Short-Term Within-Host Genomic Diversity and Clone Turnover of Carbapenem-Resistant Klebsiella pneumoniae in an Intensive Care Unit Patient

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is highly interesting and of considerable medical and scientific relevance. I only have a few minor revisions to further improve its quality, as the study is overall very well conducted and well written.

In my opinion, the manuscript should be considered a “case report” rather than a “research article.”

• L22, L45, and elsewhere: the gene names must be revised according to the current nomenclature standards. For example, “NDM-5” should appear as a subtype designation without italics, whereas the gene prefix should follow the official formatting conventions. Please review all gene names throughout the manuscript.
• L368: was it a single dose? For how long was the treatment administered?
• L379: include the doses used and the duration of treatment.
• Include the manufacturers of all culture media and laboratory reagents/materials used in the study.
• The resolution of Figure 4 should be improved, as it is difficult to visualize and read the genes identified within the plasmids.
• It would be interesting if the authors evaluated the transferability capacity of the detected plasmids, since this aspect is discussed in a speculative manner without supporting experimental evidence.
• I suggest that the authors standardize the use of the term “antimicrobials” instead of “antibiotics” throughout the manuscript, as the former encompasses natural, semi-synthetic, and synthetic compounds, whereas the latter refers only to naturally occurring substances.

Author Response

The manuscript is highly interesting and of considerable medical and scientific relevance. I only have a few minor revisions to further improve its quality, as the study is overall very well conducted and well written.

We thank the reviewer for useful comments that has led to significant improvement of our manuscript. Our answers are given below boldfaced.

In my opinion, the manuscript should be considered a “case report” rather than a “research article.”

To our opinion, case reports are focused on the patient treatment and therapy provided, while our manuscript is focused on bacterial genomic data, so a ‘research article’ seems to be more suitable classification

• L22, L45, and elsewhere: the gene names must be revised according to the current nomenclature standards. For example, “NDM-5” should appear as a subtype designation without italics, whereas the gene prefix should follow the official formatting conventions. Please review all gene names throughout the manuscript.

Gene names were revised and corrected throughout the manuscript.

• L368: was it a single dose? For how long was the treatment administered?

Yes, it was a single dose. No additional doses were administered. We added this information to the manuscript.

• L379: include the doses used and the duration of treatment.

Doses and durations were added for each drug

• Include the manufacturers of all culture media and laboratory reagents/materials used in the study.

The manufacturers were added

• The resolution of Figure 4 should be improved, as it is difficult to visualize and read the genes identified within the plasmids.

Figure 4 was updated

• It would be interesting if the authors evaluated the transferability capacity of the detected plasmids, since this aspect is discussed in a speculative manner without supporting experimental evidence.

Performing plasmid experiments lies beyond the scope of the manuscript

• I suggest that the authors standardize the use of the term “antimicrobials” instead of “antibiotics” throughout the manuscript, as the former encompasses natural, semi-synthetic, and synthetic compounds, whereas the latter refers only to naturally occurring substances.

The term use was corrected

Reviewer 2 Report

Comments and Suggestions for Authors

Please see the attached pdf for the comments.

Comments for author File: Comments.pdf

Author Response

We thank the reviewer for useful comments that has led to significant improvement of our manuscript. Our answers are given below boldfaced.

General comment

This is a good manuscript and is worth publication by this journal. However, it requires some minor reviews such as re-organizing the sections to enhance fluent flow of information to the reader. If you don t mind, organize the sections as follows after the abstract; introduction, materials & methods, results, discussion and finally conclusion at the end. Also, work the issues listed below.

Section organization follows the journal requirements for the authors, and methods should appear after Discussion according to these rules.

Parts that need revision are:

1. Introduction

L35, 38: "... of Enterobacterales..." "... Enterobacteriaceae ..." These are taxon

names. Therefore, please adhere to binomial nomenclature guidelines by

italicizing them.

According to the nomenclature guidelines, the name of order (Enterobacterales) should not be italicized (https://wwwnc.cdc.gov/eid/page/scientific-nomenclature).

L36: "...made the World Health Organization (WHO) including these bacteria to

..." typo error please rectify it.

Fixed

2. Results

2.3. Efflux systems

L163: "...Klebsiella ..." Is taxon name therefore you ought to adhere to Binomial

nomenclature guidelines by italicizing it.

Fixed

2.5. Plasmids

L208-9: "... Resistance genes are shown in black while virulence factors are

indicated in magenta." This description is not captured by diagrams in Figure 4.

Please clarify on it.

The genes are shown below the plasmids that contain them using the colors specified. We added “under the plasmids containing them” to clarify this.

L209: "... indicated for small... " Grammatical error, please rectify it.

Fixed

3. Discussion

L357-8: "Meanwhile, we can suggest that isolates obtained from the series of rectal

swabs shown K. pneumoniae lineages circulating within a specific medical unit." This

statement needs grammatical restructuring.

Fixed

Manuscript ID: antibiotics-4351059-peer-review-v1

4. Materials and Methods

4.1. Case report

L370, 378: "... hospitalization to the ICU..." Grammatical-error. Please work on

it.

Fixed

 

4.2. Sample acquisition, susceptibility testing and DNA isolation

L402-3, 406: " ... Columbia blood agar base and Urinary Tract Infections

Chromogenic Agar (UTIC) ..." "... Endo agar ..." Provide the products catalogue

numbers for these reagents or state the name and full address of the

manufacturing company (s) for each of them.

The information regarding the manufacturing companies was added to the manuscript

Reviewer 3 Report

Comments and Suggestions for Authors

The following are my comments that will help the editor's decision:

1. The antibiotic-driven selection argument for ST512 is speculative. There's no experimental or quantitative support for it; it reads more like a post hoc narrative than a tested hypothesis.
2. Calling ST512 a less virulent lineage is entirely genomics-based. Without phenotypic assays to back up those predictions, the designation is premature.
3. The plasmid remodeling story is probably overstated. The observed differences could simply reflect what's normal for each lineage rather than true within-host evolution.
4. The ICU acquisition hypothesis for ST395 and ST512 needs comparative data from isolates from other patients or environmental samples before it holds up. Right now, it's a plausible guess.
5. Hypervirulence inferred from genomic markers alone is on shaky ground. No functional validation was done, and genomic predictions can mislead.
6. One patient, a handful of isolates that's a thin base for the epidemiological and evolutionary conclusions drawn here.
7. cgMLST has real resolution limits for within-host diversification. A SNP-based phylogeny would have told a clearer story.
8. Genome coverage, assembly quality, completeness estimates — none of this is reported. It's difficult to trust the genomic analyses without knowing the sequencing fundamentals.
9. Several central conclusions overstep what the data actually shows. Within-host evolution, virulence attenuation, and antibiotic-driven selection need to be framed as possibilities, not findings.
10. The antibiotic-driven selection argument for ST512 is speculative. There's no experimental or quantitative support for it; it reads more as a post-hoc narrative than a tested hypothesis.
11. Calling ST512 a less virulent lineage is entirely genomics-based. Without phenotypic assays to back up those predictions, the designation is premature.
12. The plasmid remodeling story is probably overstated. The differences observed could just reflect what's normal for each lineage rather than true within-host evolution.
13. The ICU acquisition hypothesis for ST395 and ST512 needs comparative data from isolates from other patients or environmental samples before it holds up. Right now, it's a plausible guess.
14. Hypervirulence inferred from genomic markers alone is on shaky ground. No functional validation was done, and genomic predictions can mislead.
15. One patient and a handful of isolates that's a thin base for the epidemiological and evolutionary conclusions drawn here.
16. cgMLST has real resolution limits for within-host diversification. An SNP-based phylogeny would have told a clearer story.
17. Genome coverage, assembly quality, completeness estimates—none of this is reported. It's difficult to trust genomic analyses without understanding the fundamentals of sequencing.
18. Several central conclusions overstep what the data actually shows. Within-host evolution, virulence attenuation, and antibiotic-driven selection need to be framed as possibilities rather than findings.
19. Figures 3 and 4 can be combined. Check the numbering of Figure 1.

Author Response

We thank the reviewer for useful comments that has led to significant improvement of our manuscript. Our answers are given below boldfaced.

The points 1-9 and 10-18 were duplicated, so we removed them in our answer, thus a point 19 becomes a point 10.

1. The antibiotic-driven selection argument for ST512 is speculative. There's no experimental or quantitative support for it; it reads more like a post hoc narrative than a tested hypothesis.

We added the information regarding the limitations of our hypotheses to the manuscript.

2. Calling ST512 a less virulent lineage is entirely genomics-based. Without phenotypic assays to back up those predictions, the designation is premature.

We added the information regarding the limitations of our hypotheses to the manuscript. We also previously made a statement “This conclusion reflects genomic virulence potential inferred from the reported gene repertoire rather than a direct phenotypic measurement of virulence” in the Discussion.

3. The plasmid remodeling story is probably overstated. The observed differences could simply reflect what's normal for each lineage rather than true within-host evolution.

We changed the statement to reflect the limitations of our approach

4. The ICU acquisition hypothesis for ST395 and ST512 needs comparative data from isolates from other patients or environmental samples before it holds up. Right now, it's a plausible guess.

The epidemiological criteria for intrahospital acquisition include: the infection occurs ≥ 48 hours after hospital admission, recent exposure to invasive devices, exclusion of community acquisition, microbiological patterns specific for the clones known to cause healthcare-associated infections. ST395 and ST512 were circulating in this ICU according to epidemiological data, which is indicated in the manuscript. The detailed genomic comparison with the isolates from other patients lies beyond the scope of the manuscript.

5. Hypervirulence inferred from genomic markers alone is on shaky ground. No functional validation was done, and genomic predictions can mislead.

We had a statement “This conclusion reflects genomic virulence potential inferred from the reported gene repertoire rather than a direct phenotypic measurement of virulence” in the manuscript which provides the limitation scope for our conclusions.

6. One patient, a handful of isolates that's a thin base for the epidemiological and evolutionary conclusions drawn here.

This manuscript is dedicated to within-host evolution of the K. pneumoniae isolates, and is not intended to represent the whole clinical population change. As we noticed in the Introduction, most clinical investigations focus on broad aspects of bacterial diversity in ICUs, while the studies of the changes within a single patient are scarce, although such investigations can provide useful insights into bacterial survival under antibiotic pressure. The authors note that in a rigorous scientific review, critical comments are more constructively supported by specific comparative examples from the literature, rather than by unsupported qualitative assessments.

7. cgMLST has real resolution limits for within-host diversification. A SNP-based phylogeny would have told a clearer story.

We added the SNP data to the manuscript.

8. Genome coverage, assembly quality, completeness estimates — none of this is reported. It's difficult to trust the genomic analyses without knowing the sequencing fundamentals.

We added these data to the manuscript. The genomic data is available in public repository, so that all the analyses performed by us can be duplicated and verified by other researchers.

9. Several central conclusions overstep what the data actually shows. Within-host evolution, virulence attenuation, and antibiotic-driven selection need to be framed as possibilities, not findings.

The limitations of our hypotheses have been addressed in the Discussion section of the manuscript. While these points are not exhaustively restated in the Conclusions, the full context provided throughout the manuscript ensures that the concluding statements are interpreted appropriately.

10. Figures 3 and 4 can be combined. Check the numbering of Figure 1.

Figure 3 is rather large and contains a lot of information, so we believe that combining it with another figure will not increase the readability. The numbering of Figure 1 is correct

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

accept