Genome sequencing unveils blaKPC-2-harboring plasmids as drivers of enhanced resistance and virulence in nosocomial Klebsiella pneumoniae

ABSTRACT The threat posed by Klebsiella pneumoniae in healthcare settings has worsened due to the evolutionary advantages conferred by blaKPC-2-harboring plasmids (pKPC-2). However, the specific evolutionary pathway of nosocomial K. pneumoniae carrying pKPC-2 and its transmission between patients and healthcare environments are not yet well understood. Between 1 August and 31 December 2019, 237 ST11 KPC-2-producing-carbapenem-resistant K. pneumoniae (CRKP) (KPC-2-CRKP) were collected from patient or ward environments in an intensive care unit and subjected to Illumina sequencing, of which 32 strains were additionally selected for Nanopore sequencing to obtain complete plasmid sequences. Bioinformatics analysis, conjugation experiments, antimicrobial susceptibility tests, and virulence assays were performed to identify the evolutionary characteristics of pKPC-2. The pKPC-2 plasmids were divided into three subgroups with distinct evolutionary events, including Tn3-mediated plasmid homologous recombination, IS26-mediated horizontal gene transfer, and dynamic duplications of antibiotic resistance genes (ARGs). Surprisingly, the incidence rates of multicopy blaKPC-2, blaSHV-12, and blaCTX-M-65 were quite high (ranging from 27.43% to 67.01%), and strains negative for extended-spectrum β-lactamase tended to develop multicopy blaKPC-2. Notably, the presence of multicopy blaSHV-12 reduced sensitivity to ceftazidime/avibactam (CZA), and the relative expression level of blaSHV-12 in the CZA-resistant group was 6.12 times higher than that in the sensitive group. Furthermore, a novel hybrid pKPC-2 was identified, presenting enhanced virulence levels and decreased susceptibility to CZA. This study emphasizes the notable prevalence of multicopy ARGs and provides a comprehensive insight into the intricate and diverse evolutionary pathways of resistant plasmids that disseminate among patients and healthcare environments. IMPORTANCE This study is based on a CRKP screening program between patients and ward environments in an intensive care unit, describing the pKPC-2 (blaKPC-2-harboring plasmids) population structure and evolutionary characteristics in clinical settings. Long-read sequencing was performed in genetically closely related strains, enabling the high-resolution analysis of evolution pathway between or within pKPC-2 subgroups. We revealed the extremely high rates of multicopy antibiotic resistance genes (ARGs) in clinical settings and its effect on resistance profile toward novel β-lactam/β-lactamase inhibitor combinations, which belongs to the last line treatment choices toward CRKP infection. A novel hybrid pKPC-2 carrying CRKP with enhanced resistance and virulence level was captured during its clonal spread between patients and ward environment. These evidences highlight the threat of pKPC-2 to CRKP treatment and control. Thus, surveillance and timely disinfection in clinical settings should be practiced to prevent transmission of CRKP carrying threatful pKPC-2. And rational use of antibiotics should be called for to prevent inducing of pKPC-2 evolution, especially the multicopy ARGs.

https://pubmed.ncbi.nlm.nih.gov/35544058/https://pubmed.ncbi.nlm.nih.gov/34282943/ 4. How recombination was inferred?The authors want to use a program to properly detect recombination in the sequences analysed.5. It's not clear to me how phylogeny in Figure 2 was constructed, what this based on the relaxase or based on some Mash distance?

Minor comments
Modify the title for clarity.I would suggest something like ... "Genome sequencing unveils blaKPC-2 plasmids as drivers of enhanced resistance and virulence in nosocomial Klebsiella pneumoniae" Line 43: should be "Long-read sequencing was" Line 64: should be just "K.pneumoniae" Line 79: should be "Using long-read sequencing" Lines 218: what do you mean by recombination traces?Did you use any specific program to detect recombination?
Regarding Table S2 please highlight resistant cases in bold.
Reviewer #2 (Comments for the Author): The manuscript "Msystems00924-23" by Xinhong Han and co-authors delineates the evolutionary trajectory of blaKPC plasmids in clinical isolates of the pathogenic bacteria K. pneumoniae.Their combined approach, involving microbial isolation from both patients and ward environments, along with the utilization of Illumina and Nanopore sequencing, as well as antibiotic and virulence testing, facilitated the identification of blaKPC plasmids with distinctive structural configurations.Furthermore, the authors conducted an exhaustive sequence analysis to deduce the potential recombination mechanisms underlying the dissemination of blaKPC-2 in different plasmids.
There are limited reports focusing on tracking the evolution of antibiotic resistance plasmids in local nosocomial contexts.Additionally, it is known that assembling plasmids encoding antibiotic resistance with short sequencing reads poses a challenge.The authors effectively resolve this limitation by employing a Nanopore sequencing strategy.The study is well-executed and well-articulated.Nevertheless, some details in the text and the explanation of results could enhance its presentation.
1.I understand that the studied K. pneumoniae strains were based on the dissemination of a single clonal lineage that originated from the highest peak of sampling.The evidence presented highlights the close relationships among these isolates.Did the authors investigate the genetic or phenotypic relationships (antibiotic resistance, plasmids) of these isolates with the rest of the isolates obtained during the screening period?Is the propensity to vary a property of the clone studied, or is there a possibility of parallel evolution in other isolates?2. Since CRKP colonies were obtained at the infection sites or in the environmental ward, where did the recombinant plasmids originate?Within the patient or in any other place?3.In section 2.2, the information on Illumina and Nanopore sequencing should be complemented by adding the overall quality, sequence read length, and coverage of the plasmid assemblies.It is important to report them for a correct evaluation of the recombinant junctions and gene duplications proposed in the models.4. L. 217.What exactly does "recombination traces" mean? 5.In the section about the formation of the hybrid pKPC-2 plasmid (l.225-246), homologous recombination via identical sequences shared by the plasmids is the normal way to explain the results.However, the identity of the sequences is slightly lower than optimal for high-frequency recombination.Then the event may occur at low frequencies or the intervention of some recombinase from a transposon is needed.Would you comment on this point?6.The emergence of plasmid variants in a short period of time suggests that antibiotic selective pressures may justify their appearance at high frequencies.Are there clinical data on the antibiotic therapy applied during the sampling period that could have provoked the observed pattern? 7.While recommendations for surveillance of antibiotic-resistant strains could be drawn from this work, these are not explicitly commented on in the text.8. l. 369.What do you mean with "exact conditions if evolution"?Reviewer #3 (Comments for the Author): The current widespread blaKPC-2-bearing plasmids in Klebsiella pneumoniae constitute a great public concern.Understanding the evolution pathoways of such plasmids in clinical settings is of importance to figure out the control methods.Also, the current convergence of MDR plasmids and virulence plasmids in KP is another severe threat.This study utilized the genomic analysis including long-read sequencing data analysis to decipher the dynamic evolution pathways under the ICU ward, sheding insight on the rapid transmission of such MDR plasmids.The study is a comprehensive work covering sampling, AST, genome sequencing analysis, gene expression assay, conjugation assay and virulence detection.Several minor suggestions were provided here for reference.1.The figures were not in a right direction.2. What's the reason of picking up the isoates for long-read sequenicng? 3.For the resistance gene tandem repeats, are they stable in copy number of not for a single strain?4. Conjugation assay has been reported to facilitate the formation of hybrid plasmids.Authors could cite more referneces to highlight the wide existence of such phenomenona.More discussion in this area is suggested.5.When the resistance genes are mutiply amplified in the evolved strains, how about the MICs of corresponding antimicrobials?6. pKPC-2 plasmid seems not to be a specific plasmid, but including all plasmids harboring blaKPC-2.Authors should check the consistence of such presentation.**Thank you for your comment.In our study, we inferred the recombination of pKPC-2 plasmids through manual sequence analysis, incorporating modifications from previously described methods (1,2).We employed several bioinformatics tools, namely, Prokka, Easyfig, and CLC Genomics Workbench, during the analysis process.
First, the complete sequences of pKPC-2 were obtained by combining Illumina short-read sequencing and nanopore long-read sequencing.These sequences were then annotated using Prokka, and manual corrections were made as necessary.Subsequently, we compared the annotated plasmids using blast-2.9.0+ and visualized the results with Easyfig v2.2.2.This visualization allowed us to identify potential recombination segments between the plasmids.To further investigate the recombination events, we employed CLC Genomics Workbench for comparative analysis, which provided DNA nucleotide sequences.Within these sequences, we specifically searched for evidence of recombination around the identified recombination segments, including insertion target sites and homologous recombination fragments.
By following this approach, we inferred recombination events in the pKPC-2 plasmids without utilizing dedicated recombination detection software.In this study, there were markers that supported the manual analysis results, including the 8-bp target site of IS26, site-specific recombination markers and Tn3-related recombination.**Thank you for your review.The phylogeny in Figure 2 was constructed using the complete sequence of pKPC-2 plasmids.The alignment of the sequences was performed using MAFFT v7.310 with the maximum-likelihood method.The best-fit analysis model for the data was determined automatically based on the Bayesian Information Criterion (BIC), and the selected model was TPM2+F+G4.Once the sequence alignment was obtained, the phylogenetic tree was constructed using IQ-TREE 2.0.3.Based on this model and the aligned sequences, IQ-TREE 2.0.3 was used to construct the phylogenetic tree using the maximum likelihood method.**Thank you for your review.1.The sentence has been modified to "The two subgroups of IncFII/IncR type plasmids exhibited similar skeletons, with evidence of recombination events".Subgroup 2 contained two additional segments compared to the common segments found in both subgroups.Around these additional segments, a 28-bp marker of site-specific recombination was identified, as reported previously (3). 2. In the study, the recombination analysis was conducted manually following a previously established method for performing comparative analysis to identify recombination events.Bioinformatics tools were used during the analysis process, including Prokka, Easyfig and CLC Genomics Workbench.

Regarding Table S2 please highlight resistant cases in bold.
**Thank you for your suggestion.The resistant isolates are highlighted in bold.
Reviewer #2 (Comments for the Author): The manuscript "Msystems00924-23" by Xinhong Han and co-authors delineates the evolutionary trajectory of bla KPC-2 plasmids in clinical isolates of the pathogenic bacteria K. pneumoniae.Their combined approach, involving microbial isolation from both patients and ward environments, along with the utilization of Illumina and Nanopore sequencing, as well as antibiotic and virulence testing, facilitated the identification of bla KPC-2 plasmids with distinctive structural configurations.Furthermore, the authors conducted an exhaustive sequence analysis to deduce the potential recombination mechanisms underlying the dissemination of bla KPC-2 in different plasmids.
There are limited reports focusing on tracking the evolution of antibiotic resistance plasmids in local nosocomial contexts.Additionally, it is known that assembling plasmids encoding antibiotic resistance with short sequencing reads poses a challenge.The authors effectively resolve this limitation by employing a Nanopore sequencing strategy.
The study is well-executed and well-articulated.Nevertheless, some details in the text and the explanation of results could enhance its presentation.Therefore, we chose to focus on ST15 OXA-232-CRKP for comparative analysis.
Similar to ST11 KPC-CRKP, ST15 OXA-CRKP strains exhibited clonal spread with a close genetic relationship (with 0 to 45 SNP differences).However, the two clones differed in terms of antibiotic resistance profiles, plasmid types and the evolutionary characteristics of carbapenem resistance plasmids.Regarding the antibiotic resistance profile, the two clones displayed discrepancies in their susceptibility to meropenem, imipenem, ceftazidime/avibactam, amikacin, meropenem/vaborbactam, imipenem/relebactam and fosfomycin (Table R).ST11 KPC-CRKP contained more plasmid replicon types than ST15 OXA-CRKP.Notably, multiple evolution events were observed in the carbapenem-resistance plasmid pKPC-2 within the ST11 KPC-CRKP, including ARG duplications and homologous recombinations.In contrast, the carbapenem resistance plasmid pOXA-232 in ST15 OXA-CRKP remained stable, with a size of 6.14 kb, and no evolution events were observed in the small plasmid, as reported in our previously published study (4).**Thank you for your comments.This is a very valuable question and needs further research.In this study, we isolated 5 CRKP strains from both patients and environmental settings in bed Unit 16.Among these strains, three were found to carry recombinant plasmids.Specifically, we isolated the recombinant pKB16_E2_KPC plasmid from a micropump, which was employed for microinjection drug delivery.However, the exact origin of the plasmid could not be determined through our observational study.The presence of CRKP isolates in both patients and environmental settings suggests the possibility of a recombination process occurring either within the environment or within patients and subsequently spreading to the micropump.Previous studies have reported the isolation of recombination plasmids both from patients and through in vitro conjugation assays (5,6).Although conjugation has been shown to facilitate the formation of recombinant plasmids, the specific location of their origin has not been reported and requires further research (7,8).Based on our observations and plasmid analysis, we have described the putative recombination process in Figure 6.**Thank you for your valuable comments.We have added the assembly statistics of Illumina sequencing and nanopore sequencing in the Methods section in lines 348-352 and lines 363-364, respectively.For Illumina sequencing data, the sequence reads were de novo assembled using Shovill 0.9.0 with the options '--trim --minlen 200 --mincov 10'.The assessment of the Illumina assembly revealed an average contig N50 of approximately 169 kb and an average length of the largest contig of approximately 384 kb.The quality of the assembled data was further confirmed using Kleborate.

In section 2.2, the information on
Regarding the Nanopore sequencing data, the raw reads were de novo assembled using Raven 1.8.1, followed by error correction using Polypolish with default parameters.The plasmids were obtained by de novo assembly of nanopore sequencing, and all chromosomes and pKPC-2 plasmids of the 32 strains were circularized completely.
The quality assessment of the assembled data was performed by Quast and the results are listed in Supplementary material for review_1.occur between short homology sequences (> 20 bp) with the assistance of widely distributed bacterial strand exchange proteins, such as RecA (9).Then, we focused on the head 100 bp homology segments and found that the left segments had 100% coverage and 84% identity, while the right segments had 99% coverage and 86% identity.Similarly, Zhao et al. reported plasmid recombination between a pLVPK-like virulence plasmid and a conjugative blaKPC-2-carrying plasmid, which involved a 43 bp homology segment with 84% identity (10).This suggests that an identity of over 80% may be efficient for homologous recombination.
Furthermore, several studies have observed the involvement of recombinase enzymes from transposons in plasmid formation (11)(12)(13).Additionally, Li et al. reported plasmid recombination induced by intron reverse transcriptase and the mobile element ISShes11 (7).In our study, the homologous segments were located in TnAs1 and TnAs2, both of which belong to the Tn3 family transposons.It is possible that the recombination observed in our research involves the intervention of a site-specific recombinase called resolvase, which is derived from the Tn3 transposon.

The emergence of plasmid variants in a short period of time suggests that antibiotic
selective pressures may justify their appearance at high frequencies.Are there clinical data on the antibiotic therapy applied during the sampling period that could have provoked the observed pattern?
**During the screening period, a total of 39 patients were colonized by KPC-2-producing carbapenem-resistant Klebsiella pneumoniae (CRKP).To investigate the antibiotic factors contributing to the emergence of plasmid variants, we reviewed the medical records of these patients to identify the antibiotics prescribed prior to the isolation date.Among the 39 patients, the majority (29/39) had received treatment with piperacillin/tazobactam (TZP) and/or cefoperazone/sulbactam, while 21/39 patients had been treated with carbapenems (Supplementary material for review_2).Furthermore, we took a patient in B16 as an example, where CRKP strains carry multiple plasmid variants.A month before the isolation of the plasmid variants, this particular patient had undergone a 9-day course of TZP (4.5 g q8h), followed by a 13-day course of TZP (4.5 g q8h) and a 3-day course of biapenem (0.3 g q12h).Furthermore, TZP was still being used on the isolation day.Notably, in a separate study, TZP-resistant E. coli was isolated after a 19-day treatment with TZP, accompanied by amplification of the blaTEM-1B gene (14).
Additionally, an experimental evolution assay demonstrated the amplification of bla KPC-2 under the selective pressures of β-lactam antibiotics, including ceftazidime, meropenem, or moxalactam.(15).Based on these observations, we speculated that the overuse of β-lactam/lactamase inhibitor combinations and carbapenem antibiotics was associated with the evolution of pKPC-2.These findings highlight the potential role of antibiotic selective pressures in driving the emergence of pKPC-2 plasmid variants.However, importantly, these conclusions are based on observational data, and further research is warranted to establish a more comprehensive understanding of the relationship between antibiotic usage and the evolution of pKPC-2.

l. 369. What do you mean with "exact conditions if evolution"?
**Thank you for your comments.I apologize for the confusion of the expression.When I mentioned "exact conditions of evolution", I specifically addressed the circumstances and environmental factors that contribute to the process of pKPC-2 evolution in bacteria.These conditions include the characteristics of the host bacteria, the structure of the pKPC-2 plasmid, and exposure to antibiotics.In our study, we observed pKPC-2 evolution in ST11 CRKP.Moving forward, we plan to investigate the evolution characteristics of pKPC-2 in other ST types and explore the potential impact of mobile elements on this process.It has been reported that antibiotic usage facilitated the evolution of CRKP.However, the specific details regarding the type of antibiotic, the duration of antibiotic usage, and the concentration of the antibiotic that induces the evolution of pKPC-2 have not been confirmed.To gain a more comprehensive understanding of the conditions that promote pKPC-2 plasmid evolution, further in vitro experiments are needed.
Reviewer #3 (Comments for the Author): The current widespread blaKPC-2-bearing plasmids in K. pneumoniae constitute a great public concern.Understanding the evolution pathways of such plasmids in clinical settings is of importance to figure out the control methods.Also, the current convergence of MDR plasmids and virulence plasmids in KP is another severe threat.This study utilized the genomic analysis including long-read sequencing data analysis to decipher the dynamic evolution pathways under the ICU ward, shading insight on the rapid transmission of such MDR plasmids.The study is a comprehensive work covering sampling, AST, genome sequencing analysis, gene expression assay, conjugation assay and virulence detection.Several minor suggestions were provided here for reference.
1.The figures were not in a right direction.
**Thank you for your comments.Due to an error during the merging process, the direction of the figures became mixed.We have now rectified this mistake and made the necessary corrections.

What's the reason of picking up the isoates for long-read sequenicng?
**Thank you for your comments.Our objective was to provide a comprehensive description of the population structure and evolutionary characteristics of pKPC-2 in clinical settings.However, the use of short-read whole-genome sequencing (WGS) poses challenges in achieving complete and accurate reconstruction of plasmid structures.To overcome this limitation, we proposed the utilization of nanopore sequencing to obtain full-length plasmid sequences for a more thorough analysis of plasmid evolution.Furthermore, the widespread distribution and rapid changes in mobile genetic elements (MGEs) make it difficult to discern the specific evolutionary processes associated with pKPC-2, given its complex genetic structure.In light of this, we specifically selected genetically indistinguishable CRKP strains isolated from an ICU in the highest peak of sampling day.Along with usage of long-read sequencing, the evolution of the pKPC-2 plasmids could be tracked in a distinguishable way.

3.
For the resistance gene tandem repeats, are they stable in copy number or not for a single strain?
**Thank you for your comments.We conducted BLASTN analysis on the filtered long-read reads, comparing them to a reference containing the tandem repeat units and their respective flanking regions.The results revealed that the copy number of resistance gene tandem repeats exhibited instability within a single strain, as depicted in Figure 4C.For instance, in the case of bla KPC-2 in pKP173_KPC, the copy number varied from 1 to 8, with the majority of individual plasmids carrying 4 copies.Similarly, Schuster et al. reported that the copy numbers of the bla CTX-M genes ranged from 1 to 5 copies between individual plasmids in a single multidrug-resistant Escherichia coli isolate ( 16).

Conjugation assay has been reported to facilitate the formation of hybrid plasmids.
Authors could cite more referneces to highlight the wide existence of such phenomenona.
More discussion in this area is suggested.
**Thank you for your valuable suggestion.The relevant discussion has been added in lines 251-263."In a previous study, a hybrid pKPC-2 plasmid was reported, and its formation was inferred to be associated with IS26 based on a comparison with plasmids in GenBank (5).
Conjugation assays have been reported to facilitate the formation of hybrid plasmids.Chen et al.
conducted an experimental study, and plasmid recombination was observed during the conjugation process (6).Furthermore, fusion of the IncN1-F33:A-:B-plasmid and an mcr-1-carrying phage-like plasmid was reported with a frequency of 1.75 × 10 -4 cointegrates per transconjugant (1).Similar phenomena have been increasingly reported, indicating the important role of conjugation in the formation of hybrid plasmids, even with nonconjugative plasmids (8).For instance, Li et al. performed a conjugation assay under ceftazidime, during which they observed the fusion of a nonconjugative virulence plasmid p17-16-vir and a multidrug-resistance plasmid p17-16-CTX, resulting in the formation of a novel hybrid MDR virulence plasmid (7)."

MICs of corresponding antimicrobials?
**Thank you for your comments.The amplification of bla KPC-2 and bla SHV-12 was demonstrated to decrease the susceptibility of CRKP to certain antibiotics, including ceftazidime/avibactam (CZA), meropenem/varbobactam (MEV) and imipenem/relebactam (IMR).Specifically, conjugation assays have demonstrated that pKB16_E2_KPC and pKB16_E2_SHV, both of which carry multicopy bla SHV-12 , could increase the MIC of CZA by 4-fold in J53.Furthermore, antimicrobial susceptibility testing (AST) of novel β-lactam/lactamase inhibitor combinations was performed on the 237 KPC-2-CRKP isolates.The results revealed that CRKP strains harboring multiple copies of blaKPC-2 exhibited significantly reduced susceptibility to CZA (p < 0.01), MEV (p < 0.01) and IMR (p < 0.05).Then, to eliminate the effects of multicopy bla KPC-2 , the relationship between multicopy ESBL genes and antimicrobial resistance was investigated among isolates with fewer than 2 bla KPC-2 copy numbers, and the results suggested that multicopy bla SHV-12 significantly decreased the susceptibility of CRKP to CZA (p < 0.0001) (Fig. 5abc).
6. pKPC-2 plasmid seems not to be a specific plasmid, but including all plasmids harboring blaKPC-2.Authors should check the consistence of such presentation.
**Thank you for your suggestion.Yes, the pKPC-2 plasmid is an abbreviated representation of the bla KPC-2 -harboring plasmid in this study, encompassing all plasmids that carry the bla KPC-2 gene.
We have verified the accuracy of this presentation.Furthermore, we have made modifications to the expressions in Lines 123, 147, 195, 302, 320, and 596 as suggested.

Supplementary material for review_1
The results of quality assessment of the Illumina assembled data and average nucleotide identity analysis

Figure 2
was constructed, what this based on the relaxase or based on some Mash distance?
the title for clarity.I would suggest something like ... "Genome sequencing unveils blaKPC-2 plasmids as drivers of enhanced resistance and virulence in nosocomial Klebsiella pneumoniae" **Thank you for your valuable suggestion.The title has been modified according to your suggestion.Line 43: should be "Long-read sequencing was" **The expression has been modified.Line 64: should be just "K.pneumoniae" **The word has been modified.Line 79: should be "Using long-read sequencing" **The expression has been modified.Lines 218: what do you mean by recombination traces?Did you use any specific program to detect recombination?

1 .
I understand that the studied K. pneumoniae strains were based on the dissemination of a single clonal lineage that originated from the highest peak of sampling.The evidence presented highlights the close relationships among these isolates.Did the authors investigate the genetic or phenotypic relationships (antibiotic resistance, plasmids) of these isolates with the rest of the isolates obtained during the screening period?Is the propensity to vary a property of the clone studied, or is there a possibility of parallel evolution in other isolates??**Thank you for your comments.This is a very intriguing question and needs further investigation based on extended application of long-read nanopore sequencing.Here, we initially explored this subject using the results obtained from short-read Illumina sequencing.During the screening period, a total of 420 CRKP were isolated, among which the predominant epidemic clones were ST11 KPC-2-producing CRKP (KPC-CRKP) (237/420) and ST15 OXA-232-producing CRKP (OXA-CRKP) (106/420), as illustrated in the Figure R. CRKP strains belonging to other ST types were sporadically isolated, including 30 ST15 KPC-CRKP strains, 16 ST1 strains, 15 ST133 strains, 12 ST556 strains, 2 ST268 strains and 2 non-carbapenemase-producing ST11 strains.

Figure R .
Figure R. Molecular epidemiological characteristics of 420 strains of CRKP isolated during the screening period Illumina and Nanopore sequencing should be complemented by adding the overall quality, sequence read length, and coverage of the plasmid assemblies.It is important to report them for a correct evaluation of the recombinant junctions and gene duplications proposed in the models.

4 . 5 .
L. 217.What exactly does "recombination traces" mean?**Thank you for your review.I apologize for the confusion caused by my previous expression.Recombination traces refer to the various elements involved in the recombination process, such as recombination fragments, recombination sites, and recombination markers.A specific example is the recombination process observed in pKB16_E2_KPC, which involves the fusion of IncFII(pHN7A8)/IncR-type pKPC-2 and virulence plasmids in CRKP isolates.This fusion is mediated by the IS26 insertion of bla SHV-12 fragments and Tn3-related homologous recombination of bla KPC-2 fragments.In the section about the formation of the hybrid pKPC-2 plasmid (l.225-246), homologous recombination via identical sequences shared by the plasmids is the normal way to explain the results.However, the identity of the sequences is slightly lower than optimal for high-frequency recombination.Then the event may occur at low frequencies or the intervention of some recombinase from a transposon is needed.Would you comment on this point?**Thank you for your comments.According to a study by Lu et al. , homology recombination can

7 .
While recommendations for surveillance of antibiotic-resistant strains could be drawn from this work, these are not explicitly commented on in the text.**Thank you for your suggestion.Our study findings indicate that CRKP exhibited widespread colonization in both patients and environmental settings.Furthermore, we observed the evolution of carbapenem-resistance plasmids during the clonal spread of CRKP.Notably, our previously reported study revealed a correlation between long-term colonization and the risk of further infection (4).Considering these findings, we strongly recommend the implementation of regular surveillance measures for CRKP in both patient and environmental settings.Timely and systematic monitoring of CRKP prevalence and dynamics is crucial for effective infection control and prevention strategies.Additionally, it is important to prioritize timely decolonization interventions to curb the spread of CRKP.The recommendations for surveillance are supplied in lines 304-312.