Testing the mutant selection window hypothesis with meropenem: In vitro model study with OXA-48-producing Klebsiella pneumoniae

Kamilla N. Alieva; Maria V. Golikova; Svetlana A. Dovzhenko; Mikhail B. Kobrin; Elena N. Strukova; Vladimir A. Ageevets; Alisa A. Avdeeva; Ofeliia S. Sulian; Sergey V. Sidorenko; Stephen H. Zinner

doi:10.1371/journal.pone.0288660

Peer Review History

Original SubmissionMarch 30, 2023
16 May 2023 Decision Letter - Atef Oreiby, Editor PONE-D-23-09422Testing the mutant selection window hypothesis with meropenem: in vitro model study with OXA-48-producing Klebsiella pneumoniaePLOS ONE Dear Dr. Golikova, 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 Jun 30 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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Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. 6. 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 If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references.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 If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.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: Please address the required comments of the reviewers. It will be useful if you entered a detailed workflow figure in material and methods section. [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: 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: 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: Dear Authors Thank you for your manuscript submission. Your work is interesting; however, a Major Revision is needed as below: 1. Please do read and add the following papers to References section of the manuscript to have fruitful Introduction section: Emerging multidrug-resistant bacterial pathogens "superbugs": A rising public health threat. Front Microbiol. 2023 Feb 1;14:1135614. doi: 10.3389/fmicb.2023.1135614. PMID: 36819057; PMCID: PMC9930894. Carbapenem-Resistant Klebsiella pneumoniae: Virulence Factors, Molecular Epidemiology and Latest Updates in Treatment Options. Antibiotics (Basel). 2023 Jan 21;12(2):234. doi: 10.3390/antibiotics12020234. PMID: 36830145; PMCID: PMC9952820. Metallo-ß-lactamases: a review. Mol Biol Rep. 2020 Aug;47(8):6281-6294. doi: 10.1007/s11033-020-05651-9. Epub 2020 Jul 11. PMID: 32654052. 2. It is recommended to add the related References associated with the used protocols in Materials and Methods section. 3. Please do add a Flow chart to Materials and Methods section to show all the procedures done in the present study. 4. It is recommended to explain about the sampling: How did you isolate the bacterial strains? Where did you isolate the bacterial strains? Which clinical samples were used to isolate the mentioned bacterial strains? Please do add the process of isolation from patients (the gender and the age range of patients) to lab. 5. Please do add the place of the study (City and Country) and the duration of the sampling and the study (from MM/YYYY to MM/YYYY)? 6. It is necessary to add an effective table to show the important Results obtained from this study. 7. Please do add the figures pertaining to HPLC section. 8. It is recommended to read and add the following paper to References section of the manuscript to have fruitful Discussion section: Virulence factors, antibiotic resistance patterns, and molecular types of clinical isolates of Klebsiella Pneumoniae. Expert Rev Anti Infect Ther. 2022 Mar;20(3):463-472. doi: 10.1080/14787210.2022.1990040. Epub 2021 Oct 28. PMID: 34612762. Reviewer #2: Dear Author This is a good manuscript in the right direction to understand resistance for these bacteria and give better insight into therapeutic doses. As you have highlighted in the manuscript, it is difficult to compare your work with others since there is no standardisation of this technique for carbapenamase expressing bacteria. This is clearly going to hinder the field in making any tangible movement to better understanding of treating these infections. It would be good if you could devise a future study that will use an ATCC characterised strain of Klebsiella that all other researchers can use as a control so as to make it possible to compare results from different labs. Recently, David Nicolau gave some guidelines on how we can perform in vivo studies of these strains in a uniform way and I believe that this will help to compare results between labs and continents. ****** 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: Payam BEHZADI Reviewer #2: Yes: Tricia Naicker ******** [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.0288660.r001
Revision 1
29 Jun 2023 Author Response Response to Academic Editor and Reviewers Comments Dear Dr. Atef Oreiby, Thank you very much for sending your and referees comments/suggestions, which were addressed in the revised manuscript. Here are our comments. I. Your personal comment Please address the required comments of the reviewers. It will be useful if you entered a detailed workflow figure in material and methods section. I – Response We included the Figure (Fig 1) displaying the experimental process flowchart comprising steps used to obtain the dataset for the analysis of “concentration – response” relationships. II. Comments of Referee 1 Dear Authors Thank you for your manuscript submission. Your work is interesting; however, a Major Revision is needed as below: II.1. Please do read and add the following papers to References section of the manuscript to have fruitful Introduction section: 1. Emerging multidrug-resistant bacterial pathogens "superbugs": A rising public health threat. Front Microbiol. 2023 Feb 1;14:1135614. doi: 10.3389/fmicb.2023.1135614. PMID: 36819057; PMCID: PMC9930894. 2. Carbapenem-Resistant Klebsiella pneumoniae: Virulence Factors, Molecular Epidemiology and Latest Updates in Treatment Options. Antibiotics (Basel). 2023 Jan 21;12(2):234. doi: 10.3390/antibiotics12020234. PMID: 36830145; PMCID: PMC9952820. 3. Metallo-ß-lactamases: a review. Mol Biol Rep. 2020 Aug;47(8):6281-6294. doi: 10.1007/s11033-020-05651-9. Epub 2020 Jul 11. PMID: 32654052. II.1. – Response We agree with the Reviewer’s comment; the proposed papers will help to introduce readers to the subject of our research. We extended the introduction section (Lines 46-50) and included the suggested references in the Reference list [Refs 1-4 in the revised manuscript]. II.2. It is recommended to add the related References associated with the used protocols in Materials and Methods section. II.2 – Response We wish to clarify that all study protocols were referenced in the respective publications in the original manuscript: MIC testing procedure [ref 21], MPC testing procedure [ref 22, 23], in vitro dynamic model operational and quantitation of the antimicrobial effect procedures [ref 24-26]. II.3. Please do add a Flow chart to Materials and Methods section to show all the procedures done in the present study. II.3. – Response We are grateful to the Reviewer’s comment to include a flowchart to the manuscript; it will help readers better understand the study process. Accordingly, we added the Figure (Fig 1) to the Materials and methods section with the flowchart describing the steps used to prepare the dataset for further analysis. II.4. It is recommended to explain about the sampling: How did you isolate the bacterial strains? Where did you isolate the bacterial strains? Which clinical samples were used to isolate the mentioned bacterial strains? Please do add the process of isolation from patients (the gender and the age range of patients) to lab. II.4. – Response According to the Reviewer’s comment we added detailed information about K. pneumoniae strains used in the study (Materials and methods section; Antimicrobial agent, bacterial isolates and susceptibility testing subsection). The sources of the isolated K. pneumoniae strains were: phlegmon, blood, wound and BAL. K. pneumoniae strains were identified through standard microbial and biochemical assays. The respective K. pneumoniae collection was stored at -80 ⁰C. The carbapenemase production by K. pneumoniae isolates was detected by PCR. OXA-48-positive isolates with meropenem MICs in range from 0.5 to 32 mg/L were selected for the study. Detailed clinical and demographic data of the ICU patients are not available. II.5. Please do add the place of the study (City and Country) and the duration of the sampling and the study (from MM/YYYY to MM/YYYY)? II.5. – Response According to the Reviewer’s comment we provided information about place of isolation of bacterial strains and the duration of sampling (Materials and methods section; Antimicrobial agent, bacterial isolates and susceptibility testing subsection): Clinical specimens from ICU patients in hospitals located in Moscow and Saint Petersburg (Russia) were collected from 2015 to 2019. II.6. It is necessary to add an effective table to show the important Results obtained from this study. II.6. – Response We thank the Reviewer’s for suggesting the addition of a table to present important results obtained in the study. To improve the perception of the decline in bacterial susceptibility during meropenem exposure, we included these data in the Table (Table 1 in the results section). As for other data obtained in the study, the most important include the antibacterial effect of meropenem and resistance development (values of the ABBC and ABBCR integral parameters) that were used for further “concentration – response” relationship analyses. However, we do not think the display of ABBC and ABBCR values would be particularly informative and we prefer not to add these data to the table. II.7. Please do add the figures pertaining to HPLC section. II.7. – Response According to the Reviewer’s comment we added the representative figures with chromatograms showing the base peaks in CSMHB (S1 Fig), detection of the meropenem standard (S2 Fig) and meropenem detection (peak concentration after the first infusion) in experimental samples with two K. pneumoniae strains, 202 (S3 Fig) and 1170 (S4 Fig). II.8. It is recommended to read and add the following paper to References section of the manuscript to have fruitful Discussion section: 1. Virulence factors, antibiotic resistance patterns, and molecular types of clinical isolates of Klebsiella Pneumoniae. Expert Rev Anti Infect Ther. 2022 Mar;20(3):463-472. doi: 10.1080/14787210.2022.1990040. Epub 2021 Oct 28. PMID: 34612762. II.8. – Response According to the Reviewer’s comment we added this paper to the Reference list as well as new sentences in the introduction and discussion sections about K. pneumoniae resistance patterns among the clinical isolates (Lines 404-409 in the revised manuscript). III. Comments of Referee 2 III.1. Dear Author This is a good manuscript in the right direction to understand resistance for these bacteria and give better insight into therapeutic doses. As you have highlighted in the manuscript, it is difficult to compare your work with others since there is no standardisation of this technique for carbapenamase expressing bacteria. This is clearly going to hinder the field in making any tangible movement to better understanding of treating these infections. III.1. – Response We thank the Reviewer for this comment. First, we would explain the technique of in vitro pharmacodynamic studies, operational procedure and viable bacteria enumeration, not influenced by the bacterial species and their ability to produce carbapenemases. At the same time, we are aware that more detailed information about lab equipment, media and manufacturers might be helpful for other researchers to compare results from different labs. To address the Reviewer’s comment, we added clarifying information about equipment used to mimic meropenem pharmacokinetics in the in vitro dynamic model and perform antibiotic dosing and sampling, and we indicate the manufacturer of media used in all testing procedures and pharmacodynamic simulations (Lines 90-91, 98, 111-113, 117-118). This is in addition to data that were specified in the original manuscript (hollow-fiber cartridge source and its model, the fiber’s material, CFU data, mimicked meropenem dose, half-life and CMAX); we hope this information will be useful for readers. III.2. It would be good if you could devise a future study that will use an ATCC characterised strain of Klebsiella that all other researchers can use as a control so as to make it possible to compare results from different labs. Recently, David Nicolau gave some guidelines on how we can perform in vivo studies of these strains in a uniform way and I believe that this will help to compare results between labs and continents. III.2. – Response We absolutely agree with the Reviewer’s comment that the use of well-described bacterial strains is very important to compare study results with others. We definitely will take note at the Reviewer’s advice to use ATCC characterized bacterial strains in future studies. We support the ability to increase the reproducibility of results obtained in different labs. We hope the revised manuscript will be acceptable for publication. Thank you for your consideration. Sincerely, On behalf of the authors, Maria V. Golikova Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0288660.r002
2 Jul 2023 Decision Letter - Atef Oreiby, Editor Testing the mutant selection window hypothesis with meropenem: in vitro model study with OXA-48-producing Klebsiella pneumoniae PONE-D-23-09422R1 Dear Dr. Golikova, 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, Atef Oreiby, Ph. D. Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0288660.r003
Formally Accepted
27 Jul 2023 Acceptance Letter - Atef Oreiby, Editor PONE-D-23-09422R1 Testing the mutant selection window hypothesis with meropenem: in vitro model study with OXA-48-producing Klebsiella pneumoniae Dear Dr. Golikova: 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. Atef Oreiby Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0288660.r004

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