Whole genome sequencing of extended-spectrum β-lactamase genes in Enterobacteriaceae isolates from Nigeria

Christiana Jesumirhewe; Burkhard Springer; Franz Allerberger; Werner Ruppitsch

doi:10.1371/journal.pone.0231146

Peer Review History

Original SubmissionSeptember 26, 2019
11 Nov 2019 Decision Letter - Monica Cartelle Gestal, Editor PONE-D-19-26950 Molecular characterization of extended-spectrum β-lactamase genes in Enterobacteriaceae isolates from Nigeria Dr Christiana Jesumirhewe, Department of Pharmaceutical microbiology, Igbinedion University Okada, Edo state, Nigeria PLOS ONE Dear Dr jesumirhewe, 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. We would appreciate receiving your revised manuscript by Dec 26 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, Monica Cartelle Gestal, PhD Academic Editor PLOS ONE Additional Editor Comments: This manuscript is of great interest for us, please address the questions and suggestions offered by the reviewers. 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 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. Please amend either the title on the online submission form (via Edit Submission) or the title in the manuscript so that they are identical. 3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. 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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: 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: Jesumirhewe and collaborators present an interesting manuscript with the general objective of determine the frequency of ESBL genes in multidrug resistant human clinical Enterobacteriaceae isolates from Edo state Nigeria and to characterize the resistance mechanisms. Thank you for being able to contribute to it before your presentation to the scientific community. Therefore, I suggest that the following questions be modified, deleted, or explained. Line 1. I think authors should change in the title: Whole genome sequence of extended-spectrum β-lactamase genes in Enterobacteriaceae isolates from Nigeria.” Improve the Abstract and Introduction. ESBL concern in both healthcare and community settings Line 16, 64. “This study aimed at determining the frequency of ESBL genes in multidrug resistant human clinical Enterobacteriaceae isolates from Edo state Nigeria” using whole genome sequencing Line 33. I suggest to describe Ambler classification of beta-lactamases and groups of CTX-M on the introduction. Line 51. I would rather see table 1 summarized in a Figure instead of a table. I suppose it would be better demonstrated. The three hospitals are at a different level of care? Why did you decide to include the strains of unidentified source; Teaching Hospital, Benin n=12, and Okada n=4 Line 74. “Flurometer” Fluorometer Line 94. The authors reported that (41%) were positive for the ESBL phenotypic test and had ESBL genes detected. Please explain the results of Pret3577 and EC11, where no resistance pattern to B lactams was observed. In strains with ESBL and carbapenemase, the ESBL screening and confirmation performs well? Line 115. Table 4. Please highlight in results the presence of carbapenemases NDM and Oxa-48 genes in strains Kp_852 ST15, Kp_852K ST15, Kp_872 ST340 (First cases described?) I recommend to improve tables, columns of ST set in the beginning Have all the sequences been deposited at GenBank? Is it possible probable clonal spread of the ESBL strains Intra or inter-hospital Limitations must be included I suggest to include virulence factors and mobile genetic elements associated with ESBL and phylogeny based on core genome MLST. I recommend to highlight the WGS findings (figures), highlight the emerging global high risk clones, and to present this manuscript in a better fashion. Reviewer #2: The manuscript evaluates the frequency of ESBL genes in multidrug resistant human clinical Enterobacteriaceae isolates from Edo state Nigeria and characterize the resistance mechanisms. The research is correctly conducted, and the conclusions are supported by the data. However, results are not well exposed and are difficult to understand. Materials and method Line 66. The phenotypic method used, is not useful for microorganisms with Amp C , so some ESBL producers could not be included, and this could be considered as a limitation Table 1. Use italics to Enterobacteriaceae. This table does not present summarized data and contains partial results. Include n values Results: Include summary tables or figures Table 2. Present information by isolated microorganism or at least the predominant ones. cite the % and include the n, also include the denominator of isolates tested for each antimicrobial listed Line 93. Include n value Table 3. summarize with MIC 50 and MIC 90 values. This information must be included as supplementary material. Line 99 -114 summarize using tables or figures Line 139. How many isolates were studied by MLST? Include n values What was the resistance mechanism involved in meropenem resistance? None S. marcescens isolate had ESBL genes? ****** 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: José E Villacís 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. https://doi.org/10.1371/journal.pone.0231146.r001
Revision 1
7 Jan 2020 Author Response Additional Editor’s comments This manuscript is of great interest for us, please address the questions and suggestions offered by the reviewers. 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 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 The manuscript has been re-written to meet PLOS ONE’s style requirements including those for file naming. 2. Please amend either the title on the online submission form (via Edit Submission) or the title in the manuscript so that they are identical. The title on the online submission form and the manuscript are now identical. 3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. All necessary data are made available already and relevant accession numbers are provided in the revised 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 The supporting information files have been worked on and in-text citations updated to match accordingly Review Comments to the Author Reviewer #1: Jesumirhewe and collaborators present an interesting manuscript with the general objective of determine the frequency of ESBL genes in multidrug resistant human clinical Enterobacteriaceae isolates from Edo state Nigeria and to characterize the resistance mechanisms. Thank you for being able to contribute to it before your presentation to the scientific community. Therefore, I suggest that the following questions be modified, deleted, or explained. Line 1. I think authors should change in the title: Whole genome sequence of extended-spectrum β-lactamase genes in Enterobacteriaceae isolates from Nigeria. The title of the article has been changed to Whole genome sequencing of extended-spectrum β-lactamase genes in Enterobacteriaceae isolates from Nigeria Improve the Abstract and Introduction. ESBL concern in both healthcare and community settings The abstract and introduction has been improved on. Concern about ESBL in both healthcare and community settings have been highlighted Line 16, 64. “This study aimed at determining the frequency of ESBL genes in multidrug resistant human clinical Enterobacteriaceae isolates from Edo state Nigeria” using whole genome sequencing The sentence has been edited in the revised manuscript Line 33. I suggest to describe Ambler classification of beta-lactamases and groups of CTX-M on the introduction. Ambler classification of beta-lactamases have been described and groups of CTX-M mentioned on the introduction Line 51. I would rather see table 1 summarized in a Figure instead of a table. I suppose it would be better demonstrated. The three hospitals are at a different level of care? Table 1 is now presented as a figure. The 3 hospitals are tertiary healthcare institutions. Why did you decide to include the strains of unidentified source; Teaching Hospital, Benin n=12, and Okada n=4 I included strains of unidentified source [Data not available} because some of these strains are important i.e had the esbl resistant gene and associated resistant determinants. Removing them from the number of isolates would not give a true picture of the frequency of resistant determinants present in the sample size Line 74. “Flurometer” Fluorometer Fluorometer spelling corrected Line 94. The authors reported that (41%) were positive for the ESBL phenotypic test and had ESBL genes detected. Please explain the results of Pret3577 and EC11, where no resistance pattern to B lactams was observed. In strains with ESBL and carbapenemase, the ESBL screening and confirmation performs well? There was a mix-up in our earlier report. Isolates that were positive for the ESBL phenotypic test and had ESBL genes detected are sixty [40%]. Pret3577 was sensitive to the ESBL phenotypic test but had the ESBL gene detected by WGS. EC 11 was resistant to both ceftazidime and cefotaxime. The ESBL screening and confirmation performed well in strains with ESBL and carbapenemase Line 115. Table 4. Please highlight in results the presence of carbapenemases NDM and Oxa-48 genes in strains Kp_852 ST15, Kp_852K ST15, Kp_872 ST340 (First cases described?) I recommend to improve tables, columns of ST set in the beginning Have all the sequences been deposited at GenBank? Is it possible probable clonal spread of the ESBL strains Intra or inter-hospital Limitations must be included I suggest to include virulence factors and mobile genetic elements associated with ESBL and phylogeny based on core genome MLST. I recommend to highlight the WGS findings (figures), highlight the emerging global high risk clones, and to present this manuscript in a better fashion. The carbapenemases have been earlier reported along with a few other isolates that are not ESBL producers as indicated in the manuscript with tracked changes. The aim of this study was to determine the frequency of ESBL genes in multidrug resistant human clinical Enterobacteriaceae isolates from Edo state Nigeria. Tables have been improved on. Table S1 gives the characteristics of the ESBL isolates. Sequences have been deposited at the GenBank and accession numbers included on Table S1.Yes there is a possibility of probable clonal spread of the ESBL strains especially intra-hospital. The WGS findings have been re-presented. A limitation in this study is to conclude on clonal bacterial transmission between the ESBL Enterobacteriaceae isolates that included seven groups of different organisms. However, the presence of different plasmid replicon types in ESBL producing isolates reveals their importance in the dissemination of these resistance genes. Reviewer #2: The manuscript evaluates the frequency of ESBL genes in multidrug resistant human clinical Enterobacteriaceae isolates from Edo state Nigeria and characterize the resistance mechanisms. The research is correctly conducted, and the conclusions are supported by the data. However, results are not well exposed and are difficult to understand. The results have been represented and are now easy to understand Materials and method Line 66. The phenotypic method used, is not useful for microorganisms with Amp C , so some ESBL producers could not be included, and this could be considered as a limitation The phenotypic method used was based on CLSI guidelines. All ESBL producers detected phenotypically were confirmed in the WGS.Only one isolate had ESBL detected by WGS but not detected phenotypically Table 1. Use italics to Enterobacteriaceae. This table does not present summarized data and contains partial results. Include n values Table 1 has been re-presented as a figure Results: Include summary tables or figures Results have been re-presented Table 2. Present information by isolated microorganism or at least the predominant ones. cite the % and include the n, also include the denominator of isolates tested for each antimicrobial listed Information has been re-presented showing the % of ESBL isolates resistant to the various antibiotics with n included and the denominator of isolates tested for each antimicrobial listed Line 93. Include n value n value has been included Table 3. summarize with MIC 50 and MIC 90 values. This information must be included as supplementary material. The MIC values are included as supporting information now.The MIC values are presented only for the 60 ESBL isolates that are multidrug resistant. MIC values were not determined for the entire 218 enterobacteriaceae isolates. The E-test has been reported to be considered a reliable method to determine antimicrobial susceptibility testing and it gives results which are at least as accurate as those obtained by the broth dilution method. As our ESBL isolates are not up to 100 and the Enterobacteriaceae isolates are a collection of 7 different group of isolates calculating MIC50 and MIC90 may not be applicable here Line 99 -114 summarize using tables or figures Line 99-114 has been summarized and tables represented. The genetic characteristics of the ESBL isolates are presented in the supporting information with other details. Line 139. How many isolates were studied by MLST? Include n values All the multidrug resistant isolates that are ESBL producers were studied by MLST, n=60, The sensitive isolate that genotypically had ESBL detected was also studied by MLST, n=1.Some of the isolates did not have available sequence types. What was the resistance mechanism involved in meropenem resistance? None S. marcescens isolate had ESBL genes? No S. marcescens isolate had ESBL genes. The carbapenemases have been earlier reported along with a few other isolates that are not ESBL producers as indicated in the manuscript with tracked changes. The aim of this study was to determine the frequency of ESBL genes in multidrug resistant human clinical Enterobacteriaceae isolates from Edo state Nigeria. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0231146.r002
4 Feb 2020 Decision Letter - Monica Cartelle Gestal, Editor PONE-D-19-26950R1 Whole genome sequencing of extended-spectrum β-lactamase genes in Enterobacteriaceae isolates from Nigeria PLOS ONE Dear Dr jesumirhewe, 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. We would appreciate receiving your revised manuscript by Mar 20 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, 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: 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: N/A 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: Line 36. Enterobacteriaceae Line 38. Specie of Providencia spp Line 88. Repeated “Fig 1. Figure showing sample size of Enterobacteriaceae isolates provided by three Nigerian hospitals” Line 90. Incomplete sentence Line 94. patients’ According to the answer The ESBL screening and confirmation performed well in strains with ESBL and carbapenemase. Please consider that strains with ampC and carbapenemases can affect the performance of ESBL screening test Line 87. Please include that the 3 hospitals are tertiary healthcare institutions, number of beds Line 116- 128 Consider to rewrite the paragraph. There are words repeated “manufacturer’s instructions” Line 195. Specie of Providencia spp Line 233. The study concludes a probable clonal spread of the ESBL strains especially intra-hospital. Does WGS gave you some evidence? If not please consider the limitations. The findings in the study can support a better conclusion in the abstract and the manuscript Reviewer #2: The manuscript have been improved in quality, but there are some comments Line 77. Please use additional bibliography to support this statement Line 79. Please check the bibliography number 21 Line 87. Include that are tertiary hospitals. As a comment, Figure 1 does not improve the manuscript I have some questions about isolates: All isolates were obtained from hospitalized patients? Please specify if not Line 134: specify which isolates were further studied with MLST. The results are just for K.pneumoniae, E.coli and E. cloacae? Results Piperacillin-tazobactam for K. pneumonia n = 9 results about imcompatibility groups and ST shoould be presented in a resume table. The current version is hard to read Discussion The manuscript does not highlight well their results. Include some data about ESBL in other countries from Africa and compare them. ****** 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: Yes: José E Villacís 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. https://doi.org/10.1371/journal.pone.0231146.r003
Revision 2
25 Feb 2020 Author Response Reviewer No1 Line 36. Enterobacteriaceae Correction has been effected on the revised manuscript Line 38. Specie of Providencia spp The revised manuscript has been updated. We decided to strike out Providencia spp as part of the reported ESBL isolates as it was sensitive to the ESBL phenotypic screening but we detected CTX-M-15 by sequencing. However during our submission of the sequences to NCBI also the sequence had issues that were challenging to resolve so we decided not to include it in the study anymore. The genome submission to NCBI gave a better insight with regards to the isolates. Line 88. Repeated “Fig 1. Figure showing sample size of Enterobacteriaceae isolates provided by three Nigerian hospitals” Correction has been effected on the revised manuscript. Line 90. Incomplete sentence Sentence has been updated in the revised manuscript. Line 94. patients Correction has been effected on the revised manuscript According to the answer The ESBL screening and confirmation performed well in strains with ESBL and carbapenemase. Please consider that strains with ampC and carbapenemases can affect the performance of ESBL screening test Noted. The 60 ESBL isolates presented including the ones with carbapenemases were positive to both the phenotypic screening for ESBL and the sequencing. Alongside other resistance genes only carbapenemases were detected in the ESBL isolates. No ampC was detected in any of the ESBL isolates. Line 87. Please include that the 3 hospitals are tertiary healthcare institutions, number of beds Revised manuscript has been updated Line 116- 128 Consider to rewrite the paragraph. There are words repeated “manufacturer’s instructions” Paragraph has been revised. Line 195. Specie of Providencia spp Manuscript has been revised. Providencia isolate is no longer included in the study. Reasons have been stated earlier in the rebuttal letter. Line 233. The study concludes a probable clonal spread of the ESBL strains especially intra-hospital. Does WGS gave you some evidence? If not please consider the limitations. Using the data obtained from the MLST assignment and plasmid incompatibility grouping, the distribution of the sequence types and the plasmid replicon types especially the predominant ones of the ESBL isolates across the three hospitals suggests a probable clonal spread of the ESBL strains both intra and inter-hospital. A limitation of this study however is to conclude on clonal bacterial transmission between ESBL Enterobacteriaceae isolates that included seven groups of different organisms. The findings in the study can support a better conclusion in the abstract and the manuscript Conclusion in the abstract and manuscript have been revised Reviewer No 2 Line 77. Please use additional bibliography to support this statement Additional bibliography has been added to the revised manuscript. Line 79. Please check the bibliography number 21 Bibliography no 21 has been revised Line 87. Include that are tertiary hospitals. As a comment, Figure 1 does not improve the manuscript The manuscript has been revised indicating the hospitals are tertiary healthcare institutions. The figure was included in response to another reviewer’s comment from the previous revision. All isolates were obtained from hospitalized patients? Please specify if not Line 134: specify which isolates were further studied with MLST. The results are just for K.pneumoniae, E.coli and E. cloacae? Isolates were obtained from samples of both in-patients and out-patients of the three different hospitals. All ESBL isolates were further studied with MLST. Sequence type numbers were not available for other isolates not presented. Some isolates had unknown sequence types which are submitted for curation of sequence type number. Results Piperacillin-tazobactam for K. pneumonia n = 9 Correction has been updated in the revised manuscript results about imcompatibility groups and ST shoould be presented in a resume table. The current version is hard to read Table has been revised. Result about incompatibility groups and ST has been represented in a separate table. Discussion The manuscript does not highlight well their results. Include some data about ESBL in other countries from Africa and compare them. Discussion has been revised highlighting the results properly. Data about ESBL in other countries from Africa have been included and compared with the study Attachments Attachment Submitted filename: rebuttal letter 1.docx https://doi.org/10.1371/journal.pone.0231146.r004
18 Mar 2020 Decision Letter - Monica Cartelle Gestal, Editor Whole genome sequencing of extended-spectrum β-lactamase genes in Enterobacteriaceae isolates from Nigeria PONE-D-19-26950R2 Dear Dr. jesumirhewe, 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, Monica Cartelle Gestal, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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: Line 36-37. The report of blaCTX-M-15 in clinical isolates of Providencia spp., Citrobacter freundii and Atlantibacter hermannii is important, however the results of WGS, plasmid profiles an ST related to the dissemintation of CTX-M are more relevant. Line 79. Please include what carbapenemases were recently reported. KPC, NDM, OXA-48 ??? ****** 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: Yes: José E Villacís https://doi.org/10.1371/journal.pone.0231146.r005
Formally Accepted
31 Mar 2020 Acceptance Letter - Monica Cartelle Gestal, Editor PONE-D-19-26950R2 Whole genome sequencing of extended-spectrum β-lactamase genes in Enterobacteriaceae isolates from Nigeria Dear Dr. Jesumirhewe: 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. Monica Cartelle Gestal Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0231146.r006

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 .