<xhtml:span xmlns:xhtml="http://www.w3.org/1999/xhtml" xml:lang="en">Investigating the presence of hypervirulent carbapenemase-producing Klebsiella  pneumoniae, through three phenotypic tests, in the Maltese islands </xhtml:span>
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Hypervirulence and multi-drug resistance are two separate qualities of Klebsiella pneumoniae that have posed a public health threat in separate strains for decades now. The convergence of these two phenotypes into singular strains of Klebsiella pneumoniae have created a super bug, causing both severe infections in the young and healthy and having limited antibiotic treatment available against them. These strains were first discovered in Asia in 2013 but are now emerging all around the globe. In this study, hypervirulence was assessed in 493 known carabapenemase-producing Klebsiella pneumoniae strains isolated and cryopreserved between 2019 and 2021 from various specimen types from health institutions around the Maltese islands. Three phenotypic tests were used, namely, the string test for hypermucoviscosity, lateral flow assays for the identification of K1 and K2 capsular serotypes, and quantification of siderophore production. Since the latter test was the one that was known to be the most significant for the detection of hypervirulence, and none of the strains surpassed the set limit of a concentration of 30µg/ml, none of the 493 assayed strains were deemed hypervirulent. Hypermucoviscous, K1 and K2 capsular serotype belonging strains were found, though, so much so that a statistically significant relationship was found between hypermucoviscosity and capsular serotype. Other statistical tests carried out exploring the relationship between hypermucoviscosity and siderophore concentration and hypermucoviscosity and type of carabapenemase gene were not found to be statistically significant. According to this research, hypervirulent carabapenemase-producing Klebsiella pneumoniae strains have not yet arrived in the Maltese archipelago.
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The work presented is clear and the arguments well formed. The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
Summary: The study investigates the presence of hypervirulent carbapenemase-producing Klebsiella pneumoniae (CP-hvKp) in the Maltese islands. A collection of 466 carbapenemase-producing K. pneumoniae strains, isolated between 2019-2021, was screened using three phenotypic assays: (1) the string test for hypermucoviscosity, (2) lateral flow assays to detect K1/K2 capsular serotypes, and (3) siderophore quantification. While hypermucoviscous strains and K1/K2 serotypes were identified, none of the strains produced siderophore levels above 30 µg/ml, the cutoff set for hypervirulence. Therefore, the authors conclude that no CP-hvKp strains were detected in Malta during the study period. The discussion highlights the complexity of defining hypervirulence and compares results with international reports. The …
Comments to Author
Summary: The study investigates the presence of hypervirulent carbapenemase-producing Klebsiella pneumoniae (CP-hvKp) in the Maltese islands. A collection of 466 carbapenemase-producing K. pneumoniae strains, isolated between 2019-2021, was screened using three phenotypic assays: (1) the string test for hypermucoviscosity, (2) lateral flow assays to detect K1/K2 capsular serotypes, and (3) siderophore quantification. While hypermucoviscous strains and K1/K2 serotypes were identified, none of the strains produced siderophore levels above 30 µg/ml, the cutoff set for hypervirulence. Therefore, the authors conclude that no CP-hvKp strains were detected in Malta during the study period. The discussion highlights the complexity of defining hypervirulence and compares results with international reports. The manuscript emphasizes the importance of surveillance given Malta's high population density and history of carbapenemase outbreaks. Major Revisions 1. Definition and Assessment of Hypervirulence - The study relies exclusively on in vitro phenotypic assays, but recent literature emphasizes that no single phenotypic or genotypic test reliably defines hypervirulence. Mouse infection models remain the "gold standard" for assessing hypervirulent phenotypes. This limitation should be explicitly acknowledged in the Introduction and Discussion, to avoid over-interpretation of the negative results. - The cutoff of ≥30 µg/ml siderophore concentration as the sole criterion for hypervirulence requires stronger justification, preferably with references to standardized or consensus guidelines. 2. Scope of Isolates Analyzed: The majority of isolates came from screening (rectal/faecal swabs, \~72%), which are indicators of carriage rather than infection. This sampling bias reduces the likelihood of detecting hvKp associated with invasive disease. The manuscript should discuss how this may underestimate the true burden of hvKp and suggest that future studies focus on clinical isolates from infection sites more characteristic of hvKp (e.g., liver abscess, endophthalmitis, bacteremia). 3. Interpretation of Negative Results: The statement that "hypervirulent CP-Kp strains have not yet arrived in Malta" may be too strong, given the limitations of the assays used and the sampling strategy. A more cautious interpretation would be that "no evidence of CP-hvKp was found in this sample set using the applied assays.": The discussion should integrate more recent perspectives highlighting that hypermucoviscosity, siderophore production, and capsular serotype are "indirect virulence markers", not definitive evidence of hypervirulence. 4. Comparative Analysis: The discussion could be enriched by comparing the study's findings to more recent European and global data. For example, studies highlighting CP-hvKp carrying NDM or OXA-48 in Italy, France, or North Africa (some already cited) should be integrated to better contextualize the Maltese situation. Additionally, the absence of genomic and phylogenomic analyses is a major limitation. Whole-genome sequencing (WGS) and phylogenetic approaches have become essential in the current literature to confirm hypervirulence determinants, track plasmid dissemination, and establish evolutionary relationships among strains. Without such analyses, the study cannot fully exclude the presence of CP-hvKp or place the Maltese isolates in a broader international epidemiological framework. Incorporating genomic/phylogenomic methods in future work would considerably strengthen the robustness and impact of the findings. Minor Revisions Clarity and Focus - The Methods section is overly detailed in describing media preparation (e.g., exact weights, autoclave cycles). This could be shortened, with focus instead on assays relevant to hypervirulence testing. - Some figures (e.g., kit results) may be more appropriate as supplementary material. Terminology - Ensure consistent use of terms: "carbapenemase-producing K. pneumoniae (CP-Kp)" vs. "CPE" vs. "CP-hvKp." Standardize abbreviations throughout. - "Carabapenemase" is repeatedly misspelled; correct to "carbapenemase." Grammar and Language Overall, the English is clear and understandable, but some grammatical and stylistic improvements are needed: - Abstract: "carabapenemase" - "carbapenemase." - Introduction: "on are of major public health importance" - "are of major public health importance." - Methods: "an 10µl inoculation loop" - "a 10µl inoculation loop." - Results/Discussion: streamline overly long sentences for readability.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data The method section is divided into too many disjointed paragraph and in general they are very detailed and maybe there is an over explanation in some sections that can be considered obsolete (for example: "medium was then dispensed into sterile plastic petri dishes at 45⁰C, while being labelled with name of medium and date of preparation using the Leibinger JET2neo printer simultaneously. The media was left to settle for 24 hours.") not all the information are needed for reproducibility and even the paragraphs with all the different media are not necessary and distracting on the core of the study. Key methodological elements are missing or underexplained: - Considering the type of study, would be important to specify the …
Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data The method section is divided into too many disjointed paragraph and in general they are very detailed and maybe there is an over explanation in some sections that can be considered obsolete (for example: "medium was then dispensed into sterile plastic petri dishes at 45⁰C, while being labelled with name of medium and date of preparation using the Leibinger JET2neo printer simultaneously. The media was left to settle for 24 hours.") not all the information are needed for reproducibility and even the paragraphs with all the different media are not necessary and distracting on the core of the study. Key methodological elements are missing or underexplained: - Considering the type of study, would be important to specify the usage of control strains, unfortunately it is not clear from the methods section which controls and standards were included. - In table 1 is mentioned RTR-PCR used for detecting the genes but no explanation in the methods and the results are not discussed. - Underlying data, including a table with sample metadata (isolate ID, source, date, type of infection or colonisation, patient or environmental origin, phenotypic results, RT-PCR outcome). - Gel images or Ct value summaries, if available. 2. Presentation of results Results are described in a narrative style and summarised in tables. However, the selection of isolates included in each stage of the testing pipeline is not always clear, nor is their origin or metadata. Consider adding: - A master table with all isolates included in the study, their source, and key characteristics. This would help contextualise the results and allow the reader to follow the logic of isolate selection and downstream testing. - Currently, the manuscript includes photos of agar plates and tests, but no figures or graphical visualisations. A flow diagram of the experimental workflow or a map showing isolate distribution could aid clarity. 3. How the style and organization of the paper communicates and represents key findings The manuscript follows a logical structure overall, with clear sectioning between Introduction, Methods, Results, and Discussion. However, it would benefit from significant streamlining, especially in the Methods section, to remove outdated procedural descriptions and shift focus onto the central analytical steps. The title could be revised for clarity and conciseness, for example: "Phenotypic investigation of hypervirulent carbapenemase-producing Klebsiella pneumoniae in the Maltese islands". 4. Literature analysis or discussion The discussion provides a useful summary of hvKp, but lacks a critical evaluation of: - The limitations of the methods used, including the known variability of the string test and non-specificity of tellurite resistance. - The absence of genome-level confirmation, such as WGS or MLST. While not a requirement, acknowledging this limitation is essential for transparency. - A comparison with other European surveillance or hospital-based studies would improve the contextual value of the results. 5. Any other relevant comments Add information about future plans for typing or genome-based confirmation, if applicable.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Poor
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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