Genetic Characteristics and Microbiological Profile of Hypermucoviscous Multidrug-Resistant Klebsiella variicola Coproducing IMP-4 and NDM-1 Carbapenemases

ABSTRACT We report here a hypermucoviscous, New Delhi metallo-β-lactamase 1 (NDM-1) and imipenemase 4 (IMP-4) carbapenemases-coproducing Klebsiella variicola isolate obtained from a pediatric patient. This strain was resistant to carbapenems and most other β-lactams. Although hypermucoviscous, this strain possessed attenuated virulence according to serum killing assay and Galleria mellonella infection model. Notably, two copies of blaNDM-1 were contained on two tandem ISCR1 elements and coexisted with blaIMP-4 in a novel hybrid multidrug resistance plasmid. This is the first description of the coexistence of blaNDM-1 and blaIMP-4 in a single plasmid of hypermucoviscous K. variicola. IMPORTANCE As an important member of the Klebsiella pneumoniae complex, Klebsiella variicola is poorly studied as an emerging human pathogen. We, for the first time, report a unique K. variicola isolated from a pediatric patient in China. This isolate exhibited hypermucoviscosity, a classic hypervirulence characteristic of K. pneumoniae, and contained multiple carbapenem-resistant genes, including blaIMP-1 and blaNDM-1. Interestingly, these antimicrobial resistance genes were located on a novel hybrid plasmid, and our results suggested that this plasmid might have been introduced from K. pneumoniae and undergone a series of integration and recombination evolutionary events. Overall, our study provides more insight into K. variicola and highlights its superior capability to acquire and maintain foreign resistance genes.

explain the reason why you did not test the cefoxitin? For quality control the utilizing of only E. coli ATCC 25922 is not enough, you should add P. aeruginosa ATCC 27853 for quality control of carbapenems. In 2017, a CLSI and EUCAST joint working group recommended broth microdilution (BMD), without surfactant, as the reference method for testing colistin (rBMD), I just wonder the broth microdilution method that you used is the same or not? 3. Line 153, could you explain the reason why you must be applied both sequencer platform for WGS? Minor points 1

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To Editor: 1. The manuscript would be considered acceptable after conversion to shorter more concise format i.e. an Observations paper. Please refer to https://journals.asm.org/journal/spectrum/article-types for the article style and formatting. Briefly, the Observations format is limited to 1,200 words with a maximum of 2 figures and 25 references Response: we have rewritten our manuscript in the format of observation paper.
2. Both reviewers have brought up two areas of concern, namely, the need to demonstrate the expression of dual carbapenemases, and the transferability of carbapenemase-bearing plasmid. I would like the authors to address the comments on plasmid expression and beta-lactamase production as that is the major premise of the title. Whereas I can overlook the requirements on the conjugation work and HMV associated features. Response: thank you for your valuable comments, we have conducted experiments to give evidence to the expression of dual carbapenemases in Figure 1A in the revised version.
3. The English in the present form is not of publication quality and requires major improvement. Please carefully proof-read and spell check to eliminate grammatical errors Response: thank you for your suggestion, a native English speaker has carefully checked and polished our manuscript.  Fig.1 Can be removed.
Response: we have deleted this figure in our revised version.

Review #1
1. Wang et al provide the characterization of an interesting Klebsiella variicola strain SHET-01 with hypermucoviscosity (HMV) phenotype and carrying two types of carbapenem resistance genes: blaIMP-4 and blaNDM-1. The authors report the results of antimicrobial susceptibility testing, biofilm, serum killing and Galleria mellonella infection, as well as WGS analysis of the strain.
Although the strain carries many classes of antibiotic resistance genes, it remains susceptible to most tested antibiotics except beta-lactams. Furthermore, the authors show that the strain is avirulent to G. mellonella, even though the strain shows HMV phenotype and has a strong capacity to form biofilm. Finally, the WGS shows that the strain harbours two large plasmids, one of them (pNDM-IMP-1) carrying all plasmid-borne antibiotic genes.
The experimental design was well thought out and the data reported are of interest to the field.
However it is not clear which carbapenemase gene or both genes contribute to the carbapenem resistance. The authors do not have the data of the expression of the two carbapenemase genes or the production of the enzymes. Given that the authors claim SHET-01 co-producing both IMP-4 Response: Thank you for your valuable comments. We have added experiments to the revised manuscript to confirm the production of duel carbapenemases, as shown in Figure 1A.
About the idea that there might be some potential links between HMV phenotype and the MDR plasmid, this is a very interesting topic. We have compared this plasmid (pKV8917) with our plasmid, and found that their homology is very low. And we will sort this possibility out completely in the next research and conduct a more in-depth exploration of its HMV mechanism.

Reviewer #3
Major points 1. Related to the title, the authors used the term "extensively drug-resistant-XDR" however in the result this strain was reported susceptibility to many class of antimicrobial agents except beta-lactams and its derivatives my question is that this isolate is truly XDR or just multi-drug resistant (MDR)? In addition, the term "co-producing IMP-4 and NDM-1 carbapenemase" should be also reconsidered, I agree that this strain possesses IMP-4 and NDM-1 genes on plasmid and it has phenotypic resistant to carpapenems but how could the authors make sure that it produces both carbapenemases? Response: thank you for your valuable comments, we have reviewed our results of the antimicrobial susceptibility test, and this strain SHET-01 actually was multi-drug resistant.
We have corrected this improper description. Additionally, about the production of dual carbapenemases, we have conducted experiment to confirm this using colloidal gold method ( Figure 1A) in the revised version.
2. to Antimicrobial susceptibility testing (line 98), Which version of CLSI did you use? As I understand, from CLSI 2017 up toward fosfomycin is used for testing and reporting of E. coli and E. faecalis urinary tract isolates only. Moreover, cefoperazone and cefoperazone-sulbactam are not the antimicrobial agents should be considered for routine testing and reporting. Could you explain the reason why you did not test the cefoxitin? For quality control the utilizing of only E. coli ATCC 25922 is not enough, you should add P. aeruginosa ATCC 27853 for quality control of carbapenems. In 2017, a CLSI and EUCAST joint working group recommended broth microdilution (BMD), without surfactant, as the reference method for testing colistin (rBMD), I just wonder the broth microdilution method that you used is the same or not? Response: Thank you for your valuable comments on the antimicrobial susceptibility testing.

1)
We have used the CLSI M100 (2020) in our study and have added this reference in the revised manuscript. 2) In addition to urinary tract infections, fosfomycin has been increasingly used in the anti-infection of clinical multi-drug resistant Gram-negative bacterial in recent years, which in vitro susceptibility usually refer to the interpretation criteria of urinary tract Escherichia coli and Enterococcus faecalis. However, in our study, considering the strain SHET-01 was isolated from a pediatric patient, and fosfomycin was not recommended used in children in China, we have deleted the data of this antimicrobial sensitivity. 3) In China, cefoperazone and cefoperazone-sulbactam were widely used in the clinical anti-infections, need provide their data to give a basis for clinical treatment. About cefoxitin, we have not included this antimicrobial in our antimicrobial susceptibility testing, because we had tested another cephamycin antibiotic, cefotetan. 4) Actually, we also had used P. aeruginosa ATCC 27853 as quality control, and have added this description in line 150-151 in the revised version. 5) Colistin susceptibility test was performed using polystyrene plastic plate without tween-80 in our study.
3. Line 153, could you explain the reason why you must be applied both sequencer platform for WGS?
Response: PacBio Sequel single-molecule real-time (SMRT) sequencing platform was a third-generation sequencing platform, and the Illumina NovaSeq sequencing platform was based on second-generation sequencing technologies. The second-generation sequencing technologies have offered vast improvements over Sanger sequencing, their limitations, especially their short-read lengths (50-500bp), make them poorly suited for some particular biological problems, including assembly and determination of complex genomic regions, gene isoform detection, and methylation detection. The third-generation PacBio sequencing is a method for real-time sequencing, and it offers much longer read lengths (＞10kb) and faster runs than second-generation sequencing methods but is hindered by a lower throughput and higher error rate (~15%) [1]. Since the advantages of third-generation sequencing and second-generation sequencing are complementary, we therefore used hybrid-sequencing strategies that make use of both technologies to overcome the disadvantages of each alone.