PONE-D-21-36109Dissemination of Carbapenemase-producing Enterobacterales in the community of Rawalpindi, PakistanPLOS ONE

Dear Dr. Zahra,

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 Jan 20 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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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.

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We look forward to receiving your revised manuscript.

Kind regards,

Zhi Ruan, Ph.D.

Academic Editor

PLOS ONE

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This work was partially supported by the “Fondation pour la Recherche Médicale” (Equipe FRM 2016, grant 325 number DEQ20161136698); the Direction Générale des Armées (project FastGeneII); the Programme Hubert Curien “Peridot” (Ministère des Affaires Etrangères, France) and by the Higher Education Commission, Pakistan under Pak-France Joint Grant (PERIDOT) to RZ and ER and International Research Support Initiative Program (IRSIP) (PIN: 43 BMS 16) to AH. 

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This work was partially supported by the “Fondation pour la Recherche Médicale” (Equipe FRM 2016, grant 325 number DEQ20161136698); the Direction Générale des Armées (project FastGeneII); the Programme Hubert Curien “Peridot” (Ministère des Affaires Etrangères, France) and by the Higher Education Commission, Pakistan under Pak-France Joint Grant (PERIDOT) to RZ and ER and International Research Support Initiative Program (IRSIP) (PIN: 43 BMS 16) to AH. 

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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

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

Reviewer #3: N/A

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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

Reviewer #3: Yes

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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

Reviewer #3: Yes

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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: I have some question/comments for the author to improve their manuscript.

1. Introduction: Situation of CPE/CRE in Pakistan should be mentioned.

2. Line 110-118: How many isolates were selected? and what is the criteria to select?

3. Line 178-185: What is colistin susceptibility result? it is not mention in this part.

4. Line 230: % should be shown.

5. Discussion: Although this study showed only one case/isolate of mcr-harboring CPE but it is a signal to monitoring. The mcr-harboring CRE were documented in several literature. The author can discuss this detail to lead awareness of the clinician and infection control. Please see 'Paveenkittiporn et al., Whole-Genome Sequencing of Clinically Isolated Carbapenem-Resistant Enterobacterales Harboring mcr Genes in Thailand, 2016-2019. Front Microbiol. 2021 Jan 11;11:586368. doi: 10.3389/fmicb.2020.586368.". This might be useful to discuss because it report from the Asian country.

Reviewer #2: Reporting data on the detection of CPE in each country is necessary to understand the dissemination of CPE and to develop countermeasures. However, the description of the results is not clear in the text. It makes difficult to understand results and needs to be revised.

L. 186 - 208

The number of CPE is unclear. How many CPE strains were isolated, 73, or 78? Which genes were detected in the strains? Which species were detected? Authors should add a table summarizing the essential information from Fig. S3 to the text.

Methods, Patients and samples

Although the paper is about CPE in the Community, it includes strains detected after 72 hours of hospitalization. These should be excluded.

L. 142 - 150

There is a problem with CRE quantification because the counts are made using cultured bacteria that have been previously subjected to ertapenem selection, which overestimates the number of CRE. Also, CRE quantification is not used for discussion and I do not understand the significance of its inclusion. It would be better to delete the descriptions and figures related to CRE quantification.

L. 215

Strains named Enterobacter cloacae complex should be re-identified using ANI and the correct species name filled in. Also, do not abbreviate the name of a genus that appears for the first time.

Fig.1

In the process of processing the 262 negative patients, 4 patients (negative:262-refused:98-not-hospitalized:63-ICU:3-sampled:94) are missing. Please check.

Fig.3

It should be carefully explained where the "clade" is. Especially for the ST648 clade, it is difficult to understand where the clade is located on the tree. It would be better to add some outgroups and make root for easier reading.

In the stacked bar graph of Fig. S1, the number of samples and the results are stacked on top of each other, and the number may be twice as large. It needs to be redesigned.

In Fig. S2, S4, and S5, only the number of detections is listed, and information such as from which ST type of which bacteria species is not available. Please add this information.

Reviewer #3: In this study the authors investigated the prevalence of carbapenemase-producing Enterobacterales (CPE) in patients attending a hospital from the community in Rawalpindi, Pakistan. Rectal swabs obtained from patients attending traumatology and gynaecology services without any preceding antibiotic exposure, hospitalisation or travel history in the last 3 months were plated on screening agar. Enterobacterales were identified by MALDI-TOF MS and antibiotic susceptibility testing and whole-genome sequencing performed.

The paper is well written and all the results are clearly presented. It presents valuable information on the prevalence of community CPE in Pakistan. WGS highlights the dominant sequence types carrying CPE genes and provides epidemiological information on plasmids carrying carbapenemase genes.

I have the following comments and suggestions for the authors:

METHODS:

Page 5, line 98: Antimicrobial susceptibility testing. It would add to the manuscript if the authors could perform susceptibility testing against newer agents, like aztreonam+avibactam and cefiderocol.

RESULTS:

Sampling: Page 8, line 160-162: In this study a young patient cohort was investigated (average age 38 and 44 years). How does this compare to other studies from the region? Could this influence the finding?

Prevalence of CPE carriage: Page 8, line 169: It would be interesting to add some information on whether the high community prevalence was reflected in disease burden in the hospital. How many of the patients required treatment for CPE? Were any of the hospital acquired cases considered to be linked?

Phylogeny: Page 10, line 220: The authors comment on the maximal genetic distances but do not discuss genetic similarities. On the tree some of the sequences look like they are related? Did they find possible relatedness between community and hospital acquired strains indicating possible cross transmission? Could the authors comment on this?

Relative abundance: Page 11, line 239: The authors present data on quantification of CRE but do not discuss the finding. Did they expect a difference in the relative abundance between community-acquired CRE and hospital-associated CRE? Does this depend on whether the patient receives antibiotics? I suggest to briefly mention this in the discussion.

DISCUSSION:

Line 248: ‘CPE carriage is mostly associated with hospital environment’ Did the other studies undertaken in Pakistan focus on hospital acquired cases? It would be interesting to discuss isolates that were acquired in hospital here. On the tree they look spread out over the clades but closely related to some of the community isolates? What is known about hospital acquisition of CPE in hospitals in Pakistan? How many of the CPE positive patients did require CPE treatment? What impact does the high community carriage rate have on hospitals?

Line 278: ‘Combination of resistance genes, virulence, and ability to easily disseminate…’ The authors mention virulence here but do not discuss virulence data for the observed ST types. How frequent is ST 167 found in clinical samples?

Line 295/296: It would add to the value of the manuscript to be able to discuss cefiderocol susceptibilities here. It might not be available as an agent in Pakistan yet but with the high prevalence of metallo-b-lactamases it would be helpful to assess its activity against the isolates of the study.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Kirsten Schaffer

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PONE-D-21-36109R1Dissemination of Carbapenemase-producing Enterobacterales in the community of Rawalpindi, PakistanPLOS ONE

Dear Dr. Zahra,

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 Mar 24 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.

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: 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-emailutm_source=authorlettersutm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Zhi Ruan, Ph.D.

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.

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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)

Reviewer #3: All comments have been addressed

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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

Reviewer #3: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: No

Reviewer #3: (No Response)

**********

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

Reviewer #3: (No Response)

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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

Reviewer #3: (No Response)

**********

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: There is incorrect on lines 327-328 "A study from Thailand also reported similar findings of mcr-1 detection from asymptomatic human feces in 2012 [52]." That study is conducted on patients with variety specimens, no feces. The author should be check and carefully mentioned. Please revise this sentence again.

Reviewer #2: Overall, it has improved, but there was one point that was not answered satisfactorily because the previous my review was unclear.

L. 150-158 Methods, Quantification of CRE

The counting method regarding the total amount of Gram-negative bacteria is unclear. According to the description in the text, rectal specimens were first inoculated onto ertapenem-supplemented agar plates. Therefore, even if the specimens were cultured on agar plates without ertapenem, the total amount of Gram-negative bacteria may not have been counted accurately, and those selected for ertapenem may have been counted. In such a case, the number of Gram-negative bacteria would be low and the CRE would be overestimated, which raises questions about the reliability of the CRE-RA and is unacceptable. The method of counting total Gram-negative bacteria should be clearly explained.

Reviewer #3: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: MASAHIRO SUZUKI

Reviewer #3: 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.

PONE-D-21-36109R2Dissemination of Carbapenemase-producing Enterobacterales in the community of Rawalpindi, PakistanPLOS ONE

Dear Dr. Zahra,

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.

==============================

ACADEMIC EDITOR: A major revision is required.Please revise the manuscript according to the reviewer comments./==============================

Please submit your revised manuscript by Apr 16 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.

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: 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-emailutm_source=authorlettersutm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Abdelazeem Mohamed Algammal, Prof, Ph.D

Academic Editor

PLOS ONE

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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 #4: (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 #4: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #4: 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 #4: 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 #4: 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 #4: Comments to authors:

-The current study is interesting; however, the authors should address the following comments to improve the quality of the manuscript:

- Please write the scientific names of bacterial pathogens and genes in the correct form all over the manuscript and the references section.

Title:

I think the work would benefit from the title that contains the main conclusion of the study (should be derived from the conclusion). Please modify the title.

Abstract:

- The abstract must illustrate the used methods and the most prevalent results (give more hints about methods and results). Besides, rephrase the aim of the work and the main conclusion of your findings.

-Introduction: (it needs to be more informative):

-Give a hint about the virulence factors, different infections caused by E.coli and K. pneumoniae, and the mechanism of disease occurrence.

- The authors should illustrate the public health importance concerning the emergence of multidrug-resistant (MDR) bacterial pathogens that reflect the necessity of new potent and safe antimicrobial agents. Several studies proved the widespread MDR- bacterial pathogens;

Authors could add the following paragraph:

Multidrug resistance has been increased all over the world that is considered a public health threat. Several recent investigations reported the emergence of multidrug-resistant bacterial pathogens from different origins including humans, birds, cattle, and fish that increase the necessity of new potent and safe antimicrobial agents. Besides, the routine application of the antimicrobial susceptibility testing to detect the antibiotic of choice as well as the screening of the emerging MDR strains. You are advised to cite the following valuable studies:

1.PMID: 33177849

2.PMID: 33188216

3.PMID: 30150182

4.PMID: 33947875

5.PMID: 32994450

6. PMID: 32497922

7.PMID: 33061472

8.PMID: 34445951

9.https://doi.org/10.5114/ceji.2013.3774020 .

10.https://doi.org/10.1016/j.aquaculture.2021.737643

11.https://doi.org/10.1016/j.foodcont.2021.108066

-Rephrase the aim of the work to be clear and better sound.

Material and methods:

-Add this subtitle: Bacterial Isolation and identification:

•Discuss in detail the methods of isolation and identification of Enterobacterales, especially, E. coli and K. pneumoniae. Besides, specific references should be added.

•Add the company, city, and country of the used bacterial media and reagents that were used in the biochemical identification of isolates. Also, enumerate all used biochemical reactions.

- Modify the subtitile to be: Antimicrobial susceptibility testing:

-Illustrate the antimicrobial classes of the tested antibiotics.

-The authors are advised to classify the tested isolates to MDR , XDR, and PDR as described by Magiorakos et al.

Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012; 18:268–81. doi:10.1111/j.1469-0691.2011.03570.x.

- The correlation between phenotypic and genotypic multidrug resistance should be performed.

-Add the following subtitle to the methods section, and disscuss the method in detail ( Support the methods with specific references):

Multilocus sequence typing (MLST)

-Statistical analyses:

-Add more details about the software used in the statistical analyses.

-Results:

-Add this subtitle: Phenotypic characteristics of the recovered isolates:

•Illustrate in detail the phenotypic characteristics of the recovered E. coli and K. pneumoniae isolates.

- Illustrate in a table the results of The Antimicrobial susceptibility testing.

•Illustrate in a new table the occurrence of MDR (Multidrug resistance) among the recovered isolates as the following (illustrate the names of the antimicrobial classes and different antibiotics):

No. of strains%Type of resistance

R, MDR, and XDRPhenotypic multidrug resistance

(Antimicrobial classes and different antibiotics).The antibiotic -resistance genes

-The correlation (Correlation coefficient) between phenotypic and genotypic multidrug resistance should be performed.

- Add the supplemetary Figures to the main manuscript.

-Increase the resution of all Figures ( it should be 600 dpi).

-Discussion:

- The authors are advised to illustrate the real impact of their findings without repetition of results.

-Illustrate the different mechanisms of antimicrobial resistance in E. coli and K. pneumoniae.

-Conclusion

- Should be rephrased to be consise and sounded. A real conclusion should focus on the question or claim you articulated in your study, which resolution has been the main objective of your paper?

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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 #4: 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.

PONE-D-21-36109R3Dissemination of Carbapenemase-producing Enterobacterales in the community of Rawalpindi, PakistanPLOS ONE

Dear Dr. Zahra,

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.

Specifically, please provide your responses to the reviewer's remaining concerns.

Please submit your revised manuscript by Jun 26 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.

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: 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-emailutm_source=authorlettersutm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Jianhong Zhou

Staff 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.

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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)

Reviewer #3: All comments have been addressed

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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

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: N/A

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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: No

Reviewer #2: (No Response)

Reviewer #3: Yes

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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

Reviewer #3: Yes

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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: This revised manuscript has been address all points of the reviewer's comments. I satisfy the response of the authors.

Reviewer #2: My concerns previously noted have been corrected, and together with the corrections indicated by Reviewer 4, the manuscript is much better. I have commented on the parts of the text that the author refused in response to Reviewer 4's remarks as follows.

The comment refused by authors: Title

Comments: No modification is required. I do not believe that the title change is necessary because, as the authors claim, the study investigated the genus Enterobacterales bacteria.

The comment refused by authors: References assigned by Reviewer 4

Comments: No modification is required. The references assigned by Reviewer 4 are biased in authorship and are not impartial. I believe they should NOT be added.

The comment refused by authors: Discuss in detail the methods of isolation and identification of Enterobacterales, especially, E. coli and K. pneumoniae.

Comments: Modification is required. Selection plates and concentration of antimicrobials used will affect the detection rate. It is a good idea to add this information to the “limitation” in discussion.

The comment refused by authors: The correlation between phenotypic and genotypic multidrug resistance should be performed

Comments: Modification is required. I think, Reviewer 4 may not have pointed out the methodologies. The paper does include antimicrobial susceptibility tests and a genetic study, which suggests that the necessary experiments have been performed. However, it does not present data linking the phenotypic antimicrobial susceptibility to the genotype. In particular, a number of strains have been found to be susceptible to monobactam, aminoglycosides, and imipenem, and the correlation with genotype needs to be discussed.

The comment refused by authors: Illustrate the different mechanisms of antimicrobial resistance in E. coli and K. pneumoniae.

Comments: No modification is required. As the author claims, the drug resistance mechanism is introduced in the introduction, so there is no need to add it to the discussion again.

Reviewer #3: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Masahiro Suzuki

Reviewer #3: 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.

Dissemination of Carbapenemase-producing Enterobacterales in the community of Rawalpindi, Pakistan

PONE-D-21-36109R4

Dear Dr. Zahra,

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,

Jianhong Zhou

Staff Editor

PLOS ONE

Additional Staff Editor Comments: Please update your data availability statement on the submission details page by including the accession number PRJNA645311 as this will be used for the Data Availability Statement in the published article.  statementonthesubmissiondetailspageasthiswillbeusedfortheDASinthepublishedarticle.

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: 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 #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: 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 #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: (No Response)

**********

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: Yes: Masahiro Suzuki

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