High-Molecular-Weight Plasmids Carrying Carbapenemase Genes blaNDM-1, blaKPC-2, and blaOXA-48 Coexisting in Clinical Klebsiella pneumoniae Strains of ST39

Background: Klebsiella pneumoniae, a member of the ESKAPE group of bacterial pathogens, has developed multi-antimicrobial resistance (AMR), including resistance to carbapenems, which has increased alarmingly due to the acquisition of carbapenemase genes located on specific plasmids. Methods: Four clinical K. pneumoniae isolates were collected from four patients of a neuro-intensive care unit in Moscow, Russia, during the point prevalence survey. The AMR phenotype was estimated using the Vitec-2 instrument, and whole genome sequencing (WGS) was done using Illumina and Nanopore technologies. Results: All strains were resistant to beta-lactams, nitrofurans, fluoroquinolones, sulfonamides, aminoglycosides, and tetracyclines. WGS analysis revealed that all strains were closely related to K. pneumoniae ST39, capsular type K-23, with 99.99% chromosome identity. The novelty of the study is the description of the strains carrying simultaneously three large plasmids of the IncHI1B, IncC, and IncFIB groups carrying the carbapenemase genes of three types, blaOXA-48, blaNDM-1, and blaKPC-2, respectively. The first of them, highly identical in all strains, was a hybrid plasmid that combined two regions of the resistance genes (blaOXA-48 and blaTEM-1 + blaCTX-M-15 + blaOXA-1 + catB + qnrS1 + int1) and a region of the virulence genes (iucABCD, iutA, terC, and rmpA2::IS110). Conclusion: The spread of K. pneumoniae strains carrying multiple plasmids conferring resistance even to last-resort antibiotics is of great clinical concern.


Introduction
Healthcare-associated infections (HAIs) are major public health burdens due to higher treatment costs and more complex patient care [1,2]. They are also associated with high morbidity rates, diagnostic uncertainty, and increased mortality [3]. HAIs can be caused by either exogenous or endogenous origins; the agents can be transferred between patients, healthcare workers, contaminated objects, visitors, or various environmental sources [4]. HAIs could be caused by different microorganisms, including bacteria, fungi, and viruses. Among them, the ESKAPE group of bacterial pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) cause the most clinically significant and antimicrobial-resistant infections [5,6]. ESKAPE pathogens have developed resistance mechanisms against antibiotics gram-negative bacteria isolates were collected [26]. In this article, we characterized the complete genomes of four clonally related XDR K. pneumoniae carbapenem-resistant clinical strains harboring large hybrid plasmids and determined the location of the resistance and virulence genes.

Bioethical Requirements
The study was a point-prevalence survey. The Burdenko National Medical Research Center of Neurosurgery Review Board approved the study and granted it consent waiver status (approval code: #11/2018, approval date: 1 November 2018). Clinical isolates were labeled without the patients' names, dates of birth, addresses, disease histories, personal documents, or other personal materials.

Bacterial Strains, Growing, and Identification
Four clinical K. pneumoniae strains were isolated from rectal and tracheal swabs collected from four patients of the neuro-ICU of the Burdenko National Medical Research Center of Neurosurgery, Moscow, Russia, in October of 2019. Bacteria were growing in Luria-Bertani (LB) broth (Difco, Rockville, MD, USA) and lactose TTC agar with Tergitol-7 (SRCAMB, Obolensk, Russia) containing 50 mg/L ampicillin (Thermo Fisher Scientific, Waltham, MA, USA) at 37 • C. Bacterial identification was done using the MALDI-TOF Biotyper instrument (Bruker, Karlsruhe, Germany). The following ATCC reference strains were used as controls: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Klebsiella pneumoniae ATCC 700603. Bacterial strains were stored in 15% glycerol at −80 • C.

Whole Genome Sequencing, Assembly and Annotation
DNA isolation was performed by the CTAB method [27]. WGS was carried out using the Nextera DNA Library Preparation Kit (Illumina, San Diego, CA, USA) and MiSeq Reagent Kits v3 (Illumina, San Diego, CA, USA) for the Illumina MiSeq platform (Illumina, San Diego, CA, USA). Long reads were obtained using the Rapid Barcoding Kit RBK004 and flowcell R9.4.1 on the MinION platform (Oxford Nanopore, Oxford Science Park, GB). Basecalling was performed with Guppy ver. 5.0.16 (Oxford Nanopore, Oxford Science Park, UK) with default parameters [28]. Short and long raw reads were used to obtain the hybrid assembly of the strain using Unicycler ver. 0.4.7 software (The University of Melbourne, Melbourne, Australia) with default settings that included primary filtering and quality control [29]. Annotation was carried out by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) ver. 5.3 (National Center for Biotechnology Information, Bethesda, MD, USA) [30].

Whole Genome Analysis
The definition of resistance genes in the complete genome was performed in silico using the online resource ResFinder 2.0 [31] of the Center for Genomic Epidemiology (Technical University of Denmark, Kgs. Lyngby, Denmark). Identification of virulence genes, efflux pump genes, heavy metal resistance genes, and capsule typing was performed in silico using the online resource BIGSdb-Pasteur (https://bigsdb.pasteur.fr/cgi-  [32]. The web resource BLAST was used for homologous plasmid searching (National Center for Biotechnology Information, Bethesda, MD, USA) [33]. Whole-genome alignments were performed using Mauve ver. 26 February 2015 [34]. Snippy 4.6.0 software (https://github.com/tseemann/snippy, accessed on 5 September 2022) [35] was used to obtain variant calling into assembled chromosomes and plasmids with default parameters. Easyfig was used as a genome comparison visualizer. The prophage regions in the chromosome were identified by the online resource PHASTER (University of Alberta, Edmonton, AB, Canada) [36]. For comparative genomic analysis following reference plasmids were used: plasmid unnamed1 of K. pneumoniae strain CriePir200 (CP062994), plasmid pNDM-1_Dok01 of E. coli strain NDM-1 Dok01 (NC_018994), plasmid pKpQIL of K. pneumoniae strain 02288527-42B (MT809701), plasmid pCR14_2 of K. pneumoniae strain CR14 (CP015394), plasmid pECO-dc1b of E. coli strain ECONIH5 (CP026207), plasmid pE16KP0311-4 of K. pneumoniae strain E16KP0311 (CP052623), plasmid p53015-9.3 of K. pneumoniae strain 53015_G7 (CP098341).  15 ) belonging to the sequence type ST39 and capsular type K-23, were isolated from the rectal (n = 3) and tracheal (n = 1) swabs of four patients of the neuro-ICU. The patients were admitted to the ICU after neurosurgical operations for various diagnoses. Two patients had additional clinical manifestations of gastrointestinal dysfunction: one patient had both gastrointestinal dysfunction and a respiratory tract infection, and one patient had no such infections but was estimated to be a carrier of the antibiotic resistance genes (Table 1).

Characteristics of K. pneumoniae Strains
According to Magiorakos et al.'s [9] criteria, four strains were attributed to the XDR category; they are resistant to 6-7 functional groups of antimicrobials. All strains were resistant to beta-lactams, nitrofurans, fluoroquinolones, sulfonamides, aminoglycosides, and tetracyclines; one strain was additionally resistant to chloramphenicol; and one strain was resistant to colistin ( Table 2).

Characteristics of the Chromosoms
The complete genome assemblies of four K. pneumoniae strains contained each one chromosome and the sets of the plasmids. The chromosome sizes in the strains were comparable, and the pairwise distance between them showed a high degree of homology (99.99958-99.99943%). The total number of genes was the same in all the strains ( Table 3).
The chromosomes of all four strains carried the bla SHV-11 non-extended spectrum beta-lactamase (non-ESBL) gene and the fosA gene conferring the resistance to fosfomycin, as well as the oqxA and oqxB efflux pump genes, which are responsible for the resistance to chloramphenicol, nalidixic acid, ciprofloxacin, trimethoprim, benzylkonium chloride, and cetylpyridinium chloride. The point mutations associated with high-level fluoroquinolone resistance were found in the gyrA gene (T247A and C248T determining the amino acid substitution Ser83Ile, and G259A-the amino acid substitution Asp87Asn), and in the parC gene (G239T defining an amino acid substitution Ser80Ile). Moreover, genes of 5 efflux systems, acrABR, marAR, soxSR, ramA, and oqxABR, and genes of 5 regulators, rob, sdiA, fis, envR, and rarA, were detected in the chromosomes of all strains. Table 3. Major genome characteristics of clinical K. pneumoniae strains.

Features SCPM-O-B-8912 SCPM-O-B-8919 SCPM-O-B-8922 SCPM-O-B-8923
GenBank Analysis of virulence genetic determinants revealed the mrkABCDFIJ gene cluster coding type 3 fimbrial adhesine, the irp gene cluster of yersiniabactin biosynthesis, the ybtAEPQSTUX gene cluster of yersiniabactin biosynthesis, and the fyuA gene encoding the siderophore yersiniabactin receptor-in the chromosomes of all strains.
A comparative analysis of the chromosomes of four K. pneumoniae strains revealed ten main structural differences: eight insertions of IS-like elements, one repeat, and one inversion of three genes in the prophage region:

Characteristics of the Plasmids
Six incompatibility groups of plasmids were discovered in the genomes of four K. pneumoniae strains. Three strains contained six plasmids, while one strain contained five. Three strains, SCPM-O-B-8912, SCPM-O-B-8919, and SCPM-O-B-8923, contained three large plasmids of Inc-groups IncHI1B, IncC, and IncFIB carrying the carbapenemase genes of three types, bla OXA-48 , bla NDM-1 , and bla KPC-2 , respectively. One strain, SCPM-O-B-8922, contained two named plasmids: IncHI1B, carrying the bla OXA-48 carbapenemase gene, and IncFIB, carrying the bla KPC-2 carbapenemase gene; additionally, the IncC-group plasmid carried the bla CTX-M-55 cephalosporinase gene. Three K. pneumoniae strains, SCPM-O-B-8912, SCPM-O-B-8922, and SCPM-O-B-8923, contained the additional large plasmid of the IncFIB/IncFII group, carrying the genes conferring resistance to heavy metals as well as the genes of the toxin-antitoxin system and a conjugative transfer system. Moreover, all four strains carried two small, cryptic plasmids identified as ColRNAI and pUN1 ( Figure 1). spectively); -the PhoP/PhoQ regulator membrane protein MgrB gene was disrupted by the insertion of an IS5-like element ISKpn26 family transposase (KIF64_09410) in the strain SCPM-O-B-8922; -the inversion of three genes, KF986_013800, KF986_013805, and KF986_013810, encoding hypothetical proteins, has been detected in the strain SCPM-O-B-8919.

Characteristics of the Plasmids
Six incompatibility groups of plasmids were discovered in the genomes of four K. pneumoniae strains. Three strains contained six plasmids, while one strain contained five. Three strains, SCPM-O-B-8912, SCPM-O-B-8919, and SCPM-O-B-8923, contained three large plasmids of Inc-groups IncHI1B, IncC, and IncFIB carrying the carbapenemase genes of three types, blaOXA-48, blaNDM-1, and blaKPC-2, respectively. One strain, SCPM-O-B-8922, contained two named plasmids: IncHI1B, carrying the blaOXA-48 carbapenemase gene, and IncFIB, carrying the blaKPC-2 carbapenemase gene; additionally, the IncC-group plasmid carried the blaCTX-M-55 cephalosporinase gene. Three K. pneumoniae strains, SCPM-O-B-8912, SCPM-O-B-8922, and SCPM-O-B-8923, contained the additional large plasmid of the IncFIB/IncFII group, carrying the genes conferring resistance to heavy metals as well as the genes of the toxin-antitoxin system and a conjugative transfer system. Moreover, all four strains carried two small, cryptic plasmids identified as ColRNAI and pUN1 ( Figure  1). All four K. pneumoniae strains carried the bla OXA-48 carbapenemase gene on the large hybrid IncHI1B plasmids (323,074-327,685 bp), which are identical by 99.9% among them. A deletion of nucleotide sequence carrying 10 genes, including the EAC protein gene, the class I ribonucleotide reductase gene, the type II toxin-antitoxin system RelE/ParE genes, and hypothetical protein genes, was detected in the plasmids pB-8919_OXA-48, pB-8922_OXA-48, and pB-8923_OXA-48, compared to the plasmid pB-8912_OXA-48. Moreover, the inversion of the~112 Kb region was identified in the plasmid pB-8923_OXA-48 compared to the plasmids in the other three strains (Figure 2A).
A deletion of nucleotide sequence carrying 10 genes, including the EAC protein gene, the class I ribonucleotide reductase gene, the type II toxin-antitoxin system RelE/ParE genes, and hypothetical protein genes, was detected in the plasmids pB-8919_OXA-48, pB-8922_OXA-48, and pB-8923_OXA-48, compared to the plasmid pB-8912_OXA-48. Moreover, the inversion of the ~112 Kb region was identified in the plasmid pB-8923_OXA-48 compared to the plasmids in the other three strains (Figure 2A). The hybrid nature of the IncHI1B plasmids was postulated because they were derived from regions that carried both antimicrobial resistance (AMR) genes and virulence genes. Epidemically significant antibiotic resistance genes identified in these plasmids conferring resistance to aminoglycosides, ant(2")-Ia, aac(6 )-Ib-cr, and aadA1; to quinolones, qnrS1; to beta-lactams, bla OXA-1 and bla TEM-1 ; to cephalosporins, bla CTX-M-15 ; to carbapenems, bla OXA-48 ; to sulfamethoxazole, sul1; and to chloramphenicol, catA1 and catB3. Moreover, K. pneumoniae hypervirulence genetic determinants, the iucABCD (aerobactin-producing operon), the iutA (iron trapping receptor gene), the terC (tellurium ion resistance protein gene), and the rmpA2 gene disrupted by insertion of an IS110 family transposase were identified in these plasmids (Figure 2).
A BLAST search revealed one homologous hybrid plasmid in the GenBank database, recently submitted from the genome of K. pneumoniae strain CriePir200, isolated from the sputum of a patient at the N.I. Pirogov National Medical and Surgical Center in Moscow in 2018 and described by Shelenkov et al., 2020 [37]. The genetic environments of the bla OXA-48 carbapenemase gene were identical in four plasmids in our study, and in the plasmid pCriePir200 (Figure 2A,B). The genetic environments of the bla CTX-M-15 and bla OXA-1 betalactamase genes were identical in four plasmids in our study, including the presence of additional resistance genes (bla TEM-1 , catB3, aac(6 )-Ib-cr, and qnrS1), but such loci were missed in the plasmid CriePir200 ( Figure 2C). As for the virulence gene region, the rmpA2 gene has not been disrupted by the IS-element in the plasmid pCriePir200, in contrast to four other plasmids in our study ( Figure 2B).

Characteristics of IncC plasmids Carrying the bla NDM-1 Carbapenemase Gene
Three K. pneumoniae strains, SCPM-O-B-8912, SCPM-O-B-8919, and SCPM-O-B-8923, contained the IncC plasmid, which carried the bla NDM-1 carbapenemase gene. Additionaly, the bla OXA-10 and bla OXA-1 beta-lactamase genes, as well as genes conferring the resistance to macrolides (msr(E) and mph(E)), aminoglycosides (aac(3)-IIa, aac(6')-Ib-cr, aadA1, aadA2, and armA), sulfamethoxazole (sul1), trimethoprim (dfrA12), chloramphenicol (cmlA1 and catB3), and rifampicin (ARR-3) were located on the IncC plasmids. The disinfectant resistance gene (qacE) conferring resistance to quaternary ammonium compounds was determined in these plasmids also. The plasmid sequences were almost identical except for a few single nucleotide polymorphisms (SNPs). The bla NDM-1 gene is associated with the IS91 insertion sequences. The oxacillinase gene bla OXA-10 is located downstream of the IS110 insertion sequence as a part of the cassette array of the class 1 intergron; it is, however, associated with the Tn3 transposon. The class 1 integron is flanked by the IS91 insertion sequences and carries the set of gene cassettes conferring resistance to antibacterials. A comparison of the IncC plasmids of our study with the closest homologous plasmid, pNDM-1_Dok01of (query cover 90%, identity 99.9%), identified in E. coli ST38 collected in Japan, shows multiple rearrangements in the genetic environment of the bla NDM , bla OXA-10 , and bla OXA-1 beta-lactamase genes (Figure 3).

Characteristics of IncFIB(pQil) Plasmids Carrying the bla KPC-2 Carbapenemase Gene
All K. pneumoniae strains in our study contained the IncFIB(pQil) plasmids carrying the bla KPC-2 carbapenemase gene. The comparison of the plasmids revealed identical structures and the same sequences with a few SNPs. Additional genes for resistance determinants were not found. A comparison of the plasmid pB-8919_KPC-2 with the homologous plasmid pKpQIL of K. pneumoniae strain 02288527-42B (query cover 98%, identity 99.6%) revealed the detailed structure of transposon Tn4401 carrying the bla KPC-2 carbapenemase gene. Unlike the reference plasmid pKpQIL, the mercuric resistance gene cluster was missed in the plasmid pB-8919_KPC-2 ( Figure 4).

Characteristics of IncFIB(pQil) Plasmids Carrying the blaKPC-2 Carbapenemase Gene
All K. pneumoniae strains in our study contained the IncFIB(pQil) plasmids carrying the blaKPC-2 carbapenemase gene. The comparison of the plasmids revealed identical structures and the same sequences with a few SNPs. Additional genes for resistance determinants were not found. A comparison of the plasmid pB-8919_KPC-2 with the homologous plasmid pKpQIL of K. pneumoniae strain 02288527-42B (query cover 98%, identity 99.6%) revealed the detailed structure of transposon Tn4401 carrying the blaKPC-2 carbapenemase gene. Unlike the reference plasmid pKpQIL, the mercuric resistance gene cluster was missed in the plasmid pB-8919_KPC-2 ( Figure 4).

Characteristics of IncFIB(pQil) Plasmids Carrying the blaKPC-2 Carbapenemase Gene
All K. pneumoniae strains in our study contained the IncFIB(pQil) plasmids carrying the blaKPC-2 carbapenemase gene. The comparison of the plasmids revealed identical structures and the same sequences with a few SNPs. Additional genes for resistance determinants were not found. A comparison of the plasmid pB-8919_KPC-2 with the homologous plasmid pKpQIL of K. pneumoniae strain 02288527-42B (query cover 98%, identity 99.6%) revealed the detailed structure of transposon Tn4401 carrying the blaKPC-2 carbapenemase gene. Unlike the reference plasmid pKpQIL, the mercuric resistance gene cluster was missed in the plasmid pB-8919_KPC-2 ( Figure 4).

Characteristics of IncC Plasmids Carrying the bla CTX-M-55 Cephalosporinase Gene
One K. pneumoniae strain in our study, SCPM-O-B-8922, contained the IncC plasmid (169,151 bp) and carried the bla CTX-M-55 cephalosporinase gene. The closest homologous plasmid in the GenBank database was identified, pECO-dc1b (82% overlap, 99% identity), in the genome of an E. coli strain isolated from a hospital water supply in the United States in 2015, but this plasmid doesn't carry any bla CTX-M genes. So, we compared the plasmid pB-8922_CTX-M-55 with the plasmid pCR14_2 (79% overlap, 99% identity) of K. pneumoniae strain CR14 carrying the bla CTX-M-2 gene. The plasmids have a similar structure except for the region of gene resistance cassettes. The plasmid pB-8922_CTX-M-55 contained the genetic determinants of resistance to aminoglycosides (aac(6')-Ib-cr, aadA1, and armA), sulfamethoxazole (sul1), trimethoprim (dfrA1), and quaternary ammonium compounds (qacE) ( Figure 5).
K. pneumoniae strain CR14 carrying the blaCTX-M-2 gene. The plasmids have a similar structure except for the region of gene resistance cassettes. The plasmid pB-8922_CTX-M-55 contained the genetic determinants of resistance to aminoglycosides (aac(6')-Ib-cr, aadA1, and armA), sulfamethoxazole (sul1), trimethoprim (dfrA1), and quaternary ammonium compounds (qacE) ( Figure 5).  (171,935-173,263 bp). These plasmids carried no genes of antibiotic resistance but genes of copper and arsenical resistance, the sil cation-efflux system that confers resistance to silver, the type II toxin-antitoxin system for the RelE/ParE family toxin, Fe(3+) dicitrate transport, and the type-F conjugative transfer system. The sequences of the three plasmids were 90% identical; the difference in size was associated with the IS110 and IS5 copies. A BLAST search for homologous plasmids showed a lot of very similar plasmids in the GenBank database.

Characteristics of Low-Molecular-Weight Plasmid pColRNAI
All K. pneumoniae strains carry the identical pColRNAI plasmids (9294 bp). These plasmids carried the genes encoding colicin, cloacin, and the toxin-antitoxin system RelE/ParE. BLAST search for homologous plasmids to pColRNAI revealed a few similar plasmids of the same sizes, such as the plasmid pE16KP0311-4 (CP052623) of K. pneumoniae strain E16KP0311, isolated from the patient's blood in South Korea, and the plasmid p53015-9.3 of K. pneumoniae strain 53015_G7 (CP098341) obtained from Homo sapiens in USA.  (171,935-173,263 bp). These plasmids carried no genes of antibiotic resistance but genes of copper and arsenical resistance, the sil cation-efflux system that confers resistance to silver, the type II toxin-antitoxin system for the RelE/ParE family toxin, Fe(3+) dicitrate transport, and the type-F conjugative transfer system. The sequences of the three plasmids were 90% identical; the difference in size was associated with the IS110 and IS5 copies. A BLAST search for homologous plasmids showed a lot of very similar plasmids in the GenBank database.
3.3.6. Characteristics of Low-Molecular-Weight Plasmid pColRNAI All K. pneumoniae strains carry the identical pColRNAI plasmids (9294 bp). These plasmids carried the genes encoding colicin, cloacin, and the toxin-antitoxin system RelE/ParE. BLAST search for homologous plasmids to pColRNAI revealed a few similar plasmids of the same sizes, such as the plasmid pE16KP0311-4 (CP052623) of K. pneumoniae strain E16KP0311, isolated from the patient's blood in South Korea, and the plasmid p53015-9.3 of K. pneumoniae strain 53015_G7 (CP098341) obtained from Homo sapiens in USA.

Characteristics of Low-Molecular-Weight Plasmid pUN1
The small, identical pUN1 plasmids (4352 bp) were identified in the genomes of all K. pneumoniae strains in our study. The closest match (95% overlap, 89.94% identity) in the GenBank database was a cryptic plasmid, pSTW0522-30-5 (AP022404), of Citrobacter portucalensis isolated from hospital sewage in Japan.

Discussion
The number of infections caused by antibiotic-resistant bacteria is constantly increasing, and antibiotic resistance, especially to carbapenems, has already become a dangerous problem for health care systems around the world. The carbapenems were the last-line class of antibiotics used for the treatment of infections caused by multi-resistant bacteria, but they lost this position after the emergence and rapid dissemination of carbapenemase-producing agents of HAIs. The spread of the carbapenemase genes was associated with intra-and interspecies plasmid-mediated transfer. The plasmids of several incompatibility groups and different backbones were involved in the rapid dissemination of carbapenem-resistant (CR) Enterobacteriaceae [38,39]. The spread of carbapenemases via plasmids has been documented in Russia [24,40].
In this study, we described in detail the plasmids identified in XDR K. pneumoniae clinical strains belonging to sequence type ST39 and capsular type K-23, which were isolated at a point-prevalence study from four patients in a neuro-ICU in Moscow; two of them died [26]. Previously, strains of the same genetic line were discovered in Moscow ICUs [16,41]. The novelty of this study is the first report of three plasmids carrying carbapenemase genes of three types, bla OXA-48 , bla NDM-1 , and bla KPC-2 , being expressed simultaneously in bacterial cells on the high-molecular-weight plasmids of the IncHI1B (~326 Kb), IncC (~175 Kb), and IncFIB(pQil) (~102 Kb) incompatibility groups, respectively Mataseje et al. recently described the first complete genome sequence of a K. pneumoniae isolate carrying the three carbapenemases listed above in a strain isolated from a Canadian patient hospitalized in Romania [20]. In contrast to the plasmid's genetic background presented in our study, the bla OXA-48 , bla NDM-1 , and bla KPC-2 carbapenemase genes were located on the plasmids of IncL (64 Kb), IncFIB(pQil)/IncFII(K) (104 Kb), and IncFIB(K)/IncFII(K) (214 Kb) incompatibility groups, respectively; the strain was attributed to the ST147 cluster that is endemic in Romania. Therefore, we can conclude that there are parallel processes of the formation of multi-resistant, multi-plasmid strains in different regions of the world based on different genetic lines of K. pneumoniae.
Importantly, one of the studied plasmids, carrying the bla OXA-48 carbapenemase gene, contains not only a large set of AMR genes conferring resistance to aminoglycosides, quinolones, beta-lactams, cephalosporins, carbapenems, sulfamethoxazole, and chloramphenicol, but, additionally, the region of virulence genes inherent to hypervirulent K. pneumoniae: iucABCD, iutA, terC, and rmpA2::IS110. This hybrid plasmid has a large size (~326 Kb) and was formed as a result of an unknown event of recombination. In recent years, many studies have reported carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) [23,24].
Another important aspect of this study is the carriage of multidrug-resistant strains without clinical manifestations of infection. One of the strains described in the study, which carried three carbapenemase genes, was isolated from a patient without HAI. Aside from human-to-human transmission, the hospital environment is a major source of antibioticresistant pathogen transmission and a breeding ground for novel resistance combinations. Evolutionary changes and swapping of genetic information can occur both within populations of nosocomial infections on different surfaces and within the organisms of patients. This statement could be confirmed by detection of plasmid-mediated transfer of resistance genes on an IncFIB plasmid between K. pneumoniae isolates [42]. In comparison to outbreak investigations, the carrying of CR isolates is studied infrequently. The strategy of infection control required to reduce nosocomial infections, except for careful cleaning measures and monitoring of the microbiomes of hospital environments, should be the identification of ICU patients colonized by CR bacteria [43].
It was shown in this research that the four studied K. pneumoniae strains were undoubtedly closely related; their nucleotide sequences of chromosomes were almost identical; only insertions of IS-elements caused the differences. We defined that the strains carried various sets of plasmids, but the nucleotide sequences of the homologous plasmids were very similar. This could point to the spread of clonally related strains, particularly in the ICU, and the acquisition of differences through continuous evolution. The molecular and genomic study of bacterial pathogens is currently focused on tracking clonally evolving lineages. While surveillance of epidemic clones is important, plasmids can spread horizontally between strains and even species. The plasmids are the primary carriers of antibiotic resistance genes across many pathogens, and they should be analyzed more accurately with high-resolution techniques [44].
In research studies, it has been shown that the spread of the carbapenemase gene occurs across a small number of high-risk clones [45]. In our study, K. pneumoniae strains belonging to ST39 and K-23, which were rarely isolated from patients and poorly presented in the GenBank database, were able to accumulate plasmids carrying three carbapenemase genes, bla NDM-1 , bla KPC-2 , and bla OXA-48 , simultaneously. A detailed analysis of the genetic environments of these genes revealed the common structure of these regions. Certainly, the progenitor of these strains had the ability to accept the plasmids. Based on the fact that exact copies of the plasmids carrying bla OXA-48 , bla NDM-1 , and bla KPC-2 genes described in our study were not found in the GenBank database, it can be assumed that the plasmids have high rates of recombination and rearrangements; the structural variability is changing over time. Because plasmids can undergo rapid evolutionary change, plasmid transmission events may go undetected if only general comparisons of the strain's genomes are performed. We anticipate the need for additional comparisons of the nucleotide diversity of the plasmids and comparative analysis. A detailed study of hospital strain genomes could identify the source of HAI outbreaks. The large-scale surveillance could reveal clinical and biological insights pertaining to the hospital microbiome as a reservoir of pathogens.

Conclusions
In conclusion, the spread of K. pneumoniae strains carrying multiple plasmids conferring resistance even to last-resort antibiotics is of great clinical concern. Here we described three high-molecular-weight plasmids carrying the bla OXA-48 , bla NDM-1 , and bla KPC-2 carbapenemase genes in the strains of ST39, capsular type K-23, that were connected with high mortality. One of the plasmids, p_OXA-48, was a hybrid plasmid, carrying the genetic determinants of hyper-virulence, in addition to the large set of AMR genes. At the same time, one of the strains was associated with intestinal carriage in a patient without clinical manifestations of infection. The above indicates that continuous monitoring of high-risk clones and the detection of resistance mechanisms are necessary strategies to combat such threats. Further investigations are needed to fully understand the epidemiology of K. pneumoniae strains co-producing multiple molecular mechanisms of resistance, especially based on combining the results of whole genome sequencing, transcriptome analysis, and clinical characteristics.   (CP086665, CP086666, CP086667, CP086668, CP086669, CP086670,  CP094992, CP094993, CP094994, CP094995, CP094996, CP094364-CP094368, CP094370, CP094371,  CP094372, CP094373, CP086672, CP086673, CP086674, CP086675, CP086676, and CP086677), the chromosomes sequences have been deposited in GenBank under accessions no. (CP086664, CP094991, CP094363, and CP086671). The raw sequence MinION reads have been deposited in the SRA under accession numbers (SRR14338904, SRR14338897, SRR14493697, and SRR14493696).