Evaluation of an optimal agar medium for detecting hypervirulent Klebsiella pneumoniae using string test
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1. Abstract The string test is a screening method for detecting hypervirulent Klebsiella pneumoniae (hvKp). Agar media are used for string tests; however, the effect of the type of media on the test results remains unclear. We aimed to determine the optimal agar medium and cutoff value for the string test. We performed the string test on 99 Klebsiella strains using different agar media: sheep blood, chocolate, Drigalski's, and MacConkey. Diagnostic accuracy was calculated in concordance with the rmpA, rmpA2, or iucA gene levels. The diagnostic accuracy rates for sheep blood, chocolate, Drigalski's, and MacConkey agar were 0.79, 0.75, 0.73, and 0.64, respectively. When the cutoff was changed from 5 to 10 mm, the diagnostic accuracy rate for sheep blood agar decreased from 0.79 to 0.65. Our findings suggest that the type of agar medium impacts string test results, and sheep blood agar with a 5 mm cutoff is the optimal condition for detecting hvKp.
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I am pleased to tell you that your article has now been accepted for publication in Access Microbiology. The work presented is clear and the arguments well formed. The manuscript is well written and contributes to the literature. Thank you for addressing all reviewers comments satisfactorily and in a timely manner.
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Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
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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Thank you for submitting your manuscript for publication in Access Microbiology. It has been examined by expert reviewers who have concluded that the work is of potential interest to the readership of Access Microbiology. However, based on the comments received, it is clear that a major revision of this manuscript will be required before a decision can be made on its publication. I will be pleased to consider a revised manuscript along with a document including a point by point response to each of the reviewers comments. Your revised manuscript may be returned to one or more of the original reviewers, along with your itemised response to the reviewers’ comments.
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Comments to Author
Summary: Klebsiella pneumoniae (Kp) can be classified into classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp). HvKp is known to cause liver abscess, sepsis and invasive infection. Typically, HvKp has several known virulence genes, including the rmpA1/A2 (which are hypermucovosity capsule regulator); iucABCD, iutA (aerobactin) and iroBCDN (salmonchelin). Overall the manuscript is clearly written and of good quality. I am highlighting my comments that are mainly focused on providing more background and rationale, placing the work within existing literature, better discussion of the limitations/caveats associated with these samples and including the PCR gel results: (1) Hypervirulence seems to be correlated with hypermucoviscousity phenotype, thus why string test is commonly done. …
Comments to Author
Summary: Klebsiella pneumoniae (Kp) can be classified into classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp). HvKp is known to cause liver abscess, sepsis and invasive infection. Typically, HvKp has several known virulence genes, including the rmpA1/A2 (which are hypermucovosity capsule regulator); iucABCD, iutA (aerobactin) and iroBCDN (salmonchelin). Overall the manuscript is clearly written and of good quality. I am highlighting my comments that are mainly focused on providing more background and rationale, placing the work within existing literature, better discussion of the limitations/caveats associated with these samples and including the PCR gel results: (1) Hypervirulence seems to be correlated with hypermucoviscousity phenotype, thus why string test is commonly done. However, this association is not always clear as pointed out by others (see below). I think it's important for the authors to mention these works in the introduction, but also discuss what others have observed in the discussion. * Le, M. N. et al. Genomic epidemiology and temperature dependency of hypermucoviscous. Microb. Genom. https://doi.org/10.1099/mgen.0.000827 (2022). * Catalán-Nájera, J. C., Garza-Ramos, U. & Barrios-Camacho, H. Hypervirulence and hypermucoviscosity: Two different but complementary Klebsiella spp. phenotypes?. Virulence 8(7), 1111-1123 (2017). (2) In the introduction, the authors need to provide a bit more context on why these loci were chosen for PCR - currently it's in the methods section (3) The string test variation on media is noted by other previously. The authors should introduce this work as well as discuss how current study differs from this previous work. https://www.nature.com/articles/s41598-023-46221-w (4) I think it's important to discuss potential bias in this study as there are no genomics information or ST data, is it possible that the results are only applicable to the specific ST within these samples rather generally applicable to all Kps, especially given how diverse this is as a species? (5) Why are PCR gels not included as a figure? This should be raw data included here. (6) What are the ethics approval needed to collect these samples?
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
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?
No: No discussion on ethics approval for bacterial isolate collection from patients
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Comments to Author
The authors of this manuscript performed string tests on four different agar mediums to identify hvKP. In this present study, the authors also screened for three different hypervirulence determinants on the studied strains. Among the four different agar media, the investigators conclude that sheep blood agar with a cutoff of 5 mm is the optimal condition for detecting hvKP strains in the clinical context. However, this finding is not new as several studies have performed string tests exclusively on 5% sheep blood agar. Apart from this, there are several other aspects that I think need to be addressed. Major comments. 1. Hypermucoviscosity and hypervirulence are two different phenomena. Several studies revealed that despite carrying pLVPK-associated molecular markers (rmpA, rmpA2, iro, and iuc) K. …
Comments to Author
The authors of this manuscript performed string tests on four different agar mediums to identify hvKP. In this present study, the authors also screened for three different hypervirulence determinants on the studied strains. Among the four different agar media, the investigators conclude that sheep blood agar with a cutoff of 5 mm is the optimal condition for detecting hvKP strains in the clinical context. However, this finding is not new as several studies have performed string tests exclusively on 5% sheep blood agar. Apart from this, there are several other aspects that I think need to be addressed. Major comments. 1. Hypermucoviscosity and hypervirulence are two different phenomena. Several studies revealed that despite carrying pLVPK-associated molecular markers (rmpA, rmpA2, iro, and iuc) K. pneumoniae strains can exhibit negative string test results, and K. pneumoniae strains devoid of any such markers can also give positive string test results. Even, several cKP strains (with a positive string result) can cause invasive infections. Moreover, apart from the nature of culture media, the age of the colonies can also influence the string test result. So, assessment/identification of hvKP based on the string test result or the presence of a few markers can be confusing. Further in vivo assay (especially mouse infection model) and accurate/elaborate patient data are needed for proper identification of hvKP. 2. Line no. 84 & 136: Why were only 40 strains chosen for further analysis? In my opinion, if the same number of negative strains (n = 80) were chosen for the subsequent analysis, the statistical outcomes of the study might differ. 3. Line no. 108: Apart from such markers, Screening for several hvKP-associated capsular types (K1, K2, K5, K20, K47, K54, K57) can be performed. In addition, considering the epidemiological importance of hvKP, sequence typing analysis of the studied strains can also be carried out. It would be nice if the above-mentioned analysis could be incorporated into this existing work. 4. Line no. 110: In addition to virulence determinants, the studied K. pneumoniae strains should also assessed for AMR determinants. Since, the majority of hvKP strains are becoming antibiotic-resistant and due to the emergence and spread of carbapenem-resistant hvKP strains, the clinical landscape of hvKP is altering drastically. So, it is important to assess the antibiotic susceptibility profiles and AMR determinants of the studied strains. Please include the same in the manuscript. 5. Line no. 122: A positive string test is often an indication of the presence of a hypercapsule which is frequently associated with strong biofilm-forming ability. Despite performing string tests on different culture media, the authors of this manuscript did not perform any such experiment. Minor comments: 1. Line no. 52 & 108: Please use any one abbreviation, either HvKp or hvKp. 2. Line no. 103: Please provide a detailed clarification of inpatient and outpatient. 3. Line no. 141: The sensitivity, specificity, and diagnostic accuracy of the string tests need further clarification. 4. In Table 1: Please use the lowercase (iuc) 5. Line no. 175: This sentence needs to be revised. 6. Line no. 183: Is there any correlation between the positive string test and any specific type of virulence gene? 7. Line no. 228: This particular data is not sufficient to identify hvKP.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
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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