High-Molecular-Weight Plasmids Carrying Carbapenemase Genes bla_NDM-1, bla_KPC-2, and bla_OXA-48 Coexisting in Clinical Klebsiella pneumoniae Strains of ST39

Abstract

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, bla_OXA-48, bla_NDM-1, and bla_KPC-2, respectively. The first of them, highly identical in all strains, was a hybrid plasmid that combined two regions of the resistance genes (bla_OXA-48 and bla_TEM-1 + bla_CTX-M-15 + bla_OXA-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.

1. 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 that are the last line of defense, including carbapenems; especially, it concerns K. pneumoniae, the causative agent of invasive infections of different localizations [7]. Multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) K. pneumoniae are particularly a problem among neonates, the elderly, and immunocompromised individuals, as well as for the patients of intensive care units (ICUs) [8,9].

Until the last two decades, carbapenems were the drug of choice for the treatment of severe infections caused by MDR-K. pneumoniae. But then, the resistance to carbapenems alarmingly increased due to the acquisition of carbapenemase genes located on specific plasmids and mobile genetic elements. K. pneumoniae is the most frequently isolated pathogen from patients in ICUs, which became unprotected against infections caused by carbapenem-resistant bacteria [10].

Carbapenemases, enzymes that are able to hydrolyze carbapenems and many other beta-lactams, were divided into two structural groups: metallo-carbapenemases (class B beta-lactamases, e.g., NDM-, VIM-, and IMP-types) and “serine”-carbapenemases (classes A, C, and D beta-lactamases, e.g., KPC-, DHA-, and OXA-48-types) [11]. Carbapenemase-producing K. pneumoniae have been widely identified, and bacteria with the co-existence of two or three carbapenemases have been reported during the last decade. For example, KPC-2 and NDM-1 co-producing K. pneumoniae isolates have been identified in India, Pakistan, and China [12]. Recently, even hypervirulent K. pneumoniae isolates have been described in China [13]. Emergence of K. pneumoniae co-producing NDM-1 and OXA-48 carbapenemases have been reported from Turkey, Greece, Italy, Czech, Russia and other countries [14,15,16]. One strain was reported as a carrier of both bla_KPC-2 and bla_NDM-1 genes [17], while the other strain was reported as a carrier of both bla_KPC-2 and bla_OXA-48 genes [18]. K. pneumoniae with triple carbapenemase (KPC/NDM-1/OXA-48) genes resistance have been detected in Saudi Arabia [19]. Most recently, a K. pneumoniae isolate co-producing three carbapenemases has been collected from a Canadian patient hospitalized in Romania. Whole genome sequencing showed that the bla_KPC-2 gene was located on a 214 Kb IncFIB(K)/IncFII(K) plasmid, the bla_NDM-1 gene-on a 104 Kb IncFIB(pQil)/IncFII(K) plasmid, and the bla_OXA-48 gene-on a 64 Kb IncL plasmid. The uncommon co-carriage of genes encoding different classes of carbapenemases endows them with high carbapenem resistance [20].

Most carbapenemase genes are carried by plasmids, which are extrachromosomal DNA elements that may self-replicate and move horizontally, substantially facilitating their transmission in bacteria [21]. The transmissibility of genes located on mobile elements led to the formation of hybrid plasmids that carry both antimicrobial resistance and virulence (Vir) genes. The plasmid-mediated genetic factors conferring the hypervirulent phenotype include the rmpA and rmpA2 genes, regulators that increase capsule production, and several siderophore gene clusters [22]. The hybrid plasmids have a large size (300–400 Kb) and were formed as a result of recombination. Despite the fact that the virulence potential of isolates harboring hybrid plasmids is not well studied, it could be a new challenge for health care. The recombination and rearrangement of MDR plasmids and virulence plasmids have occurred during evolution. In recent years, many studies have reported the evolution of K. pneumoniae into carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) via the acquisition of hybrid plasmids. Many variants of Vir-KPC plasmids have been described to date in different regions [23]. Emergence of hybrid resistance and virulence plasmids harboring ND metallo-beta-lactamase in K. pneumoniae of different STs (ST15, ST147, ST395, and ST874) has been reported in Russia [24]. The bla_OXA-48 carbapenemase gene was identified in a hybrid plasmid harboring an exogenetic chromosome-located fragment and acquired the additional resistance gene bla_SHV-12 due to the recombination of the IS26-like elements in Egypt [25].

In a previous report, we described the emergence of K. pneumoniae isolates carrying three carbapenemase genes (bla_NDM-1, bla_KPC-2, and bla_OXA-48), the cephalosporinase gene (bla_CTX-M-15), and two class 1 integron genes simultaneously in the Moscow neuro-ICU at seven-point prevalence surveys, which included 60 patients in the neuro-ICU. A total of 293 clinical samples were analyzed, including 146 rectal and 147 tracheal swabs, and 344 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.

2. Materials and Methods

2.1. 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.

2.2. 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.

2.3. Susceptibility to Antimicrobials

Antimicrobial susceptibility testing was performed on the Vitek2 Compact system (BioMérieux, Marcy-l’Étoile, Auvergne-Rhône-Alpes, France) using the AST-N101 card (BioMérieux, Marcyl’Étoile, Auvergne-Rhône-Alpes, France). The interpretation was made using the requirements of the European Committee on Antimicrobial Susceptibility Testing (EUCAST), version 12.0, 2022 (http://www.eucast.org) (accessed on 5 September 2022). Escherichia coli ATCC 25922 strain was used as quality control. The category of multidrug resistance was determined according to the criteria of Magiorakos et al. [9].

2.4. 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].

2.5. 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-bin/bigsdb/bigsdb.pl?db=pubmlst_klebsiella_seqdef&page=sequenceQuery, accessed on 5 September 2022). In silico multi-locus sequence typing (MLST) was performed with the online resource MLST 2.0 of the Center for Genomic Epidemiology (Technical University of Denmark, Kgs. Lyngby, Denmark) [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].

2.6. Nucleotide Sequences Submitted into GenBank Database and Reference Plasmids

Following WGS, four K. pneumoniae clinical strains were submitted into the GenBank database:

K. pneumoniae strain SCPM-O-B-8912 chromosome (CP086664), plasmid pOXA-48 (CP086665), plasmid pNDM-1 (CP086666), plasmid pIncFIB (CP086667), plasmid pKPC-2 (CP086668), plasmid pColRNAI (CP086669), plasmid p6 (CP086670);

K. pneumoniae strain SCPM-O-B-8919 chromosome (CP094991), plasmid pB-8919_OXA-48 (CP094992), plasmid pB-8919_NDM-1 (CP094993), plasmid pB-8919_KPC-2 (CP094994), plasmid pB-8919_ColRNAI (CP094995), plasmid pB-8919_5 (CP094996);

K. pneumoniae strain SCPM-O-B-8922 chromosome (CP094363), plasmid pB-8922_OXA-48 (CP094368), plasmid pB-8922_CTX-M-55 (CP094370), plasmid pB-8922_IncFIB (CP094369), plasmid pB-8922_KPC-2 (CP094371), plasmid pB-8922_ColRNAI (CP094372), plasmid pB-8922_6 (CP094373);

K. pneumoniae strain SCPM-O-B-8923 chromosome (CP086671), plasmid pB-8923_OXA-48 (CP086672), plasmid pB-8923_NDM-1 (CP086673), plasmid pB-8923_IncFIB (CP086674), plasmid pB-8923_KPC-2 (CP086675), plasmid pB-8923_ColRNAI (CP086676), plasmid pB-8923_6 (CP086677).

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).

3. Results

3.1. Characteristics of K. pneumoniae Strains

Four carbapenem-resistant clinical strains of K. pneumoniae SCPM-O-B-8912 (bla_OXA-48, bla_NDM-1, bla_KPC-2, and bla_CTX-M-15), SCPM-O-B-8919 (bla_OXA-48, bla_NDM-1, bla_KPC-2, and bla_CTX-M-15), SCPM-O-B-8922 (bla_OXA-48, bla_KPC-2, and bla_CTX-M-55), and SCPM-O-B-8923 (bla_OXA-48, bla_NDM-1, bla_KPC-2, and bla_CTX-M-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. Patient’s information.

Information	SCPM-O-B-8912	SCPM-O-B-8919	SCPM-O-B-8922	SCPM-O-B-8923
Patient (gender, age)	Male, 36	Male, 67	Male, 59	Female, 64
ICU state, days	42	23	95	96
Outcome	Discharged	Discharged	Fatal	Fatal
Isolation source	rectal swab	rectal swab	rectal swab	tracheal swab
Collection date	18 October 2019	21 October 2019	21 October 2019	21 November 2019
Patient’s diagnosis	Diffuse cerebral and cerebellar injury, unspecified	Secondary malignant neoplasm of the brain and cerebral meninges	Nontraumatic intracerebral hemorrhage in the hemisphere, unspecified	Benign neoplasm of cranial nerves
Infectious manifestation	GD	No	GD	GD, RTI

Note: GD, gastrointestinal dysfunction; RTI, respiratory tract infection; No, there will be no GD or RTI.

).

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. Antimicrobial susceptibility of K. pneumoniae strains (MIC, mg/L).

Antimicrobials	SCPM-O-B-8912	SCPM-O-B-8919	SCPM-O-B-8922	SCPM-O-B-8923
Ampicillin	>16	>16	>16	>16
Amoxicillin-clavulanic acid	>128	>128	>128	>128
Cefoperazone	64	64	32	64
Cefotaxime	>32	>32	>32	>32
Ceftazidime	>256	>256	>256	>256
Cefepime	>16	>16	>16	>16
Aztreonam	32	64	64	32
Imipenem	>64	>64	>64	>64
Meropenem	128	>256	>256	256
Ertapenem	>4	>4	>4	>4
Tigecycline	>4	>4	4	>4
Ciprofloxacin	256	256	256	>256
Chloramphenicol	8	8	4	16
Gentamicin	>256	>256	>256	>256
Netilmicin	>16	>16	>16	>16
Amikacin	>32	>32	>32	>32
Trimethoprim-sulfamethoxazole	>160	>160	>160	>160
Fosfomycin	128	128	>128	128
Colistin	≤0.5	≤0.5	>8	≤0.5

).

3.2. 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. 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 chromosome	CP086664	CP094991	CP094363	CP086671
Chromosome size, bp	5,351,360	5,350,432	5,348,898	5,351,820
Genes (total)	6079	5902	6079	6091
Genes (coding)	5788	5626	5790	5801
Genes (RNA)	126	126	126	126
rRNA genes (5S, 16S, 23S)	9, 8, 8	9, 8, 8	9, 8, 8	9, 8, 8
tRNA genes	88	88	88	88
Pseudo Genes (total)	165	150	163	164
- frameshifted	67	61	65	66
- incomplete	102	88	101	102
- internal stop	25	22	25	26
- multiple problems	24	18	23	25
Plasmids	6	5	6	6

).

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.

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:

-

the lysophospholipid acyltransferase family protein gene was functional in the strain SCPM-O-B-8922 (KIF64_00650), but disrupted by insertion of an IS5-like element ISKpn26 family transposase in the strains SCPM-O-B-8912, SCPM-O-B-8919, and SCPM-O-B-8923 (KFX87_000655, KF986_000655, and KIF80_000655, respectively);

-

the O-antigen ligase family protein gene in the strain SCPM-O-B-8922 (KIF64_00770) was disrupted by insertion of an IS5-like element ISKpn26 family transposase (KIF64_00765), unlike this gene in the other three strains;

-

the gene glpT encoding glycerol-3-phosphate transporter in the strain SCPM-O-B-8923 (KIF80_007475) was disrupted by the insertion of an IS4-like element by an ISVsa5 family transposase;

-

the insertion of an IS5-like element ISKpn26 family transposase (KIF64_08135) was detected in the intergenic space between the phosphatase PAP2 family protein gene (KIF64_08130) and hypothetical protein gene (KIF64_08140) in the strain SCPM-O-B-8922, in contrast to other three strains;

-

four genes, homologues to gmlABC (providing the structural modification of D-galactan I) and kfoC (unknown function), in the rfb cluster were deleted and replaced by the insertion of an IS5-like element ISKpn26 family transposase in the strain SCPM-O-B-8922; the rfb cluster in the chromosomes of the other three strains was complete and homologous to the K. pneumoniae type O1/O2-antigen gene cluster;

-

the IV secretion system protein gene was disrupted by the insertion of an IS5-like element, ISKpn26 family transposase (KFX87_008725), in the strain SCPM-O-B-8912;

-

the aspartate/glutamate racemase family protein gene was functional in the strain SCPM-O-B-8922 (KIF64_08860), while it was disrupted by the insertion of an IS1-like element by the IS1B family transposase in the strains SCPM-O-B-8912, SCPM-O-B-8919, and SCPM-O-B-8923 (KFX87_008890, KF986_008880, and KIF80_008890, respectively);

-

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.

3.3. 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).

3.3.1. Characteristics of IncHI1B Plasmids Carrying the bla_OXA-48 Carbapenemase Gene

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).

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 beta-lactamase 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).

3.3.2. 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).

3.3.3. 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).

3.3.4. 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).

3.3.5. Characteristics of IncFIB(K)/IncFII(K) Plasmids

Three K. pneumoniae strains, SCPM-O-B-8912, SCPM-O-B-8922, and SCPM-O-B-8923, contained the plasmid of the IncFIB(K)/IncFII(K) group (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.

3.3.7. 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.

4. 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 antibiotic-resistant 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.

5. 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.