Molecular and Epidemiological Analysis of Carbapenem-Resistant Klebsiella pneumoniae in a Greek Tertiary Hospital: A Retrospective Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I have read your manuscript with great interest and find it highly relevant for publication. I have some comments and concerns that I hope will be useful in improving your manuscript.

Line 3:

Please verify the use of italics for microorganism names following scientific nomenclature guidelines. In particular, Klebsiella pneumoniae should be correctly formatted throughout the text.

Lines 87-103:

The CLSI no longer recommends the Modified Hodge Test (MHT) for carbapenemase detection due to its low specificity, particularly the possibility of false positives caused by other resistance mechanisms, such as porin mutations or AmpC overexpression.

The most recent CLSI versions (2024 or 2025) recommend using mCIM (Modified Carbapenem Inactivation Method), eCIM (Extended Carbapenem Inactivation Method), and Carba NP Test as more reliable methods for carbapenemase detection.

While this should not be a reason for manuscript rejection, it would be important for the authors to clarify this point and consider discussing it as a potential limitation of the study. It is suggested to justify the use of MHT and, if possible, complement it with other phenotypic or molecular tests.

Lines 95-98:

It is recommended to clarify what is meant by "same phenotypic profile." Does this refer to antibiotic resistance, carbapenemase production, or another microbiological aspect? Specifying this would help avoid ambiguity in result interpretation.

Lines 104-108:

The authors state that an informed consent letter is not required due to the nature of the study. However, since the study involves human clinical isolates and patient clinical data, bioethical committee approval might be necessary to ensure compliance with ethical regulations.

The manuscript states that "the institutional scientific board" approved the study's publication and results, but it is unclear whether this committee has bioethical functions. It is recommended to clarify this in the text, specifying if this is a formally established bioethics committee and, if necessary, include the ethical approval reference number.

Line 116:

The cited reference corresponds to a very outdated version of CLSI, which may affect result interpretation. It is important to consider that breakpoints for classifying isolates based on antibiotic susceptibility change annually, and some antibiotics are no longer recommended for evaluation.

It is recommended to review and update the breakpoints using the most recent CLSI version (2024 or 2025) to ensure that antibiotic resistance interpretation aligns with current standards.

Lines 119-120:

The cited reference is not appropriate, as the protocol used for antibiotic susceptibility interpretation originates directly from CLSI. It is recommended to cite the most recent CLSI version instead of the current reference to ensure the methodology is properly supported by the original and official source.

Line 124:

It is recommended to specify which blaCTX-M variant was detected in the isolates. Given the existence of multiple variants (blaCTX-M-1, blaCTX-M-2, blaCTX-M-15, etc.), identifying the specific variant is relevant for epidemiological analysis and comparison with other studies. If differentiation was not performed, it should be mentioned as a study limitation.

Line 145:

Please verify the use of italics for microorganism and gene names according to scientific nomenclature guidelines. Ensure that Klebsiella pneumoniae and other species names are correctly formatted in italics throughout the manuscript.

Line 203:

Since KPC-2 isolates present a higher number of resistance-associated genes compared to KPC-9, it would be interesting to analyze whether there are differences in the demographic characteristics of patients infected with each variant.

Was KPC-9 more prevalent in patients with severe or less severe comorbidities compared to KPC-2? Including this information could provide greater clinical and epidemiological context to the study.

Line 230:

Did the authors consider implementing the Magiorakos et al. criteria for classifying isolates based on multidrug resistance? This article establishes that "non-susceptible" includes both resistant and intermediate isolates, which could modify the reported prevalence data.

Here is the article doi for reference: doi: 10.1111/j.1469-0691.2011.03570.x.

Adopting this classification could also help organize Figure 1 more effectively, allowing the presentation of only non-susceptible isolates and highlighting prevalence per antibiotic more clearly.

Line 240:

Including MIC data for each antibiotic and separating them by KPC groups adds value to the study.

However, to improve data visualization, it is suggested to consider using a grouped bar chart comparing each antibiotic between KPC groups.

Additionally, the current table could be moved to supplementary material, while keeping the graphical representation in the main manuscript to enhance readability. This is merely a suggestion, but it could improve data presentation and text flow.

Line 250:

It is recommended to consider using the Magiorakos et al. criteria for antimicrobial resistance classification in the comparative analysis. According to this approach, "non-susceptible" includes both resistant and intermediate isolates, which could influence data interpretation and KPC group comparisons.

If this criterion has not yet been evaluated, it is suggested to analyze it and incorporate it if relevant, to ensure that the results align with international standards.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Review of the Manuscript ID: microbiolres-3526066

 

Dear Authors,

I read your article with interest, the changing epidemiology of Klebsiella pneumoniae requires constant evaluation, and for this reason, the presented studies are of interest.

I have a few questions and remarks regarding your manuscript, which I present below.

 

Title

Biomolecular […]—This suggests that aspects other than molecular characteristics will be presented. Of course, drug susceptibility is a characteristic; however, in my opinion, the title is a little bit misleading. Consider changing the title to one that will reflect more the scope of your study.

Abstract

In this section, please, add information about the study period – 2014-15, and 2021-22.

In line 32, you state that the KPC-2 outbreak is still ongoing, however, your study ends in 2022, so it is only possible to suspect that the currently isolated strains are the same as the ones from 2022 (belonging to the same clone).

 

Introduction section

This section presents the general characteristics of the studied pathogen, providing the reader with a basic understanding of carbapenem resistance.

In line 69, I suggest changing the word “variants” to “alleles”.

I recommend adding information that KPC genes also are found in other bacteria, not only in K. pneumoniae.

 

Materials and methods

In line 116, please explain why the CLISI and the EUCAST criteria were used?

In lines 146-7, please explain if the purification of the PCR products with NucleoSpin is enough to subject the product for sequencing, or a treatment with ExoSap is required, followed by purification on a column (e.g. NucleoSpin). This explanation will help readers unfamiliar with molecular techniques better understand how a sample is prepared for sequencing.

 

Results

This section is presented clearly, and in a logical way.

In Figure 3, I recommend changing the word “histogram” to “graph” or “line graph with marks” in Figure 1a, and “chart” or “bar chart” in Figure 1b.

In both Figure 1a and Figure 1b, the axis Y should have the same scale ending with value 5.

 

Discussion and conclusions

The discussion is written properly, has a proper structure, study findings are referred to properly selected publications and reference databases, and supported with conclusions reflecting the study's scope and indicating further direction of studies and action to be taken to minimize the spread of the drug resistant microorganisms.

 

Overall, I found the manuscript well written and interesting for microbiologists and clinicians. The abovementioned remarks, if considered, will make the manuscript clearer to the readers.  

Kind regards,

/-/ reviewer

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This study provides a well-structured, thorough epidemiological and molecular characterization of KPC-producing Klebsiella pneumoniae, particularly focusing on KPC-2 and KPC-9 alleles within a Greek hospital setting. The research is timely and significant, addressing antimicrobial resistance—a major global health concern. The methodology is sound, with robust microbiological and genetic analyses, and the results contribute valuable insights into the persistence and evolution of carbapenem-resistant K. pneumoniae.

However, while the study is scientifically strong, there are areas that require revision before it can be accepted for publication:

1. The study mentions two distinct sampling periods (2014–2015 and 2021–2022) but does not clearly explain how the isolates from these periods were compared. It would be beneficial to add a brief rationale for revisiting the prevalence of KPC-9 in 2021–2022 and how these data integrate with the initial outbreak analysis.

2. The mortality comparison between KPC-2 and KPC-9 carriers is useful but lacks further statistical context. Could additional regression analysis or hazard ratios clarify the potential independent impact of KPC-9 on patient outcomes? The antibiotic susceptibility data (especially in Figure 1) should explicitly mention whether trends over time were evaluated (e.g., is resistance increasing?).

3. The study includes patients who were colonized or infected with KPC-producing K. pneumoniae, but it would be helpful to clarify how colonization was defined and whether outcomes differed between colonized and infected individuals.

4. The demographic and clinical data are highly detailed, but a brief summary of key trends in patient outcomes would help the reader digest the findings more efficiently. It might also be worth including a column indicating whether patients had prior exposure to antibiotics or hospital transfers, as this could be relevant to transmission dynamics.

5. The discussion effectively places the findings in the broader context of carbapenem resistance in Greece and globally. However, it would benefit from:

 

- A comparison to more recent studies (post-2020) to assess whether KPC-9 outbreaks are becoming more common in other settings.

- A deeper exploration of infection control measures—what specific steps should be taken to prevent further spread within hospitals?

 

- An expanded mention of how plasmid dynamics and horizontal gene transfer might contribute to the persistence of these strains.

6. Some figures, such as the MIC distribution graphs, could be labeled more clearly to ensure immediate interpretation. Consider emphasizing key differences (e.g., KPC-9’s higher ceftazidime resistance) with annotations.

This is a high-quality study that provides crucial insights into antimicrobial resistance patterns. With minor revisions to improve clarity, statistical interpretations, and discussion depth, this paper will be well-suited for publication in Microbiological Research.

Author Response

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Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The present manuscript summarizes the results of a retrospective study of clinical samples from patients hospitalized at the University General Hospital of Ioannina, Greece, to reveal the epidemiological and molecular characteristics of carbapenemase-producing K. pneumoniae strains. These strains were examined for KPC production potential by PCR and were further typed for the presence of bla-alleles. Using the ARMS method, the presence of the KPC-2 allele and the KPC-9 allele was interrogated. For the KPC-9-allele strains responsible for a distinct outbreak, significant increase in MIC for ceftazidime, as well as resistance to amikacin and colistin in comparison with KPC-2 allele strains were confirmed. Mortality rates for the patients infected with each of the two bacterial types were compared. The study confirms the possibility of spread of carbapenem-resistant K. pneumoniae with heterogenous resistance genes within one hospital setting. The topic of the study is highly important as it underlines the importance of proper containment of nosocomial infections, and supports the combat of bacterial multidrug resistance, one of the central  WHO “One Health” goals.  

My main comment would be that two groups of patients, infected with either KPC-2 or KPC-9 strains, are compared (Figure 4), but the demographic data are presented either for all patients infected with KPC strains (Table 2), or only for those infected with KPC-9 strains (Supplementary material). These data should be given as a side-to-side comparison.

Statistical tests used are not revealed in detail (method and measure of significance should be presented with every evaluation).  

For the discussion, a comparison of the findings on other KPCs but 2 and 9 (e.g. 93), which also exhibit altered substrate spectra, would be appropriate.

Please find below a list of minor remarks which I hope you will find helpful.

 

Line 145: names of the genes are typically in italics

Line 165 and throughout the manuscript: decimal point should be used instead of comma

Line 176: “blaKPC and blaNDM genes, in only one strain.”- no comma between the sentences

Table 2: decimal point should be used instead of comma. As the groups of the KPC-2 and KPC-9 are compared later, demographic and clinical description of both groups should be given.

Figure 2: the significance is usually presented with asterisks in the diagrams. As the Figures should be self-explanatory, the Figure title should contain a short description of the measure of significance. Why is p for ceftazidime with 16 mg/ml not shown, surely there is a large difference? Also, the category I for amikacin should be labelled with n.s.

Figures 3 a and b: something wrong with line numbers positioned within the display elements. Again, 3a should be described better: y-axis only says “number of patients”, and the byline should be specifying the contents more precisely.

Figure 4: The byline should explain the Figure more precisely, including what the “censored” stands for. Although the curves look similar, the censoring events in the KPC-9 group do not impact the behaviour of the curve, while the censoring events in the KPC-2 mostly do so – please explain what these events were (although only two reasons for censoring are mentioned in the text).

Line 373: “that TEM and SHV enzymes dominated during the 1980s and 1990s” – this sentence is too colloquial, please rewrite so it is understood that these genotypes (and/or phenotypes) were predominantly found in the nosocomial outbreak

Line 392: slightly increasing trend

Line 411: “strains belonging to different alleles”: surely, you mean strains harbouring the different alleles

Line 422-423: same sentence as in the lines 328-329

Author Response

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Author Response File: Author Response.pdf