Characterisation of a Novel Hybrid IncFII_pHN7A8:IncR:IncN Plasmid Co-Harboring bla_NDM-5 and bla_KPC-2 from a Clinical ST11 Carbapenem-Resistant Klebsiella pneumoniae Strain

Introduction

Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a global threat to clinical practice because of its ability to cause severe infections with high mortality rates.^1,2 Carbapenemase production is the dominant cause of carbapenem resistance in carbapenem-resistant Enterobacteriaceae, with KPC and NDM being the most common. KPC-2 carbapenemase, encoded by bla_KPC-2 is predominant in Klebsiella pneumoniae and can be potently inhibited by new β-lactamase inhibitors such as avibactam, vaborbactam, and relebactam. Thus, ceftazidime/avibactam is a valuable treatment option to combat CRKP-producing KPC carbapenemase.³ However, metallo-β-lactamase NDM encoded by bla_NDM, which is most common in Escherichia coli in China,^4,5 cannot be overcome using clinically available β-lactamase inhibitors, including ceftazidime/avibactam.⁶ CRKP, which produces both KPC and NDM, confers resistance to nearly all β-lactams and β-lactamase inhibitors, and presents a significant challenge in clinical treatment owing to extremely limited options. In recent years, the co-production of KPC and NDM (mainly KPC-2 and NDM-1) has become a severe threat to public health, with increasing reports worldwide,^7–12 and has been documented in other Enterobacteriaceae.^13–15 A nationwide survey in China revealed an increased prevalence of CRKP co-producing KPC-2 and NDM-1 in recent years.¹⁶ According to previous reports, bla_KPC and bla_NDM are usually located on separate plasmids that can be transferred to new recipient bacteria through conjugation. There are few reports on plasmids co-carrying bla_KPC and bla_NDM. Recently, an IncR plasmid known as pCF2075-1 carrying both bla_KPC-2 and bla_NDM-1 was identified in Citrobacter freundii.¹⁷ Additionally, an IncHI5 plasmid named pK254-KPC_NDM co-carrying bla_KPC-2 and bla_NDM-1 was discovered in Klebsiella michiganensis.¹⁸ Furthermore, a conjugative hybrid plasmid containing bla_KPC-2 and bla_IMP-4 that co-expressed serine- and metallo-carbapenemases was documented in a clinical Klebsiella quasipneumoniae isolate.¹⁹ Here, we describe the co-occurrence of NDM-5 as the most common metallo-carbapenemase and KPC-2 as the most prevalent serine β-lactamase on a chimeric plasmid from a clinical K. pneumoniae ST11 isolate.

Materials and Methods

Strain Identification and Antimicrobial Susceptibility Testing

Strain ZY27320 was obtained from an abdominal drainage sample of a man aged 61 with severe abdominal infection who was hospitalized in a tertiary hospital in February 2021. The isolate was identified as K. pneumoniae using a Bruker Microflex MALDI-TOF (Bruker, Germany).

The minimum inhibitory concentrations (MICs) of colistin, tigecycline, ceftazidime/avibactam, piperacillin/tazobactam, ceftolozane/tazobactam, imipenem/relebactam, meropenem/vaborbactam, meropenem, imipenem, ertapenem, levofloxacin, ciprofloxacin, amikacin, fosfomycin, and aztreonam were determined using the microbroth dilution method with E. coli ATCC 25922 as a quality control. The results were interpreted based on the MIC breakpoints of the Clinical and Laboratory Standards Institute (CLSI M100-S32, 2022). The MIC breakpoint of tigecycline was based on the standards of the US FDA and National Medical Products Administration (NMPA) in China.

S1 -PFGE and Southern Blot Hybridization

To clarify the plasmid location of bla_KPC-2 and bla_NDM-5, we performed S1-PFGE and Southern hybridization as described previously¹⁹ using digoxigenin-labelled bla_KPC-2 and bla_NDM-5-specific probes with an NBT/BCIP color detection kit (Roche, Mannheim, Germany).

Conjugation Assay

To assess plasmid transferability, we performed conjugation experiments by filter mating and broth mating using different recipient bacteria (E. coli J53, E. coli C600, and induced rifampin-resistant K. pneumoniae ATCC13883). E. coli J53 transconjugants were selected on MH agar plates containing 100 mg/L of sodium azide and 4 mg/L of meropenem. E. coli EC600 transconjugants were selected on MH agar plates containing 600 mg/L of rifampicin and 4 mg/L of meropenem. K. pneumoniae ATCC13883 transconjugants were selected on MH agar plates containing 300 mg/L of rifampicin and 4 mg/L of meropenem.

Whole Genome Sequencing and Analysis

The whole genome of ZY27320 was sequenced using the Illumina NovaSeq6000 (Illumina, San Diego, CA, USA) and Oxford Nanopore MinION (Nanopore, Oxford, UK) platforms (Digital-Micro Biotech Co., Ltd, Hangzhou, China). The hybrid assembly of Illumina and Nanopore reads was performed using Unicycler v0.4.8.²⁰ Complete plasmid sequences were annotated using the RAST server.²¹ Multilocus sequence typing (MLST), resistance genes, virulence genes, and plasmid replicons were performed using MLST 2.0, ResFinder 4.1, VirulenceFinder 2.0, and PlasmidFinder 2.1 web databases (http://www.genomicepidemiology.org/services), respectively. Insertion sequence elements (ISs) were identified using the ISFinder.²² The self-transmissible mobile genetic elements (MGE) and origin of the transfer site were identified using the OriTfinder web server.²³ Sequence alignment and visualization were performed using Proksee web server v1.1.4 and Easyfig v2.2.3.²⁴

Accession Numbers

The complete chromosome and plasmid sequences of Klebsiella pneumoniae ZY27320 were deposited in GenBank under BioProject No. PRJNA975440; accession numbers CP128564-CP128565.

Results

Strain ZY27320 was resistant to all β-lactamase inhibitors (ceftazidime/avibactam, piperacillin/tazobactam, ceftolozane/tazobactam, imipenem/relebactam, and meropenem/vaborbactam), carbapenems (ertapenem, meropenem, and imipenem), quinolones (ciprofloxacin and levofloxacin), aminoglycosides (amikacin), fosfomycin, and aztreonam; it was susceptible only to tigecycline and colistin (Supplementary Table 1).

S1-PFGE detected one plasmid approximately 167 kb in size in ZY27320 cells. Southern blotting with KPC and NDM probes revealed bands at the same location, suggesting that bla_KPC-2 and bla_NDM-5 were located on the same plasmid (Supplementary Figure 1).

To better understand the genetic environment of bla_KPC-2 and bla_NDM-5, the complete genome of ZY27320 was successfully sequenced by Illumina and Nanopore sequencing. The hybrid assembly using Unicycler revealed a circular chromosome of 5,578,590 bp (accession no. CP128564) with a GC content of 57.3%, and one circular plasmid of 167,277bp, designated pKPC2-NDM5 (accession no. CP128565) with a GC content of 53.0%. MLST typing of the assembled genome showed that ZY27320 belonged to the ST11. The chromosome of ZY27320 carries three antimicrobial resistance (AMR) genes (conferring resistance to fosfomycin (fosA), β-lactams (bla_SHV-182), and aminoglycosides (aadA2)) and three virulence factors, including fyuA encoding a siderophore receptor, iutA encoding a ferric aerobactin receptor, and an irp2 gene.

pKPC2_NDM5 is a multiple drug resistance (MDR) plasmid that carries multiple AMR genes conferring resistance to β-lactams (bla_KPC-2, bla_NDM-5, bla_CTX-M-65, and bla_TEM-1B), fosfomycin (fosA3), streptomycin (aadA2), sulfamethoxazole (sul1), aminoglycosides (rmtB), and trimethoprim (dfrA12). In addition, plasmid pKPC2_NDM5 showed no sequence similarity to the virulence plasmid pLVPK²⁵ and contained no virulence factors. We compared the sequence of pKPC2_NDM5 with the plasmid sequences co-carrying bla_KPC-2 and bla_NDM-1 registered in GenBank.^17,18 BLASTN results showed that pKPC2_NDM5 exhibited low coverage (20%) with an IncR plasmid pCF2075-1 (accession no. CP119166) from an Citrobacter freundii strain, and shared 24% coverage with an IncHI5 plasmid pK254-KPC_NDM (accession no. OM938013) from an Klebsiella michiganensis strain. Three replicons, IncFII_pHN7A8, IncR, and IncN, were identified in pKPC2_NDM5 by using PlasmidFinder. IncFII_pHN7A8:IncR bla_KPC-bearing plasmids are widely disseminated in K. pneumoniae strains from China and are typically associated with ST11 strains.²⁶ Plasmid pKPC2_NDM5 had high coverage (73–90%) and sequence similarity (99.93– 99.97%) with several IncFII_pHN7A8:IncR:IncN bla_KPC2-carrying plasmids of K. pneumoniae strain from Zhengzhou City, China, such as pKP18-41-KPC2 (accession no. CP082012) and pKP19-2196-KPC2 (accession no. CP082042) and pKP18-2172-KPC2 (accession no. CP082039) and pKP18-1-KPC2 (accession no. CP082003) and pKPN-hnqyy-kpc (accession no. CP074118).²⁷ Subsequently, a series of bla_NDM-5-carrying plasmids with high similarity (99.53–100%) appeared to carry complementary sequences to form the backbone of pKPC2_NDM5. A bla_NDM-5-carrying plasmid pKPN-hnqyy-ndm (accession no. CP074117) exhibited 33% coverage and 100% identity, coincidentally originating from the same NDM-5 and KPC-2 co-producing K. pneumoniae isolate K9 as the bla_KPC-2-carrying plasmid pKPN-hnqyy-kpc (accession no. CP074118).²⁷ Interestingly, these two plasmids from a single NDM-5 and KPC-2 coproducing K. pneumoniae appeared to integrate into the backbone of pKPC2_NDM5 (Figure 1). Detailed sequence analysis revealed that pKPC2_NDM5 was a cointegrate consisting of four regions: an ~116 kb IncFII_pHN7A8:IncR:IncN plasmid-derived KPC-module (Region-1), an ~ 2.4 kb fosA carrying region (Region-2), an ~ 43.5kb bla_NDM-5 carrying NDM-module (Region-3), and an ~ 4.6 kb bla_TEM-1B and rmtB carrying region (Region-4). The Region-1 was perfectly aligned with the plasmid pKPN-hnqyy-kpc and Region-3 shares 100% sequence identity with that of plasmid pKPN-hnqyy-ndm. All four regions are bracketed by two IS26 elements (Figure 2). The formation of pKPC2_NDM5 appeared to be a recombination event in which an IS26-flanked NDM-fragment, IS26-bounded fosA-fragment and an IS26-bounded bla_TEM-1B - rmtB-fragment were inserted into an IncFII_pHN7A8:IncR:IncN KPC-plasmid (plasmid pKPN-hnqyy-kpc) using IS26 as the insertion site.

Figure 1 Schematic map of plasmid pKPC2_NDM5 and alignment with other similar plasmids from GenBank. The visualization map was generated by Proksee web server v1.1.4.

Figure 2 Sequence alignment of plasmid pKPC2_NDM5 with sequences of pKPN-hnqyy-ndm (GenBank accession no. CP074117), and pKPN-hnqyy-kpc (GenBank accession no. CP074118). The visualization map was created by EasyFig software.

We further analyzed the environments of bla_KPC-2 and bla_NDM-5 (Figure 3). bla_KPC-2 is located on a non-Tn4401 element with the following structures: IS26, ΔtnpR, ISKpn27, bla_KPC-2, and ISKpn6-korC-klcA-ΔrepA-tnpR-ΔtnpA-IS26. The gene bla_KPC has typically been found in classical Tn4401 elements,²⁶ as well as non-Tn4401 elements (NTE_KPC), including NTE_KPC-I, NTE_KPC-II, and NTE_KPC-III.^28,29 The bla_KPC-bearing genetic elements in pKPC2-NDM5 were assigned to the NTE_KPC-I group because Δbla_TEM or Tn5563/IS6100 were not inserted upstream of bla_KPC-2.³⁰ Compared with the bla_KPC-carrying fragment with the NTE_KPC-Ib structure in pKPHS2 (Accession no. CP003224), a large part of Tn1721 tnpA was absent and was replaced by an IS26 element in the bla_KPC-2 genetic context of pKPC2_NDM5. In addition, the genetic environment of bla_KPC-2 in pKPC2_NDM5 had 92% query coverage compared to that of the plasmid pKPC2_020009 (accession no. CP038003) carrying a truncated tnpA of Tn1721.

Figure 3 (A) Linear comparison of genetic environment of blaNDM-5 of pKPC2_NDM5 and that of pNDM_2_P21_SE1_04.20. (B) Linear comparison of the genetic environment of blaKPC-2 among pKP048, pKPHS2, pKPC2_NDM5, and pKPC2_020009. The plasmid comparison figure was generated by EasyFig software. Regions with identical sequences are shown in gray shadow between the different plasmids.

The bla_NDM-5 gene is located in a 16.7-kb multi-resistance module carrying dfrA12, aadA2, sul1, bla_NDM-5, rmtB, and bla_TEM-1B flanked by IS26 elements. The bla_NDM-bearing element of pKPC2_NDM5 carries the common feature of ΔISAba125-bla_NDM-ble_MBL⁶ and has the same genes as classical Tn125 including trpF and dsbC downstream of ble_MBL. In contrast to Tn125, the bla_NDM-carrying element in pKPC2_NDM5 lost several genes, including cutA1, groES-groEL, and ISCR27, but had one additional IS26 element upstream of ΔISAba125 and carried an additional AGR gene cluster (ISCR1-sul1-aadA2-dfrA12-intI1-IS26) downstream of dsbC. BLAST analysis revealed that the bla_NDM-bearing element in pKPC2_NDM5 was highly similar (99.99% identity and 94% coverage) to that of pNDM_2_P21_SE1_04.20 (accession no. CP085076.1) of one clinical Escherichia coli isolate from the United Kingdom (Figure 3) and many other plasmids, such as pM217_FII from Myanmar (not shown).

To understand the transmission capability of pKPC2_NDM5, we performed conjugation assays. We attempted filter mating and broth mating with different recipient bacteria (K. pneumoniae ATCC13883, E. coli strains EC600 and ECJ53) and tested various donor: recipient ratios. We did not obtain a transconjugant under any conditions. Plasmid extraction and electroporation were both unsuccessful. These results suggest that pKPC2_NDM5 is nontransmissible. To test this hypothesis, we submitted the plasmid sequence to the OriTfinder web server.^23,31 The plasmid pKPC2_NDM5 sequence contained an oriT region (153,968–154,068 bp) with a conserved nick region (154,025–154,034 bp), relaxase-encoding gene, type IV coupling protein-encoding gene trwB, and two T4SS gene clusters: traB-traK-traE-traL-traA-traY-traM (80,324–85,114 bp) and trwN-trwM-trwK-trwJ-trwI-trwG-trwF-trwE (135,941–153,401 bp). The conjugative region of the self-transmissible MGE seems to be intact, but pKPC2_NDM5 lacks transfer ability which still presents a puzzle.

Discussion

bla_KPC-2 is the predominant carbapenemase gene widely spread among Klebsiella pneumoniae ST11 and is usually carried by IncFII-like plasmids in China.³² In our study, the backbone of pKPC2-NDM5 displayed a multi-replicon IncFII_pHN7A8:IncR:IncN chimera structure, which may have evolved from several recombination events as described in previous study.²⁷ NDM-5, as a variant of NDM-1, was found to exhibit enhanced carbapenemase activity and hydrolysis ability of ceftazidime/avibactam,³³ and was carried by plasmids with diverse replicon types, including IncX3, IncFII, IncC, IncN, and IncR. Co-harboring bla_NDM-5 and bla_KPC-2 on a multidrug-resistant plasmid confers high-level resistance to both carbapenems and ceftazidime/avibactam and therefore becomes a significant challenge in clinical treatment. The plasmids co-carrying bla_KPC-2 and bla_NDM-1 were recently reported in Citrobacter freundii and Klebsiella michiganensis.^17,18 The emergence of this kind of plasmid co-carrying bla_KPC-2 and bla_NDM-5 in the most prevalent ST clone of K. pneumoniae, poses a significant public health threat and deserves significant attention.

Plasmid pKPC2-NDM5 appeared to be a hybrid plasmid generated by the movement of an IS26-associated translatable unit. The high copy number of IS26 in pKPC2_NDM5 and its presence in each composition module suggest that IS26 plays an essential role in the mobilization and recombination of AMR genes.^34,35 However, we did not recognize matching pairs of target site duplications (TSD) at the junction of each IS26, which are usually considered as the “tracers” of transposition. The lack of flanking TSDs was not uncommon, as reported previously, perhaps because of homologous recombination following IS26 transposition.³⁶ Another explanation is that IS26 transposes through replicative transposition, which can generate IS26 copies without two-sided TSDs.³⁴

pKPC2-NDM5 was non-conjugative, although it carried conjugal modules of the self-transmissible MGE. Interestingly, as the backbone of pKPC2_NDM5, the plasmid pKPN-hnqyy-kpc was proved to have high transfer ability to E. coli J53, however, plasmid pKPN-hnqyy-ndm was a non-conjugative plasmid.²⁷ Previous studies have shown that the expression of conjugative systems is tightly controlled, because the conjugation process is energetically expensive.³⁷ Although we could not measure the fitness cost of this plasmid, we suspected that the loss of horizontal transfer ability was a manifestation of fitness cost. Furthermore, although there have been many reports on the concurrence of the bla_NDM-plasmid and bla_KPC-plasmid in the same K. pneumoniae isolate,^9,16,38 the same plasmid co-harboring bla_NDM and bla_KPC in K. pneumoniae has not yet been reported. However, with the widespread use of ceftazidime/avibactam and frequent combined with carbapenems in clinical practice, plasmids co-carrying these two critical carbapenemase genes might gradually gain a selection advantage during continuous evolution. Therefore, KPC-NDM-plasmid could become a great challenge in the treatment of CRE infections and is worth strengthening monitoring in the future.

Conclusion

In this study, we characterized a novel hybrid MDR plasmid co-harboring bla_KPC-2 and bla_NDM-5 obtained from an ST11 clinical K. pneumoniae isolate. This chimeric plasmid is likely a consequence of a recombination event involving the insertion of a bla_NDM-5 fragment into an IncFII_pHN7A8:IncR:IncN bla_KPC2-carrying plasmid, probably mediated by IS26 elements. Despite the lack of plasmid self-transmissibility, integration of these two critical carbapenemase genes, bla_NDM-5 and bla_KPC-2, into the same plasmid in K. pneumoniae presents great challenges for clinical treatment and requires stringent surveillance.