Identification of a Novel KPC Variant, KPC-204, Conferring Resistance to Both Carbapenems and Ceftazidime–Avibactam in an ST11 Klebsiella pneumoniae Strain

This study describes KPC-204, a novel variant of Klebsiella pneumoniae carbapenemase, characterized by a Lys-Asp-Asp (KDD) amino acid insertion at Ambler position 269 deviates from KPC-2. This variant was identified in an ST11-type clinical isolate of carbapenem-resistant Klebsiella pneumoniae from China. Notably, KPC-204 exhibits resistance to both ceftazidime-avibactam and carbapenems. Genetic analysis revealed that blaKPC-204 was located on a highly mobile IncFII/IncR plasmid within a complex genetic structure that facilitates its spread. Functional analysis, achieved through cloning into E. coli DH5α, validates KPC-204’s contribution to increased resistance to ceftazidime-avibactam. The kinetic parameters showed that KPC-204 exhibited similar affinity to KPC-2 toward ceftazidime and reduced sensitivity to avibactam. Docking simulations revealed a weaker interaction between KPC-204 and avibactam compared to KPC-2. Mating experiments demonstrated the resistance’s transmissibility. This investigation underscores the evolving diversity of KPC variants affecting ceftazidime-avibactam resistance, highlighting the necessity for continuous monitoring.


The Strains and In Vitro Susceptibility
The K. pneumoniae strain, designated 130125, was isolated from the respiratory tract secretion of a patient in 2017 in the intensive care unit (ICU) at West China Hospital.The patient received treatment with penicillin and meropenem for a duration of 12 days, followed by a 3-day course of cefoperazone-sulbactam before the sample was taken.Preliminary species identification was performed by Vitek II (bioMérieux, Marcy-l'Étoile, France).The MICs of antimicrobial agents were determined using the microdilution method of the Clinical and Laboratory Standards Institute (CLSI) [15].Avibactam was added at a fixed concentration of 4 mg/L, tazobactam at 4 mg/L, relebactam at 4 mg/L, and vaborbactam at 8 mg/L.

qRT-PCR
The expression of the bla KPC gene was assessed using qRT-PCR.RNA was extracted from late-exponential-phase cultures using the TRIzol reagent (Invitrogen China Limited, Beijing, China).Genomic DNA was removed, and cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa Biotechnology (Dalian), Dalian, China).qRT-PCR was conducted on a LightCycler 96 (Roche, Basel, Switzerland) using SYBR Premix Ex Taq II (Tli RNaseH plus kit) (TaKaRa Biotechnology (Dalian), Dalian, China) according to the manufacturer's instructions.The assays utilized primers for bla KPC and the housekeeping gene rpoB listed in Table S1.The expression of bla KPC was normalized to the housekeeping gene rpoB.The relative expression was calibrated against isolate 015093.Relative transcript levels were calculated using the 2 −∆∆CT formula based on the mean values.For each strain, three independent cultures were used to extract RNA as three biological replicates, and for each RNA sample, the whole process of qRT-PCR was repeated in triplicate as technical replicates.

Cloning Experiment
To evaluate the KPC-204 variant's role in CZA resistance in K. pneumoniae 130125, the bla KPC-204 and bla KPC-2 genes, along with their promoter regions, were amplified from strains 130125 and 015093 using primers KPC_NdeI_F and KPC_EcoRI_R listed in Table S1.The amplified products and pET-28a vector were digested with NdeI and EcoRI enzymes, ligated with T4 ligase, and transformed into E. coli DH5α (Tsingke, Beijing, China), as described before [25].Transformants were selected on Luria-Bertani agar plates containing 50 mg/L kanamycin, confirmed through PCR-employing primers KPC-F and KPC-R listed in Table S1, followed by Sanger sequencing validation.A control utilizing the empty vector pET-28a was similarly established in E. coli DH5α.

Kinetic Assay and Determination of IC 50 Values
The bla KPC gene sequence (residues 25-293) was cloned into the pET-28a vector using In-Fusion HD Cloning Kits (Takara Bio, Kusatsu, Japan).The bla KPC gene sequence (residues 25-293) and the pET-28a vector were amplified with the primers listed in Table S1.The resultant recombinants were then transformed into E. coli Transetta (DE3)-competent cells (Novagen, Sacramento, CA, USA).Protein purification was achieved via Ni-NTA affinity chromatography, as described previously [26].Extinction coefficients and wavelengths were adopted from earlier studies [11].The enzyme kinetics assay was performed in triplicate using a SPECTROstar Nano microplate reader (BMG Labtech, Ortenberg, Germany) for 15 min in each round.Kinetic parameters (K m and k cat ) were determined using non-linear regression analysis with GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA).
The IC 50 values for the inhibition of KPC-2 and KPC-204 proteins by avibactam, tazobactam, and clavulanic acid were determined using nitrocefin as the substrate.The enzymes were mixed with these inhibitors at concentrations ranging from 0 to 30 µM in PBS and incubated for 10 min, after which 100 µM of nitrocefin was added.Absorbance at 482 nm was recorded after 30 min and analyzed with GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA).This procedure was replicated in three independent experiments.

Structure Prediction
The crystal structure of the KPC-2 protein was retrieved from the PDB database (PDB ID: 2OV5, https://doi.org/10.2210/pdb2OV5/pdb,accessed on 28 April 2024) [27].AlphaFold v2.2.3 [28] was employed to generate a structural model for the KPC-204 mutant.All software utilized, including AlphaFold v2.2.3 for CC312 modeling and the openMM Amber force field for protein relaxation, ran on a local server equipped with a powerful dual-channel E5-2697 Intel 24-core CPU, Nvidia A100 (40 GB) GPU, 96 GB of memory, for efficient computation.Notably, AlphaFold's "monomer" model searched all genetic databases used in CASP14 to create the most accurate model, with the final selection based on the highest plddt confidence score.This combined approach ensured the availability of reliable protein structures for further analysis.

Comparative Secondary and Stereoscopic Structures
Amino acid sequence alignments and secondary structure predictions were conducted using ESPript 3 [29], with the crystal structure of KPC-2 (PDB ID: 2OV5) as a reference for secondary structure depiction.Stereoscopic structures of KPC-204 and KPC-2 were aligned using PyMOL v2.5.2 software.

Molecular Docking
AutoDock4.2.6 [30] software was used for the molecular docking simulations.Threedimensional protein structures were retrieved from the Protein Data Bank (PDB) database and AlphaFold modeling.Ligand structures were built using Chem3D v20.1 software.The protein and ligand structures were then prepared for docking calculations by converting them into the pdbqt file format according to the AutoDock protocol.All docking parameters were maintained at their default settings, except for the maximum number of energy evaluations (eval) and the number of genetic algorithm (GA) runs.The docking simulations were executed using the prepareCovalent.pyPython script, and the resulting interactions were analyzed using PyMOL software.A site-specific approach ensured ligands explored the relevant binding pocket of the KPC enzymes.The grid box, with a spacing of 0.375 Å and dimensions of 60 × 60 × 60 points, was centered at coordinates (59.28, −23.36, −4.18) Å.

Mating Experiments
Mating experiments were performed in broth and on filters, using E. coli J53 AziR (an azide-resistant variant of J53) as the recipient, at 25 • C and 37 • C, as described previously [31].Transconjugants were selected on LB agar with 4 mg/L of ceftazidime and 150 mg/L of sodium azide.The bla KPC-204 gene and plasmid replicons in transconjugants were verified via PCR using primers listed in Table S1, followed by confirmation through Sanger sequencing.

Genomic Analysis of Clinical K. pneumoniae Isolate 130125
The complete genome sequence of strain 130125 was obtained by de novo hybrid assembly of both short (Illumina) and long (Nanopore) reads, and had a 5.4 Mb circular chromosome and three plasmids (Table 2).Strain 130125 harbored the bla KPC-204 gene, encoding KPC-204, a novel variant with a three-amino-acid insertion (Lys-Asp-Asp) between amino acids 268 and 269 within loop 267-275, diverging from KPC-2.Strain 130125 was identified as K. pneumoniae, exhibiting 99.67% average nucleotide identity (ANI) with reference strain HS11286 (GCF_000240185.1),exceeding the classification threshold of ≥96% ANI for bacterial species.Strain 130125 was classified as ST11, a prevalent type of CRKP in China, and exhibited the KL64 capsule type.The genetic assessment of ompK35 and ompK36 genes identified characteristic mutations in ST11 K. pneumoniae strains included a truncation in ompK35 and an insertion of GD amino acids at positions 134-135 in ompK36 [32].

Relative blaKPC Gene's Expression Levels
Quantitative Real-Time PCR (qRT-PCR) was employed to assess the relative blaKPC gene's expression levels using K. pneumoniae 015093 [35] (an ST11 isolate that produces blaKPC-2 and exhibits susceptibility to CZA, sourced from our institution, with MIC values presented in Table 1) as a reference.The expression of blaKPC in isolate 130125 showed a (1.104 ± 0.135)-fold increase compared to isolate 015093, a difference that was statistically insignificant (p > 0.05) (Figure 2).Alignment of pKPC204_130125 with pKPC2_015093.pKPC2_015093 is used as a reference.The alignment, conducted as a pairwise BLASTn comparison using the BLAST Ring Image Generator (BRIG) [23], between plasmid pKPC204_130125 and pKPC2_015093 (GenBank accession no.CP036301) demonstrated a 100% coverage and 99.97% identity.The bla KPC-204 gene is located within a composite transposon, flanked downstream by ISKpn27 and upstream by ISKpn6.Moreover, bla KPC-204 , in conjunction with bla CTX-M-65 , is part of a 10 kb integrative composite transposon, bounded by IS26 sequences.The locations of tra genes, pivotal for conjugation [34], are indicated.

Relative bla KPC Gene's Expression Levels
Quantitative Real-Time PCR (qRT-PCR) was employed to assess the relative bla KPC gene's expression levels using K. pneumoniae 015093 [35] (an ST11 isolate that produces bla KPC-2 and exhibits susceptibility to CZA, sourced from our institution, with MIC values presented in Table 1) as a reference.The expression of bla KPC in isolate 130125 showed a (1.104 ± 0.135)-fold increase compared to isolate 015093, a difference that was statistically insignificant (p > 0.05) (Figure 2).

Enzyme Kinetic Parameters and IC50 Values
The enzymatic kinetics analyses revealed that KPC-204 exhibits comparable catalytic efficiencies with substrates such as nitrocefin, ceftazidime, and meropenem to those of KPC-2, as shown in Table 3.The hydrolytic profile of KPC-204 was consistent with the MIC observations presented above (Table 1).Moreover, the IC50 value, defined as the concentration required to achieve 50% inhibition of avibactam, was approximately 16 times greater for KPC-204 compared to KPC-2.This suggests that the insertion at position 269 (ins_269_KDD) in KPC-204 correlates with a reduced affinity and diminished sensitivity to avibactam.In contrast, the inhibitory effects of tazobactam and clavulanic acid on KPC-204 were markedly stronger than on KPC-2, displaying approximately 21-fold and 7-fold lower IC50 values, respectively (Table 4).

Enzyme Kinetic Parameters and IC 50 Values
The enzymatic kinetics analyses revealed that KPC-204 exhibits comparable catalytic efficiencies with substrates such as nitrocefin, ceftazidime, and meropenem to those of KPC-2, as shown in Table 3.The hydrolytic profile of KPC-204 was consistent with the MIC observations presented above (Table 1).Moreover, the IC 50 value, defined as the concentration required to achieve 50% inhibition of avibactam, was approximately 16 times greater for KPC-204 compared to KPC-2.This suggests that the insertion at position 269 (ins_269_KDD) in KPC-204 correlates with a reduced affinity and diminished sensitivity to avibactam.In contrast, the inhibitory effects of tazobactam and clavulanic acid on KPC-204 were markedly stronger than on KPC-2, displaying approximately 21-fold and 7-fold lower IC 50 values, respectively (Table 4).Table 3. Kinetic parameters of purified β-lactamases KPC-2 and KPC-204 a .

Comparative Secondary and Stereoscopic Structures of KPC-204 and Related Variants
The secondary structures of KPC-204 and other KPC variants with insertions at Ambler position 269 were aligned using data from the NCBI database (April 2024) (Figure 3a).This alignment focused on the Omega loop (residues 164-179) and loop 267-275 regions, both crucial for enzyme activity and antibiotic resistance.Notably, KPC-204 and KPC-29 share an identical KDD insertion at position 269 despite their distinct evolutionary origins (KPC-2 and KPC-3, respectively), indicating potential convergent evolution.KPC-67 contains a KDDKDD insertion at the same position.KPC-29 and KPC-67 were previously reported to exhibit resistance to both CZA and MEM [36], warranting further studies on their enzyme kinetics and structure.
Stereoscopic structural analysis between KPC-204 and KPC-2 reveals a high degree of similarity in the Omega loop (residues 164-179).However, significant divergence is observed in loop 267-275 (Figure 3b).In KPC-204, the insertion of three amino acids (DDK) at position 269 extends the loop 267-275 range.A comparison of key hydrogen bonds in loop regions 237-243 and 267-275 between KPC-2 and KPC-204 shows that in KPC-2, Tyr241 and Lys270 form a hydrogen bond with a bond length of 3.2 Å, Gly242 and Asp272 form a hydrogen bond with a bond length of 3.0 Å, and Tyr241 and Ala267 form a hydrogen bond with a bond length of 2.8 Å.In KPC-204, Gly242 and Asp275 form a hydrogen bond with a bond length of 2.9 Å, and Tyr241 and Ala267 form a hydrogen bond with a bond length of 2.7 Å. The hydrogen bond between Tyr241 and Lys270 is no longer present (Figure 3c).

Molecular Docking of KPC-204 and KPC-2 with Ceftazidime, Avibactam and Meropenem
We then performed molecular docking simulations to visualize the interactions between KPC-204 and KPC-2 enzymes with ceftazidime, avibactam and meropenem (Figure 4).Interestingly, both KPC-204 and KPC-2 display similar binding scores for ceftazidime (−11.26kcal/mol and −11.62 kcal/mol, respectively), with slight variations in the specific hydrogen bonding residues (Asn170 vs. Leu169).In contrast, avibactam lacks the hydrogen bond with the side chain of Thr235 in KPC-204, which is present in KPC-2.Moreover, the binding free energy is significantly weaker in KPC-204 (−5.09 kcal/mol) compared to KPC-2 (−8.92 kcal/mol), indicating a reduced affinity for the protein.This diminished interaction may contribute to the resistance observed in KPC-204.KPC-204's KDD insertion at position 269 likely weakens its meropenem resistance compared to KPC-2.This is suggested by both the weaker binding affinity observed in docking analysis (−6.33 kcal/mol vs. −8.72 kcal/mol) and the absence of a key hydrogen bond with Glu166, which is present in KPC-2.3.9.bla KPC-204 Was Located in a Self-Transmissible Plasmid The transfer frequencies of plasmids pKPC2_015093 and pKPC204_130125 to E. coli J53 AziR were 2.4 × 10 −5 and 8.6 × 10 −4 , respectively, based on the ratio of transconjugants to recipients, highlighting that pKPC204_130125 is readily self-transmissible.MICs for meropenem in E. coli J53 AziR carrying the respective plasmids were 32 mg/L and 64 mg/L.MICs for CZA were 0.5 mg/L for J53 with pKPC2_015093 and 64 mg/L for J53 with pKPC204_130125 (Table 1).

Discussion
Carbapenem antibiotics are the last-resort drugs for treating infections caused by multidrug-resistant Gram-negative bacteria, and CZA is crucial for treating CRKP infections.KPC-2 and KPC-3 are the most prevalent KPC enzymes [4].KPC-2-producing ST11-type CRKP strains have emerged as a prevalent clonal lineage in China, posing significant clinical challenges [37,38].With the global utilization of CZA, resistance to CZA has increased, primarily due to novel mutations in the genes encoding the KPC enzyme [4].Reports of CZA-resistant KPC variants are common.194 KPC variants were been identi-fied in the NCBI Reference Sequences (RefSeq) database (https://www.ncbi.nlm.nih.gov/pathogens/refgene/#KPC; accessed on 28 April 2024).
Simultaneous resistance to CZA and MEM is rare.Muresu et al. reported [39] a K. pneumoniae strain harboring bla KPC-31 and bla OXA-181 that was resistant to both CZA and MEM, but the cloning of bla KPC-31 [40] and bla OXA-181 [41] did not show MEM resistance.Arcari et al. reported [42] a K. pneumoniae strain carrying bla KPC-154 that showed MEM resistance, but resistance was not observed when the bla KPC-154 gene was cloned.This suggests the involvement of bla KPC gene over-expression and porin modifications, reflecting the complexity of resistance mechanisms.
We identified an ST11-type K. pneumoniae strain simultaneously resistant to CZA and MEM, with no prior exposure to CZA.Compared to the control strain 015093, there were no significant differences in bla KPC gene expression levels in strain 130125.Our study identified KPC-204, featuring a KDD insertion at position 269 within loop 267-275, which demonstrated resistance to both CZA and MEM after cloning.Notably, instances of CZA resistance in K. pneumoniae have been reported even in the absence of prior CZA exposure [36,39,43].Given the high genomic similarity between this variant and the ST11 KPC2 strain 015093, it suggests a single case arising from a spontaneous mutation.
Enzyme kinetic analyses revealed that KPC-204 maintains similar catalytic activity towards ceftazidime and meropenem compared to KPC-2.However, KPC-204 displays a significantly reduced affinity for avibactam, reflected in a 16-fold increase in the IC50 value.Docking simulations support these findings, revealing a weaker interaction between KPC-204 and avibactam compared to KPC-2.The DDK insertion at position 269 of the KPC-204 is situated in the loop 267-275 region, which is peripheral and does not directly interact with the binding pocket or the substrate.KPC-204 may alter the conformation of the binding pocket and substrate affinity by modifying loop 267-275, subsequently affecting loop 237-243.
The KPC-type carbapenemase gene frequently resides on self-conjugative plasmids, facilitating its spread across bacterial populations [4,[47][48][49].Specifically, bla KPC-204 is harbored on an IncFII/IncR plasmid, known for its capability to transfer horizontally via conjugation, thus highlighting the need for rigorous monitoring.The genetic context of bla KPC-204 and plasmid pKPC2_015093 shows a high degree of similarity, positioned within a composite transposon, flanked downstream by ISKpn27 and upstream by ISKpn6.This arrangement is similar to that found in pKP048 from K. pneumoniae isolates in China, yet it diverges from Tn4401 [50,51].Additionally, bla KPC-204 , together with bla CTX-M-65 , forms part of a 10 kb integrative composite transposon, enclosed by IS26 sequences, indicating the potential for mobility and spread that warrants heightened attention.On a positive note, recent studies have demonstrated the efficacy of novel inhibitor combinations, such as imipenem-relebactam and meropenem-vaborbactam, in addressing these resistant strains.

Conclusions
In conclusion, our study is notable for several reasons.Firstly, we reported an ST11type clinical CRKP isolate that produces KPC-204, a novel plasmid-borne KPC variant that confers CZA resistance.Secondly, we documented a rare antimicrobial resistance profile, demonstrating resistance to both CZA and meropenem.Thirdly, we investigated the enzymatic changes induced by the KDD insertion at position 269, which diminishes the inhibitory efficacy of avibactam, leading to resistance.Docking simulations support these findings, revealing a weaker interaction between KPC-204 and avibactam compared to KPC-2.Lastly, we examined the genetic context of KPC-204, located on a highly transmissible IncFII/IncR plasmid within a composite transposon, presenting a potential for mobility and spread that warrants significant attention.These findings emphasize the need for the vigilant monitoring and development of novel therapeutic strategies to manage such resistant bacterial strains effectively.

Figure 1 .
Figure 1.Alignment of pKPC204_130125 with pKPC2_015093.pKPC2_015093 is used as a reference.The alignment, conducted as a pairwise BLASTn comparison using the BLAST Ring Image Generator (BRIG)[23], between plasmid pKPC204_130125 and pKPC2_015093 (GenBank accession no.CP036301) demonstrated a 100% coverage and 99.97% identity.The blaKPC-204 gene is located within a composite transposon, flanked downstream by ISKpn27 and upstream by ISKpn6.Moreover, blaKPC-204, in conjunction with blaCTX-M-65, is part of a 10 kb integrative composite transposon, bounded by IS26 sequences.The locations of tra genes, pivotal for conjugation[34], are indicated.

Figure 1 .
Figure1.Alignment of pKPC204_130125 with pKPC2_015093.pKPC2_015093 is used as a reference.The alignment, conducted as a pairwise BLASTn comparison using the BLAST Ring Image Generator (BRIG)[23], between plasmid pKPC204_130125 and pKPC2_015093 (GenBank accession no.CP036301) demonstrated a 100% coverage and 99.97% identity.The bla KPC-204 gene is located within a composite transposon, flanked downstream by ISKpn27 and upstream by ISKpn6.Moreover, bla KPC-204 , in conjunction with bla CTX-M-65 , is part of a 10 kb integrative composite transposon, bounded by IS26 sequences.The locations of tra genes, pivotal for conjugation[34], are indicated.

Figure 2 .
Figure 2. The relative expression level of bla KPC compared to strain 015093.The relative expression of bla KPC mRNA in isolate 130125 was 1.104 ± 0.135 times that of isolate 015093 (p > 0.05); ns, p > 0.05 (Student's t-test).

Microorganisms 2024 , 15 Figure 3 .Figure 3 .
Figure 3.The secondary structure of KPC-204 compared with closely related KPC variants and the stereoscopic structure alignment of KPC-204 with KPC-2.(a) The alignment of secondary structures.Closely related KPC variants are those with insertions at Ambler position 269, available in the NCBI database (April 2024).The image shows the Omega loop (residues 164-179) and loop 267-275 regions.Secondary structure elements, α helixes, β sheets, and 310-helixes (represented by η), are indicated.β-strands are rendered as arrows, and strict β-turns are shown as TT letters.(b) Stereoscopic structure alignment.KPC-204 and KPC-2 are colored in blue and green, respectively.The Omega loop and loop 267-275 of both KPCs are highlighted in red.(c) The comparison of key hydrogen bonds in loop regions 237-243 and 267-275 between KPC-2 and KPC-204.In KPC-2, Tyr241 and Lys270 form a hydrogen bond with a bond length of 3.2 Å, Gly242 and Asp272 form a hydrogen Figure 3.The secondary structure of KPC-204 compared with closely related KPC variants and the stereoscopic structure alignment of KPC-204 with KPC-2.(a) The alignment of secondary structures.

Microorganisms 2024 ,
12,  x FOR PEER REVIEW 10 of 15 the binding free energy is significantly weaker in KPC-204 (−5.09 kcal/mol) compared to KPC-2 (−8.92 kcal/mol), indicating a reduced affinity for the protein.This diminished interaction may contribute to the resistance observed in KPC-204.KPC-204's KDD insertion at position 269 likely weakens its meropenem resistance compared to KPC-2.This is suggested by both the weaker binding affinity observed in docking analysis (−6.33 kcal/mol vs. −8.72 kcal/mol) and the absence of a key hydrogen bond with Glu166, which is present in KPC-2.

Author Contributions:
Writing-original draft: Y.G.; writing-review and editing: Y.G. and X.L.; conceptualization: Y.G. and X.L.; methodology: Y.G.; software: Y.G. and Y.F.; data analysis: Y.G. and Y.F.; project administration: X.L.; funding acquisition: X.L.All authors have read and agreed to the published version of the manuscript.Funding: This research was funded by the Elderly Health Project of Sichuan Provincial Health Commission, grant number GBKT20021.

Table 2 .
The complete genome and antimicrobial resistant genes of isolate 130125.

Table 4 .
IC 50 of β-lactamases inhibitors against KPC-2 and KPC-204 a .Data are the means of three independent experiments.Standard deviations were within 15% of the mean value. a