Frequency and diversity of carbapenemase-producing Enterobacterales recovered from untreated wastewater impacted by selective media containing cefotaxime and meropenem in Ohio, USA

Rael J. Too; George C. Gitao; Lilly C. Bebora; Dixie F. Mollenkopf; Samuel M. Kariuki; Thomas E. Wittum

doi:10.1371/journal.pone.0281909

Peer Review History

Original SubmissionOctober 4, 2022
24 Oct 2022 Decision Letter - Mabel Kamweli Aworh, Editor PONE-D-22-27391Frequency and diversity of Carbapenemase-producing Enterobacteriaceae recovered from untreated wastewater impacted by β-lactam antibiotics Cefotaxime and Meropenem in Ohio, USAPLOS ONE Dear Dr. TOO, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Dec 08 2022 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. 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Please renumber the tables so that they correspond to the information already provided in the text. In the methods section, kindly reference "Table 1" in the text preceding the table as this is currently missing. 2. The discussion section needs to be rewritten. Please do not repeat the results in the discussion section, rather interpret the results in the light of previous studies and provide possible explanations for your findings. 3. Please highlight the limitations of this present study. [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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: N/A ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes 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: 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: Good publication, however, it seems like the author at least from the introduction fails to state the full objective of the study, on line 85 the author should consider revising and including that she/they were testing the concentration of the drugs in addition to the different types of drugs. On line 187, the author provides how different Meropenem concentrations faired but fails to provide how different concentrations of cefotaxime faired on the study. Were they just testing the concentration of Meropenem? This conflicts a finding on line 228 where they state the specific concentrations of Meropenem, and Cefotaxime showed good results without providing data on cefotaxime. Lastly, the author should provide more details on the DNA extraction method used Reviewer #2: This is a very important paper as the AMR threat to global health persists. The more curated data we have to combat this threat from all fronts, the more efficient and fine-tuned our efforts would be. The value of this particular work might be improved by providing background data on antimicrobial consumption in the hospitals/areas contributing to the waste water tested in the study. Such data can then be compared to the results of the present study. The authors stated "Thus for areas where there is high antibiotic consumption, like hospitals and animal agriculture facilities, active and targeted surveillance of wastewater should be carried out, to establish the dynamics of the situation". Data that actually shows the area studied actually has high antibiotic consumption will be helpful. The authors may wish to know that the table labeling in the manuscript is overtly confusing. The genesis of this confusion remains unknown to the reviewer and may not enhance readers' experiences. Are some tables missing? The sic regular pattern by which this error is also bewildering. Even numbered tables (2, 4, 6 and 8) referred to the text are missing in the downloaded manuscript. Odd numbered tables not referred to in the text are available. ****** 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". 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Revision 1
24 Dec 2022 Author Response I hereby appreciate all the PLOS ONE Secretariat and the reviewers; one (1) and two (2) for the good review comments raised and suggestions to improve this manuscript. They were very helpful. Thank you. The comments raised were addressed as follows; Note: Corresponding email; changed to too.4@osu.edu 1. Plos -One style requirements- All the files are named accordingly. 2. PLOS questionnaire on Inclusivity – included as an attachment. 3. Financial disclosure; a. I am a Ph.D. student at the University of Nairobi, OHEART – GOHi Ph.D. fellow at the Ohio State University (OSU) through the National Institutes of Health (NIH) Fogarty international center (grant number TW008650) This was a mentored Training through Ph.D. fellowship exchange program involving the three institutes (OSU, KEMRI and the University of Nairobi). b. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript c. 1st Author student stipend (National Institutes of Health (NIH) Fogarty international center (grant number TW008650) d. The authors received no specific funding for this work 4. Financial information: a. Rael Too (1st and corresponding author) b. grant number TW008650 (NIH D43 Fogarty PhD Fellow GOHi-OHEART-OSU) c. Student stipend 5. Data Availability statement; • The original contribution presented in this study is included in the study manuscript, further inquiries can be directed to the corresponding author. 6. Figure 1&2. No figures, (All deleted) 7. There is no figure 2, and any references to it are deleted and revised 8. Tables -The Tables are corrected and captioned correctly see (Tables 1, 2, 3, 4, 5) 9. Reference list The reference list (font changed -Times Roman 12), duplicate references edited additional references added and the list updated; 1. Azuma T, Otomo K, Kunitou M, Shimizu M, Hosomaru K, Mikata S, et al. Environmental fate of pharmaceutical compounds and antimicrobial-resistant bacteria in hospital effluents, and contributions to pollutant loads in the surface waters in Japan. Sci Total Environ. 2019 Mar;657:476–84. 2. Proia L, Anzil A, Borrego C, Farrè M, Llorca M, Sanchis J, et al. Occurrence and persistence of carbapenemases genes in hospital and wastewater treatment plants and propagation in the receiving river. J Hazard Mater. 2018 Sep;358:33–43. 3. Cahill N, O’Connor L, Mahon B, Varley Á, McGrath E, Ryan P, et al. Hospital effluent: A reservoir for carbapenemase-producing Enterobacterales? Sci Total Environ. 2019 Jul;672:618–24. 4. Suzuki Y, Nazareno PJ, Nakano R, Mondoy M, Nakano A, Bugayong MP, et al. Environmental Presence and Genetic Characteristics of Carbapenemase-Producing Enterobacteriaceae from Hospital Sewage and River Water in the Philippines. Elkins CA, editor. Appl Environ Microbiol. 2020 Jan 7;86(2):e01906-19. 5. Pauwels B, Verstraete W. The treatment of hospital wastewater: an appraisal. J Water Health. 2006 Dec 1;4(4):405–16 6. Cornaglia G, Akova M, Amicosante G, Cantón R, Cauda R, Docquier JD, et al. Metallo-β-lactamases as emerging resistance determinants in Gram-negative pathogens: open issues. Int J Antimicrob Agents. 2007 Apr;29(4):380–8. Additional Editor comments 1. Tables There were only five tables presented in the manuscript and all are renumbered and now correspond to the information in the text. “Table 1” in the text preceding included (page 4 line-150) 2. Discussion section - rewritten 3. Limitations of this present study included (page 9 lines 281-296) 4. Review comments to the author A. Reviewer #1: the full objective of the study, i. “Testing the concentration of the drugs in addition to different types of drugs” Only two β-lactam antibiotics were being tested (meropenem and cefotaxime) ii. ‘How did Meropenem concentrations fair but fail to provide different (Line 243-249) concentrations of cefotaxime, were they testing meropenem concentration?’ Yes, we tested Meropenem concentration at 0.5 μg/mL and 1.0 μg/mL meropenem modified with 70µg/mL ZnSO4 to check what concentration was ideal to recover more CPEs. iii. Line 228 specific concentrations of Meropenem, and cefotaxime showed good results without providing data on cefotaxime. Cefotaxime had low recovery rate compared to Meropenem in MacConkey broth (Line 243-249) iv. Details on DNA extraction method included (page 4, lines 139-144) B. Reviewer #2: AMR threats i. Background data on AMR consumption in Hospitals/areas contributing to the wastewater tested in the study. (Done- Introduction section lines 61-65) ii. Data that shows the area studied has high antibiotic consumption will be helpful. (Included lines 216-222) iii. Table labeling in the manuscript Addressed NB: Additional comments raised in recent submissions R1 and edits from the Supervisor About the mismatch in financial disclosure and financial information, this has been addressed as follows; Financial disclosure; a) I am a Ph.D. student at the University of Nairobi, OHEART – GOHi Ph.D. fellow at the Ohio State University (OSU) through the National Institutes of Health (NIH) Fogarty international center (grant number TW008650) This was a mentored Training through Ph.D. fellowship exchange program involving the three institutes (OSU, KEMRI and the University of Nairobi). b) The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript c) 1st Author student stipend (National Institutes of Health (NIH) Fogarty international center (grant number TW008650) d) The authors received no specific funding for this work Financial information: a. Rael Too (1st and corresponding author) b. grant number TW008650 c. Student stipend Other comments raised by the last author and have been addressed includes; a. Title edited to read; “Frequency and diversity of Carbapenemase-producing Enterobacterales recovered from untreated wastewater impacted by selective media containing cefotaxime and meropenem in Ohio, USA.” b. replaced Enterobacteriaceae- Enterobacterales through out the manuscript. c. Minor typos in throughout the Manuscript (Final Marked up copy with track changes) Attachments Attachment Submitted filename: Response to the Reviewers. 24-12-2022. docx.docx https://doi.org/10.1371/journal.pone.0281909.r002
8 Jan 2023 Decision Letter - Mabel Kamweli Aworh, Editor PONE-D-22-27391R1Frequency and diversity of Carbapenemase-producing Enterobacterales recovered from untreated wastewater impacted by selective media containing cefotaxime and meropenem in Ohio, USAPLOS ONE Dear Dr. TOO, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by January 27, 2023 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. 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Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Mabel Kamweli Aworh, DVM, MPH, PhD. FCVSN Academic Editor PLOS ONE 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. Additional Editor Comments: 1. In discussing your results, please do not repeat the values as these have already been presented in the results section, for example you do not need to mention Tables in your discussion section rather provide the interpretation of your results and possible explanations for your findings while comparing your results with reports of other published works. The discussion section needs to be revised please. [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: N/A ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: 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: No additional comments. The author has responded to all the comments raised adequately and as such, I propose that the document is pulished Reviewer #2: The authors have rectified the issues around the tables as earlier raised. However, the new references added by the authors are general and did not seem to reflect the specific area where their research was carried out. The original request was ...."The value of this particular work might be improved by providing background data on antimicrobial consumption in the hospitals/areas contributing to the waste water tested in the study. Such data can then be compared to the results of the present study. .... Data that actually shows the area studied actually has high antibiotic consumption will be helpful." Are previous studies on AMR and antibiotic consumption lacking for that area? ****** 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.0281909.r003
Revision 2
14 Jan 2023 Author Response Response to Reviewers Manuscript R2. Greetings! I hereby appreciate all of you the PLOS ONE Secretariat and the reviewers; one (1) and two (2) for the good review comments raised to improve this paper. They were very helpful. Thank you. 1. Laboratory protocols- N/A 2. Discussion - Done i. Repeated values were deleted and others used percentages to discuss. ii. Mentioned tables deleted iii. The discussion section is revised 3. Reference list The reference list is complete and correct Additional Editor comments 1. Review comments i. Reviewer #2: i. There was no background data collected on AMR consumption in hospitals/areas contributing to the wastewater tested in the study. ii. This is one of the limitations where by background data is missing or would have been collected and compare the results to ascertain the source (Line 286-290). However, this is a research gap and is part of the ongoing study in Kenya. iii. Discussion section revised. N/B: The findings from this study informed on the ideal method to use in implementing the current study in Kenya. Attachments Attachment Submitted filename: Response to the Reviewers -14th January 2023.docx https://doi.org/10.1371/journal.pone.0281909.r004
6 Feb 2023 Decision Letter - Mabel Kamweli Aworh, Editor Frequency and diversity of Carbapenemase-producing Enterobacterales recovered from untreated wastewater impacted by selective media containing cefotaxime and meropenem in Ohio, USA PONE-D-22-27391R2 Dear Dr. TOO, 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, Mabel Kamweli Aworh, DVM, MPH, PhD. FCVSN Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 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: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: N/A ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: 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: No additional comments. The author has responded to all the comments raised adequately and as such, I propose that the document is pulished Reviewer #2: The authors of the manuscript have addressed all the concerns earlier raised. The manuscript is now acceptable ****** 7. 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Formally Accepted
10 Feb 2023 Acceptance Letter - Mabel Kamweli Aworh, Editor PONE-D-22-27391R2 Frequency and diversity of Carbapenemase-producing Enterobacterales recovered from untreated wastewater impacted by selective media containing cefotaxime and meropenem in Ohio, USA Dear Dr. Too: 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. Mabel Kamweli Aworh Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0281909.r006

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