Extensively Drug-Resistant ST16 Klebsiella pneumoniae Co-Harboring bla_KPC-2, bla_OXA-181, and Chromosomal bla_NDM-4: Genomic Features and Therapeutic Challenges

Abstract

Purpose

Carbapenem-resistant Klebsiella pneumoniae (CRKP) represents a major global public health threat due to its high-level antimicrobial resistance and limited therapeutic options. The emergence of CRKP isolates co-harboring multiple carbapenemase genes further complicates clinical management and infection control. This study aimed to characterize the antimicrobial resistance phenotype, complete genomic features, and carbapenemase gene environments of an extensively drug-resistant ST16 CRKP bloodstream isolate co-harboring class A, B, and D carbapenemases.

Methods

A CRKP isolate, designated KPY1, was recovered from the bloodstream of an intensive care unit patient. Antimicrobial susceptibility testing was performed to determine the resistance phenotype. Whole-genome sequencing was conducted using a hybrid Illumina–Nanopore approach. Genome assembly, antimicrobial resistance gene detection, plasmid replicon typing, comparative plasmid analysis, and genetic environment analysis of carbapenemase genes were performed using established bioinformatic tools.

Results

KPY1 belonged to sequence type 16 and exhibited an extensively drug-resistant phenotype, including resistance to carbapenems, β-lactam/β-lactamase inhibitor combinations, ceftazidime–avibactam, and colistin. Genomic analysis revealed the coexistence of three carbapenemase genes: bla_KPC-2, bla_NDM-4, and bla_OXA-181. Notably, bla_NDM-4 was located on the chromosome and flanked by two IS26 elements, suggesting a possible IS26-associated rearrangement event. In contrast, bla_KPC-2 was located on an IncFII(K) plasmid within a Tn3-family transposon structure, whereas bla_OXA-181 was carried by a conserved ColKP3-IncX3 plasmid backbone.

Conclusion

This study describes a rare and clinically challenging ST16 CRKP bloodstream isolate co-harboring class A, B, and D carbapenemases. The chromosomal integration of bla_NDM-4 may contribute to stable maintenance of carbapenem resistance, while the coexistence of multiple carbapenemase determinants highlights the value of complete genome sequencing for resolving complex resistance configurations and supporting surveillance of high-risk CRKP lineages.

Introduction

Klebsiella pneumoniae (K. pneumoniae) is a common Gram‑negative opportunistic pathogen responsible for a wide spectrum of infections, including pneumonia, bloodstream infections, and urinary tract infections. Since the 1990s, carbapenem‑resistant K. pneumoniae (CRKP) has emerged as a major global public health threat, with associated mortality rates reaching as high as 37.2%.¹ A recent global meta-analysis further highlighted the widespread dissemination of carbapenem-resistant Enterobacteriaceae, with K. pneumoniae being one of the predominant colonizing species.² In China, the incidence of CRKP increased from 6.4% to 11.3% between 2014 and 2021, underscoring that CRKP remains a major multidrug‑resistance problem.³

The primary resistance mechanism of CRKP is the production of carbapenemases. Based on the Ambler classification, carbapenemases are divided into three major classes: class A serine carbapenemases, such as Klebsiella pneumoniae carbapenemase (KPC); class B metallo-β-lactamases, such as New Delhi metallo-β-lactamase (NDM), imipenemase (IMP), and Verona integron-encoded metallo-β-lactamase (VIM); and class D serine carbapenemases, such as OXA-48-like oxacillinases.⁴ Among these, KPC enzymes are the most common carbapenemases in Chinese CRKP isolates, followed by metallo-β-lactamases. These carbapenemases exhibit distinct enzymatic properties and inhibitor susceptibility profiles, contributing to diverse resistance phenotypes and therapeutic challenges.^4,5

Worryingly, an increasing number of K. pneumoniae isolates co‑carry two or even three different types of carbapenemases, rendering resistance phenotypes more complex and leaving extremely limited therapeutic options. Common co‑production combinations include KPC together with a metallo‑β‑lactamase, dual metallo‑β‑lactamases, or a metallo‑β‑lactamase together with an OXA‑48‑like enzyme. Several studies have reported CRKP isolates co-producing different combinations of carbapenemases, including NDM-1 and KPC-2 in China⁶ and KPC/NDM or KPC/OXA-48 co-production in Turkey.⁷ Although such co‑producing isolates remain uncommon, they complicate resistance phenotypes and pose substantial challenges for infection control. More frequently reported are K. pneumoniae strains co‑producing NDM and OXA‑48‑like enzymes. It has been shown that ST16 isolates can carry both bla_NDM‑1 and bla_OXA‑48 and were associated with high mortality in an ICU outbreak in Iran.⁸

A recent study from China reported a K51-ST16 CRKP isolate co-producing NDM-4 and OXA-181, with both resistance genes located on plasmids.⁹ ST16 has been increasingly reported worldwide and is emerging as a potential high-risk clone.^9–11

To date, K. pneumoniae isolates co‑producing all three major types of carbapenemases (KPC, NDM and OXA) have been rarely reported, and their genomic organization and evolutionary mechanisms remain insufficiently characterized. Here, we describe a clinical ST16 K. pneumoniae isolate that simultaneously harbors bla_KPC‑2, bla_OXA‑181, and bla_NDM‑4, with a notable distribution pattern: bla_NDM‑4 is integrated into the chromosome, bla_KPC‑2 resides on an IncFII(K) plasmid, and bla_OXA‑181 is carried on a ColKP3-IncX3 plasmid. Using whole-genome sequencing, we characterized the complete genomic features, genetic contexts, and potential evolutionary mechanisms of these carbapenemase determinants, and further discussed their clinical implications. Our findings provide insights into the emergence and dissemination of CRKP isolates co-producing multiple carbapenemases.

Materials and Methods

Bacterial Isolation and Storage

A total of 15 multidrug-resistant K. pneumoniae isolates were collected between October and December 2025 from clinical specimens processed by the Department of Clinical Laboratory at the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China. KPY1, one of these isolates, was recovered from a bloodstream specimen from an intensive care unit (ICU) patient. The isolate was cultured on Columbia blood agar plates at 35 °C under aerobic conditions to obtain pure colonies. Species identification was performed using matrix‑assisted laser desorption/ionization time‑of‑flight mass spectrometry (MALDI‑TOF MS; Bruker Daltonik, Bremen, Germany). The isolate KPY1 was stored at –80 °C in Luria‑Bertani (LB) broth (Oxoid, UK) supplemented with 20% glycerol.

Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing was performed using the agar dilution method (for most agents) and the broth microdilution method (for tigecycline, colistin, and ceftazidime‑avibactam) to determine minimum inhibitory concentrations (MICs). All tests were carried out on Mueller‑Hinton media (agar: Oxoid, UK; broth: BD Diagnostics, USA) and incubated at 35 °C for 16–20 h. Results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (M100, 35th edition). For tigecycline, which lacks CLSI breakpoints, the MICs were interpreted using the US Food and Drug Administration (FDA) breakpoints. Escherichia coli ATCC 25922 was used as the quality control strain.

Phenotypic carbapenemase detection assays, including the modified carbapenem inactivation method (mCIM), EDTA-carbapenem inactivation method (eCIM), serine carbapenemase detection assay (SCARB), and metallo-β-lactamase (MBL) assay, were additionally performed according to standard procedures.

Genomic DNA Extraction and Sequencing

Genomic DNA was extracted from a fresh overnight culture using the TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0 (TaKaRa, Japan) following the manufacturer’s instructions. Whole‑genome sequencing was performed using a hybrid strategy combining short‑read and long‑read technologies. Short‑read sequencing was carried out on an Illumina MiSeq platform with a 2 × 150 bp paired‑end protocol, generating approximately 361 Mb of high‑quality data. Long‑read sequencing was performed on an Oxford Nanopore MinION platform, yielding approximately 1.20 Gb of raw data with an average read length of approximately 2.1 kb.

Genome Assembly and Annotation

Raw reads were quality‑controlled using FastQC v0.11.9 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) and trimmed with Trimmomatic v0.39.¹² Hybrid assembly was performed using Unicycler v0.5.0.^13,14 Genome completeness was assessed with CheckM v1.2.5.¹⁵ Manual verification confirmed a single circular chromosome and five circular plasmids. The assembled genome was annotated using Prokka v1.14.6 on the Galaxy platform with default parameters.^16,17

Resistance Gene and Plasmid Analyses

Multilocus sequence typing (MLST) was performed using the MLST 1.0 tool available at the Center for Genomic Epidemiology (CGE) website (https://www.genomicepidemiology.org). Plasmid replicon typing was conducted with PlasmidFinder 2.1,¹⁸ and antimicrobial resistance genes were identified using ResFinder 4.7.2.¹⁹ Default thresholds were applied for all analyses (≥95% identity and ≥60% coverage for plasmid replicon typing; ≥90% identity and ≥90% coverage for resistance gene identification). Insertion sequences and transposons were manually curated and annotated using the ISfinder and TnCentral databases.

Comparative Analysis of Resistance Plasmids

Plasmid circular comparison maps were generated using BRIG v1.0.²⁰ For the bla_KPC‑2‑carrying plasmid, designated KPY1p4_KPC-2, it was used as the reference against six highly similar plasmid sequences retrieved from NCBI (GenBank accession numbers CP128030.1, CP188761.1, ON111451.1, CP133348.1, MN904741.1, MF072963.1). BLASTn alignment was performed with a minimum similarity of 70% and an E‑value cutoff of 1e^‑5. Key genes, including bla_KPC‑2, were highlighted on the resulting ring image, which was exported as an SVG file. The same procedure was followed for the bla_OXA‑181‑carrying plasmid, using five homologous plasmids (GenBank accession numbers CP024806.1, CP103490.1, KT005457.1, MK412920.1, KX523903.1) as queries.

Comparative Plasmid and Genetic Context Analyses

The genetic environments of the three carbapenemase genes were analyzed using Easyfig v2.2.5.²¹ Genomic regions containing each carbapenemase gene and approximately 8–10 kb of upstream and downstream flanking sequences were extracted from the complete genome assembly and aligned with homologous reference sequences downloaded from GenBank. All sequences were annotated with Prokka prior to import into Easyfig. BLASTn comparisons were performed with a minimum identity of 70% and a minimum hit length of 200 bp. A 2‑kb scale bar was added to each figure. The resulting images were exported as SVG files.

Validation of Chromosomal bla_NDM-4 Localization

PCR amplification was performed using primers targeting the left and right junction regions of the bla_NDM-4-containing chromosomal region. The primer sequences used for this validation are listed in Supplementary Table 1. The PCR products were purified and subjected to Sanger sequencing. The obtained sequences were compared with the hybrid genome assembly for validation.

Results

Clinical Origin and Selection of the KPY1 Isolate

During the study period, a total of 15 multidrug-resistant K. pneumoniae isolates were collected from clinical specimens processed by the Department of Clinical Laboratory at the First Affiliated Hospital of Zhengzhou University. Preliminary antimicrobial susceptibility testing and genomic screening revealed that one isolate, subsequently designated KPY1, harbored three clinically important carbapenemase genes, including bla_KPC-2, bla_NDM-4, and bla_OXA-181. Due to the rarity and clinical significance of this resistance configuration, KPY1 was selected for further comprehensive genomic characterization. The isolate was recovered from a bloodstream specimen of a critically ill intensive care unit (ICU) patient. The other 14 isolates did not exhibit comparable carbapenemase combinations or genomic characteristics and were not included in the present genomic analysis.

Antimicrobial Resistance Phenotype of KPY1

Antimicrobial susceptibility testing was performed for KPY1, and the results are summarized in Table 1. KPY1 exhibited resistance to most antimicrobial agents tested, including all β-lactam antibiotics, such as penicillins, cephalosporins, and β-lactam/β-lactamase inhibitor combinations. The isolate was also resistant to all tested carbapenems (imipenem, meropenem, and ertapenem), fluoroquinolones, most aminoglycosides, tetracyclines, and trimethoprim–sulfamethoxazole. In contrast, KPY1 remained susceptible to gentamicin and chloramphenicol and showed intermediate susceptibility to tigecycline. Notably, KPY1 was resistant to colistin. Phenotypic carbapenemase detection assays revealed positive mCIM, SCARB, and MBL results, whereas eCIM remained negative (Table 2). Whole-genome sequencing identified three carbapenemase genes, including bla_KPC-2, bla_NDM-4, and bla_OXA-181. The negative eCIM result persisted after repeated testing and may be explained by the coexistence of multiple carbapenemases in KPY1, which could mask the inhibitory effect of EDTA on NDM-4 activity.

Table 1.

Antimicrobial Susceptibility Profile of K. pneumoniae Isolate KPY1

No.	Antimicrobial Agent	Method	MIC (μg/mL)	Interpretation
1	Amoxicillin–clavulanate	MIC	>32/16	R
2	Ampicillin–sulbactam	MIC	>16/8	R
3	Piperacillin–tazobactam	MIC	>64/4	R
4	Cefoperazone-sulbactam	MIC	>32/8	R
5	Cefazolin	MIC	>16	R
6	Cefuroxime	MIC	>16	R
7	Cefuroxime axetil	MIC	>16	R
8	Ceftriaxone	MIC	>32	R
9	Ceftazidime	MIC	>32	R
10	Cefepime	MIC	>16	R
11	Cefoxitin	MIC	>16	R
12	Aztreonam	MIC	>32	R
13	Imipenem	MIC	>8	R
14	Meropenem	MIC	>8	R
15	Ertapenem	MIC	>2	R
16	Gentamicin	MIC	≤2	S
17	Tobramycin	MIC	>8	R
18	Amikacin	MIC	16	R
19	Chloramphenicol	MIC	8	S
20	Ciprofloxacin	MIC	>4	R
21	Levofloxacin	MIC	>8	R
22	Tetracycline	MIC	>8	R
23	Minocycline	MIC	>16	R
24	Trimethoprim–sulfamethoxazole	MIC	>4/76	R
25	Tigecycline	MIC	4	I
26	Colistin	MIC	>4	R

Note: MIC interpretation followed CLSI M100 (35th edition) guidelines, except for tigecycline, for which FDA breakpoints were applied.

Abbreviations: R, resistant; S, susceptible; I, intermediate.

Table 2.

Phenotypic Carbapenemase Detection Assays of K. pneumoniae Isolate KPY1

Test	Method	Result
Modified carbapenem inactivation method (mCIM)	KB	Positive
EDTA-carbapenem inactivation method (eCIM)	KB	Negative
Serine carbapenemase detection (SCARB)	KB	Positive
Metallo-β-lactamase detection (MBL)	KB	Positive

Based on the criteria proposed by Magiorakos et al,²² KPY1 was classified as an extensively drug-resistant (XDR) isolate. The coexistence of multiple carbapenemase determinants and resistance to most antimicrobial classes resulted in extremely limited remaining therapeutic options.

Complete Genomic Features of the KPY1 Isolate

To characterize the genomic architecture of KPY1, whole‑genome sequencing was performed using a hybrid strategy combining short‑read (Illumina MiSeq) and long‑read (Oxford Nanopore MinION) technologies. Hybrid assembly yielded a complete genome consisting of one circular chromosome and five circular plasmids, with a total genome size of 5,722,379 bp. Genome assembly quality assessment demonstrated high reliability, with 100.0% completeness and 0.82% contamination according to CheckM analysis. Detailed assembly statistics and chromosome/plasmid-level sequencing coverage information are summarized in Supplementary Tables 2 and 3.

The chromosome had a total length of 5,380,807 bp with a GC content of 57.2%. Multilocus sequence typing (MLST) identified KPY1 as sequence type 16 (ST16), an emerging high-risk clone that has been increasingly reported worldwide and is frequently associated with multidrug resistance and clinically important resistance determinants. The chromosome carried multiple resistance genes, including bla_NDM-4, bla_SHV-28, fosA, and aac(6′)-Ib, as well as efflux pump- and porin-related genes such as the acrAB-tolC operon and mdtK. The complete list of resistance determinants and their corresponding antimicrobial susceptibility profiles are summarized in Supplementary Table 4. Notably, although the aminoglycoside resistance gene rmtB was detected in KPY1, the isolate remained susceptible to gentamicin, indicating a genotype–phenotype discrepancy. The chromosomal localization of bla_NDM-4 and its surrounding genetic context are further analyzed in the following sections.

Analysis of colistin resistance-associated loci identified an ISAeme19 insertion sequence (ISL3 family) within the mgrB coding sequence. Comparison with publicly available K. pneumoniae mgrB sequences from the NCBI database revealed that ISAeme19 was inserted after the fourth nucleotide of the mgrB coding sequence. Since mgrB disruption by mutations or insertion sequences is frequently associated with colistin resistance in K. pneumoniae,²³ this insertion may impair MgrB function and potentially contribute to the colistin-resistant phenotype of KPY1; however, further experimental validation is required.

In addition to antimicrobial resistance determinants, virulence-associated genes were screened using Kleborate and VFDB. KPY1 harbored an ICEKp3-associated yersiniabactin cluster and genes related to fimbriae, enterobactin, and T6SS, whereas major hypervirulence-associated determinants, including aerobactin (iucABCD-iutA), salmochelin (iroBCDN), colibactin (clb cluster), and rmpA/rmpA2, were not detected (Supplementary Table 5).

The five plasmids ranged in size from 13,254 bp to 121,549 bp. Plasmid replicon typing showed that KPY1p1 (112,549 bp) belonged to the IncFIB(K) type; KPY1p2 (121,549 bp) was a hybrid type comprising IncFII(K) and repB(R1701); KPY1p3 (13,254 bp) carried a ColRNAI replicon; KPY1p4_KPC-2 (42,742 bp), which harbored bla_KPC‑2, belonged to the common IncFII(K) type; and KPY1p5_OXA-181 (51,478 bp) was a ColKP3‑IncX3 composite plasmid carrying bla_OXA‑181 (Table 3). In addition to the carbapenemase-carrying plasmids KPY1p4_KPC-2 and KPY1p5_OXA-181, KPY1p2 carried the extended-spectrum β-lactamase genes bla_CTX-M-15 and bla_TEM-1C.

Table 3.

Characteristics of the Plasmids and the Chromosome

Replicon	Size (bp)	Replicon Type	Resistance Gene(s)	Note
KPY1p1	112,549	IncFIB(K)	—
KPY1p2	121,549	IncFII(K)-repB(R1701)	blaCTX-M-15, blaTEM-1C
KPY1p3	13,254	ColRNAI	—
KPY1p4_KPC-2	42,742	IncFII(K)	blaKPC-2
KPY1p5_OXA-181	51,478	ColKP3-IncX3	blaOXA-181
Chromosome_NDM-4	5,380,807	—	blaNDM-4	ST16

The genetic contexts of the three carbapenemase genes are described in the subsequent sections.

Comparative Plasmid Analysis

Using KPY1p4_KPC-2 as the reference plasmid, BLASTn comparisons were performed against the query sequences CP128030.1, CP188761.1, ON111451.1, CP133348.1, MN904741.1, and MF072963.1. KPY1p4_KPC-2 exhibited 100% coverage and >99.9% nucleotide identity with MN904741.1 (originally identified in Serratia marcescens) and MF072963.1 (from Citrobacter freundii), suggesting the potential for cross-species dissemination of this plasmid backbone. CP133348.1 showed 75% coverage with 100% identity in the aligned region, while CP128030.1, CP188761.1, and ON111451.1 showed approximately 30% coverage, again with 100% identity in the aligned portions (Figure 1A). Notably, the aligned regions corresponded precisely to the location of the resistance gene. Both CP128030.1 and ON111451.1 were reported in K. pneumoniae, indicating that within the same species, the same resistance gene can reside on different plasmid backbones. Most of these query sequences originated from China, highlighting the widespread circulation of KPC‑2‑producing strains in the country.

Figure 1.

Comparative genomic analysis of the bla_KPC-2- and bla_OXA-181-carrying plasmids in KPY1. (A) Circular comparison of KPY1p4_KPC-2 with six homologous plasmids retrieved from the NCBI database: CP128030.1, CP188761.1, ON111451.1, CP133348.1, MN904741.1, and MF072963.1. (B) Circular comparison of KPY1p5_OXA-181 with five homologous plasmids retrieved from the NCBI database: CP024806.1, CP103490.1, KT005457.1, MK412920.1, and KX523903.1. The outermost circle represents the reference plasmid from KPY1, and the inner rings represent BLASTn comparisons with homologous plasmids. Colored regions indicate homologous sequences, while blank regions indicate sequence absence or divergence. The positions of bla_KPC-2 and bla_OXA-181 are indicated on the corresponding reference plasmids.

Using KPY1p5_OXA-181 as the reference, BLASTn comparisons were performed against the query sequences CP024806.1, CP103490.1, KT005457.1, MK412920.1, and KX523903.1. KPY1p5_OXA-181 showed 100% coverage and >99.99% nucleotide identity with all these plasmids, indicating that this plasmid backbone is highly conserved during dissemination (Figure 1B). Analysis of the origins of these plasmids revealed a global distribution: CP024806.1 (from E. coli in Denmark), CP103490.1 (from Klebsiella quasipneumoniae in the United States), KT005457.1 (from Klebsiella variicola in Switzerland), MK412920.1 (from K. pneumoniae in the United Arab Emirates), and KX523903.1 (from K. pneumoniae in the Czech Republic). These findings suggest that plasmids highly similar to KPY1p5_OXA-181 have disseminated across multiple bacterial species and geographic regions, indicating the global circulation of this conserved OXA-181-associated plasmid backbone.

Genetic Environments of Carbapenemase Genes

To elucidate the genetic contexts of the three carbapenemase genes, genomic regions containing each carbapenemase gene and approximately 8–10 kb of upstream and downstream flanking sequences were extracted and comparatively analyzed using Easyfig, with homologous reference sequences retrieved from GenBank.

Genetic Environment Analysis of bla_NDM‑4

Based on the complete hybrid genome assembly, bla_NDM-4 was located on the chromosome at positions 4,702,521–4,703,333. Oxford Nanopore long reads were mapped to the assembled genome, and multiple long reads spanning the bla_NDM-4-containing region and the adjacent chromosomal sequences were identified, further supporting the chromosomal localization of bla_NDM-4 (Supplementary Figure 1).

To further validate the chromosomal localization of bla_NDM-4, PCR assays were designed to amplify the left and right junction regions between the bla_NDM-4-containing fragment and the adjacent chromosomal sequences (Figure 2A). Two expected fragments (2071 bp for the left junction and 4469 bp for the right junction) were successfully amplified from independent colonies (Figure 2B). The PCR products were further confirmed by Sanger sequencing, showing complete agreement with the corresponding regions in the hybrid genome assembly.

Figure 2.

Chromosomal localization and genetic environment analysis of bla_NDM-4 in KPY1. (A) Schematic representation of PCR primer design for validation of the chromosomal insertion junctions. Two primer pairs were designed to amplify the left and right junction regions between the bla_NDM-4-associated sequence and the flanking chromosomal regions. The left junction PCR fragment was expected to be 2071 bp (Chr-L), and the right junction PCR fragment was expected to be 4469 bp (Chr-R). The positions and orientations of primers are indicated by arrows. (B) PCR amplification of the predicted chromosomal junction fragments. The expected products corresponding to the left junction (2071 bp) and right junction (4469 bp) were successfully amplified from KPY1 colonies. The PCR products were further validated by Sanger sequencing, and the obtained sequences were consistent with the corresponding regions in the hybrid genome assembly. M, DNA marker; KPY1 colony 1 and KPY1 colony 2, independent bacterial colonies used for PCR validation. (C) Linear comparison of the bla_NDM-4-containing chromosomal region of KPY1 with homologous regions from CP041930.1, CP080366.1, and PV023152.1. Conserved genomic regions and surrounding genetic structures are shown. Open reading frames (ORFs) are shown as arrows, with arrowheads indicating the direction of transcription. Different colors indicate different functional categories of genes or mobile genetic elements. Shaded regions between sequences represent nucleotide homology identified by BLASTn comparison.

Analysis of the approximately 10 kb upstream and downstream regions showed that only the central segment was completely identical when compared with highly homologous sequences (Figure 2C). This central segment was flanked by two IS26 elements in the same orientation. The upstream IS26 was located only 225 bp upstream of bla_NDM‑4, while the downstream IS26 was located 2787 bp downstream. Comparison with a reference sequence (the NDM‑4‑carrying plasmid pSECR18‑2374C, GenBank accession CP041930.1) revealed that the region from the upstream IS26 through bla_NDM‑4 to the region immediately before the downstream IS26 shared >99% nucleotide identity with the plasmid. In contrast, the downstream IS26 and its flanking sequences did not match any part of the plasmid (Figure 2C), suggesting that this IS26 is likely a chromosomal resident element. These findings indicate that the bla_NDM-4-containing region is located within a chromosomal IS26-associated structure. The presence of flanking IS26 elements and the high similarity of the bla_NDM-4-containing segment to plasmid-borne NDM-4 regions suggest that IS26-associated genetic rearrangement may have contributed to the acquisition or mobilization of bla_NDM-4 (Figure 2C). However, the precise mechanism underlying this chromosomal acquisition remains to be experimentally confirmed.

Genetic Environment Analysis of bla_KPC‑2

The bla_KPC‑2 gene was located on plasmid KPY1p4_KPC-2 at positions 7,718‑8,599. Genomic regions including bla_KPC-2 and approximately 9 kb of upstream and downstream flanking sequences were analyzed. The bla_KPC‑2 gene was embedded in a typical Tn3 family transposon structure, with the core arrangement ISKpn27–bla_KPC‑2–ISKpn6. Upstream of this core, the tnpA and tnpR genes of the Tn3 transposon were present, followed by the insertion sequence ISKpn27. Downstream, the insertion sequence ISKpn6 was followed by another copy of tnpR and tnpA of the Tn3 family (Figure 3A). Comparison with homologous regions from other highly similar plasmids revealed that this region is highly conserved, with only limited sequence variation.

Figure 3.

Comparative analysis of the genetic environments of bla_KPC-2 and bla_OXA-181 in KPY1. (A) Linear comparison of the bla_KPC-2-containing region of plasmid KPY1p4_KPC-2 with homologous regions from CP047392.1, CP188761.1, and MF072963.1. (B) Linear comparison of the bla_OXA-181-containing region of plasmid KPY1p5_OXA-181 with homologous regions from AP018837.1, CP024806.1, and CP103490.1. Open reading frames are shown as arrows, with arrowheads indicating the direction of transcription. Different colors indicate different functional categories of genes or mobile genetic elements. Shaded regions between sequences represent nucleotide homology identified by BLASTn comparison.

Genetic Environment Analysis of bla_OXA‑181

The bla_OXA‑181 gene was located on plasmid KPY1p5_OXA-181 at positions 44,665‑43,868 on the complement strand. Genomic regions including bla_OXA-181 and approximately 8 kb of upstream and downstream flanking sequences were analyzed. The bla_OXA‑181 gene was situated in a complex genetic environment composed of multiple insertion sequences (Figure 3B). It was flanked by two IS26 elements in the same orientation, forming a typical composite transposon structure, suggesting that IS26 plays a major role in the mobilization of this resistance gene. In addition, an ISEC63 element was located immediately upstream, which may be involved in the initial mobilization or expression regulation of bla_OXA‑181, while ISKPN19 downstream likely exists as an accompanying insertion sequence. The region carrying bla_OXA‑181 was highly conserved among the different reference sequences examined, with virtually identical gene arrangement and structure and no obvious structural variations (Figure 3B). This high conservation suggests that bla_OXA‑181 is disseminated by widely disseminated plasmid backbones, but the conserved genetic background limits precise tracing of its origin.

In summary, the genetic environments of the three carbapenemase genes each have distinct features: bla_NDM‑4 is integrated into the chromosome via two IS26 elements; bla_KPC‑2 resides on a plasmid carrying a Tn3 family transposon; and bla_OXA‑181 is carried on a typical ColKP3‑IncX3 composite plasmid backbone, which is very common among bla_OXA‑181‑carrying plasmids. These different genetic backgrounds likely reflect their respective transmission and evolutionary pathways.

Discussion

ST16: An Emerging High‑Risk Clone

ST16 has recently emerged as a high-risk K. pneumoniae clone with considerable dissemination potential.²⁴ A comprehensive genomic comparison between ST16 and the well‑established high‑risk clone ST11 showed that both lineages possess a marked capacity to acquire antimicrobial resistance and virulence determinants.³ However, whereas ST11 exhibits substantial diversity in capsular and lipopolysaccharide types,²⁵ ST16 is characterized by a highly conserved core genome, with fewer than 1% of core genome sites being polymorphic and a median pairwise single-nucleotide polymorphism (SNP) difference of only 87 among global isolates.²⁶ Notably, the number and diversity of antimicrobial resistance genes in ST16 have increased steadily over time, whereas its virulence gene repertoire has remained relatively stable.²⁶

In the present study, the ST16 isolate KPY1 co‑harbored bla_KPC‑2, bla_OXA‑181, and bla_NDM‑4, further demonstrating the ability of this lineage to accumulate multiple carbapenemase genes. The convergence of class A, class B, and class D carbapenemases in a single ST16 isolate is particularly concerning, as it may substantially limit therapeutic options and facilitate the persistence of extensively drug-resistant phenotypes. Given its conserved genetic backbone, global dissemination potential, and ongoing acquisition of resistance determinants, ST16 may represent an increasingly important public health threat.²⁷

Chromosomal Localization of bla_NDM-4: A Possible IS26-Associated Rearrangement

A striking feature of KPY1 is the chromosomal location of bla_NDM-4. The chromosomal region harboring bla_NDM-4 was flanked by two IS26 elements. The segment between the two IS26 copies shared >99% nucleotide identity with the NDM-4-carrying plasmid pSECR18-2374C (GenBank accession no. CP041930.1), whereas the sequences outside the IS26-flanked region showed >99% identity to chromosomal sequences of K. pneumoniae. These findings suggest that the bla_NDM-4-containing region may have been acquired through an IS26-associated genetic rearrangement event.

Based on these observations, we propose a plausible evolutionary model (Supplementary Figure 2). The structure flanked by two directly oriented IS26 elements resembles a pseudo-compound transposon-like arrangement, suggesting that IS26 may have contributed to the formation or rearrangement of the bla_NDM-4-containing region. One possible scenario is that a translocatable unit containing IS26 and the adjacent bla_NDM-4 gene was initially formed in a plasmid context and subsequently integrated into a pre-existing chromosomal IS26 site. The apparent different origins of the two flanking IS26 elements—one associated with a plasmid-derived resistance region and the other embedded in the chromosomal background—support a two-step evolutionary process.

Such chromosomal integration may have important biological implications. Unlike plasmid-borne resistance genes, chromosomally integrated carbapenemase genes may be stably maintained through vertical inheritance and may be less dependent on plasmid stability or selective pressure. This process may therefore contribute to the long-term persistence of carbapenem resistance in high-risk clones. However, the impact of this localization on long-term persistence requires further investigation.

The observed structure is consistent with previously described IS26-associated rearrangement mechanisms, in which recombination between IS26 copies can facilitate movement or restructuring of resistance regions.^28,29 In KPY1, the pre-existing chromosomal IS26 may have served as a recombination hotspot, facilitating the site-specific integration of the IS26–bla_NDM-4-associated resistance module.

Clinical Implications and Therapeutic Challenges

KPY1 exhibited an extensively drug-resistant phenotype. The isolate was resistant to all tested β-lactam antibiotics, including carbapenems and ceftazidime–avibactam. It was also resistant to colistin, with an ISAeme19 insertion within mgrB representing a potential genetic alteration associated with this phenotype. In addition, KPY1 showed intermediate susceptibility to tigecycline, further limiting available therapeutic options. Only gentamicin and chloramphenicol retained in vitro activity; however, neither is routinely used as a first-line treatment for severe CRE bloodstream infections. These findings indicate that therapeutic options for infections caused by this isolate were extremely limited.

KPY1 was isolated from the bloodstream of a critically ill patient with severe underlying conditions. Due to the complexity of the clinical course and concurrent infections, the contribution of KPY1 to the clinical outcome cannot be independently determined. Nevertheless, the recovery of a triple-carbapenemase-producing CRKP strain from bloodstream infection highlights the therapeutic challenges associated with infections caused by highly resistant K. pneumoniae isolates.

In clinical practice, treatment options for infections caused by extensively drug-resistant K. pneumoniae remain extremely limited.³⁰ Combination regimens, such as tigecycline plus aminoglycosides or colistin plus carbapenems, have been used in selected cases; however, their clinical efficacy remains uncertain and is influenced by infection site, drug exposure, toxicity, and host immune status.^31,32 Therefore, early detection, strict infection-control measures, active surveillance, and antimicrobial stewardship are essential to prevent the spread of such high-risk strains.

The limited therapeutic options available for triple-carbapenemase-producing isolates also highlight the need for alternative treatment strategies. Recently, Roscioli et al demonstrated that human monoclonal antibodies targeting the KL64 capsule protected against pandrug-resistant K. pneumoniae ST147 in a mouse infection model.³³ Although those monoclonal antibodies were specific to the KL64 capsular type, whereas KPY1 belongs to ST16 with KL51 capsular type, the study provides a proof of concept for antibody-based strategies against high-risk K. pneumoniae clones. Future work should explore whether monoclonal antibodies or other immunotherapeutic approaches targeting conserved or clone-specific surface antigens could be developed for ST16 and other carbapenemase-producing lineages.

Limitations

This study has several limitations. First, only a single clinical isolate was analyzed, and therefore our findings may not fully represent the regional epidemiology of triple-carbapenemase-producing K. pneumoniae. Nevertheless, given the rarity of isolates co-harboring class A, B, and D carbapenemases, timely reporting of such strains is important for raising awareness and supporting infection prevention and control efforts.

Second, although comparative genomic analysis suggested that IS26 may have contributed to the chromosomal acquisition of bla_NDM-4, functional validation experiments were not performed. Therefore, the proposed IS26-associated rearrangement mechanism remains to be experimentally confirmed.

Third, although the bla_KPC-2-carrying IncFII(K) plasmid and the bla_OXA-181-carrying ColKP3-IncX3 plasmid belong to plasmid types that have frequently been associated with antimicrobial resistance dissemination, their actual transferability in KPY1 was not experimentally evaluated. Conjugation experiments were not performed in this study; therefore, the horizontal transfer capability of these resistance plasmids remains to be confirmed.

Fourth, detailed clinical treatment information, including pharmacokinetic/pharmacodynamic data, was not available. Therefore, the impact of antimicrobial exposure and other clinical factors on the patient’s outcome could not be fully assessed. In addition, given the complexity of the patient’s clinical condition, the contribution of KPY1 to the unfavorable outcome cannot be independently determined.

Future studies should include additional clinical isolates, functional validation of the proposed IS26-mediated integration mechanism, conjugation experiments to evaluate plasmid transferability, and prospective clinical investigations to better define the epidemiology, transmission dynamics, and clinical impact of triple-carbapenemase-producing K. pneumoniae.

Conclusion

In conclusion, this study characterized an extensively drug-resistant ST16 K. pneumoniae bloodstream isolate co-harboring three clinically important carbapenemase genes, bla_KPC-2, bla_NDM-4, and bla_OXA-181. The coexistence of class A, B, and D carbapenemases in a single isolate was associated with broad resistance to carbapenems, β-lactam/β-lactamase inhibitor combinations, ceftazidime–avibactam, and colistin, leaving very limited remaining in vitro treatment options. Notably, bla_NDM-4 was integrated into the chromosome and flanked by IS26 elements, suggesting a possible mechanism for stable maintenance and vertical transmission of this resistance determinant. In contrast, bla_KPC-2 and bla_OXA-181 were located on distinct plasmid backbones with high similarity to previously reported resistance plasmids, indicating the contribution of both chromosomal integration and plasmid-mediated dissemination to the complex resistance profile of this isolate. These findings highlight the clinical and epidemiological importance of continuous genomic surveillance for high-risk CRKP lineages and underscore the value of complete genome sequencing in resolving complex carbapenemase gene configurations.

Funding Statement

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China [grant number 82502626] and the Postdoctoral Research Start-up Fund from the First Affiliated Hospital of Zhengzhou University.

Abbreviations

AMR, Antimicrobial resistance; AST, Antimicrobial susceptibility testing; BLASTn, Basic Local Alignment Search Tool for nucleotide sequences; BRIG, BLAST Ring Image Generator; CGE, Center for Genomic Epidemiology; CLSI, Clinical and Laboratory Standards Institute; CRE, Carbapenem-resistant Enterobacteriaceae; CRKP, Carbapenem-resistant Klebsiella pneumoniae; eCIM, EDTA-carbapenem inactivation method; FDA, US Food and Drug Administration; GC, Guanine-cytosine; ICU, Intensive care unit; IS, Insertion sequence; KPC, Klebsiella pneumoniae carbapenemase; LB, Luria–Bertani; MBL, Metallo-β-lactamase; mCIM, Modified carbapenem inactivation method; MIC, Minimum inhibitory concentration; MLST, Multilocus sequence typing; NDM, New Delhi metallo-β-lactamase; OXA, Oxacillinase; SCARB, Serine carbapenemase; SNP, Single-nucleotide polymorphism; ST, Sequence type; SVG, Scalable Vector Graphics; XDR, Extensively drug-resistant.

Data Sharing Statement

The complete genome sequence of Klebsiella pneumoniae strain KPY1 has been deposited in GenBank under BioProject accession number PRJNA1291995 and BioSample accession number SAMN62135400. The chromosome and plasmid sequences are available under accession numbers JCBPDJ010000001–JCBPDJ010000006, including the Chromosome_NDM-4 (JCBPDJ010000001) and plasmids KPY1p1 (JCBPDJ010000002), KPY1p2 (JCBPDJ010000003), KPY1p3 (JCBPDJ010000004), KPY1p4_KPC-2 (JCBPDJ010000005), and KPY1p5_OXA-181 (JCBPDJ010000006). Raw Illumina and Oxford Nanopore sequencing reads have been deposited in the NCBI Sequence Read Archive under accession numbers SRR39969794 and SRR39969793, respectively.

Ethics Approval and Informed Consent

This study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Approval No. 2025-KY-1365-001). The study was conducted in accordance with the principles of the Declaration of Helsinki. Written informed consent for publication of the clinical information presented in this study was obtained from the patient’s next of kin. All clinical data used in this study were de-identified to protect patient privacy. All bacterial isolates were obtained from de-identified clinical specimens collected during routine diagnostic procedures, including blood, urine, and respiratory samples.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The author(s) report no conflicts of interest in this work.