Alfonso E. Molecular epidemiology and resistance profiles among healthcare- and community-associated Staphylococcus aureus keratitis isolates. Infect Drug Resist. 2019 Apr 11;12:831-843. doi: 10.2147/IDR.S190245. 2. Senok A, Somily AM, Nassar R, Garaween G, Kim Sing G, Müller E, Reissig A, Gawlik D, Ehricht R, Monecke S. Emergence of novel methicillin-resistant Staphylococcus aureus strains in a tertiary care facility in Riyadh, Saudi Arabia. Infect Drug Resist. 2019 Sep 3;12:2739-2746. doi: 10.2147/IDR.S218870. 3. Senok A, Nassar R, Celiloglu H, Nabi A, Alfaresi M, Weber S, Rizvi I, Müller E, Reissig A, Gawlik D, Monecke S, Ehricht R. Genotyping of methicillin-resistant Staphylococcus aureus from the United Arab Emirates. Sci Rep. 2020 Oct 29;10(1):18551. doi: 10.1038/s41598-020-75565-w. 4. Udo EE, Boswihi SS, Mathew B, Noronha B, Verghese T, Al-Jemaz A, Al Saqer F. Emergence of Methicillin-Resistant Staphylococcus aureus Belonging to Clonal Complex 15 (CC15-MRSA) in Kuwait Hospitals. Infect Drug Resist. 2020 Feb 21;13:617-626. doi: 10.2147/IDR.S237319. 5. Boswihi SS, Udo EE, Mathew B, Noronha B, Verghese T, Tappa SB. Livestock-Associated Methicillin-Resistant Staphylococcus aureus in Patients Admitted to Kuwait Hospitals in 2016-2017. Front Microbiol. 2020 Jan 8;10:2912. doi: 10.3389/fmicb.2019.02912. In our study, data on the proportion of isolates positive or negative for the antibiotic resistance genes (microarray) and antibiotic susceptibility testing (AST) was essential in determining the percentage and level of agreement of the two methods as described in Table 1 (revised manuscript). In conclusion, the previous Table 1 is now presented as Supplementary data (S2 Table). 3) The full nucleotide sequence of each gene in the microarray needs to be provided. Currently no sequence or length of the sequence is provided. Length of the sequence is important because in some instances the length for a microarray target might be only 75 nucleotides. Any search or table for a study of this nature, needs to include the full nucleotide sequence as part of the minimum requirements for publishing! Response: We appreciate the comment of the reviewer. The list of primer/probe sequences for the microarray has been published previously (Monecke et al., 2011), and this reference is included in the revised manuscript. Monecke S, Coombs G, Shore AC, Coleman DC, Akpaka P, Borg M, Chow H, Ip M, Jatzwauk L, Jonas D, Kadlec K, Kearns A, Laurent F, O'Brien FG, Pearson J, Ruppelt A, Schwarz S, Scicluna E, Slickers P, Tan HL, Weber S, Ehricht R. A field guide to pandemic, epidemic and sporadic clones of methicillin-resistant Staphylococcus aureus. PLoS One. 2011 Apr 6;6(4):e17936. doi: 10.1371/journal.pone.0017936. Yours sincerely, Professor Adebayo Shittu Corresponding author https://doi.org/10.1371/journal.pone.0237124.r002
27 Jan 2021 Decision Letter - Monica Cartelle Gestal, Editor PONE-D-20-22325R1 DNA microarray analysis of Staphylococcus aureus from Nigeria and South Africa PLOS ONE Dear Dr. Shittu, 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. ============================== Reviewer 2 still has some concerns about the methodology and results that are included in this study. Please make sure you carefully revised the manuscript and address all the reviewer comments prior to resubmission. ============================== Please submit your revised manuscript by Mar 13 2021 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Monica Cartelle Gestal, PhD 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: All comments have been addressed Reviewer #2: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: No ****** 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 authors have addressed all my comments in a satisfactory manor and I have nothing further to suggest. Reviewer #2: This is the second review for the manuscript by Shittu et al., which describes the screening of S. aureus isolates collected in South Africa (n=38) and Nigeria (n=2) from 2001-2004 for a variety of virulence factors and antimicrobial elements using DNA microarray analysis. This paper basically summarizes the findings of the screen and the concludes that “DNA microarray assay provides information on antibiotic resistance and virulence gene determinants and can be a useful tool to identify gene markers of specific S. aureus clones in Africa”. While the authors have made some revisions to accommodate some of the suggestions posed by reviewers, overall, the authors fail to adequately address some key points raised by the reviewers. The manuscript in its current form is in need of revision to adequately address these key points to improve its suitability for publication. Major key points: 1) The manuscript in its current form fails to meet PLoS One Data availability requirements. Specifically, “Data: PLOS journals require authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception.” Also, the instructions to authors state that all manuscripts incorporating Microarray experiments must meet MIAME (Minimum Information About a Microarray Experiment) requirements. To help aid the authors, MIAME describes the Minimum Information About a Microarray Experiment that is needed to enable the interpretation of the results of the experiment unambiguously and potentially to reproduce the experiment. [Brazma et al. (2001), Nature Genetics]. The six most critical elements contributing towards MIAME are: a. The raw data for each hybridisation (e.g., CEL or GPR files) b. The final processed (normalised) data for the set of hybridisations in the experiment (study) (e.g., the gene expression data matrix used to draw the conclusions from the study) c. The essential sample annotation including experimental factors and their values (e.g., compound and dose in a dose response experiment) d. The experimental design including sample data relationships (e.g., which raw data file relates to which sample, which hybridisations are technical, which are biological replicates) e. Sufficient annotation of the array (e.g., gene identifiers, genomic coordinates, probe oligonucleotide sequences or reference commercial array catalog number) f. The essential laboratory and data processing protocols (e.g., what normalisation method has been used to obtain the final processed data) The current manuscript fails to provide any of this required information. While references are given, they do not disclose the DNA sequence information of the probes. The manuscript needs to be revised to include all of the above required information! Additionally, providing a supplementary table listing target or gene names, along with the actual DNA sequence of the probes and primers is essential along with MIAME compliance. 2) Lines 81-82: Provide the precise CLSI standard version applied in the phenotpical assay. 3) The authors claim that the objective of the current was to determine the percentage and level of agreement between antibiotic sensitive testing and DNA microarray, which alone is not suitable for publication. The authors also claim that the objective of the current was to provide detailed information on antibiotic resistance and virulence-related genes, and the population structure of S. aureus isolates from Africa. So, which is it? Please clarify! If the second objective is the correct one, then please address the original comments made both reviewers. ****** 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 [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.0237124.r003
Revision 2
21 Feb 2021 Author Response Professor Monica Cartelle Gestal Academic Editor PLoS One Dear Professor Gestal, Re: Revised manuscript: PONE-D-20-22325-R2 – DNA microarray analysis of Staphylococcus aureus from Nigeria and South Africa On behalf of other co-authors, I forward herewith the revised manuscript for your kind consideration. The comments of reviewer 2 have been examined and we provide a point-by-point response as indicated below. Reviewer’s comments Reviewer #2: This is the second review for the manuscript by Shittu et al., which describes the screening of S. aureus isolates collected in South Africa (n=38) and Nigeria (n=2) from 2001-2004 for a variety of virulence factors and antimicrobial elements using DNA microarray analysis. This paper basically summarizes the findings of the screen and the concludes that “DNA microarray assay provides information on antibiotic resistance and virulence gene determinants and can be a useful tool to identify gene markers of specific S. aureus clones in Africa”. While the authors have made some revisions to accommodate some of the suggestions posed by reviewers, overall, the authors fail to adequately address some key points raised by the reviewers. The manuscript in its current form is in need of revision to adequately address these key points to improve its suitability for publication. Major key points: 1) The manuscript in its current form fails to meet PLoS One Data availability requirements. Specifically, “Data: PLOS journals require authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception.” Also, the instructions to authors state that all manuscripts incorporating Microarray experiments must meet MIAME (Minimum Information About a Microarray Experiment) requirements. To help aid the authors, MIAME describes the Minimum Information About a Microarray Experiment that is needed to enable the interpretation of the results of the experiment unambiguously and potentially to reproduce the experiment. [Brazma et al. (2001), Nature Genetics]. The six most critical elements contributing towards MIAME are: a. The raw data for each hybridisation (e.g., CEL or GPR files) b. The final processed (normalised) data for the set of hybridisations in the experiment (study) (e.g., the gene expression data matrix used to draw the conclusions from the study) c. The essential sample annotation including experimental factors and their values (e.g., compound and dose in a dose response experiment) d. The experimental design including sample data relationships (e.g., which raw data file relates to which sample, which hybridisations are technical, which are biological replicates) e. Sufficient annotation of the array (e.g., gene identifiers, genomic coordinates, probe oligonucleotide sequences or reference commercial array catalog number) f. The essential laboratory and data processing protocols (e.g., what normalisation method has been used to obtain the final processed data) The current manuscript fails to provide any of this required information. While references are given, they do not disclose the DNA sequence information of the probes. The manuscript needs to be revised to include all of the above required information! Additionally, providing a supplementary table listing target or gene names, along with the actual DNA sequence of the probes and primers is essential along with MIAME compliance. Response: We appreciate the comments of the reviewer. A description of the microarray assay including the raw data (matrix in excel) has been provided in the revised manuscript. Moreover, in response to the reviewer’s comments, we cite recent publications (kindly see below) that utilized the Staphtype genotyping kit 2.0 and refer to the methodology and data provided in the Supplementary section of these publications. These papers (including those in PLoS One) provided the raw data (in MS-Excel) and clearly showed that the methodology including the DNA sequence for the primers/probes has been published previously. Consequently, we do not agree with the reviewer that a supplementary table with the DNA sequence of the probes and primers should be provided in our manuscript. In conclusion, the description of the genes in S1 Table 1 is stated in S1 Table 3. Monecke S, Slickers P, Gawlik D, Müller E, Reissig A, Ruppelt-Lorz A, Akpaka PE, Bandt D, Bes M, Boswihi SS, Coleman DC, Coombs GW, Dorneanu OS, Gostev VV, Ip M, Jamil B, Jatzwauk L, Narvaez M, Roberts R, Senok A, Shore AC, Sidorenko SV, Skakni L, Somily AM, Syed MA, Thürmer A, Udo EE, Vremerǎ T, Zurita J, Ehricht R. Molecular Typing of ST239-MRSA-III From Diverse Geographic Locations and the Evolution of the SCCmec III Element During Its Intercontinental Spread. Front Microbiol. 2018 Jul 6;9:1436. doi: 10.3389/fmicb.2018.01436. Earls MR, Shore AC, Brennan GI, Simbeck A, Schneider-Brachert W, Vremerǎ T, Dorneanu OS, Slickers P, Ehricht R, Monecke S, Coleman DC. A novel multidrug-resistant PVL-negative CC1-MRSA-IV clone emerging in Ireland and Germany likely originated in South-Eastern Europe. Infect Genet Evol. 2019 Apr;69:117-126. doi: 10.1016/j.meegid.2019.01.021. Epub 2019 Jan 21. Morach M, Käppeli N, Hochreutener M, Johler S, Julmi J, Stephan R, Etter D. Microarray based genetic profiling of Staphylococcus aureus isolated from abattoir byproducts of pork origin. PLoS One. 2019 Sep 6;14(9):e0222036. doi: 10.1371/journal.pone.0222036. Alfouzan W, Udo EE, Modhaffer A, Alosaimi A. Molecular Characterization of Methicillin- Resistant Staphylococcus aureus in a Tertiary Care hospital in Kuwait. Sci Rep. 2019 Dec 6;9(1):18527. doi: 10.1038/s41598-019-54794-8. Boswihi SS, Udo EE, Mathew B, Noronha B, Verghese T, Tappa SB. Livestock-Associated Methicillin-Resistant Staphylococcus aureus in Patients Admitted to Kuwait Hospitals in 2016-2017. Front Microbiol. 2020 Jan 8;10:2912. doi: 10.3389/fmicb.2019.02912. Etter D, Corti S, Spirig S, Cernela N, Stephan R, Johler S. Staphylococcus aureus Population Structure and Genomic Profiles in Asymptomatic Carriers in Switzerland. Front Microbiol. 2020 Jun 24;11:1289. doi: 10.3389/fmicb.2020.01289. Gawlik D, Ruppelt-Lorz A, Müller E, Reißig A, Hotzel H, Braun SD, Söderquist B, Ziegler-Cordts A, Stein C, Pletz MW, Ehricht R, Monecke S. Molecular investigations on a chimeric strain of Staphylococcus aureus sequence type 80. PLoS One. 2020 Oct 14;15(10):e0232071. doi: 10.1371/journal.pone.0232071. Senok A, Nassar R, Celiloglu H, Nabi A, Alfaresi M, Weber S, Rizvi I, Müller E, Reissig A, Gawlik D, Monecke S, Ehricht R. Genotyping of methicillin resistant Staphylococcus aureus from the United Arab Emirates. Sci Rep. 2020 Oct 29;10(1):18551. doi: 10.1038/s41598-020-75565-w. 2) Lines 81-82: Provide the precise CLSI standard version applied in the phenotpical assay. Response: The observation is noted and the amendment is in the revised manuscript. 3) The authors claim that the objective of the current was to determine the percentage and level of agreement between antibiotic sensitive testing and DNA microarray, which alone is not suitable for publication. The authors also claim that the objective of the current was to provide detailed information on antibiotic resistance and virulence-related genes, and the population structure of S. aureus isolates from Africa. So, which is it? Please clarify! If the second objective is the correct one, then please address the original comments made both reviewers. Response: We appreciate the comment of the reviewer. The statement on the percentage and level of agreement between antibiotic susceptibility testing and DNA microarray has been removed in the revised manuscript (discussion section). The objective of the study is to provide detailed information on antibiotic resistance and virulence-related genes, and the population structure of S. aureus isolates (from Africa) based on the microarray assay. Yours sincerely, Professor Adebayo Shittu Corresponding author https://doi.org/10.1371/journal.pone.0237124.r004
6 Apr 2021 Decision Letter - Monica Cartelle Gestal, Editor PONE-D-20-22325R2 DNA microarray analysis of Staphylococcus aureus from Nigeria and South Africa PLOS ONE Dear Dr. Shittu, 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. ============================== There are some minor changes that need to be address, please send us the revised version as early as your convenience ============================== Please submit your revised manuscript by May 21 2021 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. 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If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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 #2: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? 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 #2: No ****** 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 #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 #2: This is the third review for the manuscript by Shittu et al., which describes the screening of 40 S. aureus isolates collected in South Africa from 2001-2004 for a variety of virulence factors and antimicrobial elements using DNA microarray analysis. This paper basically summarizes the findings of the screen and the concludes that “DNA microarray assay provides information on antibiotic resistance and virulence gene determinants and can be a useful tool to identify gene markers of specific S. aureus clones in Africa”. While the authors have made revisions to accommodate suggestions posed by reviewers, the authors fail to accommodate the request to provide a list of all target or gene names, along with the actual DNA sequence of the probes and primers. Therefore, the manuscript in its current form fails to meet PLoS One Data availability requirements. Instead of simply complying with the policy, the authors choose to argue that 1) previous published papers have not met the policy requirements and 2) that references listed provide the requested information. This reviewer’s response to these points are: 1) This reviewer was not part of the peer-review process of the noncompliant previous published papers. In contrast, this reviewer is part of the peer-review process for the manuscript by Shittu et al., which in its current form, is NOT compliant. 2) The authors are correct in that one of the cited references provides the required information however, it is provided in a convoluted style. For example, the gene aacA-aphD, a bifunctional enzyme Aac/Aph conferring gentamicin resistance, the probe name is “hp_aacA-59243_PM1” and the probe sequence is “AB049452.1[5151:5178]”. This reviewer is unsure why the authors persist to refuse to provide a list of all target or gene names, along with the actual DNA sequence of the probes and primers, which would fulfill PLoS One Data availability requirements. These requirements are not meant to be a cumbersome obstacle. They are meant to help make all aspects of the scientific process accessible to everyone. ****** 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 #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0237124.r005
Revision 3
24 Apr 2021 Author Response Re: Revised manuscript: PONE-D-20-22325-R3 – DNA microarray analysis of Staphylococcus aureus from Nigeria and South Africa On behalf of other co-authors, I forward herewith the revised manuscript for your kind consideration. The Journal requirements and comments of reviewer 2 have been examined and we provide a point-by-point response (in italics) as indicated below. Journal Requirements Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Response: Based on comments/suggestions from the reviewers, the under-listed references were included in the revised manuscript which was not in the original version. 9. Franklin R. Cockerill, III M, Jean B. Patel, PhD D. M100-S25 Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fifth Informational Supplement. Clin Lab Stand Inst. 2015. 10. Al-Haqan A, Boswihi SS, Pathan S, Udo EE. Antimicrobial resistance and virulence determinants in coagulase-negative staphylococci isolated mainly from preterm neonates. PLoS One. 2020. doi:10.1371/journal.pone.0236713. 13. Monecke S, Coombs G, Shore AC, Coleman DC, Akpaka P, Borg M, et al. A field guide to pandemic, epidemic and sporadic clones of methicillin-resistant Staphylococcus aureus. PLoS One. 2011. doi:10.1371/journal.pone.0017936. 14. Monecke S, Jatzwauk L, Müller E, Nitschke H, Pfohl K, Slickers P, et al. Diversity of SCCmec elements in staphylococcus aureus as observed in south-eastern Germany. PLoS One. 2016. doi:10.1371/journal.pone.0162654. 15. Landis JR, Koch GG. The Measurement of Observer Agreement for Categorical Data. Biometrics. 1977. doi:10.2307/2529310. The reference number 10 in the original manuscript: Monecke S, Jatzwauk L, Weber S, Slickers P, Ehricht R. DNA microarray-based genotyping of methicillin-resistant Staphylococcus aureus strains from Eastern Saxony. Clinical Microbiology and Infection. 2008. doi:10.1111/j.1469-0691.2008.01986.x was replaced with the reference below. 12. Monecke S, Slickers P, Ehricht R. Assignment of Staphylococcus aureus isolates to clonal complexes based on microarray analysis and pattern recognition. FEMS Immunol Med Microbiol. 2008. doi:10.1111/j.1574-695X.2008.00426.x. The reference list is complete and correct in the revised manuscript. Reviewer’s comments Reviewer #2: This is the third review for the manuscript by Shittu et al., which describes the screening of 40 S. aureus isolates collected in South Africa from 2001-2004 for a variety of virulence factors and antimicrobial elements using DNA microarray analysis. This paper basically summarizes the findings of the screen and the concludes that “DNA microarray assay provides information on antibiotic resistance and virulence gene determinants and can be a useful tool to identify gene markers of specific S. aureus clones in Africa”. While the authors have made revisions to accommodate suggestions posed by reviewers, the authors fail to accommodate the request to provide a list of all target or gene names, along with the actual DNA sequence of the probes and primers. Therefore, the manuscript in its current form fails to meet PLoS One Data availability requirements. Instead of simply complying with the policy, the authors choose to argue that 1) previous published papers have not met the policy requirements and 2) that references listed provide the requested information. This reviewer’s response to these points are: 1) This reviewer was not part of the peer-review process of the noncompliant previous published papers. In contrast, this reviewer is part of the peer-review process for the manuscript by Shittu et al., which in its current form, is NOT compliant. 2) The authors are correct in that one of the cited references provides the required information however, it is provided in a convoluted style. For example, the gene aacA-aphD, a bifunctional enzyme Aac/Aph conferring gentamicin resistance, the probe name is “hp_aacA-59243_PM1” and the probe sequence is “AB049452.1[5151:5178]” This reviewer is unsure why the authors persist to refuse to provide a list of all target or gene names, along with the actual DNA sequence of the probes and primers, which would fulfill PLoS One Data availability requirements. These requirements are not meant to be a cumbersome obstacle. They are meant to help make all aspects of the scientific process accessible to everyone. Response: We have included the primer and probe names, GenBank coordinates and the corresponding DNA sequences in the revised manuscript (Supplementary data: S1 Table 3). The DNA sequences for the primers and probes were retrieved from the NCBI website https://www.ncbi.nlm.nih.gov/nuccore through their accession numbers and coordinates, as published previously. Monecke S, Coombs G, Shore AC, Coleman DC, Akpaka P, Borg M, et al. A field guide to pandemic, epidemic and sporadic clones of methicillin-resistant Staphylococcus aureus. PLoS One. 2011. doi: 10.1371/journal.pone.0017936. Monecke S, Jatzwauk L, Müller E, Nitschke H, Pfohl K, Slickers P, et al. Diversity of SCCmec elements in Staphylococcus aureus as observed in south-eastern Germany. PLoS One. 2016. doi: 10.1371/journal.pone.0162654. Yours sincerely, Professor Adebayo Shittu Corresponding author Attachments Attachment Submitted filename: Response to Reviewers_R3.doc https://doi.org/10.1371/journal.pone.0237124.r006
18 Jun 2021 Decision Letter - Monica Cartelle Gestal, Editor DNA microarray analysis of Staphylococcus aureus from Nigeria and South Africa PONE-D-20-22325R3 Dear Dr. Shittu, 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, Monica Cartelle Gestal, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0237124.r007
Formally Accepted
24 Jun 2021 Acceptance Letter - Monica Cartelle Gestal, Editor PONE-D-20-22325R3 DNA microarray analysis of Staphylococcus aureus from Nigeria and South Africa Dear Dr. Shittu: 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. Monica Cartelle Gestal Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0237124.r008

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