Preplanned Studies: Co-harboring bla_KPC-2 and bla_IMP-4 on an IncP Plasmid in A Clinical Isolate of Klebsiella pneumoniae — Shanghai Municipality, China, 2023

Introduction: Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a significant global public health threat. The dissemination of resistance is accelerated by plasmids harboring multiple carbapenemase genes, posing a particular challenge to the limited treatment options, including ceftazidime-avibactam.

Methods: In this study, a CRKP strain, KpBSI024, was isolated from a patient with bloodstream infection in the intensive care unit of a tertiary hospital in China. The whole-genome sequencing combined with bioinformatic analysis was used to investigate the structural features of plasmids and associated resistance genes. In addition, conjugation experiments were conducted to assess the transferability of the resistance plasmid.

Results: KpBSI024 exhibited resistance to carbapenems and ceftazidime-avibactam and was identified as sequence type ST1514. Whole-genome sequencing revealed that two carbapenemase genes, bla_KPC-2 and bla_IMP-4, coexisted on a 53 kb IncP6-type plasmid. This plasmid exhibited a complex structure, likely formed through multiple recombination events mediated by IS26 between plasmids of different Inc types. Although the resistance plasmid encodes a type IV secretion system, it lacks a relaxase gene and is therefore non-self-transmissible; however, it could be transferred at low frequency to Escherichia coli with the assistance of a conjugative plasmid. The growth of the transconjugants was not affected by the acquisition of the resistance plasmid, and they displayed resistance profiles to carbapenems and ceftazidime-avibactam similar to the donor strain.

Conclusions: The coexistence of bla_KPC-2 and bla_IMP-4 on an IncP-type plasmid in a clinical K. pneumoniae isolate highlights the critical role of recombination events in the dissemination of resistance genes. The emergence of such multidrug-resistant plasmids underscores the urgent need for genomic surveillance and the development of innovative antimicrobial strategies to control the spread of high-risk resistance plasmids.

The worldwide prevalence of carbapenem-resistant Klebsiella pneumoniae (CRKP) presents a significant public health challenge. The major carbapenemases include KPC (class A), IMP, VIM, and NDM (class B), and OXA-48 (class D), all of which contribute to nosocomial outbreaks. Recent findings have highlighted the emergence of clinical isolates co-producing multiple carbapenemases, further complicating the already limited therapeutic options (1). This phenomenon is associated with various plasmid-borne carbapenemase genes. Among them, IncP plasmids are notable for their broad host range, high conjugation efficiency, and frequent carriage of resistance genes, facilitating widespread dissemination and microbial adaptation through mobile genetic elements and recombination hotspots (2). In this study, a novel IncP6 plasmid that concurrently harbors the bla_KPC-2 and bla_IMP-4 genes in a K. pneumoniae clinical isolate was identified and designated as KpBSI024.

The strain KpBSI024 was isolated in 2023 from a patient with a bloodstream infection admitted to an intensive care unit (ICU) at a tertiary hospital in Shanghai, China, as part of a surveillance study. Antimicrobial susceptibility testing revealed resistance to β-lactam/β-lactam inhibitors and carbapenems, with MICs of 16 μg/mL for both meropenem and imipenem, and reduced susceptibility to ceftazidime-avibactam (Table 1). Whole-genome sequencing using Illumina and Nanopore platforms (Supplementary Methods) revealed that KpBSI024 belongs to ST1514 and KL109, a rare lineage previously associated only with carbapenem-susceptible K. pneumoniae (3). The genome comprised a chromosome and five plasmids (IncFIB, IncFII, IncQ1, IncP6, and Col440I), with bla_KPC-2 and bla_IMP-4 co-located on the IncP6 plasmid pKpBSI024-3 (Figure 1A). In addition, KpBSI024 harbors the aminoglycoside resistance genes aac(3)-IId, aac(6')-Ib-cr, aadA16, aph(3')-Ia, aph(3'')-Ib, and aph(6)-Id; β-lactam resistance genes bla_CTX-M-3, bla_SHV-61, bla_TEM-1A, and bla_TEM-1B; rifamycin resistance gene ARR-3; amphenicol resistance gene floR; quinolone resistance genes qnrS1, qnrB91, OqxA, OqxB, and mdf(A); folate pathway antagonist resistance genes sul1, sul2, and dfrA27; tetracycline resistance gene tet(A); macrolide resistance gene mph(A); and fosfomycin resistance gene fosA.

Figure 1. 

Organizational schematic of the bla_KPC-2 and bla_IMP-4-carrying plasmid pKpBSI024-3 from the K. pneumoniae clinical isolate KpBSI024. (A) Plasmid map annotated by gene function; (B) Linear comparison of pKpBSI024-3 with reference plasmids p121SC21-KPC2 and p20389-IMP; (C) Bacterial hosts carrying similar plasmids with >90% coverage and >99% identity.

Note: For (A), orange: replication; green: mobile elements; blue: transfer; red: resistance; For (C), based on NCBI nr database analysis.

VRprofile2 analysis indicated that pKpBSI024-3 encodes a type IV secretion system but lacks the relaxase gene and chaperone protein, which are essential for conjugation (Figure 1B). Attempts to transfer pKpBSI024-3 from K. pneumoniae KpBSI024 into E. coli (C600-pA) via conjugation were unsuccessful. However, the mobilization of pKpBSI024-3 was achieved at a frequency of (2.51±2.00)×10⁻⁷ using the helper conjugative plasmid pKP2648-34 (Supplementary Material, Supplementary Tables S1–S2, and Supplementary Figure S1). The self-transmissible pKP2648-34, which is 34 kb in size and lacks any antibiotic resistance genes (4), was first introduced into KpBSI024. Following acquisition of the pKpBSI024-3 plasmid, E. coli (C600-pA) exhibited resistance to carbapenems and ceftazidime-avibactam similar to the donor strain KpBSI024 (Table 1). Growth curve analysis demonstrated that transfer of the 53-kb plasmid pKpBSI024-3 did not impose a substantial metabolic burden on the recipient strain (Supplementary Figure S2).

Comparative genomic analysis revealed that pKpBSI024-3 originated through recombination between two plasmids: IncP6 plasmid p121SC21-KPC2 (55% coverage, 100% identity) (5) and IncN plasmid p20389-IMP (43% coverage, 99.99% identity) (6) (Figure 1B). Sequence analysis indicated a complex mosaic arrangement, wherein the bla_KPC-2 gene resides within a Tn3-based transposon exhibiting a disrupted structure (ΔISEc33-Tn3-ISApu1-orf-ISApu2-ISKpn27-Δbla_TEM-1-bla_KPC-2-ΔISKpn6-korC-klcA-ΔrepB). This structure resembles the Tn1722 transposon unit, a major vehicle for bla_KPC-2 dissemination frequently identified in China, characterized by the conserved ISKpn27-bla_KPC-2-ΔISKpn6 core. However, unlike typical Tn1722, this variant lacks the tnpR and tnpA transposition genes, and includes multiple additional insertion sequences (e.g., ISApu1, ISApu2, and ISEc33). These findings suggest that sequential insertion events and extensive structural rearrangements contributed to the current mosaic architecture of pKpBSI024-3. Meanwhile, the bla_IMP-4 gene is embedded within a class 1 integron associated with IS26. Additional mobile genetic elements, including Tn5045 and IS4321, further emphasize the complexity of the plasmid’s recombination processes. Based on these findings, it was hypothesized that pKpBSI024-3 was formed through a multi-step recombination process. Initially, the IS26-flanked region containing bla_IMP-4 was excised from p20389-IMP via IS26-mediated homologous recombination, forming a circular intermediate. This structure subsequently integrated into the backbone of p121SC21-KPC2. Notably, this insertion event appeared to be accompanied by deletion of a contiguous region associated with conjugative transfer, resulting in a rearranged backbone.

Furthermore, it is notable that the two plasmids involved in the recombination event leading to pKpBSI024-3 exhibit differing transfer capabilities. The plasmid p20389-IMP is self-transmissible, whereas p121SC21-KPC2 contains a mobilization module (mobA-mobE) and may transfer with the assistance of a conjugative plasmid (Figure 1B). Additionally, genomic comparisons with p121SC21-KPC2 and p20389-IMP from the NCBI nr database revealed highly similar plasmids across multiple bacterial hosts and datasets. p20389-IMP has 56 similar plasmids, while p121SC21-KPC2 has 62. These exhibited >90% query coverage and >99% nucleotide identity (Figure 1C). The bacterial hosts involved primarily include K. pneumoniae, E. coli, and C. freundii, spanning more than 20 species. Their distribution is geographically diverse, encompassing more than nine countries, with a significant prevalence in China and Spain (Supplementary Table S3). The widespread presence of these highly similar plasmids highlights the potential for recombination and the associated risk of broad dissemination.

The identification of K. pneumoniae KpBSI024 harboring an IncP6 plasmid containing both bla_KPC-2 and bla_IMP-4 represents a notable advancement in our understanding of multidrug resistance evolution. Although KPC and IMP carbapenemase genes have been individually associated with various plasmid replicon types, including IncP-type plasmids, this is the first report of their co-localization within a single IncP plasmid. In contrast to prior studies documenting IncP plasmids carrying bla_IMP in Pseudomonas or bla_KPC variants in Klebsiella (7–8), this study highlights a novel evolutionary convergence, underscoring the increased recombination capacity and heightened risk of multidrug resistance. Furthermore, this finding differs from the recently characterized IncN-IncR plasmid co-harboring bla_KPC and bla_IMP in K. pneumoniae ST1393, where recombination was facilitated by ISKpn19 and ISKpn27 (9). These observations emphasize the evolutionary pressures driving plasmid recombination and the emergence of complex resistance mechanisms. Despite the requirement for a helper conjugative plasmid to facilitate mobilization of pKpBSI024-3, its genomic resemblance to plasmids identified across diverse bacterial hosts suggests the potential for widespread dissemination.

Plasmid pKpBSI024-3 exemplifies the importance of recombination in promoting genetic diversity among resistance determinants. The interplay of Tn3-based transposons, IS26-associated integrons, and multiple IS elements likely contributed to the integration of resistance genes from various plasmid sources. Such mosaic plasmids pose a considerable threat to antimicrobial treatment, as the co-expression of serine- and metallo-carbapenemases can synergistically compromise the efficacy of available therapies, including next-generation inhibitors such as ceftazidime-avibactam.

From an epidemiological standpoint, the ST1514 lineage remains relatively rare, with prior reports predominantly involving carbapenem-susceptible isolates. The acquisition of a high-risk plasmid such as pKpBSI024-3 may signify a potential shift toward multidrug resistance within this lineage. The limited conjugation observed under laboratory conditions contrasts with the widespread detection of similar plasmids in diverse bacterial hosts, implying possible alternative mechanisms of horizontal gene transfer or environmental adaptation.

The extensive repertoire of resistance genes present on pKpBSI024-3, including those mediating resistance to β-lactams, aminoglycosides, quinolones, and fosfomycin, illustrates the therapeutic difficulties associated with such plasmids. The observed resistance to ceftazidime-avibactam is particularly concerning, as this agent constitutes one of the few remaining treatment options for CRKP infections. The potential for interspecies plasmid transfer further exacerbates these concerns.

The public health implications of such plasmids are profound, as they undermine the efficacy of critical antimicrobial agents and complicate infection control strategies. Continuous surveillance of high-risk plasmids and their derivatives should be prioritized. Moreover, there is an urgent need for novel therapeutic strategies to combat plasmid-mediated multidrug resistance. One promising approach involves the development of CRISPR-Cas-based systems engineered to selectively eliminate resistant plasmids (10).

In conclusion, the identification of the IncP6 plasmid co-harboring bla_KPC-2 and bla_IMP-4 exemplifies the dynamic nature of resistance gene dissemination in K. pneumoniae. The public health implications of such plasmids are substantial. Genomic surveillance efforts should prioritize the detection of plasmid-mediated resistance genes, particularly those capable of co-harboring multiple carbapenemases. Rigorous monitoring of plasmid dynamics and resistance gene transmission is essential to prevent the emergence of untreatable infections. Additionally, the development of novel therapeutic strategies targeting plasmid stability and mobility may offer promising approaches to mitigate the spread of multidrug resistance.

Approved by the Ethics Committee of Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University (No. RJ2019NO1-3).