Review Reports - Extensively Drug-Resistant (XDR) and Pandrug-Resistant (PDR) Acinetobacter baumannii as Sentinel Indicators of Cumulative System-Level Antimicrobial Pressure in Iraqi Burn and High-Risk Hospital Units

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

In your manuscript titled „ Pandrug-Resistant Acinetobacter baumannii as a Sentinel Indicator of Cumulative System-Level Antimicrobial Pressure in Iraqi Burn and High-Risk Hospital Units” you set out to investigate the distribution of extremely resistant phenotypes (XDR and PDR) of Acinetobacter baumannii in high-risk hospital units in Iraq and assess whether these phenotypes can be interpreted as sentinel indicators of cumulative antimicrobial pressure at the healthcare system level. It is an ambitious goal, with an epidemiological and conceptual component (interpretation as a marker of systemic stress). Your study describes the distribution of XDR/PDR phenotypes in 80 non-duplicate isolates collected from high-risk units (ICU, burn units, surgical, etc.) and correlates the presence of PDR with clinical and organizational factors (admission to burn unit, wound infection, invasive devices, hospitalization longer than 14 days, exposure to broad-spectrum antibiotics). The topic is original, current and critical for low- and middle-income countries. Data from Iraq are scarce, and the explicit link between PDR and systemic (not just microbiological) stress is original and well-argued. The tables are well structured and easy to analyze. The references are well integrated into the manuscript and the analysis.

The study has several issues that need to be answered in order to potentially improve your manuscript:

Why didn't you use a multivariate analysis to control for major confounders (length of hospital stay, invasive devices)?
How do you explain the total absence of classic multidrug resistant isolates (only XDR+PDR)? Is there a possible selection bias in the processing of the ~1000 samples (only isolates that grew well on BD Phoenix were included)?
You propose PDR as a sentinel indicator of systemic stress. What would be a concrete operational threshold (e.g. %PDR >10% in burn unit) that would trigger a national alert and immediate intervention by the Iraqi Ministry of Health?
How can you generalize these study results from a single governorate to a national or international level?
Why didn't you include detailed clinical data in the manuscript (mortality, clinical outcome, severity, timing of infection vs colonization, etc.)?
Add a short section “Molecular limitations and future directions” in the limitations chapter (1–2 paragraphs) explaining why genotypic characterization was not possible and what it would bring.

Minor issue

Reducing the length of sentences in the Abstract and Introduction.
Reduce repetition in the Discussion chapter.
Check and standardize references (some have DOIs, some don't).
Write the Latin words in italics (ex in vitro)

Comments on the Quality of English Language

Many cumbersome sentences, repetitions, non - idiomatic constructions in English. Fully revise the English with a native editor or use a professional service.

Author Response

Dear Reviewer,

We sincerely thank you for your careful reading of our manuscript and for your insightful and constructive comments. We greatly appreciate your recognition of the originality, relevance, and policy importance of our study. We have carefully revised the manuscript in response to all comments. Our point-by-point responses are provided below.

1) Why didn’t you use a multivariate analysis to control for major confounders (length of hospital stay, invasive devices)?

Thank you for this important comment. We fully agree that multivariable analysis would have strengthened the analytical framework by allowing simultaneous adjustment for potential confounders such as prolonged hospitalization and invasive device exposure. However, in our dataset, all PDR isolates occurred exclusively among patients with invasive device exposure and prolonged hospital stay ≥14 days, resulting in complete separation (zero-cell problem). Under these conditions, standard logistic regression produces unstable or non-estimable coefficients and inflated standard errors. Because of this structural limitation and the relatively small sample size (20 PDR events), we considered unadjusted odds ratios with Fisher’s exact test to be the most transparent and statistically appropriate exploratory approach.

To address the reviewer’s concern, we have now clarified this methodological issue explicitly in the Methods and Limitations sections. We have also added that future studies with larger multicenter datasets should apply penalized logistic regression or exact logistic regression to better estimate independent associations in the presence of sparse data and complete separation.

2) How do you explain the total absence of classic multidrug-resistant isolates (only XDR + PDR)?

We appreciate this valuable observation. The absence of conventionally MDR-only isolates in our study is most likely explained by the clinical ecology and surveillance design of the study rather than by a true absence of such phenotypes in the broader hospital system. Sampling was intentionally restricted to high-risk hospital units (ICUs, burn units, surgical and medical wards, and emergency departments) where antimicrobial exposure, invasive procedures, referral of severe cases, and prolonged hospitalization are concentrated. These settings are characterized by particularly intense selective pressure, making recovery of advanced resistance phenotypes more likely than recovery of less resistant strains.

In addition, only non-duplicate clinical isolates judged clinically relevant in routine diagnostic workflows were included. Therefore, the study was not designed to estimate the full hospital-wide resistance spectrum of A. baumannii, but rather to characterize the most clinically consequential resistance phenotypes circulating in high-risk units. We have clarified this point in the Discussion and Limitations sections to avoid overinterpretation.

3) Is there a possible selection bias in the processing of the ~1000 samples (only isolates that grew well on BD Phoenix were included)?

Thank you for this important methodological point. We agree that selection bias is a relevant concern in laboratory-based surveillance studies. The approximately 1,000 processed clinical samples represented the broader surveillance frame for suspected healthcare-associated infections, but only non-duplicate A. baumannii isolates recovered through routine microbiological workflows and confirmed by the BD Phoenix system were included in the final analysis. Thus, the study population represents culture-positive and diagnostically confirmed isolates rather than all submitted clinical samples.

We have clarified that the study does not evaluate culture negativity, mixed-growth samples, or isolates that may not have been recoverable under routine diagnostic conditions. Therefore, our findings should be interpreted as applying to confirmed clinical A. baumannii isolates from high-risk units, not to all suspected infections. We have now explicitly acknowledged this as a potential source of selection bias in the revised manuscript.

4) You propose PDR as a sentinel indicator of systemic stress. What would be a concrete operational threshold (e.g. %PDR >10% in burn unit) that would trigger a national alert and immediate intervention by the Iraqi Ministry of Health?

We thank the reviewer for this highly valuable translational comment. We agree that the concept of PDR as a sentinel indicator gains practical value only if linked to operational thresholds. Because our study was not designed as a national threshold-validation study, we cannot claim to define an evidence-based universal cutoff for Iraq. However, based on the observed clustering of PDR in burn units and the severity of therapeutic exhaustion associated with these isolates, we now propose a pragmatic preliminary framework for surveillance action rather than a definitive national benchmark.

In the revised manuscript, we suggest that the detection of any sustained PDR cluster in a high-risk unit should trigger immediate local review, and that a PDR proportion exceeding 10% among A. baumannii isolates in a burn unit or other high-risk unit may reasonably be treated as an early warning threshold requiring intensified infection-control audits, antimicrobial stewardship review, environmental decontamination assessment, and laboratory reporting escalation. We emphasize that this threshold is hypothesis-generating and should be prospectively validated in multicenter Iraqi surveillance systems before adoption as formal national policy.

5) How can you generalize these study results from a single governorate to a national or international level?

We appreciate this important comment and agree that the findings should not be overgeneralized. We have revised the manuscript to make clear that this was a single-governorate study and that the results should primarily be interpreted as a sentinel description of resistance dynamics in resource-constrained, high-risk hospital environments rather than as a nationally representative estimate.

At the same time, we believe the study has broader relevance at the conceptual level. The mechanisms identified—burn-unit concentration, prolonged hospitalization, invasive device exposure, cumulative broad-spectrum antibiotic pressure, and constrained infection-control capacity—are not unique to one hospital or one governorate. Therefore, while the numerical prevalence values should not be generalized directly, the observed pattern and the proposed interpretation of extreme resistance phenotypes as warning signals of structural stress may be transferable to comparable settings in Iraq and other low- and middle-income healthcare systems. This distinction between statistical generalizability and conceptual transferability has now been clarified in the revised Discussion and Limitations sections.

6) Why didn’t you include detailed clinical data in the manuscript (mortality, clinical outcome, severity, timing of infection vs colonization, etc.)?

Thank you for this important point. We agree that patient-level clinical data such as mortality, severity of illness, timing of infection versus colonization, and treatment outcomes would have substantially strengthened the clinical interpretation of the findings. However, the present study was designed as a laboratory-based surveillance study centered on isolate-level resistance phenotypes and their distribution across high-risk units, rather than as a clinical outcomes cohort.

Because anonymized microbiological surveillance data were prioritized, detailed individual clinical variables were not consistently available in a standardized form across participating hospitals. We have now clarified this more explicitly in the manuscript and highlighted it as a major limitation. We also state that future prospective studies should integrate microbiological, epidemiological, and outcome-level data in order to better distinguish colonization from infection and to quantify the effect of PDR on mortality, length of stay, and therapeutic failure.

Add a short section “Molecular limitations and future directions” in the limitations chapter (1–2 paragraphs)

Thank you for this important point. Done dear doctor.

8) Minor issue: reducing the length of sentences in the Abstract and Introduction

We thank the reviewer for this suggestion. The Abstract and Introduction have been carefully revised to reduce sentence length, improve readability, and remove cumbersome phrasing.

9) Reduce repetition in the Discussion chapter

We agree and have revised the Discussion substantially to reduce repetition, improve paragraph structure, and sharpen the distinction between interpretation, policy implications, and limitations.

10) Check and standardize references (some have DOIs, some don’t)

Thank you. We have carefully standardized the reference list and unified formatting across all references, including DOI presentation where available.

11) Write Latin words in italics (in vitro, etc.)

Thank you. Latin expressions throughout the manuscript, including in vitro, have been italicized and standardized.

12) English language needs major revision

We appreciate this important comment. The manuscript has undergone thorough language revision to improve clarity, idiomatic accuracy, sentence structure, and overall readability. Repetitions, cumbersome constructions, and overly long sentences were carefully edited throughout the Abstract, Introduction, Discussion, and policy sections.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have submitted a cross-sectional laboratory-based surveillance study that was carried out between January 2024 and January 2025 in hospitals in Kirkuk, Iraq, about pandrug-resistant Acinetobacter baumanii.

Although antibiotic resistance is an important public health problem and Acinetobacter baumanii is one of the critical-priority pathogens, there are many aspects that raise questions in this study.

The introduction section should contain data on the prevalence of Acinetobacter baumanii and on antibiotic resistance at the national level, which supports the hypothesis behind the study.

The title refers only to Pandrug-Resistant Acinetobacter baumannii, although inside the article XDR strains are analyzed, and the number of PDRs is very small.

The period indicated in the abstract (between January 2024 and January 2025) should also be included in the Material and Methods section.

The authors note that the study included about 1,000 clinical specimens of suspected healthcare-associated infections, without indicating the exact number of samples processed and the hospitals involved.

The authors should specify exactly how many clinical specimens were analyzed (not about 1,000), their distribution among the hospitals analyzed, how many were positive, how many showed the presence of Gram-negative pathogens, and the percentage of Acinetobacter baumanii. Also, the article does not specify the number of patients from whom samples were collected or the distribution by sex and age groups. The analysis of possible statistical differences in the prevalence of multidrug-resistant strains by age group and gender would contribute to increasing the value of the study.

Although they mentioned that the samples were collected from patients admitted to several hospitals, at the end he specifies within the limits of the study that the data was confined to a single governorate hospital. This must be clarified. The possible difference between the percentages of PDR strains existing in different hospitals and the factors that would explain this must be analyzed. The authors should mention whether the analysis carried out refers only to healthcare-associated infections and include definitions and specific references.

Author Response

Dear Reviewer,

We sincerely thank you for your careful reading of our manuscript and for your constructive and detailed comments. We greatly appreciate your observations, which helped us improve the clarity, methodological transparency, and overall scientific quality of the manuscript. All comments were carefully considered, and the manuscript has been revised accordingly. Our point-by-point responses are provided below.

1. The introduction section should contain data on the prevalence of Acinetobacter baumannii and on antibiotic resistance at the national level, which supports the hypothesis behind the study.

Response:

Thank you for this important comment. We agree that stronger contextualization at the national level improves the rationale of the study. Accordingly, the Introduction has been revised to better situate the study within the Iraqi healthcare context by emphasizing the burden of antimicrobial resistance in Iraqi hospitals, particularly in high-risk units where broad-spectrum antibiotic exposure, infection-control limitations, and constrained laboratory capacity may facilitate the emergence and persistence of highly resistant Acinetobacter baumannii. We also clarified that the study is intended to explore how extreme resistance phenotypes may reflect cumulative antimicrobial and system-level pressure in such settings.

2. The title refers only to pandrug-resistant Acinetobacter baumannii, although inside the article XDR strains are analyzed, and the number of PDRs is very small.

Response:

We appreciate this important observation and agree with the reviewer. Since the manuscript analyzes both extensively drug-resistant (XDR) and pandrug-resistant (PDR) phenotypes, the title has been revised to more accurately reflect the scope of the study. The revised title is:

“Extensively Drug-Resistant and Pandrug-Resistant Acinetobacter baumannii as Sentinel Indicators of Cumulative System-Level Antimicrobial Pressure in Iraqi Burn and High-Risk Hospital Units.”

This revised title better aligns with the analytical content of the manuscript.

3. The period indicated in the abstract (between January 2024 and January 2025) should also be included in the Material and Methods section.

Response:

Thank you for this comment. The study period has now been explicitly stated in the Materials and Methods section. We revised the text to clearly indicate that the study was conducted in public hospitals within Kirkuk governorate, Iraq, between January 2024 and January 2025, ensuring consistency between the Abstract and the Methods section.

4. The authors note that the study included about 1,000 clinical specimens of suspected healthcare-associated infections, without indicating the exact number of samples processed and the hospitals involved.

Response:

We appreciate this important point. To avoid overstatement and improve methodological precision, the manuscript has been revised to clarify that the study was designed as an isolate-based laboratory surveillance study rather than a denominator-based prevalence study. We removed wording that could imply that the study was intended to quantify prevalence across all processed specimens. The revised manuscript now clearly states that the broader surveillance frame consisted of routine clinical specimens processed in public hospitals within Kirkuk governorate, but the present analysis included only 80 non-duplicate A. baumannii isolates recovered from high-risk hospital units and confirmed through routine diagnostic workflows.

5. The authors should specify exactly how many clinical specimens were analyzed, their distribution among the hospitals, how many were positive, how many showed Gram-negative pathogens, and the percentage of Acinetobacter baumannii.

Response:

Thank you for this valuable comment. We agree that denominator-based microbiological data would add epidemiological depth. However, the present study was not structured as a hospital-wide denominator-based prevalence analysis. Rather, it was designed to characterize resistance phenotypes among confirmed, non-duplicate A. baumannii isolates recovered from high-risk hospital units in routine laboratory practice. Complete harmonized denominator data across participating hospitals, including total positive cultures, total Gram-negative isolates, and hospital-level specimen distributions, were not available in a sufficiently standardized form for robust comparative presentation. We have now clarified this point explicitly in the Methods and Limitations sections and emphasized that the study is not intended to estimate prevalence, but rather to describe clinically consequential resistance phenotypes in a defined surveillance context.

6. The article does not specify the number of patients from whom samples were collected or the distribution by sex and age groups. The analysis of possible statistical differences in the prevalence of multidrug-resistant strains by age group and gender would contribute to increasing the value of the study.

Response:

We appreciate this important suggestion. We agree that patient-level demographic analysis would strengthen the manuscript. However, because the study was designed as an anonymized isolate-based laboratory surveillance study, a complete linked demographic dataset was not consistently available for all included isolates across participating hospitals. For this reason, age- and sex-specific subgroup analyses could not be performed reliably. We have now explicitly acknowledged this limitation in the revised manuscript and stated that future prospective studies integrating microbiological and patient-level clinical data should examine the distribution of resistance phenotypes by age, sex, and other epidemiologically relevant factors.

7. Although they mentioned that the samples were collected from patients admitted to several hospitals, at the end they specify within the limits of the study that the data was confined to a single governorate hospital. This must be clarified.

Response:

Thank you for identifying this ambiguity. We agree that this wording required clarification. The manuscript has been revised throughout to consistently state that the study was conducted in public hospitals within Kirkuk governorate, not in a single hospital. We also revised the Limitations section to make clear that the geographic limitation relates to one governorate, rather than to one hospital. This change was made to eliminate confusion and improve consistency across the manuscript.

8. The possible difference between the percentages of PDR strains existing in different hospitals and the factors that would explain this must be analyzed.

Response:

We thank the reviewer for this important observation. We agree that hospital-level comparison of PDR distribution would be valuable. However, the sample size of the present study, particularly the limited number of PDR isolates (n = 20), was not sufficient to support statistically stable hospital-level comparative analysis across participating hospitals. We have now clarified this in the revised Limitations section and emphasized that larger multicenter datasets will be needed to evaluate hospital-specific differences and their explanatory factors more robustly.

9. The authors should mention whether the analysis carried out refers only to healthcare-associated infections and include definitions and specific references.

Response:

Thank you for this important methodological comment. We have revised the Materials and Methods section to clarify that the surveillance context of the study focused on patients managed in high-risk hospital units where healthcare-associated infections were clinically suspected in routine practice. We also added a brief clarifying definition of the healthcare-associated infection context within the methodological framework to improve precision and consistency. This clarification has been incorporated into the revised manuscript.

We thank the reviewer again for these thoughtful comments, which helped us improve the scientific clarity and transparency of the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript describes investigations of pandrug-resistant A. baumannii clinical isolates. The topic of this manuscript is an interesting issue, however, the methodology part and the study design should be strengthend.

In this study only phenotypic analysis was performed. Antibitoic resistance determinants must be analyzed by PCR. I suggest to authors to perform PCR for OXA-type carbapenemases, NDM, VIM beta-lactamases.
Efflux pumps could be also analyzed by PCR.
Aminoyglycoside and fluoroquinolone resistance determinants should be analyzed by PCR and sequencing.
Clonality analysis of pandrug-resistant A. baumannii should be performed by multilocus sequence typing.
MIC distribution of different antibiotics should be presented in a table or figure.

Author Response

Dear Reviewer,

We sincerely thank you for your careful evaluation of our manuscript and for your valuable and technically insightful comments. We agree that molecular and deeper microbiological characterization would further strengthen the study. We have revised the manuscript to better acknowledge these methodological limitations and to clarify the scope of the present work. Our responses are provided below.

1. In this study only phenotypic analysis was performed. Antibiotic resistance determinants must be analyzed by PCR. I suggest to authors to perform PCR for OXA-type carbapenemases, NDM, VIM beta-lactamases.

Response:

Thank you for this important and scientifically relevant suggestion. We fully agree that molecular characterization of resistance determinants would provide important mechanistic insight into the resistance profiles observed in this study. However, the present work was designed as a phenotypic laboratory-based surveillance study conducted in a resource-constrained setting, with the primary objective of describing clinically relevant extreme resistance phenotypes recovered through routine diagnostic workflows in high-risk hospital units. Molecular testing for carbapenemase genes, including OXA-type enzymes, NDM, and VIM, was beyond the operational scope of the current study.

To address the reviewer’s concern, we have now explicitly acknowledged this limitation in the revised manuscript and added a dedicated subsection entitled “Molecular Limitations and Future Directions” in the Limitations section. In that subsection, we specifically note that future studies should incorporate PCR-based detection of major carbapenemase determinants to strengthen the biological interpretation of phenotypic resistance.

2. Efflux pumps could be also analyzed by PCR. Aminoglycoside and fluoroquinolone resistance determinants should be analyzed by PCR and sequencing.

Response:

We appreciate this valuable comment and agree that molecular investigation of efflux pump-related mechanisms, aminoglycoside resistance genes, and fluoroquinolone resistance determinants would substantially improve mechanistic understanding. These analyses were not feasible within the design and infrastructure capacity of the present study, which was focused on isolate-level phenotypic surveillance rather than genotypic resistance profiling. We have now added explicit acknowledgment of this limitation in the manuscript and stated that future multicenter studies should integrate phenotypic data with PCR- and sequencing-based resistance mechanism analysis.

3. Clonality analysis of pandrug-resistant A. baumannii should be performed by multilocus sequence typing.

Response:

Thank you for this important recommendation. We agree that clonality analysis by multilocus sequence typing (MLST) would be highly informative, particularly for distinguishing local emergence from transmission or outbreak-related spread. However, clonality analysis was beyond the scope of the present surveillance study. We have now explicitly stated in the revised manuscript that MLST or other genomic epidemiology approaches were not performed and should be incorporated in future studies to better understand the transmission dynamics of PDR A. baumannii in Iraqi hospital settings.

4. MIC distribution of different antibiotics should be presented in a table or figure.

Response:

We thank the reviewer for this constructive suggestion. We agree that detailed minimum inhibitory concentration (MIC) distribution data would increase the microbiological depth of the manuscript. However, full standardized MIC distribution outputs were not consistently available in an extractable format across all isolates in the current dataset. Therefore, we did not present a detailed MIC distribution table or figure in the manuscript. To address this concern transparently, we have now acknowledged this issue in the revised Limitations section and stated that future studies should incorporate detailed MIC distribution analysis in addition to phenotypic categorization.

5. The methodology part and the study design should be strengthened.

Response:

We appreciate this general but important comment. In response, we have revised the Materials and Methods section extensively to improve clarity and methodological transparency. Specifically, we have:

clarified that the study was conducted in public hospitals within Kirkuk governorate between January 2024 and January 2025;

clarified that the study is an isolate-based laboratory surveillance study rather than a denominator-based prevalence study;

refined the explanation of resistance phenotype definitions;

strengthened the description of susceptibility interpretation using CLSI criteria;

clarified the rationale for using unadjusted odds ratios and Fisher’s exact test in the presence of complete separation;

and expanded the Limitations section to include both clinical and molecular constraints.

We believe these revisions have strengthened the methodological framing of the manuscript while remaining faithful to the design and data actually available.

We sincerely thank the reviewer again for these thoughtful comments, which helped us improve the rigor, transparency, and interpretive balance of the manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have adequately addressed all my comments and revised it accordingly. The manuscript can be published.

Author Response

Greetings,

The language of the manuscript has been carefully improved and reviewed by an English language editor from the Department of English at our college. In addition, the tables have been reorganized and strengthened according to the valuable comments and suggestions provided by the respected reviewers.

All required revisions have been addressed carefully in order to improve the quality of the manuscript and meet the reviewers’ expectations.

Thank you very much for your time and consideration.

Kind regards,

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for the revised manuscript.

The authors have revised the manuscript according to the recommendations.

Author Response

Greetings,

All required revisions have been addressed carefully in order to improve the quality of the manuscript and meet the reviewers’ expectations.

Thank you very much for your time and consideration.

Kind regards,

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript describes investigations of antibiotic resistant A. baumannii. The topic of this manuscript is an interesting issue, however, the methodology part of this manuscript should be improved to get to an in-depth analysis.

I suggest to authors to perform PCR for OXA-type carbapenemases, NDM, VIM beta-lactamases on A. baumannii strains of this study.
Efflux pumps could be also analyzed by PCR.
Aminoglycoside and fluoroquinolone resistance determinants should be analyzed by PCR and sequencing.
Clonality analysis of pandrug-resistant A. baumannii should be performed by multilocus sequence typing

Author Response

Greetings,

All required revisions have been addressed carefully in order to improve the quality of the manuscript and meet the reviewers’ expectations.

Thank you very much for your time and consideration.

Reviewer 3 – Comment 1

Comment: I suggest authors to perform PCR for OXA-type carbapenemases, NDM, VIM beta-lactamases on A. baumannii strains of this study.

Response: We sincerely thank the reviewer for this important and valuable suggestion. We fully agree that molecular characterization of major carbapenemase genes such as OXA-type carbapenemases, NDM, and VIM would significantly strengthen the microbiological interpretation of extreme resistance phenotypes in Acinetobacter baumannii. However, the present work was designed as a laboratory-based phenotypic surveillance study focusing on clinically relevant resistance patterns in high-risk hospital units rather than a molecular epidemiology study. Due to resource limitations and the multicenter routine diagnostic setting, PCR-based molecular detection was not performed during the study period. To address this important point, we have explicitly added this limitation in the revised manuscript under the “Limitations and Perspectives” section and emphasized that future multicenter studies should incorporate molecular characterization of carbapenemase genes to better define the genetic basis of XDR and PDR phenotypes.

Reviewer 3 – Comment 2

Comment: Efflux pumps could be also analyzed by PCR.

Response: We appreciate the reviewer’s valuable observation. Efflux pump systems are indeed important contributors to multidrug and extreme drug resistance in A. baumannii, particularly regarding carbapenem and tigecycline resistance. We agree that molecular analysis of efflux pump-associated genes would provide additional mechanistic insight. However, since the present study focused primarily on phenotypic resistance surveillance within routine hospital laboratory workflows, these analyses were beyond the scope of the current investigation. This limitation has now been acknowledged in the revised manuscript, and future studies integrating phenotypic and molecular resistance profiling have been recommended.

Reviewer 3 – Comment 3

Comment: Aminoglycoside and fluoroquinolone resistance determinants should be analyzed by PCR and sequencing.

Response: We thank the reviewer for this constructive recommendation. We agree that characterization of aminoglycoside-modifying enzymes and fluoroquinolone resistance determinants through PCR and sequencing would provide stronger mechanistic understanding of the observed resistance profiles. Nevertheless, the current study was intentionally structured as a phenotypic surveillance investigation aimed at identifying clinically significant resistance phenotypes and their association with hospital-unit level risk factors. Because of resource constraints and the surveillance-based design, molecular sequencing analyses were not included. We have now clarified this limitation in the revised manuscript and highlighted the importance of incorporating molecular resistance determinant analysis in future studies.

Reviewer 3 – Comment 4

Comment: Clonality analysis of pandrug-resistant A. baumannii should be performed by multilocus sequence typing.

Response: We thank the reviewer for this highly relevant suggestion. We fully agree that clonality analysis using multilocus sequence typing (MLST) would provide valuable epidemiological insight by distinguishing between local emergence and possible transmission of PDR A. baumannii strains within high-risk hospital units. However, MLST was beyond the scope of the present isolate-based phenotypic surveillance study and was not feasible within the available laboratory resources during the study period. To address this important point, we have added this limitation to the revised manuscript and emphasized that future multicenter investigations should include MLST and molecular epidemiological analysis to better understand transmission dynamics and strengthen infection control strategies.

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript describes investigations of extensively-drug resistant and pandrug-resistant A. baumannii clinical isolates. The topic of this manuscript is an interesting issue, however, molecular methods are needed to strengthen obtained results.

PCR should be performed to detect carbapenemases NDM, VIM, OXA-48
Multilocus sequence typing should be performed for clonality analysis of extensively-drug resistant and pandrug-resistant A. baumannii
All these molecular methods are needed to make your results comparable with international data and to interpret your results in the international context.

Author Response

Greetings,

All required revisions have been addressed carefully in order to improve the quality of the manuscript and meet the reviewers’ expectations.

Thank you very much for your time and consideration.

Reviewer 3 – Comment 1

Comment: I suggest authors to perform PCR for OXA-type carbapenemases, NDM, VIM beta-lactamases on A. baumannii strains of this study.

Reviewer 3 – Comment 2

Comment: Efflux pumps could be also analyzed by PCR.

Reviewer 3 – Comment 3

Comment: Aminoglycoside and fluoroquinolone resistance determinants should be analyzed by PCR and sequencing.

Reviewer 3 – Comment 4

Comment: Clonality analysis of pandrug-resistant A. baumannii should be performed by multilocus sequence typing.