Skip to main content

Carbapenem-resistant hypervirulent ST23 Klebsiella pneumoniae with a highly transmissible dual-carbapenemase plasmid in Chile



The convergence of hypervirulence and carbapenem resistance in the bacterial pathogen Klebsiella pneumoniae represents a critical global health concern. Hypervirulent K. pneumoniae (hvKp) strains, frequently from sequence type 23 (ST23) and having a K1 capsule, have been associated with severe community-acquired invasive infections. Although hvKp were initially restricted to Southeast Asia and primarily antibiotic-sensitive, carbapenem-resistant hvKp infections are reported worldwide. Here, within the carbapenemase production Enterobacterales surveillance system headed by the Chilean Public Health Institute, we describe the isolation in Chile of a high-risk ST23 dual-carbapenemase-producing hvKp strain, which carbapenemase genes are encoded in a single conjugative plasmid.


Phenotypic and molecular tests of this strain revealed an extensive resistance to at least 15 antibiotic classes and the production of KPC-2 and VIM-1 carbapenemases. Unexpectedly, this isolate lacked hypermucoviscosity, challenging this commonly used hvKp identification criteria. Complete genome sequencing and analysis confirmed the K1 capsular type, the KpVP-1 virulence plasmid, and the GIE492 and ICEKp10 genomic islands carrying virulence factors strongly associated with hvKp. Although this isolate belonged to the globally disseminated hvKp clonal group CG23-I, it is unique, as it formed a clade apart from a previously reported Chilean ST23 hvKp isolate and acquired an IncN KPC-2 plasmid highly disseminated in South America (absent in other hvKp genomes), but now including a class-I integron carrying blaVIM−1 and other resistance genes. Notably, this isolate was able to conjugate the double carbapenemase plasmid to an E. coli recipient, conferring resistance to 1st -5th generation cephalosporins (including combinations with beta-lactamase inhibitors), penicillins, monobactams, and carbapenems.


We reported the isolation in Chile of high-risk carbapenem-resistant hvKp carrying a highly transmissible conjugative plasmid encoding KPC-2 and VIM-1 carbapenemases, conferring resistance to most beta-lactams. Furthermore, the lack of hypermucoviscosity argues against this trait as a reliable hvKp marker. These findings highlight the rapid evolution towards multi-drug resistance of hvKp in Chile and globally, as well as the importance of conjugative plasmids and other mobile genetic elements in this convergence. In this regard, genomic approaches provide valuable support to monitor and obtain essential information on these priority pathogens and mobile elements.


The rise of bacterial pathogens displaying multi-drug resistance and increased virulence is currently one of the most pressing threats to global health. Among them is the Gram-negative rod-shaped bacteria Klebsiella pneumoniae (Enterobacterales), one of the most prevalent agents causing multi-drug resistant infections worldwide and a major trafficker of antibiotic resistance genes [1, 2]. In particular, hypervirulent K. pneumoniae (hvKP) causing severe community-acquired metastatic infections in healthy individuals, and especially hvKp strains resistant to last-resort carbapenem antibiotics (CR-hvKp), are a critical concern [3, 4]. Although the number of cases is still relatively limited, these convergent CR-hvKp strains have been shown to arise through different evolutionary pathways, including hvKp acquiring carbapenemase plasmids, CR-Kp acquiring virulence determinants, or by the acquisition of hybrid plasmids encoding carbapenemases and hypervirulence determinants [5, 6].

Phylogenomic analyses showed that most hvKp strains are from sequence type (ST) and clonal group (CG) 23, mainly from the globally disseminated CG23-I subgroup. Also, they tend to have K1 or K2 capsular serotypes and an expanded array of virulence factors encoded in mobile genetic elements [7,8,9]. Among them, the large virulence plasmid (KpVP) carries genes for the synthesis, secretion, and uptake of aerobactin and salmochelin, two siderophores for iron scavenging, the rmpACD genes linked to the hypermucoviscous capsule, and genes involved in metal resistance [10]. Additionally, they harbor the integrative-conjugative element ICEKp10, including genes for producing yersiniabactin siderophores and the genotoxin colibactin. Also, most hvKp have the GIE492 genomic island carrying the genes for producing the antibacterial peptide microcin E492 and salmochelin [11, 12].

While ST23 CR-hvKP strains from several countries, including China [13], Singapore [14], Germany [15], and the USA [16], have been reported and characterized, there are limited reports from other regions, such as Latin America [17, 18], especially at a genomic level. Therefore, monitoring the emergence and spread of these strains is essential to develop public health strategies and guide interventions for their prevention and control. Towards this direction, we report the isolation of an ST23 dual-carbapenemase-producing hvKp strain from a respiratory tract infection in Chile. We combined genomics analyses and phenotypic tests to obtain relevant information on this high-risk isolate.

Results and discussion

Phenotypic antibiotic resistance profile of K. pneumoniae VA585-22

The VA585-22 strain was isolated on September 29, 2022, in a hospital in Santiago, Chile, from the tracheal aspirate of a 31-year-old gun-injured male after a prolonged hospitalization in the intensive care unit with pneumonia and mechanical ventilation. Antimicrobial susceptibility testing showed resistance to 3rd, 4th, and 5th -generation cephalosporins, aminopenicillins, carbapenems, and aminoglycosides (Table 1). Also, this isolate showed intermediate resistance to colistin and levofloxacin. The presence of blaKPC−2 and blaVIM−1 genes, encoding the KPC-2 serine carbapenemase and the VIM-1 metallo-carbapenemase, was detected by PCR. Furthermore, the expression of the carbapenemases was confirmed by immunochromatography (Table 1).

Table 1 Antimicrobial susceptibility profile and detection of carbapenemase genes and its expression in K. pneumoniae VA585-22 and E. coli K12 transconjugants that acquired pVA585-22_54

Genomic virulence, antibiotic resistance, and mobile genetic elements

The VA585-22 complete genome was sequenced and assembled (Table S1), which consisted of three circular replicons, a 5,334,759 bp chromosome, and two plasmids. One corresponded to a KpVP-1-like large virulence plasmid (~ 227 kbp, IncFIBK) encoding several iron acquisition systems, including the siderophore gene clusters iucABCDiutA (aerobactin) and iroBCDN (salmochelin), the fepBCD ABC-type iron transporter, and the Fur-dependent regulatory system for iron uptake fecIRA (Fig. 1, left side). Also, this plasmid included the sil, pco, and ter genes linked with resistance to copper, silver, lead, and tellurite [10, 19] and the rmpADC genes linked to hypermucoviscosity [20]. Unexpectedly, VA585-22 lacked hypermucoviscosity, as revealed by low-speed sedimentation assays and the string test (Fig. S1). We used Kp SGH10, a hypermucoviscous strain proposed as an hvKp representative [7], and the DwcaJ capsule-null mutant lacking hypermucoviscosity [21] as controls. A closer examination of the rmpADC locus indicated 100% amino acid identity of RmpA and RmpC, comparing VA585-22 with SGH10. Moreover, the gene organization of this operon and its immediate surroundings are conserved between both strains, including a 100% nucleotide identity in the promoter region (Fig. S2). Conversely, RmpD had a D3E substitution, which, along with the lack of rmpA2, a second copy of this gene found in the SGH10 virulence plasmid, could explain the lack of hypermucoviscosity in VA585-22. Previous reports indicated that not all the hypervirulent strains are hypermucoviscous, with deletions in rmpA or rmpA2 a common feature, and thus, this trait is not a good hvKp predictor [8, 22, 23].

Fig. 1
figure 1

Main features of the plasmids present in the hypervirulent ST23 K. pneumoniae strain VA585-22. The carbapenemase genes are shown in bold

The second plasmid, pVA585-22_54 (~ 54 kbp, IncN) (Fig. 1, right side), was highly similar to a potentially conjugative plasmid carrying blaKPC−2 reported in a multispecies outbreak in Chile [24], which was also found with a high prevalence in other K. pneumoniae clinical isolates from Chile and South America [25]. However, pVA585-22_54 differed as it included a class-I integron carrying the resistance genes blaVIM−1 (VIM-1 carbapenemase), aacA7 (aminoglycoside acetyltransferase), dfrA1 (dihydrofolate reductase), and aadA1 (aminoglycoside nucleotidyltransferase). This corresponds to the first report in Chile of hvKp producing two carbapenemases encoded in a single plasmid, along with other resistance determinants.

Phylogenomic relationships with other CG23 hvKp and carbapenemase plasmids mediating convergence

We investigated the phylogenetic relationships of VA585-22 with other 434 genomes of CG23 K. pneumoniae (most of the available from the NCBI database) isolated from more than 35 countries (Table S2), including the KPC-2-producing hvKp strain K-2157 isolated recently in Chile [26]. Although a few other reports described CR-hvKp from South America [17, 18, 27], no genome sequences of these isolates were published. Classical seven-gene and core-genome multilocus sequence analysis (using the 629-loci scgMLSTv2 scheme [28]) indicated that VA585-22 belongs to ST23, specifically to the globally disseminated CG23-I, clustering with isolates from diverse geographical origins, mainly from the USA, Australia, Japan, China, Taiwan, and Singapore (Fig. 2). The K-2157 strain clustered in a separate branch from VA585-22 (diverging by 17 allelic mismatches), suggesting it would correspond to a different clone. For clarity, distances in the tree shown in Fig. 2 are omitted. A tree including the distances and the accession numbers of the genomes is shown in Fig. S3.

Fig. 2
figure 2

Phylogenomic relationships, antimicrobial resistance, and carbapenemase-encoding plasmids among VA585-22 and other CG23 hvKp. Phylogenetic tree inferred from the core genome multiple sequence alignment (cg-MSA) using 629 loci from the scgMLSTv2 scheme. The distances were ignored for more clarity. The tracks show (inner to outer) the country of origin, the presence/class of carbapenemases (CP), the number of plasmids identified in each isolate, the number of plasmid-encoded antibiotic resistance genes (pARGs), the carbapenemases plasmid (pCP) cluster (as defined by the MOB_typer tool), and its predicted mobility. The two carbapenem-resistant ST23 K. pneumoniae isolated in Chile are indicated with a red triangle

Given the relevance of carbapenem resistance in hvKp, we compared the carbapenemase-coding genes and plasmids found in VA585-22 and our set of 435 CG23 isolates. Fifty-eight strains (13.3%) encoded one carbapenemase, while three strains (0.69%) encoded two (including VA585-22, the only producing KPC-2 and VIM-1) (Fig. 2). In 58 of these 61 isolates (95%) the carbapenemase was encoded in a plasmid, and isolates with more plasmids tended to have more acquired ARGs, highlighting the relevance of these mobile elements in hvKp multi-drug resistance.

Using the MOB-Suite tools, we typed and clusterized the plasmids encoding carbapenemases. The most frequent carbapenemase was KPC-2, carried by several plasmid clusters (11) and CG23-I subclades from different countries, leading the cluster AA448 (IncU), also carrying the mercury resistance genes mer, mainly from Chinese isolates (Fig. 2, Fig. S4). On the other hand, VA585-22 and a 2017 Chinese isolate (NZ_CP096241) were the only CG23 strains carrying an AA552 (IncN) plasmid encoding KPC-2. Conversely, the K-2157 KPC-2 plasmid (cluster AA018; ~114-kbp; IncFIB, IncFII), also including blaTEM−1, blaOXA−9, and the mer operon, was highly similar to pDHQP17016, previously found in an ST23 isolate from the USA [16, 18]. We found a similar plasmid in a 2018 CG23 isolate from Poland (GCF_022748855). Other relevant KPC-2 plasmids identified in CG23 hvKp included the cluster AC978, which is highly stable and transmissible and has become dominant among Enterobacterales in Singapore [29].

Other carbapenemases showed a narrower distribution among CG23 hvKp. VIM-1 was found only in three isolates, in VA585-22, in one from China, and one from Poland, the two latter bearing AA621 plasmids (IncA). OXA-48 was found only in AA002 plasmids (IncL/M), mainly from Russian isolates, OXA-232 was found exclusively in AC129 plasmids (rep_cluster_1195) from India, while NDM-1 was found in four plasmid clusters, whileIMP-4 was found in only one isolate bearing an AA552 plasmid. Most of the carbapenemase plasmids described above were predicted as conjugative.

Dissemination of the KPC-2 VIM-1 plasmid VA585_22–54 by conjugation

We tested the possible conjugative dissemination of pVA585-22_54, setting up a conjugation assay using an E. coli K12 GmR strain as the recipient. Eighteen transconjugants were obtained, and two were selected for characterization (K12-1 and K12-2). Remarkably, the acquisition of this single plasmid conferred to the E. coli recipients resistance to all the beta-lactams tested, pointing out the broad spectrum of beta-lactam antibiotics targeted by this carbapenemases combination. Accordingly, PCR amplification confirmed the acquisition of blaKPC−2 and blaVIM−1 genes by K12-1 and K12-2, while their expression was confirmed by immunochromatography (Table 1). Thus, contrary to that observed with the other hvKp strain isolated in Chile (K-2157) [26], VA585-22 could conjugate the plasmid pVA585-22_54 to E. coli, conferring dual carbapenemase production and multi-drug resistance.


We characterized phenotypically and at the genomic level the CG23-I (ST23) hvKp strain VA585-22 isolated in Chile (main features summarized in Fig. 3), corresponding to the first report of hvKp co-producing KPC-2 and VIM-1 carbapenemases encoded in a single conjugative plasmid. This plasmid likely arose from incorporating a class-I integron carrying blaVIM−1 and other resistance genes into an IncN KPC-2 plasmid highly disseminated in South America and previously found in Chile. Considering its high transmissibility and that it confers resistance to most beta-lactams, this double carbapenemases plasmid, and especially convergent hvKp comprising it, are of utmost concern.

Fig. 3
figure 3

Schematic representation of the main resistance genes, virulence factors, and mobile genetic elements in the CR-hvKp strain K. pneumoniae VA585-22. The virulence and metal resistance factors encoded in GIE492 (mce, MccE492 and salmochelin), ICEKp10 (ybt, yersiniabactin; clb, colibactin), and pKPVP-1 (iuc, aerobactin; iro, salmochelin; fep, iron transporter; fec, Fur-dependent system for iron uptake) are colored in magenta. For simplicity, only one export and one import protein complexes are depicted. The virulence plasmid also includes the sil, pco, pbr, and ter genes linked with resistance to copper, silver, lead, and tellurite, respectively, and the rmp genes linked to capsule upregulation. The antibiotic resistance proteins encoded in pVA585-22_54 are presented in dark blue (VIM-1 and KPC-2 carbapenemases, DfrA1 trimethoprim-resistant dihydrofolate reductase, ANT(3’’)-Ia aminoglycoside nucleotidyltransferase, AAC(6’)-II aminoglycoside acetyltransferase, and Sul1 sulfonamide-resistant dihydropteroate synthase). Created with


Bacterial strains and culture conditions

K. pneumoniae VA585-22 was collected by the Chilean Public Health Institute. K. pneumoniae SGH10 was kindly provided by Prof. Yunn Hwen Gan (National University of Singapore). The SGH10 DwcaJ mutant derivative was constructed following the scarless site-directed mutagenesis procedure described previously [21]. The K. pneumoniae strains were grown overnight at 37 °C in LB, Mueller-Hinton, or blood agar plates, supplemented with meropenem (4 µg/mL) when required. The E. coli K12 GmR recipient strain (spontaneous mutant from A. Marcoleta’s laboratory collection) for conjugation experiments was grown at 37 °C in LB or Mueller-Hinton agar plates supplemented with gentamicin (10 µg/mL).

Mucoviscosity assessment

Mucoviscosity was evaluated using the string test and a low-speed sedimentation assay, as described previously [21]. For both assays, bacterial isolates were grown overnight in blood agar at 37 °C. For sedimentation, an appropriate amount of biomass was suspended in sterile PBS and then diluted to obtain 5 mL of bacterial suspensions adjusted to an OD600 nm = 4, poured into sterile 15 mL conical tubes. Then, the tubes were centrifuged for 5 min at 1000 xg, measuring the OD600 nm of the supernatant (diluting when required). A higher OD600 nm (close to the value before centrifuging) indicated hypermucoviscosity, as these strains tend to float and remain in the supernatant. For the string test, isolated colonies from VA585-22, or the control strains SGH10 and SGH10 DwcaJ, were stretched with a bacteriology inoculation loop. The test was positive if a viscous string with a length > 5 mm is obtained [30].

Bacterial conjugation

A standard conjugation assay in E. coli was carried out as previously described [29]. Briefly, the donor VA585-22 and recipient E. coli K12-GmR strains were grown overnight in LB broth supplemented with meropenem and gentamicin, respectively. Conjugation was performed in a 0.22 μm sterile cellulose ester membrane filter (Merck, Germany) deposited over an LB agar plate, pouring a 1:1 ratio (50 µL) of donor and recipient strains. The plate with the filter was incubated for 3 h at 37ºC, and then half of the membrane was mixed with 3 mL of LB broth and vortexed briefly. Then, 100 µL of this suspension were plated onto LB-Gm-Mem and incubated at 37 °C overnight to select transconjugants (E. coli K12- GmR-MemR).

Detection of carbapenemase genes and carbapenemase production

Transconjugants were analyzed by PCR using Platinum™ Taq DNA polymerase (Invitrogen, ThermoFischer Scientific, USA) and specific primers for blaKPC−2 and blaVIM−1 (Table S3), as previously described [31]. Carbapenemase expression was confirmed by immunochromatography using NG-Test Carba 5 assay, according to the manufacturer’s instructions [32].

Antimicrobial susceptibility test

The Kp VA585-22 isolate, E. coli K12-GmR and two E. coli K12 transconjugant clones (GmR-MemR) were studied by epsilometry (E-test, Biomerieux) and broth microdilution using BD Phoenix™ System (Becton Dickinson, USA), according to M100 Performance Standards for Antimicrobial Susceptibility Testing, 33rd edition [33].

Genome sequencing, assembly and annotation

Genomic DNA was extracted using the GeneJET Kit (Thermo Scientific) and quantified using a Qubit fluorometer (Invitrogen). Illumina sequencing (100-bp paired-end) was done with the TruSeq Nano DNA kit and a Hiseq4000 machine (hired to Seqcenter, Inc., USA). Nanopore sequencing was done using the Rapid Barcoding kit (SQK-RBK004) and a FLO-MIN106 flow cell in a MinION device.

Illumina reads were trimmed and quality filtered using fastp v0.23.2 [34]. Nanopore sequencing data was base-called with Guppy v6.5.7 + ca6d6af with the dna_r9.4.1_450bps_sup.cfg model, and the ONT reads were then quality-filtered using Filtlong v0.2.1 (, and subsampled into 12 read sets using Trycycler v0.5.4 [35]. Four read sets were assembled using Flye v2.9.2-b1786 [36], four using raven v1.8.1 [37], and four using minimap2 v2.24-r1122 [38], miniasm v0.3-r179 [39] and minipolish v0.1.2 [40]. All 12 assemblies were used as input to generate a consensus long-read assembly following the trycycler v0.5.4 pipeline. The long-read consensus assembly was polished using Medaka v1.7.2 ( with the r941_min_sup_g507 model. The Medaka-polished assembly was then polished using the filtered Illumina reads with Polypolish v0.5.0 [41] and POLCA (MaSuRCA v4.0.5) [42]. The assembly was evaluated using QUAST v5.0.2 [43] and CheckM [44], and annotated with Bakta v1.8.1 [45]. Gene organization analysis was performed with Clinker [46]. Nucleotide sequence alignment and visualization was performed with Clustal Omega [47] and Jalview [48].

CG23 K. pneumoniae database construction

17,612 K. pneumoniae species complex (KpSC) genomes were downloaded from the NCBI RefSeq database on April 5th, 2023. Upon filtering out genomes of poor quality, with ambiguous nucleotide bases, and non-standard Klebsiella genomes (≥ 1,000 contigs, genomic size ≤ 4.5 Mbp or ≥ 6.5 Mbp, > 59% GC content, < 96% Average Nucleotide Identity to reference KpSC genomes), 11,817 genomes were selected for further analysis. Additionally, we included K. pneumoniae VA585-22, 119 genomes from the Antibiotics for Klebsiella Liver Abscess (A-KLASS) cohort (PRJNA956314) [49], 365 K. pneumoniae isolates from a bloodstream infection cohort (BSI) from Asia [50], 34 Klebsiella genomes from the Murray Collection in the pre-antibiotic era [51], and 97 CG23 genomes analyzed by Lam et al. (2018) [7]. The resulting 12,433 K. pneumoniae sensu stricto species genome set was subjected to multilocus sequence typing (MLST) and screened for relevant information using Kleborate v2.3.2 [52]. The 629-loci cgMLST scheme, scgMLSTv2 [28], was used to search for alleles in all 12,433 Kp1 genomes using the BLASTn v2.13.0 algorithm [53] under a 95% identity and 95% coverage threshold. The best hit for each allele was selected according to the local alignment reported bitscore. Selected alleles were used to generate cgMLST profiles for each genome. The cgMLST profiles of the 12,433 Kp1 genomes and 34,055 reference profiles were used as input for the LINcoding algorithm [28] to assign cgLIN codes to each genome. 435 genomes belonging to CG23 according to the cgLIN code were kept for further analysis.

Phylogenomic analysis

A core genome multiple sequence alignment (cg-MSA) was constructed using 629 loci previously defined [28] for the 435 CG23 genomes. The individual gene sequences were aligned in global pair mode using MAFFT v7.471 [54]. A phylogenetic tree was inferred using IQ-TREE v2.2.2.3 [55] with 139,808 set as seed and 1,000 non-parametric bootstraps. The best nucleotide substitution model, GTR + F + I, was predicted using ModelFinderPlus [56].

Plasmid typing, clustering, and prediction of mobile genetic elements

The 435 CG23 genomes were used as input for the MOB-suite v3.1.4 [57] mob_recon module to reconstruct and type plasmids with default parameters. All plasmid sequences were used as input for NCBI AMRFinderPlus v3.11.14 [58] to identify antimicrobial, metal, and biocide resistance genes (Database version: 2023-08-08.2). Insertion sequences were predicted in reconstructed plasmids using ISEScan v1.7.2.3 [59]. Complete integrons, CALINs and integron integrases were predicted using IntegronFinder v2.0.2 [60].

Data availability

The complete genome assembly of K. pneumoniae VA585-22 was deposited in the NCBI genome database under the Bioproject number PRJNA1020101 and the assembly accession GCF_032253935.1.


  1. Wyres KL, Holt KE. Klebsiella pneumoniae as a key trafficker of drug resistance genes from environmental to clinically important bacteria. Curr Opin Microbiol [Internet]. The Authors. 2018;45:131–9.

  2. Clegg S, Murphy CN. Epidemiology and virulence of Klebsiella pneumoniae. Microbiol Spectr. 2016;4.

  3. Choby JE, Howard-Anderson J, Weiss DS. Hypervirulent Klebsiella pneumoniae – clinical and molecular perspectives. J Intern Med. 2020;287:283–300.

    Article  PubMed  CAS  Google Scholar 

  4. Lan P, Jiang Y, Zhou J, Yu Y. A global perspective on the convergence of hypervirulence and carbapenem resistance in Klebsiella pneumoniae. J Glob Antimicrob Resist [Internet]. Elsevier Ltd. 2021;25:26–34.

  5. Han YL, Wen XH, Zhao W, Cao XS, Wen JX, Wang JR, et al. Epidemiological characteristics and molecular evolution mechanisms of carbapenem-resistant hypervirulent Klebsiella pneumoniae. Front Microbiol. 2022;13:1–13.

    Article  Google Scholar 

  6. Tang M, Kong X, Hao J, Liu J. Epidemiological characteristics and formation mechanisms of Multidrug-Resistant Hypervirulent Klebsiella pneumoniae. Front Microbiol. 2020;11:1–10.

    Article  Google Scholar 

  7. Lam MMC, Wyres KL, Duchêne S, Wick RR, Judd LM, Gan YH et al. Population genomics of hypervirulent Klebsiella pneumoniae clonal-group 23 reveals early emergence and rapid global dissemination. Nat Commun. 2018;9.

  8. Wyres KL, Lam MMC, Holt KE. Population genomics of Klebsiella pneumoniae. Nat Rev Microbiol [Internet]. Springer: US.;2020.

  9. Russo TA, Marr CM. Hypervirulent Klebsiella pneumoniae. 2019;32:1–42.

    Google Scholar 

  10. Chen YT, Chang HY, Lai YC, Pan CC, Tsai SF, Peng HL. Sequencing and analysis of the large virulence plasmid pLVPK of Klebsiella pneumoniae CG43. Gene. 2004;337:189–98.

    Article  PubMed  CAS  Google Scholar 

  11. Marcoleta AE, Berríos-Pastén C, Nuñez G, Monasterio O, Lagos R. Klebsiella pneumoniae asparagine tDNAs are integration hotspots for different genomic islands encoding microcin E492 production determinants and other putative virulence factors present in hypervirulent strains. Front Microbiol. 2016;7.

  12. Lam MMC, Wick RR, Wyres KL, Gorrie CL, Judd LM, Jenney AWJ et al. Genetic diversity, mobilisation and spread of the yersiniabactin-encoding mobile element ICEKp in Klebsiella pneumoniae populations. Microb Genomics [Internet]. 2018;1–14. Available from:

  13. Zhang R, Lin D, Chan EW, Gu D, Chen G, Chen S. Emergence of Carbapenem-Resistant Serotype K1 Hypervirulent Klebsiella pneumoniae Strains in China. Antimicrob Agents Chemother [Internet]. 2016;60:709–11.

  14. Chen Y, Marimuthu K, Teo J, Venkatachalam I, Pei B, Cherng Z, et al. Acquisition of plasmid with Carbapenem-Resistance Gene blaKPC2 in Hypervirulent Klebsiella pneumoniae, Singapore. Clin Infect Dis. 2020;26:549–59.

    CAS  Google Scholar 

  15. Becker L, Kaase M, Pfeifer Y, Fuchs S, Reuss A, von Laer A, et al. Genome-based analysis of carbapenemase-producing Klebsiella pneumoniae isolates from German hospital patients, 2008–2014. Antimicrob Resist Infect Control Antimicrob Resist Infect Control. 2018;7:1–12.

    Google Scholar 

  16. Karlsson M, Stanton RA, Ansari U, McAllister G, Chan MY, Sula E et al. Identification of a carbapenemase-producing hypervirulent klebsiella pneumoniae isolate in the United States. Antimicrob Agents Chemother. 2019;63.

  17. Cejas D, Canigia LF, Cruz GR, Elena AX, Maldonado I, Gutkind GO, et al. First isolate of KPC-2-producing Klebsiella pneumonaie sequence type 23 from the Americas. J Clin Microbiol. 2014;52:3483–5.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Morales-León F, Matus-Köhler M, Araya-Vega P, Aguilera F, Torres I, Vera R et al. Molecular Characterization of the Convergent Carbapenem-Resistant and Hypervirulent Klebsiella pneumoniae Strain K1-ST23, Collected in Chile during the COVID-19 Pandemic. Lincopan N, editor. Microbiol Spectr [Internet]. American Society for Microbiology; 2023;2019:1–7.

  19. Passet V, Brisse S. Association of tellurite resistance with hypervirulent clonal groups of Klebsiella pneumoniae. J Clin Microbiol. 2015;53:1380–2.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Mikei LA, Starki AJ, Forsyth VS, Vornhagen J, Smith SN, Bachman MA et al. A systematic analysis of hypermucoviscosity and capsule reveals distinct and overlapping genes that impact Klebsiella pneumoniae fitness [Internet]. PLoS Pathog. 2021.

  21. Tan YH, Chen Y, Chu WHW, Sham LT, Gan YH. Cell envelope defects of different capsule-null mutants in K1 hypervirulent Klebsiella pneumoniae can affect bacterial pathogenesis. Mol Microbiol. 2020;1–17.

  22. Russo TA, Olson R, Fang C-T, Stoesser N, Miller M, MacDonald U et al. Identification of Biomarkers for Differentiation of Hypervirulent Klebsiella pneumoniae from Classical K. pneumoniae. Diekema DJ, editor. J Clin Microbiol [Internet]. 2018;56.

  23. Lee CH, Liu JW, Su LH, Chien CC, Li CC, Yang KD. Hypermucoviscosity associated with Klebsiella pneumoniae-mediated invasive syndrome: A prospective cross-sectional study in Taiwan. Int J Infect Dis [Internet]. International Society for Infectious Diseases; 2010;14:e688–92.

  24. Wozniak A, Figueroa C, Moya-Flores F, Guggiana P, Castillo C, Rivas L, et al. A multispecies outbreak of carbapenem-resistant bacteria harboring the bla KPC gene in a non-classical transposon element. BMC Microbiol BMC Microbiol. 2021;21:1–10.

    Google Scholar 

  25. Veloso M, Arros P, Acosta J, Rojas R, Berríos-Pastén C, Varas M et al. Antimicrobial resistance, pathogenic potential, and genomic features of carbapenem-resistant Klebsiella pneumoniae isolated in Chile: high-risk ST25 clones and novel mobile elements. Lincopan N, editor. Microbiol Spectr [Internet]. American Society for Microbiology; 2023;0.

  26. Morales-León F, Matus-Köhler M, Araya-Vega P, Aguilera F, Torres I, Vera R et al. Molecular Characterization of the Convergent Carbapenem-Resistant and Hypervirulent Klebsiella pneumoniae Strain K1-ST23, Collected in Chile during the COVID-19 Pandemic. Lincopan N, editor. Microbiol Spectr [Internet]. American Society for Microbiology; 2023;11:1–7.

  27. Coutinho RL, Visconde MF, Descio FJ, Nicoletti AG, Pinto FCL, da Silva ACR, et al. Community-acquired invasive liver abscess syndrome caused by a K1 serotype Klebsiella pneumoniae isolate in Brazil: a case report of hypervirulent ST23. Mem Inst Oswaldo Cruz. 2014;109:973–4.

    Article  Google Scholar 

  28. Hennart M, Guglielmini J, Bridel S, Maiden MCJ, Jolley KA, Criscuolo A et al. A dual Barcoding Approach to bacterial strain nomenclature: genomic taxonomy of Klebsiella pneumoniae strains. Mol Biol Evol. 2022;39.

  29. Yong M, Chen Y, Oo G, Chang KC, Chu WHW, Teo J, et al. Dominant carbapenemase-encoding plasmids in clinical Enterobacterales isolates and Hypervirulent Klebsiella pneumoniae, Singapore. Emerg Infect Dis. 2022;28:1578–88.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Shon AS, Bajwa RPS, Russo TA. Hypervirulent (hypermucoviscous) Klebsiella Pneumoniae: a new and dangerous breed. Virulence. 2013;4:107–18.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Araya I, Roach-Poblete F, Tapia T, Rodas PI, Villamil A, Aguero R, et al. Caracterización fenotípica y molecular de cepas de Klebsiella pneumoniae productores de carbapenemasas tipo OXA-48 circulantes en Chile. Rev Chil infectología. 2022;39:551–8.

    Article  Google Scholar 

  32. Han R, Guo Y, Peng M, Shi Q, Wu S, Yang Y, et al. Evaluation of the Immunochromatographic NG-Test Carba 5, RESIST-5 O.O.K.N.V., and IMP K-SeT for Rapid Detection of KPC-, NDM-, IMP-, VIM-type, and OXA-48-like carbapenemase among Enterobacterales. Front Microbiol. 2021;11:1–7.

    Article  Google Scholar 

  33. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing. 33rd ed. CLSI supplement M100. Clin. Lab. Stand. Institue. 2023.

  34. Chen S, Zhou Y, Chen Y, Gu J. Fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34:i884–90.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Wick RR, Judd LM, Cerdeira LT, Hawkey J, Méric G, Vezina B, et al. Trycycler: consensus long-read assemblies for bacterial genomes. Genome Biol Genome Biology. 2021;22:1–17.

    Google Scholar 

  36. Kolmogorov M, Yuan J, Lin Y, Pevzner PA. Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol [Internet]. Springer US; 2019;37:540–6.

  37. Vaser R, Šikić M. Time- and memory-efficient genome assembly with Raven. Nat Comput Sci [Internet]. Springer US; 2021;1:332–6.

  38. Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018;34:3094–100.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Li H. Minimap and miniasm: fast mapping and de novo assembly for noisy long sequences. Bioinformatics. 2016;32:2103–10.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Wick RR, Holt KE. Benchmarking of long-read assemblers for prokaryote whole genome sequencing. F1000Research. 2019;8:1–23.

    Article  Google Scholar 

  41. Wick RR, Holt KE, Polypolish. Short-read polishing of long-read bacterial genome assemblies. PLoS Comput Biol [Internet]. 2022;18:1–13.

  42. Zimin AV, Marçais G, Puiu D, Roberts M, Salzberg SL, Yorke JA. The MaSuRCA genome assembler. Bioinformatics. 2013;29:2669–77.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Gurevich A, Saveliev V, Vyahhi N, Tesler G. QUAST: Quality assessment tool for genome assemblies. Bioinformatics. 2013;29:1072–5.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 2015;25:1043–55.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Schwengers O, Jelonek L, Dieckmann MA, Beyvers S, Blom J, Goesmann A, Bakta. Rapid and standardized annotation of bacterial genomes via alignment-free sequence identification. Microb Genomics. 2021;7.

  46. Gilchrist CLM, Chooi YH. Clinker & clustermap.js: automatic generation of gene cluster comparison figures. Bioinformatics. 2021;37:2473–5.

    Article  PubMed  CAS  Google Scholar 

  47. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol. 2011;7:1–6.

    Article  Google Scholar 

  48. Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ. Jalview Version 2-A multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009;25:1189–91.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Molton JS, Chan M, Kalimuddin S, Oon J, Young BE, Low JG, et al. Oral vs intravenous antibiotics for patients with klebsiella pneumoniae liver abscess: a randomized, controlled noninferiority study. Clin Infect Dis. 2020;71:952–9.

    Article  PubMed  CAS  Google Scholar 

  50. Wyres KL, Nguyen TNT, Lam MMC, Judd LM, Van Vinh Chau N, Dance DAB, et al. Genomic surveillance for hypervirulence and multi-drug resistance in invasive Klebsiella pneumoniae from South and Southeast Asia. Genome Med Genome Med. 2020;12:1–16.

    Google Scholar 

  51. Baker KS, Burnett E, McGregor H, Deheer-Graham A, Boinett C, Langridge GC et al. The Murray collection of pre-antibiotic era Enterobacteriacae: A unique research resource. Genome Med [Internet]. Genome Medicine; 2015;7:1–7.

  52. Lam MMC, Wick RR, Watts SC, Cerdeira LT, Wyres KL, Holt KE. A genomic surveillance framework and genotyping tool for Klebsiella pneumoniae and its related species complex. Nat Commun [Internet]. Springer US; 2021;12.

  53. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol [Internet]. 1990;215:403–10. Available from:

  54. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30:772–80.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  55. Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, Von Haeseler A, et al. IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol. 2020;37:1530–4.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  56. Kalyaanamoorthy S, Minh BQ, Wong TKF, Von Haeseler A, Jermiin LS, ModelFinder. Fast model selection for accurate phylogenetic estimates. Nat Methods [Internet]. Nature Publishing Group; 2017;14:587–9.

  57. Robertson J, Nash JHE. MOB-suite: software tools for clustering, reconstruction and typing of plasmids from draft assemblies. Microb Genomics. 2018;4.

  58. Feldgarden M, Brover V, Gonzalez-Escalona N, Frye JG, Haendiges J, Haft DH et al. AMRFinderPlus and the Reference Gene Catalog facilitate examination of the genomic links among antimicrobial resistance, stress response, and virulence. Sci Rep [Internet]. Nature Publishing Group UK; 2021;11:1–9.

  59. Xie Z, Tang H. ISEScan: automated identification of insertion sequence elements in prokaryotic genomes. Bioinformatics. 2017;33:3340–7.

    Article  PubMed  CAS  Google Scholar 

  60. Néron B, Littner E, Haudiquet M, Perrin A, Cury J, Rocha EPC. IntegronFinder 2.0: identification and analysis of integrons across Bacteria, with a Focus on Antibiotic Resistance in Klebsiella. Microorganisms. 2022;10.

Download references


P Arros would like to acknowledge the Maria Ghilardi Venegas Foundation (Chile) for their continuous support. Computational resources were provided by the supercomputing infrastructure of Soroban (SATREPS MACH – JPM/JSA1705) at Centro de Modelación y Computación Cientı́fica at Universidad de La Frontera, Chile. We would like to thank Hospital San José (Santiago) and all the clinical laboratories in Chile that contribute to the national antimicrobial resistance surveillance system, and also to the Antimicrobial Resistance Special Program, Pan American Health Organization (PAHO).


Grants FONDECYT 1221193 (Marcoleta AE), FONDECYT 1211852 (Chavez FP).

Author information

Authors and Affiliations



According to CRediT criteria: MG-S and PA: Conceptualization, methodology, validation, formal analysis, investigation, data curation, software, visualization, writing-original draft preparation, writing-review and editing. CB-P: Investigation, formal analysis, software, writing-review and editing. AV, PIR, IA, RI, CB and YC: Investigation, methodology, validation, formal analysis. PA, JCH, Y-HG, FPC, RL: Conceptualization, methodology, resources, writing-review and editing, supervision, project administration. AM: Conceptualization, methodology, validation, formal analysis, investigation, resources, data curation, writing-original draft preparation, writing-review and editing, visualization, supervision, project administration, funding acquisition.

Corresponding author

Correspondence to Andrés E. Marcoleta.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1:

Table S1. VA585-22 genome sequence stats. Table S3. Primers used for carbapenemase genes detection. Figure S1. Hypermucoviscosity evaluation through low-speed sedimentation and the string test. Figure S2. Gene organization and sequence conservation of the rmpADC locus and its surroundings. Figure S3. Phylogenetic relationships among VA585-22 and other 434 K. pneumoniae CG23 genomes. Figure S4. Plasmids carrying carbapenemase genes present in CG23 hvKp genomes

Supplementary Material 2:

Table S2. Accession numbers and relevant information of the CG23 K. pneumoniae isolates included in the phylogenomic analysis

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gálvez-Silva, M., Arros, P., Berríos-Pastén, C. et al. Carbapenem-resistant hypervirulent ST23 Klebsiella pneumoniae with a highly transmissible dual-carbapenemase plasmid in Chile. Biol Res 57, 7 (2024).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: