PONE-D-24-53922An investigation of carbapenemase-encoding genes in Burkholderia cepacia and Aeromonas sobria nosocomial infections among Iraqi patientsPLOS ONE

Dear Dr. Al-Ouqaili,

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5. We note that you have referenced (Hussein RA, Al-Ouqaili MTS, Majeed YH. Association between alcohol consumption, cigarette smoking, and Helicobacter pylori infection in Iraqi patients submitted to gastrointestinal endoscopy. J Emerg Med Trauma Acute Care [Internet]. 2022 Dec 22 [cited 2022 Dec 23];2022(6):12. Available from: https://www.qscience.com/content/journals/10.5339/jemtac.2022.aimco.12) which has currently not yet been accepted for publication. Please remove this from your References and amend this to state in the body of your manuscript: (ie “Bewick et al. [Unpublished]”) as detailed online in our guide for authors

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Additional Editor Comments:

The manuscript provides important information on antibiotic sensitivity pattern of Burkholderia spp. and Aeromonas isolates from Iraq and resistance related marker genes. However authors need to revise manuscript as per reviewers comments! Adress all comments from reviewers!

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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

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

Reviewer #1: No

Reviewer #2: No

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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: Yes

Reviewer #2: 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

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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: This study investigates the prevalence of antibiotic resistance, including carbapenem resistance, in Burkholderia cepacia and Aeromonas sobria isolates from clinical specimens in Iraq. It reported significant resistance rates to multiple antibiotics, with a predominance of blaKPC and blaGES carbapenemase producers, highlighting the need for rapid and accurate bacterial identification methods and better treatment strategies for multi-drug resistant infections.

The manuscript can be accepted only after incorporating following points:

Major comments

1.Please revise the title as Investigation of Carbapenemase-Encoding Genes in Burkholderia cepacia and Aeromonas sobria Isolates from Nosocomial Infections in Iraqi Patients

2.In abstract,

-The methods section is vague. Important details such as patient population, or sampling method are missing.

-There is some inconsistency in reporting numbers and percentages. For example You say “75 isolates of A. sobria and B. cepacia were assessed. A. sobria made up 16.6% (n=20), B. cepacia 45.8% (n=55)” — this totals 100% (75 isolates), but the calculation is different 20/75 = 26.6%, 55/75 = 73.3%. There is also a line that mentions “55/57 recA gene positive,” but earlier it says only 55 isolates of B. cepacia were included.

3.Introduction

- Several sentences repeat similar points (e.g., B. cepacia’s resistance and persistence are mentioned multiple times).

"Burkholderiaceae family (previously known as Pseudomonas cepacian)" is misleading. Pseudomonas cepacia was the former name of the species, not the family.

- Reference [2] on B. cepacia persistence and transmission seems dated or generalized; newer sources might better reflect current understanding of nosocomial spread.

- Consider briefly stating why A. sobria (less frequently reported than A. hydrophila) is the focus, especially when A. hydrophila is mentioned as more common.

-The study objective appears abruptly at the end. The authors are suggested for the transition into the aim more clearly by summarizing the knowledge gap.

4.Methodology Section:

-Although the study employs both VITEK 2 System and recA gene-based PCR for identification, more details on the specific conditions under which these methods were conducted would improve reproducibility. For example, did the VITEK 2 System settings or parameters vary from standard procedures? Similarly, the exact primers used for PCR should be included, along with details on the PCR conditions and any potential limitations of these methods.

-The description of carbapenemase testing through both phenotypic and genotypic approaches is valuable, but further clarification is needed on how results were interpreted in case of discrepancies between the two methods. This would help readers understand the rationale behind the final conclusions on resistance.

5.Discussion of Results:

-The discussion regarding the resistance profiles, especially with respect to B. cepacia and A. sobria, could be expanded to address the clinical implications of these findings more comprehensively. For example, how do these resistance patterns correlate with patient outcomes or impact therapeutic decisions? Also, how do the findings compare with other studies in the region or globally in terms of therapeutic options?

-The prevalence of carbapenemase-producing strains is discussed, but the manuscript could benefit from more in-depth analysis on the mechanisms of carbapenem resistance. For instance, are there any particular resistance mechanisms (e.g., porin loss, overproduction of β-lactamases) that could explain the observed high levels of resistance in B. cepacia and A. sobria? The manuscript suggests that other resistance mechanisms could be at play, but this could be explored in more detail.

6.Statistical Analysis:

While the study includes some breakdowns of resistance patterns (e.g., percentage of resistance), the manuscript lacks a detailed statistical analysis of the data. Including confidence intervals, significance testing (e.g., chi-square tests for categorical variables like gender and resistance patterns), and comparative analysis would improve the robustness of the findings.

Minor Comments and Typos:

1.Typo/Grammar:

o"No A. sobria was found in any of the cerebrospinal fluid specimens." → "No A. sobria isolates were found in any of the cerebrospinal fluid specimens."

o"This is consistent with Pineda-Reyes et al. (2024), who observed that A. sobri is linked to a variety of human infections..." → "A. sobria" (should match the species name format consistently).

oPlease be consistent in naming species, especially with the genus and species names like B. cepacia and A. sobria. Use italics throughout and ensure the first letter of the genus is capitalized.

oEnsure uniform citation formatting throughout the manuscript. There are a few inconsistencies in the way references are presented (e.g., reference numbering).

2.Figures/Tables:

oThe manuscript could benefit from including figures or tables that summarize the major findings, such as the resistance rates for each antibiotic tested, the types of specimens sampled, and a comparison of resistance patterns between B. cepacia and A. sobria. This would make the data more accessible and visually impactful.

3.Conclusion:

oThe conclusion is well-written but could be more focused on specific recommendations for future research and clinical practice. A few key research questions or clinical practice guidelines based on the findings would strengthen this section. For example, should clinicians be more cautious when using specific antibiotics for B. cepacia and A. sobria infections based on the resistance patterns observed?

oThere seems to be some inconsistency in the way data is reported. For example, you mention percentages in some cases but then refer to absolute numbers in others (e.g., "58.7% male" vs. "44 males"). It would be clearer and more standardized to report either both absolute numbers and percentages, or just one for consistency.

Reviewer #2: Review Report of the manuscript entitled An investigation of carbapenemase-encoding genes in Burkholderia cepacia and Aeromonas sobria nosocomial infections among Iraqi patients

This manuscript explores the prevalence of carbapenemase-encoding genes (blaKPC, blaGES, and blaIMP) among Burkholderia cepacia and Aeromonas sobria isolated from nosocomial infections in Iraq, using both phenotypic and molecular techniques. The topic is more relevant, especially in regions where antibiotic surveillance data are limited. The manuscript provides valuable local AMR data that may contribute to broader global antimicrobial resistance mapping. However, while the research question is relevant and the methods broadly appropriate, the manuscript further requires major revisions before it can be considered for publication.

1. Lack of clarity about the sampling method and inclusion/exclusion criteria. Please clarify.

2. Justification for selecting only three carbapenemase genes (blaKPC, blaGES, blaIMP) is insufficient—why you exclude blaNDM, blaOXA, blaVIM Please give the reasons?

3. Please give Controls for PCR and phenotypic testing (positive/negative) are not mentioned.

4. Only basic chi-square testing is used. A more robust analysis (e.g., multivariate regression) should be considered to correlate resistance genes with demographics or clinical variables.

5. Figures (e.g., gel electrophoresis) are low resolution and poorly labeled.

6. Tables need improved formatting; define all abbreviations and ensure legends are self-explanatory.

7. Sample size reporting is occasionally inconsistent (e.g., 75 vs 56 isolates).

8.Several results are repeated multiple times across the text, tables, and figures.

9.Lack of MIC50/MIC90 or ranges diminishes the quality of antibiotic resistance interpretation.

10.The discussion is largely descriptive and lacks critical evaluation of findings.

11. Clinical implications of findings are weakly addressed (e.g., impact on treatment policy or infection control practices).

12. Mentions “genomic sequencing” without this being part of the study.

13.Inconsistent use of scientific names (italicization of B. cepacia, A. sobria must be needed throughout the manuscript).

14. Abstract needs grammatical and syntactical revisions (e.g., “92.8%%” should be “92.8%”).

15 Some references are outdated, and certain statements require more recent supporting evidence.

16. In methodology: Study design (cross-sectional) and sampling methods are acceptable but

Was the sampling random or convenience-based? What were the inclusion/exclusion criteria?Ethical approval and consent are reported appropriately. Primer sequences and thermocycling protocols should be summarized more clearly in one table. Justification for excluding genes like blaNDM, blaOXA, or blaVIM is missing. The phenotypic test (double disk synergy) is correctly described but lacks proper validation or controls explanation.

17. There is confusion in sample size reporting: The abstract mentions 75 isolates, with 20 A. sobria and 55 B. cepacia, totaling 75, yet elsewhere “56 carbapenem-resistant isolates” are analyzed without clear criteria. Please give Justification??

18. Use of vague terms like “high”, “little resistance”, or “more accurate” should be replaced with quantified expressions.

19. Not all abbreviations are defined e.g., MEM, IMP are missing.

20.Some values are redundant across tables and figures.

21.The absence of sequencing data for PCR amplicons is a major limitation. Without sequencing, false-positive bands from non-specific amplification cannot be ruled out. Lack of controls (positive and negative) for each PCR gene detection is not mentioned.

22. According to CLSI guidelines, the double-disk synergy test (DDST) is not the gold standard for carbapenemase detection; tests like Carba NP or modified Hodge test should be used.

23. The study donot correlate clinical outcomes (e.g., mortality, hospital stay) with resistance profiles. Host factors, such as patient comorbidities, prior antibiotic exposure, and ICU admission, are not considered in the study. Please consdider for these factors.

24.The authors state adherence to the “Helsinki Declaration of 1979,” which is outdated; the 2013 version is recent Helsinki Declaration.

Final Recommendation: Major Revisions

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Reviewer #1: No

Reviewer #2: Yes:  Komal Raj Rijal

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PONE-D-24-53922R1Investigation of Carbapenemase-Encoding Genes in Burkholderia cepacia and Aeromonas sobria Isolates from Nosocomial Infections in Iraqi PatientsPLOS ONE

Dear Dr. Al-Ouqaili,

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 Aug 27 2025 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-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Hideo Kato

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 #3: All comments have been addressed

Reviewer #4: All comments have been addressed

Reviewer #5: (No Response)

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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 #3: Yes

Reviewer #4: Partly

Reviewer #5: Yes

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

Reviewer #3: Yes

Reviewer #4: N/A

Reviewer #5: Yes

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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 #3: Yes

Reviewer #4: Yes

Reviewer #5: 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 #3: Yes

Reviewer #4: Yes

Reviewer #5: No

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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 #3: The authors have done well in addressing the comments raised and the work is technically sound, it is a commendable work which can make significant impact in the field of medical and pharmaceutical microbiology

Reviewer #4: Authors have made a significant changes in the manuscripts. However, before final remarks, some are needs to be improved for better clarification.

Abstract:

1. You have mentioned that you collected 120 samples, and assessed 75 isolates for further identification. did you mean that 75 isolates were positive in culture out of 120 samples? If so then add it and clarify it or if not so then mention how did you choose 75 isolates?

2. You have mentioned "The study isolates showed

highest antimicrobial resistance to piperacillin, cefepime, ceftriaxone (100%), ceftazidime

(97.3%), and lowest antimicrobial resistance to imipenem (36%)". it is better to add the resistance percentage of all antibiotics for scientific clarity.

3. In a few areas of the whole paper, you have mentioned a data like only number. For as an example " The 42 B. cepacia isolates that tested positive for carbapenem

resistance were constituted of 38 blaKPC (n = 38) and two blaGES (n = 2); in contrast, four blaKPC

(n = 4) and eight blaGES (n = 8) were present in the A. sobria isolates that tested positive for

carbapenems resistance" However, it is better to add percentage as well with the number.

4. You have added "None of isolates studied tested positive for the blaIMP gene. The recent study concluded that recA gene identification was more sensitive and specific technique for detection B. cepacia complex isolates. There was a notable predominance of blaKPC and blaGES carbapenemase producers among the isolates under investigation. The blaIMP gene was not found in any of the research isolates". Please remove the last two line from " There was a notable predominance of blaKPC and blaGES carbapenemase producers among the isolates under investigation. The blaIMP gene was not found in any of the research isolates" as you mentioned earlier. Instead of this, add a remarks which steps should be considered to reduce this the threat during treatment by a clinician or something like that.

Materials and Methods

5. you have mentioned " Many of the samples were cultivated on culture media such as MacConkey

agar, Blood agar, and Mannitol agar (Oxoid, UK), as obtained from CSF, urine, ear swabs, burns,

wounds and diabetic foot ulcer". But it's not clear how many have you done and add the result of this in the result section. Otherwise its too vague to understand.

6. You have described that you have identified these isolates "According to MacFaddin's (2000) procedures" but did not cite this reference, please add the reference for reader to find clearly.

7. You have done Antibiotic Susceptibility by CLSI guildlike but did not mention the year for CLSI, as it is changing day by day or CLSI is publishing new reference to use for researcher.

8. You have mentioned "A total of 56 strains resistant to at least one carbapenem were investigated" but ther number you have found did not included in previous section as it is in result section. So it is better to the number in only result section and escribe it like " The isolates that was resistant to at least one carbapenem were investigated for MBL production.

9. In DNA extraction and PCR sectionS, you have mentioned " the entire DNA of 75 B. cepacia and 20

A. sobria isolates was extracted" is it 75 B. cepacia or it would be 55?? As earlier you have added in the asbtract that you found 55 B. cepacia!!!!. It is also better to skip the number in methods, just add in result section like, "in molecular detecction of 55 B. cepacia and 20 A. sobria, we found...........

10. In statistical methods, you did not mention how did you calculate 95% CI. Please add.

Results:

11. You have outlined " Among the 75 isolates assessed 20 (26.6%) were A. sobria and 55 (73.3%) were B.

cepacia" it is better to say like "Among the 75 culture positive isolates assessed in this study, 20 (26.6%) were A. sobria and 55 (73.3%) were B. cepacia. or add from you have selected or found 75 isolates.

12. You have added "The average age of the study participants, who ranged in age from 10 to 70,

was 40.13 ± 22.5 years" but did not included in table 2. Please add for all the categories for better understanding.

13. Pleas add percentage here for each antibiotics "The isolates showed resistance to cefepime, piperacillin, and ceftriaxone (100%),ceftazidime (97.3%), and imipenem (36%)"

14. In figure 2, it seems L3 is negative or absence of positive bands but you did not added this information the the figure legend.

15. In the "Phenotypic and Genotypic Validation" section you described : There was no gene present in two

isolates that were resistant to carbapenem. None of the isolates contained the blaIMP gene, as can

be seen in Figures 4, 5. There was no gene present in the two isolates resistant to carbapenem. It seems like there is repetition. Please clarify.

16. In a previous question "The prevalence of carbapenemase-producing strains is discussed, but the manuscript

could benefit from more in-depth analysis on the mechanisms of carbapenem

resistance. For instance, are there any particular resistance mechanisms (e.g., porin

loss, overproduction of β-lactamases) that could explain the observed high levels of

resistance in B. cepacia and A. sobria? The manuscript suggests that other resistance

mechanisms could be at play, but this could be explored in more detail", you have added the description in the result section and discussion both. But it is better to add only in discussion section to make the result section more clear and concise.

17.The abbreviation of PCR should only be "*PCR: Polymerase Chain Reaction"

18. Result section seems to large. It is better to make it concise by adding only the findings from your study and the information like your theoritical observation would be kept in discussion section only.

19. Please add reference for below information in discussion parrt "Carbapenemases are enzymes that have various different hydrolytic profiles; all are categorized as �-lactamases. All the �-lactam antibiotics, including the penicillins,

cephalosporins, monobactams, and the carbapenems, are acted upon by these enzymes. Due to

carbapenemase activity, a majority of �-lactam drugs can become inactive against the severe

infections that result from bacteria that produce such �-lactamases. Enzyme superfamilies’

comprise carbapenemases from the A, B, and D classes of �-lactamases. Zinc is present in the

active region of class B enzymes, which are metallo-�-lactamases; class A and D enzymes, on

the other hand, hydrolyze serine. The Guiana-Extended-Spectrum and Klebsiella pneumoniae

18carbapenemase families are amongst the Class A carbapenemases

20. If possible reduce the discussion and keep only relevant information though it is well described. But reducing will enhance the clarify for better understanding.

21. In conclusion, you have added "The main objectives of this work were to identify the resistance genes in isolates

of B. cepacia and A. sobria molecularly and to detect antibiotic resistance phenotypically" in the middle part, but it would be better to add in the first line then followed by your major findings and conclusion remarks.

22. You also added "The analysis did not include clinical outcome data, such as ICU admission, comorbidities, length of hospital stay, prior antibiotic exposure, or patient mortality. Although we acknowledge the significance of these host-related factors in comprehending the clinical consequences of antibiotic resistance, they were outside the purview of the current microbiological study" in the conclusion but it would be better to add in the end part of the discussion to illustrate your limitation and for better understanding of your conclusion remarks.

Reviewer #5: Thank you for your efforts in revising the manuscript. While I acknowledge that some reviewer comments have been addressed, several critical issues remain that require your attention to improve the manuscript’s quality and clarity.

General Comments:

Manuscript Formatting: The absence of line numbers made it difficult to conduct an efficient review. In future resubmissions, please ensure that all revisions are clearly indicated and line numbers are included.

Language and Style: The revised manuscript still includes numerous typographical errors and lacks an academic tone in several sections. It is strongly recommended to have the manuscript professionally edited or reviewed by a fluent English speaker.

Discussion Section: The beginning of the discussion section closely mirrors the results, without providing critical analysis. The discussion should succinctly highlight key findings, contextualize them with relevant literature, and explain their broader implications.

Specific Comments:

Introduction:

Line 12: The phrase “The bacterial isolates of B. cepacian” should be revised to “B. cepacian isolates” for conciseness and grammatical accuracy.

Page 4, Line 6: Please correct the phrase “by using the random sampling procedure..” by removing the extra period.

Page 4, Lines 8–20: The inclusion of general precautions for handling B. cepacian and A. sobria seems misplaced in the methodology section. The methods should focus on detailing the specific procedures you followed, not general laboratory guidelines.

Discussion:

Page 20, Lines 1–8: The current text—“In ultimately, these resistance profiles have significant clinical implications. They need the use of combination or second-line treatments, which could be more expensive, hazardous, or less effective.”—should be revised for clarity and academic tone. Suggested revision:

"Ultimately, these resistance profiles carry significant clinical implications, often necessitating combination therapies or the use of second-line antibiotics, which may be more costly, associated with higher toxicity, or have reduced efficacy."

References: Several citations are outdated (e.g., references 3, 5, 17, 18, 25). It is advised to incorporate recent and relevant literature to strengthen the scientific foundation of the manuscript.

I hope these comments are helpful in guiding further improvements. I look forward to reviewing a revised version that addresses these concerns more thoroughly.

Best regards

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Reviewer #3: Yes:  Dr. Salim Faruk Bashir

Reviewer #4: No

Reviewer #5: No

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Investigation of Carbapenemase-Encoding Genes in Burkholderia cepacia and Aeromonas sobria Isolates from Nosocomial Infections in Iraqi Patients

PONE-D-24-53922R2

Dear Dr. Al-Ouqaili,

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.

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Kind regards,

Hideo Kato

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 #4: All comments have been addressed

Reviewer #5: 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 #4: Yes

Reviewer #5: Yes

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

Reviewer #4: Yes

Reviewer #5: Yes

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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 #4: Yes

Reviewer #5: 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 #4: Yes

Reviewer #5: 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 #4: The revised manuscript reflects substantial improvements, and all reviewer comments have been satisfactorily addressed. The findings presented are likely to contribute significantly to the relevant area of research.

Reviewer #5: While the revised manuscript addresses many of the previously raised concerns, there are still recurring issues related to the use of scientific nomenclature and abbreviations. For example, species names such as Burkholderia cepacia and Aeromonas sobria must be consistently italicized throughout the manuscript in accordance with standard taxonomic conventions. In particular, Burkholderia species appear multiple times without proper italicization—this should be carefully corrected.

Moreover, when bacterial species are mentioned repeatedly, it is advisable to use abbreviated genus names after the first full mention (e.g., B. cepacia instead of Burkholderia cepacia). This not only aligns with scientific writing standards but also improves clarity and conciseness.

Additionally, abbreviations such as PCR (polymerase chain reaction) should only be defined in full at first mention. Subsequent mentions should use the abbreviation alone. For example, the sentence “The gene technique PCR (Polymerase Chain Reaction) results are highly accurate…” (Lines 409–410) is redundant and non-standard. A more appropriate and concise version would be: “PCR results are highly accurate and reliable [26].”

I recommend a thorough manuscript review to correct these formatting and consistency issues in line with scientific writing norms.

Thanks

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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

Reviewer #5: Yes:  Muhammad Haris Raza Farhan

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