Co-occurrence of blaIMP-4 and blaKPC-2 in a Clinical Isolate and Global Evolutionary Genomics of Klebsiella grimontii

Co-occurrence of blaIMP-4 and blaKPC-2 in a Clinical Isolate and Global Evolutionary Genomics of Klebsiella grimontii | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Co-occurrence of blaIMP-4 and blaKPC-2 in a Clinical Isolate and Global Evolutionary Genomics of Klebsiella grimontii yali Zheng, Zhouguang Jiao, Xin Lin, Yu Zhang, Jianbo Ye, Jian Lan, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9296935/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Background Klebsiella grimontii is a recently confirmed member of the Klebsiella oxytoca complex with significant clinical importance. As an opportunistic pathogen, reports of infections it causes are increasing, and the associated threats warrant attention. However, its phylogeny and epidemiological patterns remain poorly studied. Methods In this study, we identified a strain of Klebsiella grimontii , designated K767, simultaneously carrying the carbapenemase genes bla IMP−4 and bla KPC−2 through phenotypic assays and sequencing techniques from a tertiary hospital in Zhejiang Province, China. After performing whole-genome analysis of this strain, we integrated data from the NCBI public database, incorporating genomic information from a total of 235 Klebsiella grimontii strains for in-depth bioinformatics analysis. Results Genomic analysis revealed that in strain K767, bla IMP−4 is located on an IncHI5-type plasmid. This plasmid harbors multiple resistance modules and is highly conserved among similar plasmids originating from China. bla KPC−2 is located on an IncFII:IncFIA-type hybrid plasmid, a type predominantly found in Klebsiella species within China. Analysis of the 235 global strains indicated spatiotemporal heterogeneity in their prevalence, with Europe being the primary region. A total of 76 sequence types were identified, with Cluster III being the predominant evolutionary clade. The strains carried 75 acquired antimicrobial resistance genes, including various carbapenemase genes, and a significant co-occurrence was observed between certain resistance genes and specific plasmid replicons. Conclusion This study reports a strain of Klebsiella grimontii simultaneously carrying bla KPC−2 and bla IMP−4 . These genes are carried by an IncFII:IncFIA-type hybrid plasmid and an IncHI5-type conjugative plasmid, respectively, both of which are widely disseminated in China. Global population analysis suggests that this species exhibits high genetic diversity, with spatiotemporal heterogeneity in its prevalence, and that plasmids play a key role in the transmission of multidrug resistance. The findings provide an important basis for the clinical prevention and control of such resistant pathogens. Klebsiella grimontii carbapenemase genes bla KPC−2 bla IMP−4 sequence types Global population analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Several species within the genus Klebsiella have evolved into significant clinical and public health threats worldwide due to their virulence and antimicrobial resistance[ 1 ]. Among these, Klebsiella pneumoniae has attracted considerable attention owing to its high pathogenicity and extensive drug resistance [ 2 , 3 ]. However, the clinical significance and transmission risks of another important yet long overlooked group, the Klebsiella oxytoca ( K. oxytoca ) complex, are becoming increasingly apparent. This complex represents the second most clinically relevant group of Klebsiella species associated with human infections, after Klebsiella pneumoniae [ 4 ]. Due to the high phenotypic similarity among members of the complex, conventional biochemical methods often fail to accurately distinguish them, and precise identification relies on genomic analysis. Genome-based taxonomic studies have clarified that the K. oxytoca complex is a genetically diverse group comprising at least nine species. These include the recently identified species Klebsiella grimontii , Klebsiella huaxiensis , Klebsiella michiganensis , Klebsiella oxytoca sensu stricto, Klebsiella pasteurii , and Klebsiella spallanzanii , along with three additional unnamed species [ 5 ]. Klebsiella grimontii ( K. grimontii ) was formally established as a new species in 2018. Its taxonomic status originated from an in-depth analysis of the original K. oxytoc phylogroups Ko6[ 6 ] and B2[ 7 ], and was confirmed based on average nucleotide identity analysis, core gene sequences, and distinctive biochemical characteristics[ 5 , 8 ]. This bacterium has been confirmed as an opportunistic pathogen with clear clinical significance, capable of causing various infections such as bacteremia, soft tissue infections, urinary tract infections, and antibiotic-associated hemorrhagic colitis[ 8 ]. Particularly noteworthy is that recent epidemiological surveillance has revealed the detection of K. grimontii in multiple locations worldwide, along with the emergence of resistant strains carrying carbapenemase genes (such as KPC and VIM types). This suggests its potential to become a new reservoir and transmission vector for resistance genes[ 9 – 13 ]. Despite its increasing clinical and epidemiological importance, significant gaps remain in the current understanding of K. grimontii . Systematic studies are particularly lacking regarding its resistance profiles co‑harboring multiple carbapenemase genes, the plasmid genetic contexts that mediate the horizontal transfer of these critical resistance determinants, as well as the population genetic structure, evolutionary dynamics, and transmission patterns of this species on a global scale. Therefore, this study reports a clinical isolate of K. grimontii , designated K767, which co-produces KPC-2 and IMP-4 carbapenemases. Through whole-genome sequencing and in-depth analysis, we elucidate the genetic basis of multidrug resistance in strain K767. Furthermore, by integrating global K. grimontii genomic data from public databases, this work presents, for the first time on a global scale, the population structure, characteristics of the resistome, and evolutionary transmission patterns of K. grimontii . The findings aim to provide crucial scientific evidence and molecular epidemiological data for the effective clinical surveillance and control of this emerging multidrug-resistant pathogen. 2 Materials and methods 2.1 Bacterial Strain Collection, Identification, Antimicrobial Susceptibility Testing, and Enzyme Production Assays Strain K767 was isolated in 2015 from an abdominal drainage specimen of a patient with rectal malignancy at a tertiary hospital in Zhejiang, China. The bacterial strain was preliminarily identified using matrix‑assisted laser desorption/ionization time‑of‑flight mass spectrometry (MALDI‑TOF MS). Further species‑level identification was accomplished based on average nucleotide identity (ANI) analysis of the genomic sequence [ 14 ]. Antimicrobial susceptibility testing (AST) was performed using the bioMérieux VITEK 2 system, and results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (2024 edition). Carbapenemase production was detected using the NG‑Test CARBA 5, a rapid immunochromatographic assay. 2.2 Sequencing and Sequence Assembly Genomic DNA of the strain was extracted using the Gentra Puregene Yeast/Bacteria Kit (Qiagen, Valencia, CA, USA). Libraries were constructed with the TruePrep DNA Library Prep Kit V2 and the SQK‑LSK109 Ligation Sequencing Kit, followed by sequencing on the Illumina HiSeq X Ten platform (Illumina, San Diego, CA, USA) and the GridION X5 platform (Oxford Nanopore Technologies, Oxford, UK), respectively. Raw data from the HiSeq X Ten and GridION X5 platforms were trimmed using Canu (version 1.8; https://canu.readthedocs.io/en/latest/index.html ) to obtain high‑quality clean reads. De novo assembly was performed by combining Illumina paired‑end short reads and Nanopore long reads using Unicycler (version 0.4.5; https://github.com/rrwick/Unicycler ). 2.3 Phylogenetic Tree Construction and Average Nucleotide Identity (ANI) Analysis Publicly available K. grimontii genome data up to April 23, 2025, were downloaded from the GenBank database ( https://www.ncbi.nlm.nih.gov/datasets/genome/ ). All datasets had an assembly level of contig or higher. Genome quality was assessed using Quast 5.0.2, with an N50 value of at least 20 kb required for inclusion. Subsequently, CheckM v1.2.2 was used to evaluate genome completeness and contamination, retaining only genomes with completeness ≥ 95% and contamination ≤ 5%. For strains that passed quality control, average nucleotide identity analysis was performed using FastANI v1.33 to calculate the ANI values between each strain and the reference strain 2750 (GenBank accession: GCA_042137965.1). Based on the species threshold (ANI ≥ 95%), a total of 235 high-quality K. grimontii genomes, including strain K767, were finally selected for subsequent analysis. Furthermore, the FastANI software was employed to calculate pairwise average nucleotide identity values among the K. grimontii genomes, and an ANI heatmap was generated. Additionally, using strain 2750 (GenBank accession: GCA_042137965.1) as the reference sequence and K. oxytoca NCTC13727 (accession: NZ_LR134333.1) as the outgroup, the genomic sequences of all strains were aligned against the complete chromosome sequence of reference strain 2750. Core single nucleotide polymorphisms were identified using MUMmer v3.2 ( https://mummer.sourceforge.net/ ). Single-nucleotide polymorphisms (SNPs) located in all repetitive DNA regions were identified and filtered out using RepeatMasker ( http://www.repeatmasker.org/ ). Finally, based on 86,032 filtered, non-recombinant core SNP sites derived from the chromosomal sequences of the 235 strains, a maximum likelihood phylogenetic tree was constructed using RAxML under the GTR model with 1000 bootstrap replicates, and the tree was visualized using iTOL ( https://itol.embl.de ). 2.4 Screening for Antibiotic Resistance Genes (ARGs), Multi-Locus Sequence Typing (MLST), and Plasmid Typing A local sequence database was constructed using the software abricate (v1.2.0) based on the ResFinder database (dated 2024-03-22), with thresholds for both identity and coverage set at ≥ 90%. The detected resistance genes were organized into a binary presence/absence matrix and visualized as a heatmap using the pheatmap package (v1.0.12) in R (v3.27.1; https://www.r-project.org ). Multi-locus sequence typing (MLST) for the 235 K. grimontii strains was performed via the PubMLST website ( https://pubmlst.org/ ). To facilitate plasmid typing, a custom plasmid sequence database was built by integrating data from the PlasmidFinder database ( https://cge.food.dtu.dk/services/PlasmidFinder/ ) with plasmid sequences obtained from laboratory sequencing. 2.5 Genome Annotation and Comparative Analysis Functional annotation of the sequences was performed using the RAST 2.0 online platform. Annotation of resistance genes, mobile genetic elements, and other genomic features was conducted with online databases including CARD[ 15 ], ResFinder[ 16 ], ISFinder[ 17 ], DANMEL[ 18 ]. Finally, Inkscape ( https://inkscape.org/en/ ) was used to visualize the comparative structure of plasmid mobile elements and the characteristics of resistance regions. 2.6 Nucleotide sequence accession number The complete genome sequences of the chromosome and the four plasmids (pK767-IMP, pK767-KPC, pK767-sul, and pK767-NR) of strain K767 have been deposited in the GenBank database under the accession numbers CP198962, CP198963, CP198964, CP198966, and CP198965, respectively. 3 Results 3.1 Species Identification and Antimicrobial Susceptibility Testing Strain K767 was initially identified as K. oxytoca by mass spectrometry. To verify this result, ANI analysis was further performed, which revealed 99.37% identity with the reference genome of K. grimontii (GenBank accession: GCA_042137965.1), confirming its identity as K. grimontii at the genomic level. Antimicrobial susceptibility testing demonstrated that strain K767 was resistant to multiple antimicrobial agents, including penicillin/beta-lactamase inhibitor combinations (ticarcillin/clavulanate, piperacillin/tazobactam), cephalosporins (ceftazidime, cefoperazone/sulbactam, cefepime), monobactams (aztreonam), carbapenems (imipenem, meropenem), aminoglycosides (amikacin, tobramycin), fluoroquinolones (ciprofloxacin, levofloxacin), and trimethoprim/sulfamethoxazole. However, the strain remained susceptible to tetracyclines (doxycycline, minocycline) and tigecycline (Table 1 ). Furthermore, the NG Test CARBA 5 immunochromatographic assay confirmed the production of KPC type and IMP type carbapenemases by this strain (Fig S1 ). Table 1 Antimicrobial drug susceptibility profiles Antibiotics MIC (mg/L)/antimicrobial susceptibility K767 Ticarcillin/Clavulanic Acid ≥ 128/R Piperacilin/Tazobactam ≥ 128/R Ceftazidime ≥ 64/R Cefoperazone/Sulbactam ≥ 64/R Cefepime ≥ 32/R Aztreonam ≥ 64/R Imipenem ≥ 16/R Meropenem ≥ 16/R Amikacin ≥ 64/R Tobramycin ≥ 16/R Ciprofloxacin ≥ 4/R Levofloxacin 4R Doxycycline 1S Minocycline ≤ 1/S Tigecycline ≤ 0.5/S Colistin ≤ 0.5/I Trimethoprim/Sulfamethoxazole ≥ 320/R S=sensitive; R=resistant. 3.2 Overview of the Genomic Information of K. grimontii K767 Genomic sequencing analysis revealed that strain K767 comprises a chromosome of 6,025,577 bp in length (accession number: CP198962.1), with a GC content of 55.64%, and encodes 5,676 predicted open reading frames (ORFs) (Table 2 ). The resistance genes dfrA1 and bla OXY−6−2 were identified on the chromosome. Multilocus sequence typing via the PubMLST website ( https://pubmlst.org/ ) assigned this strain to sequence type ST172. Furthermore, K767 harbors four circular plasmids, designated pK767-IMP, pK767-KPC, pK767-sul, and pK767-NR. Among these, plasmid pK767-IMP is 339,206 bp in length, has a GC content of 47.38%, contains 369 ORFs, and carries 19 different resistance genes, including bla IMP−4 ; its replicon type is IncHI5. Plasmid pK767-KPC is 126,694 bp long with a GC content of 53.50%, contains 152 ORFs, carries the resistance gene bla KPC−2 , and belongs to the replicon type IncFII:FIA. Plasmid pK767-sul measures 182,801 bp, has a GC content of 52.14%, comprises 223 ORFs, and carries the resistance gene sul1 . The remaining plasmid, pK767-NR, did not contain any identified resistance genes (Table 2 ). Table 2 Whole genome information of Klebsiella grimontii K767 Sequence Mean G + C content (%) Length (bp) Total number of ORFs MLST Inc type Accession number Resistance genes cK767 55.64% 6,025,577 5,676 ST172 - CP198962 dfrA1 , bla OXY−6−2 pK767-IMP 47.38% 339,206 369 - IncHI5 CP198963 bla IMP−4 , fosA3 , bla SHV−12 , sul2 , strA , strB , qnrS1 , aacA4 , arr3 , bla TEM−1 , dfrA12 , aadA2 , qacEDl , armA , msr (E), mph (E), aacC2, catA2 , sul1 pK767-KPC 53.50% 126,694 152 - IncFII:FIA CP198964 bla KPC−2 pK767-sul 52.14% 182,801 223 - Unknown CP198966 sul1 pK767-NR 48.12% 6,156 8 - Unknown CP198965 - -, not available 3.3 Genetic Characterization of pK767-IMP and Comparative Analysis with Related Plasmids To further elucidate the genetic structure of plasmid pK767-IMP, which carries the bla IMP−4 gene, we performed an in-depth analysis. This plasmid belongs to the IncHI5 incompatibility (Inc) group. Its structure can be divided into a backbone region and ten accessory insertion regions. The backbone region (approximately 168.9 kb) contains functional modules for replication, maintenance, and conjugative transfer: the replication region includes the replication initiation protein (Rep) and its specific binding site, regulating plasmid replication; the maintenance region includes genes such as parA and parB ; and the conjugative transfer region contains core components of a typical type F IV secretion system (a series of tivF genes). The accessory modules include a Tn 6535 -associated multidrug resistance (MDR) region of approximately 67.8 kb, which harbors a Tn 1696 -derived resistance transposon and multiple insertion sequences (IS Ecl1 , IS 903B , an IS 3 family element, IS Ec33 , IS Kpn28 , IS Kpn28 , IS 5 , and IS Kpn37 ) (Table S1 , Fig. 1 ). To investigate the structural relationships between pK767-IMP and similar plasmids within the same Inc group, we screened the GenBank database using the replication initiation gene repA1 and the carbapenemase gene bla IMP−4 , identifying 30 related plasmids: p12208-IMP, p13450-IMP, p19051-IMP, among others. Analysis revealed that these 31 plasmids, including pK767-IMP from this study, are predominantly distributed in the genus Klebsiella (27/31, 87.1%), followed by Raoultella (4/31, 12.9%). For example, plasmids such as p12208-IMP and p13450-IMP were isolated from Klebsiella quasipneumoniae , pKP18-31-IMP from Klebsiella quasipneumoniae , while p208355-IMP and pMT136604 were isolated from Raoultella species. Notably, with the exception of one strain for which the geographical origin was not specified, all strains were isolated from different regions of China (Table S2). A comparative genomics analysis was conducted on these 31 plasmids. The results revealed that, with the exception of p19051-IMP (accession: MF344565), which lacks a conjugation transfer region, all other plasmids exhibited a highly conserved backbone in regions related to conjugation transfer and plasmid maintenance when compared to the reference plasmid pK767-IMP. However, significant variations were observed in their accessory modules. Specifically, plasmids p13450-IMP (accession: MF344564), p19051-IMP (accession: MF344565), and pRo24724 (accession: CP021328) showed a high degree of similarity to the approximately 67.8 kb multidrug resistance region found in pK767-IMP. The accessory modules in the majority of the remaining plasmids displayed varying degrees of divergence. Similarly, regions highly homologous to the Tn 1696 -derived resistance transposon were present only in pRo24724 (accession: CP021328), p12208-IMP (accession: MF344562), p13450-IMP (accession: MF344564), and p19051-IMP (accession: MF344565). In other plasmids, this module was either partially missing or structurally incomplete. Notably, these structurally similar plasmids were all derived from strains isolated in Zhejiang Province, China (Fig. 1 ). 3.4 Genetic Characterization of pK767-KPC and Comparative Analysis with Related Plasmids Concurrently, we characterized the genetic structure of plasmid pK767-KPC, which carries the bla KPC−2 gene. This plasmid belongs to the IncFII:IncFIA type and its structure can be divided into a backbone region and four accessory modules. The backbone region (approximately 91.64 kb) contains functional units for replication, maintenance, and conjugative transfer: the replication region is centered around the replication initiation protein; the maintenance region includes genes such as parA and stbD / stbE ; and the conjugative transfer region comprises core components of a typical type F IV secretion system (a series of tivF genes) along with the regulatory protein FinO . The accessory modules include a 15.86 kb bla KPC−2 resistance region and the insertion sequences IS Ror3 , IS 903B , and a truncated ΔIS Pa38b (Table S1 ). Similarly, to investigate the relationship between pK767-KPC and related plasmids within the same Inc group, we screened the GenBank database based on the replication initiation gene repA1 and the carbapenemase gene bla KPC−2 , identifying 10 additional plasmids. Analysis showed that these 11 plasmids, including pK767-KPC from this study, are predominantly distributed in the genus Klebsiella (8/11, 72.7%). However, this screening strategy does not capture related plasmid backbones that lack bla KPC−2 or utilize alternative replication initiation genes. Notably, all strains harboring these plasmids were isolated from various regions within China (Table S3). Structural comparison of the 11 plasmids revealed that the conjugative transfer regions of all plasmids were highly conserved compared to that of pK767-KPC. Regarding the plasmid maintenance region, plasmids pBKPC18-1 (accession: CP022275), pFAHZZU5885-2 (accession: CP135254), pK516_KPC (accession: CP022349), pK518_KPC (accession: CP023186), and pK92-KPC (OL828742) exhibited a highly similar maintenance region to pK767-KPC. The remaining plasmids showed varying degrees of insertions, deletions, or truncations in this region. Furthermore, the plasmids with highly similar maintenance regions mentioned above carried identical bla KPC−2 multidrug resistance modules (Fig. 2 ). 3.5 Genetic Structure of the Antimicrobial Resistance Modules in pK767-KPC and pK767-IMP The Tn 1696 -derived transposon carried by plasmid pK767-IMP is 49.9 kb in length and exhibits a complex structure. Tn 1696 was originally identified in plasmid R1033, isolated from a clinical Pseudomonas aeruginosa strain in Spain in 1975 [ 19 ]. Compared to the prototype transposon, the derivative in pK767-IMP contains an insertion of IS 5075 within its backbone, which splits the left inverted repeat into two segments. The remainder of the backbone is identical to the prototype. Furthermore, the accessory regions of this derivative are more complex than those of the prototype, primarily consisting of four resistance modules: an integron, a truncated aacC2-tmrB region, a truncated IS26-catA2-IS26 unit, an ars region, and ΔTn 1548 . The integron contains a resistance gene cassette harboring the carbapenem resistance gene bla IMP−4 . The truncated aacC2 - tmrB region features the gene arrangement aacC2-tmrB-orf192-orf228-Δorf1158 . The truncated IS 26 - catA2 -IS 26 unit mainly comprises a truncated IS26 element. ΔTn 1548 only includes two modules: an IS Ec29-mph(E) - IS 26 unit and an IS CR1-armA unit, while the right-side integron In 27 and the IS 26 element are absent (Fig. 3 a). The approximately 67.8-kb MDR region associated with Tn 6535 in plasmid pK767-IMP is flanked on its left side by the transposase gene of Tn 6535 , suggesting its likely origin from Tn 6535 or a variant-derived structure. The 16 resistance genes it carries are located within nine distinct resistance modules: an IS 26 - fosA3 -IS 26 unit, an IS 26 - bla SHV−12 -IS 26 unit, the unit transposon ∆Tn 5393c (carrying sul2 , strA , and strB genes), an IS Kpn19 - qnrS1 -IS 26 unit, integron In 792 (with the gene cassette array aacA4-arr3), unit transposon Tn6320 (carrying the bla TEM−1 gene), a truncated integron In 27 (with the gene cassette array dfrA12 - gcuF - aadA2 ), an IS CR1 - armA unit, and an IS Ec29 - mph(E) -IS 26 unit. Collectively, these modules constitute a vast multidrug resistance region (Fig. 3 b). The unit transposon Tn 6296 was originally identified in the IncFII plasmid pKP048 from Klebsiella pneumoniae . Its canonical structure has been described as a genetic platform harboring bla KPC−2 , spanning from Tn 6376 to Δ repB , which is integrated within the Tn 1722 transposon [ 19 ]. In comparison, plasmid pK767-KPC retains only a 15.9 kb segment of this bla KPC−2 genetic platform. Its structure is arranged as Δ repB - orf396 - orf279 - klcA - korC -ΔIS Kpn6 - bla KPC−2 -Tn 6376 . Notably, the backbone structure of Tn 6296 ( tnpA - tnpR - res ) as well as a truncated mcp gene are absent in this plasmid (Fig. 3 c). 3.6 Global Spatiotemporal Distribution Characteristics of K. grimontii Strains We retrieved and downloaded all available K. grimontii genome data from the GenBank database ( https://www.ncbi.nlm.nih.gov/datasets/genome/ ) up to April 23, 2025. After quality control screening, high-quality genomes were selected. Combined with one newly sequenced strain from this study, a total of 235 genomes were included in the analysis. The isolation years of these strains spanned from 1997 to 2024, and they were distributed across six continents and 22 countries/regions worldwide. Regarding sample sources, isolates from clinical origins were relatively dominant (60.85%, 143/235), environmental isolates accounted for 24.68% (58/235), and the source information for 34 strains (14.47%) was unknown (Table S4). Spatially, European strains constituted the highest proportion (156/235, 66.38%), primarily collected from the United Kingdom (92/235, 39.15%) and Switzerland (34/235, 14.47%) in Europe. The remaining strains were mostly distributed across Asia and the Americas. Temporal dynamic analysis revealed that the prevalence of K. grimontii displayed distinct phase characteristics. The highest number of isolate data uploaded to NCBI occurred between 2017 and 2018, totaling 133 strains, which accounted for 56.60% of all strains. In contrast, the prevalence intensity weakened before 2017 and after 2018 but persisted (Fig. 4 , TableS5). 3.7 Spatiotemporal Distribution and Phylogenetic Analysis of Global K. grimontii Using MLST, a key tool in bacterial epidemiology, we identified 76 distinct STs among all 235 strains. MLST typing revealed that ST408 was the predominant type (15/235, 6.38%), followed by ST186 (14 strains) and ST517 (10 strains). To gain deeper insight into the genetic background of K. grimontii , a maximum likelihood phylogenetic tree was constructed based on filtered, non-recombinant core SNP sites from the chromosomal sequences of the 235 strains (Fig. 5 ). Phylogenetic analysis showed that all strains could be clearly divided into three evolutionary clusters (Cluster I–III). The ANI heatmap (Fig S2, Table S5) and the SNP heatmap (Fig S3, Table S6) further validated the reliability of this clustering structure. Strains formed three distinct, well-separated genomic clusters along the diagonal with high homology (ANI > 98.5%), which was highly consistent with the topological structure of the phylogenetic tree branches. In terms of cluster size, Cluster III was the largest, containing 221 strains (94.04%), followed by Cluster I with 12 strains (5.1%). The strain sequenced in this study is located within Cluster III. Strains in Cluster I were isolated in 2017, 2018, 2019, and 2022 and included various STs such as ST351 and ST386. Cluster II contained only two strains, both from European clinical samples. Cluster III had a broad temporal span (1997–2024) and exhibited a prevalence pattern characterized by coexistence across multiple countries and diverse STs. Strains within this cluster originated from several countries including the United Kingdom, the United States, and Australia. The STs in this cluster were highly diverse, comprising 66 types including ST186, ST168, and ST215. The dominant ST in this cluster was ST186 (6.33%, 14/221). Notably, potential clonal expansion was observed among European strains; for example, isolates from Europe in 2018 were predominantly of specific STs (ST408, ST577). Overall, this bacterial population exhibited high genetic diversity (Fig S4, Table S4). 3.8 Plasmid and Antimicrobial Resistance Gene Distribution in Global K. grimontii Screening of plasmid replicons in the strain genomes identified 17 replicon types, including IncHI2, IncQ1, IncFII, IncX3, and IncFIB. Among these, the IncFIB type was the most prevalent (150/235, 63.83%), followed by IncFII (146/235, 62.13%) and IncFIA (72/235, 30.64%), while types such as IncN1 and IncHI5 occurred at lower frequencies (Fig. 6 ). Analysis of antimicrobial resistance genes in the 235 K. grimontii genomes revealed that these strains harbor a substantial number of acquired resistance genes. A total of 75 distinct resistance genes were identified, covering 10 major classes of antimicrobial agents: β‑lactams, polymyxins, tetracyclines, aminoglycosides, macrolides, phenicols, quinolones, rifampicin, sulfonamides, and trimethoprim. β‑Lactam and aminoglycoside resistance genes showed the highest diversity, with 31 and 13 different genes detected, respectively (Fig. 6 ; Table S7). Regarding detection frequency, the quinolone resistance gene oqxB was detected in the majority of strains, showing the highest prevalence (231/235, 97.88%), suggesting it is likely located on the chromosome. This was followed by the β-lactamase genes bla OXY−6−4 and bla OXY−6−2 , detected in 32.63% (77/235) and 31.78% (75/235) of strains, respectively. A total of eight carbapenemase genes were identified, including two bla KPC variants ( bla KPC−2 , bla KPC−3 ), one bla NDM variant ( bla NDM−1 ), four bla IMP variants ( bla IMP−1 , bla IMP−4 , bla IMP−22 , bla IMP−38 ), and one bla VIM variant ( bla VIM−1 ). Strains carrying these carbapenemase genes predominantly belonged to STs 186 and 131 (Fig. 7 ; Table S8). Within Cluster III, six ST371 strains isolated in 2019 all carried an identical set of multiple resistance genes, including bla KPC−2 , bla OXY−6 , oqxB , and aac(3)-IIa , and all harbored IncFIB and IncFII type plasmids, indicating a clear clonal spread event (Table S4). Additionally, multiple ST431 strains isolated in Portugal in 2023 carried resistance genes such as bla IMP−22 and bla KPC−3 , and all contained IncFIB, IncFII, and IncFIA type plasmids (Fig. 7 ). Due to current technical limitations, we cannot directly confirm that resistance genes and plasmid replicons reside on the same DNA fragment. Our statistical analysis, based on Spearman correlation, revealed that certain resistance genes and plasmid replicons exhibit similar prevalence patterns across genomes (P < 0.05). While this indicates a trend of co-occurrence at the population level, it does not provide direct evidence of physical co-localization on the same plasmid. For instance, bla IMP−22 , bla KPC−3 , aph(3'')-Ib , and tet(A) show correlated prevalence patterns with each other and with the plasmid replicon IncHI3; IncHI2 is correlated with genes such as mcr-9.1 and bla LAP−2 , and IncC correlates with dfrA19 (Fig. 7 ; Table S9). These results reflect statistical co-occurrence rather than physical co-location, and plasmids may serve as potential vectors for these resistance genes. 4 Discussion In this study, we performed genomic sequencing and in-depth characterization of K. grimontii K767, a clinical isolate co-harboring the carbapenemase genes bla KPC−2 and bla IMP−4 . By further integrating resources from the GenBank public database, we conducted a systematic epidemiological analysis of 235 K. grimontii genomes from a global collection. This comprehensive approach elucidated the resistome profile, transmission mechanisms, and global epidemiological characteristics of this species. Strain K767 demonstrated resistance to multiple classes of critical antimicrobial agents, including carbapenems and cephalosporins, but remained susceptible to tetracyclines and tigecycline. This resistance phenotype closely aligns with its carriage of corresponding resistance genes. Of particular significance is the concurrent production of both KPC and IMP type carbapenemases by K767. The co-production of two carbapenemases belonging to different Ambler classes (Class A and Class B) within a single strain may lead to the failure of various therapeutic regimens, including those involving novel β-lactam/β-lactamase inhibitor combinations. This severely limits treatment options and substantially increases the difficulty of infection control [ 20 , 21 ]. As crucial mobile genetic elements, plasmids serve as the primary vectors mediating the spread of carbapenemase genes [ 22 , 23 ]. Among these, IncFII-type plasmids are common and significant carriers facilitating the dissemination of carbapenemase genes such as bla KPC and bla NDM in Enterobacteriaceae [ 24 ]. When IncFII and IncFIA replicons coexist, they often form hybrid plasmids, which can enhance plasmid stability within the host and broaden the host range [ 25 ].In this study, pK767-KPC, which carries bla KPC−2 , belongs to the IncFII:IncFIA plasmid type. Hybrid IncFII:IncFIA plasmids are frequently reported to mediate the spread of bla KPC genes in Enterobacteriaceae [ 26 – 28 ]. Further analysis revealed that all strains included in this study carrying structurally similar IncF-KPC-2 plasmids were isolated from various regions of China. Notably, some of these plasmids (e.g., pBKPC18-1) exhibited a highly consistent backbone and resistance module with pK767-KPC, further substantiating the significant role of this plasmid type in mediating the cross-regional transmission of bla KPC−2 . IncHI5-type plasmids constitute a class of large, broad-host-range conjugative plasmids in Enterobacteriaceae , typically exceeding 200 kb in size [ 29 ]. Studies indicate that bacteria harboring IncHI5 plasmids are primarily isolated from human specimens, and these plasmids possess a wide dissemination capability among Enterobacteriaceae [ 30 ]. Notably, most IncHI5 plasmids are closely associated with multidrug resistance, with nearly all carrying various types of antibiotic resistance genes [ 31 ]. These resistance genes are often linked to transposons such as Tn 1696 or Tn 6535 [ 30 ]. The plasmid pK767-IMP identified in this study belongs to the IncHI5 type. Its structure comprises a conserved backbone and two highly variable multidrug resistance regions: a 49.9 kb Tn 1696 -derived transposon and a 67.8 kb Tn 6535 -associated multidrug resistance region. These two large resistance regions function like dual "resistance gene toolkits," significantly enhancing the strain's ability to acquire and accumulate diverse resistance determinants. Comparative genomic analysis revealed that strains carrying structurally highly similar IncHI5-IMP-4 plasmids (e.g., p13450-IMP, p19051-IMP) were almost exclusively from the genus Klebsiella (27/31, 87.1%) and were all isolated in China, suggesting this plasmid type mediates the horizontal transfer and dissemination of the bla IMP−4 gene among Klebsiella species within China. Whole-genome screening for resistance genes confirmed that K. grimontii constitutes a significant reservoir of resistance determinants, harboring a wide variety of acquired resistance genes. Statistical analysis revealed that the IncFIB (63.83%) and IncFII (62.13%) plasmid replicon types were the most prevalent, underscoring their important role in mediating the dissemination of resistance genes among these strains. Furthermore, statistical analysis identified significant correlations between specific resistance genes and particular plasmid replicon types (e.g., bla IMP−22 with IncHI3, and mcr-9.1 with IncHI2). Although physical linkage cannot be directly confirmed from the assembled data, this co-occurrence strongly suggests that plasmids serve as key vectors driving the co-dissemination of these resistance genes across different strains. The isolation of strains showed distinct spatiotemporal clustering. Temporally, 2017 to 2018 marked a peak period for genome data submissions, which may reflect heightened clinical attention or outbreak events during that time. Spatially, Europe, particularly the United Kingdom and Switzerland, is currently the primary source of genome data. This likely relates to robust microbial surveillance and genome sequencing initiatives in these regions and does not necessarily indicate the true epicenter of prevalence. Notably, within specific timeframes and locations (e.g., Europe in 2018), clonal expansion dominated by STs such as ST408 and ST577 was observed, suggesting potential local transmission chains within healthcare or community settings. Furthermore, the ST371 and ST431 clonal groups identified within Cluster III provide compelling evidence for clonal spread co-occurring with the dissemination of plasmids and resistance genes. Members of these groups not only shared identical STs but also carried nearly identical resistance gene and plasmid profiles. This study has several main limitations. First, the genomic data were primarily sourced from public databases, which introduces potential geographical and temporal sampling biases. Therefore, the data may not fully represent the true global distribution of this bacterial species. Second, the analysis was largely based on short-read sequencing data. While bioinformatics methods allowed for the accurate identification of resistance genes and plasmid replicons and confirmed their statistical associations, the limitations of short read lengths hindered the complete assembly of plasmids and the precise localization of resistance genes. Consequently, direct physical evidence confirming that resistance genes and plasmids reside on the same DNA molecule could not be obtained, which may affect the precision of inferences regarding plasmid transmission dynamics. Future research should involve more prospective, standardized surveillance. This should be combined with long-read sequencing technologies to accurately resolve the complete structure of resistance plasmids, alongside in vitro and in vivo conjugation experiments to empirically validate plasmid transferability. Such approaches will provide a stronger scientific foundation for developing more effective infection prevention and control strategies. 5 Conclusion This study reports a clinical strain of K. grimontii , K767, co-harboring the carbapenemase genes bla KPC−2 and blaIMP-4. It exhibits resistance to multiple classes of antimicrobial agents, including carbapenems and cephalosporins, representing a clinically high-risk case of accumulated resistance. Our analysis confirmed that the bla KPC−2 gene is carried by an IncFII:IncFIA-type hybrid plasmid (pK767-KPC). This plasmid type is widely disseminated across different regions in China and serves as a primary vector driving the cross-regional spread of bla KPC−2 . The bla IMP−4 gene is carried by a large IncHI5-type conjugative plasmid (pK767-IMP). This plasmid contains two large and variable "resistance gene toolkits," including a Tn 1696 -derived transposon and a Tn 6535 -associated element, which substantially enhance the multidrug resistance capacity of the host bacterium. Strains carrying similar IncHI5-IMP-4 plasmids are predominantly Klebsiella species of Chinese origin, indicating this plasmid type mediates the dissemination of bla IMP−4 within this genus in China. Whole-genome analysis confirmed that this bacterial species harbors a wide array of acquired resistance genes, highlighting the key role of plasmids in driving their co-dissemination. Phylogenetic analysis revealed high genetic diversity within the global K. grimontii population, with its prevalence demonstrating spatiotemporal heterogeneity. The observed clonal spread events underscore the complexity of its transmission mechanisms. Abbreviations K. oxytoca Klebsiella oxytoca ANI Average Nucleotide Identity AST Antimicrobial susceptibility testing CLSI Clinical and Laboratory Standards Institute SNPs Single-nucleotide polymorphisms ARGs Antibiotic Resistance Genes MLST Multi-Locus Sequence Types STs Sequence types ORFs Open reading frames Rep Replicon Inc Incompatibility Declarations Ethics approval and consent to participate This study was conducted in accordance with the Declaration of Helsinki. The use of human specimens and all related experimental protocols was reviewed and approved by the Ethics Committee of Taizhou Municipal Hospital, Zhejiang, China, in accordance with the medical research regulations of the Ministry of Health, China. Research and all related procedures involving biohazardous materials were approved by the Biosafety Committee of Taizhou Municipal Hospital. The Ethics Committee of Taizhou Municipal Hospital, Zhejiang, China granted a waiver of the requirement for informed consent for this study. This research was conducted in China. Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. Funding This work was supported by the Medical and Health Science and Technology Project of Zhejiang Province (2025KY461), and the Natural Science Foundation of Zhejiang Province (LTGY23H190003). Author Contribution Conceptualization, X.H.L., Y.L.Z., and Z.G.J.; methodology, Y.L.Z., Y.Z., J.B.Y., H.M.C., Z.G.J., and J.L.; data analysis, X.H.L., Y.L.Z., X.L., J.L., Y.Z., and H.M.C.; resources, X.L., J.B.Y. and H.M.C.; writing-original draft, Y.L.Z. and Z.G.J.; writing-review and editing, X.H.L. All authors read and approved the final manuscript. Acknowledgements Not applicable Data Availability The datasets generated and analyzed during the current study are available in the public NCBI GenBank database, under the following accession numbers: CP198962, CP198963, CP198964, CP198966, and CP198965. References Dong N, et al. Klebsiella species: Taxonomy, hypervirulence and multidrug resistance. EBioMedicine. 2022;79:103998. Lei TY, et al. Hypervirulent and carbapenem-resistant Klebsiella pneumoniae: A global public health threat. Microbiol Res. 2024;288:127839. Kocsis B. Hypervirulent Klebsiella pneumoniae: An update on epidemiology, detection and antibiotic resistance. Acta Microbiol Immunol Hung. 2023;70(4):278–87. Neog N, et al. Klebsiella oxytoca and Emerging Nosocomial Infections. Curr Microbiol. 2021;78(4):1115–23. Yang J, et al. Klebsiella oxytoca Complex: Update on Taxonomy, Antimicrobial Resistance, and Virulence. Clin Microbiol Rev. 2022;35(1):e0000621. Fevre C, et al. 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Infect Genet Evol. 2025;130:105740. Chen R, et al. First documentation of a clinical multidrug-resistant Enterobacter chuandaensis ST2493 isolate co-harboring bla(NDM-1) and two bla(KPC-2) bearing plasmids. Sci Rep. 2024;14(1):26817. Maher D, Taylor DE. Host range and transfer efficiency of incompatibility group HI plasmids. Can J Microbiol. 1993;39(6):581–7. Yu L, et al. Epidemiology, molecular characterization, and drug resistance of IncHI5 plasmids from Enterobacteriaceae. Int Microbiol. 2023;26(2):371–8. Zhang X, et al. High-Level Aminoglycoside Resistance in Human Clinical Klebsiella pneumoniae Complex Isolates and Characteristics of armA-Carrying IncHI5 Plasmids. Front Microbiol. 2021;12:636396. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.zip Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 19 May, 2026 Reviews received at journal 18 May, 2026 Reviews received at journal 22 Apr, 2026 Reviews received at journal 20 Apr, 2026 Reviewers agreed at journal 18 Apr, 2026 Reviewers agreed at journal 16 Apr, 2026 Reviewers agreed at journal 13 Apr, 2026 Reviewers invited by journal 13 Apr, 2026 Editor invited by journal 06 Apr, 2026 Editor assigned by journal 04 Apr, 2026 Submission checks completed at journal 04 Apr, 2026 First submitted to journal 01 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9296935","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":626105921,"identity":"da061b13-0330-4100-94f6-acfd36535b73","order_by":0,"name":"yali Zheng","email":"","orcid":"","institution":"Shanghai Institute of Materia Medica, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"yali","middleName":"","lastName":"Zheng","suffix":""},{"id":626105922,"identity":"e43ec41d-5d3c-4a82-997d-d165d62c6518","order_by":1,"name":"Zhouguang Jiao","email":"","orcid":"","institution":"Key Laboratory of Pathogenesis Mechanism and Control of Inflammatory-Autoimmune Disease of Hebei Province, College of Basic Medical Sciences, Hebei University","correspondingAuthor":false,"prefix":"","firstName":"Zhouguang","middleName":"","lastName":"Jiao","suffix":""},{"id":626105924,"identity":"8530f7c1-7257-4b0f-b513-b626705ba5e5","order_by":2,"name":"Xin Lin","email":"","orcid":"","institution":"Shanghai Institute of Materia Medica, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Lin","suffix":""},{"id":626105926,"identity":"59d3259a-3d2b-4c05-b5ec-3cb6c9642971","order_by":3,"name":"Yu Zhang","email":"","orcid":"","institution":"School of Life Sciences, Suzhou Medical College of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Zhang","suffix":""},{"id":626105929,"identity":"8ff1b233-7e9d-4caa-bbe0-d960471d6b47","order_by":4,"name":"Jianbo Ye","email":"","orcid":"","institution":"Department of Critical Care Medicine, Taizhou Municipal Hospital (Taizhou University Affiliated Municipal Hospital), School of Medicine, Taizhou University","correspondingAuthor":false,"prefix":"","firstName":"Jianbo","middleName":"","lastName":"Ye","suffix":""},{"id":626105932,"identity":"c80f9534-bf9d-4e41-90e5-8e049c7515af","order_by":5,"name":"Jian Lan","email":"","orcid":"","institution":"Department of Critical Care Medicine, Taizhou Municipal Hospital (Taizhou University Affiliated Municipal Hospital), School of Medicine, Taizhou University","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Lan","suffix":""},{"id":626105934,"identity":"e1607a43-fb36-44d8-9ce7-aa9d4130b7fc","order_by":6,"name":"Huimin Chen","email":"","orcid":"","institution":"Department of Critical Care Medicine, Taizhou Municipal Hospital (Taizhou University Affiliated Municipal Hospital), School of Medicine, Taizhou University","correspondingAuthor":false,"prefix":"","firstName":"Huimin","middleName":"","lastName":"Chen","suffix":""},{"id":626105936,"identity":"d19e8e72-ae62-4378-9a07-8366a7c90dfd","order_by":7,"name":"Xinhua Luo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIie2RMWrDMBRABQGng1JvxcZDrvBNoHQoyVVkBJ1MyQE6JEum0K42vUS30k3hgyaHrIEWaihk8mAIFE+lX6FemshdO+gN4gn+Q0hizOH4h5ybRfxsembxGSpVN/bEO0rCuU5W+fKPpOWQABYjPPNOjx+SQOqgvHsbP/fXq/2UJF8UNTLOhv6FsiQ3EoTeyZflrYwykge+fsLpFYvzR2FJ0rgUHkpQ6WWPk4QZJRlnAl6tCSjxRcmmosTIe1Ui9zqTuEwWOIYtnTIwogrWnfCdhOQeBWyrUTQgCWca6JED6138vtRh84kT2KTxnpPQV37UdXM99KPTSUsy+yVB57hhciQOh8PhaPkGLlxoywSakDAAAAAASUVORK5CYII=","orcid":"","institution":"Department of Critical Care Medicine, Taizhou Municipal Hospital (Taizhou University Affiliated Municipal Hospital), School of Medicine, Taizhou University","correspondingAuthor":true,"prefix":"","firstName":"Xinhua","middleName":"","lastName":"Luo","suffix":""}],"badges":[],"createdAt":"2026-04-02 02:08:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9296935/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9296935/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107577099,"identity":"590c97ad-64f8-48d3-8e35-b9a9d2bf7e99","added_by":"auto","created_at":"2026-04-22 20:51:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":17103718,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative genetic map of 31 \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebla\u003c/strong\u003e\u003c/em\u003e\u003csub\u003e\u003cstrong\u003eIMP\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e carrying plasmids using pK767 IMP as the reference.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis figure presents a circular alignment comparison of pK767 IMP and 30 other \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u003c/sub\u003e positive plasmids. The inner ring displays the GC content and GC skew of the reference plasmid pK767 IMP. A series of concentric outer rings illustrate the homologous structural relationships between the other 30 \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u003c/sub\u003e positive plasmids and pK767 IMP. The outermost ring annotates the functional modules of pK767 IMP itself, including the plasmid replication backbone region (blue), the conjugative transfer region (orange), and other accessory structures.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-9296935/v1/783262a5c614d0d2c5927578.png"},{"id":107706520,"identity":"4bdb2b2c-925b-49db-b737-6a2200dd01be","added_by":"auto","created_at":"2026-04-24 09:18:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":967642,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative genetic map of 11 \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebla\u003c/strong\u003e\u003c/em\u003e\u003csub\u003e\u003cstrong\u003eKPC\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e carrying plasmids using pK767 KPC as the reference.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis figure presents a circular alignment comparison of pK767 KPC and the 10 other \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e positive plasmids. The inner ring displays the GC content and GC skew of the reference plasmid pK767 KPC. A series of concentric outer rings illustrate the homologous structural relationships between the other 10 \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e positive plasmids and pK767 KPC. The outermost ring annotates the functional modules of pK767 KPC itself, including the plasmid replication backbone region (blue), the conjugative transfer region (orange), and other accessory structures.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-9296935/v1/096da0ec1888de7e156437a6.png"},{"id":107706131,"identity":"0b40c67a-ff5c-4397-9e2c-ed22dd1c2243","added_by":"auto","created_at":"2026-04-24 09:17:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2232632,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenetic structure of the carbapenemase-associated regions in plasmids pK767-IMP and pK767-KPC and comparison with related regions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenes are represented by arrows and color-coded according to their functional categories (e.g., transposase, resistance gene). Shaded areas indicate regions with ≥95% nucleotide sequence identity.\u003c/p\u003e\n\u003cp\u003e(a) Structure of the Tn\u003cem\u003e1696\u003c/em\u003e-derived transposon from pK767-IMP.\u003c/p\u003e\n\u003cp\u003e(b) Structure of a 67.8 kb multidrug resistance region in pK767-IMP.\u003c/p\u003e\n\u003cp\u003e(c) Genetic context of the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-2\u003c/sub\u003e gene in pK767-KPC.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-9296935/v1/f7079a83570619866592277f.png"},{"id":107705665,"identity":"5c29eb14-acfb-4d88-ac1d-906d1b01964a","added_by":"auto","created_at":"2026-04-24 09:14:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1553948,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatiotemporal distribution and characteristics of 235 globally collected \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eK. grimontii \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eisolates.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Geographical distribution of isolates by country/region. Color intensity represents the number of isolates.\u003c/p\u003e\n\u003cp\u003e(b) Spatiotemporal heatmap of 235 global \u003cem\u003eKlebsiella grimontii\u003c/em\u003e isolates distributed by year and country. The heatmap colors reflect the number of isolates per country per year. The bar plots at the top and on the right show the total number of isolates per year and per country, respectively. The colored bar at the bottom represents different continents.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-9296935/v1/9671dde4965706fffdccf4e1.png"},{"id":107706883,"identity":"2bd7dc36-decd-4ac6-a49a-b61eca4467d3","added_by":"auto","created_at":"2026-04-24 09:18:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":11096662,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree of 235 global \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eK. grimontii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e strains.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe reference strain used was 2750 (GenBank accession GCA_042137965.1). \u003cem\u003eK. oxytoca\u003c/em\u003e NCTC13727 (GenBank accession NZ_LR134333.1) was selected as the outgroup. Bootstrap values are indicated by circles on the branches.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-9296935/v1/f84c2a750d6585901a391679.png"},{"id":107705446,"identity":"892348c1-f53f-4b01-8fca-f88a5f745618","added_by":"auto","created_at":"2026-04-24 09:12:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4748354,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of antibiotic resistance genes and plasmid types among the 235 strains.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe left panel shows the phylogenetic tree, the middle panel displays the antibiotic resistance gene matrix, and the right panel shows the plasmid type matrix, all aligned in the same order. The top of the figure uses color coding to distinguish antibiotic resistance gene classes and plasmid markers. Colored cells in the matrices indicate the presence or absence of the corresponding antibiotic resistance gene cluster or plasmid type.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-9296935/v1/a2bc5d8bf248a2e60402e3b9.png"},{"id":107706795,"identity":"3cbfa095-8669-41af-abbd-bc2f17cb12cc","added_by":"auto","created_at":"2026-04-24 09:18:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":10128017,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation heatmap (lower triangle) between plasmids and resistance genes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrelations were analyzed using Spearman's rank correlation analysis. Significance (p \u0026lt; 0.05) is marked with an asterisk (*). Blue indicates a negative correlation, and brown indicates a positive correlation. Plasmid names are highlighted in red.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-9296935/v1/50f25d2da136fd68693b7925.png"},{"id":107706868,"identity":"b77df3d5-c2ca-4a3d-b2b1-cacb841c7c56","added_by":"auto","created_at":"2026-04-24 09:18:57","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6564503,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.zip","url":"https://assets-eu.researchsquare.com/files/rs-9296935/v1/04d90842852d6027967df811.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Co-occurrence of blaIMP-4 and blaKPC-2 in a Clinical Isolate and Global Evolutionary Genomics of Klebsiella grimontii","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSeveral species within the genus \u003cem\u003eKlebsiella\u003c/em\u003e have evolved into significant clinical and public health threats worldwide due to their virulence and antimicrobial resistance[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among these, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e has attracted considerable attention owing to its high pathogenicity and extensive drug resistance [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, the clinical significance and transmission risks of another important yet long overlooked group, the \u003cem\u003eKlebsiella oxytoca\u003c/em\u003e (\u003cem\u003eK. oxytoca\u003c/em\u003e) complex, are becoming increasingly apparent. This complex represents the second most clinically relevant group of \u003cem\u003eKlebsiella\u003c/em\u003e species associated with human infections, after \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Due to the high phenotypic similarity among members of the complex, conventional biochemical methods often fail to accurately distinguish them, and precise identification relies on genomic analysis. Genome-based taxonomic studies have clarified that the \u003cem\u003eK. oxytoca\u003c/em\u003e complex is a genetically diverse group comprising at least nine species. These include the recently identified species \u003cem\u003eKlebsiella grimontii\u003c/em\u003e, \u003cem\u003eKlebsiella huaxiensis\u003c/em\u003e, \u003cem\u003eKlebsiella michiganensis\u003c/em\u003e, \u003cem\u003eKlebsiella oxytoca\u003c/em\u003e sensu stricto, \u003cem\u003eKlebsiella pasteurii\u003c/em\u003e, and \u003cem\u003eKlebsiella spallanzanii\u003c/em\u003e, along with three additional unnamed species [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eKlebsiella grimontii\u003c/em\u003e (\u003cem\u003eK. grimontii\u003c/em\u003e) was formally established as a new species in 2018. Its taxonomic status originated from an in-depth analysis of the original \u003cem\u003eK. oxytoc\u003c/em\u003e phylogroups Ko6[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and B2[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and was confirmed based on average nucleotide identity analysis, core gene sequences, and distinctive biochemical characteristics[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This bacterium has been confirmed as an opportunistic pathogen with clear clinical significance, capable of causing various infections such as bacteremia, soft tissue infections, urinary tract infections, and antibiotic-associated hemorrhagic colitis[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Particularly noteworthy is that recent epidemiological surveillance has revealed the detection of \u003cem\u003eK. grimontii\u003c/em\u003e in multiple locations worldwide, along with the emergence of resistant strains carrying carbapenemase genes (such as KPC and VIM types). This suggests its potential to become a new reservoir and transmission vector for resistance genes[\u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite its increasing clinical and epidemiological importance, significant gaps remain in the current understanding of \u003cem\u003eK. grimontii\u003c/em\u003e. Systematic studies are particularly lacking regarding its resistance profiles co‑harboring multiple carbapenemase genes, the plasmid genetic contexts that mediate the horizontal transfer of these critical resistance determinants, as well as the population genetic structure, evolutionary dynamics, and transmission patterns of this species on a global scale.\u003c/p\u003e \u003cp\u003eTherefore, this study reports a clinical isolate of \u003cem\u003eK. grimontii\u003c/em\u003e, designated K767, which co-produces KPC-2 and IMP-4 carbapenemases. Through whole-genome sequencing and in-depth analysis, we elucidate the genetic basis of multidrug resistance in strain K767. Furthermore, by integrating global \u003cem\u003eK. grimontii\u003c/em\u003e genomic data from public databases, this work presents, for the first time on a global scale, the population structure, characteristics of the resistome, and evolutionary transmission patterns of \u003cem\u003eK. grimontii\u003c/em\u003e. The findings aim to provide crucial scientific evidence and molecular epidemiological data for the effective clinical surveillance and control of this emerging multidrug-resistant pathogen.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Bacterial Strain Collection, Identification, Antimicrobial Susceptibility Testing, and Enzyme Production Assays\u003c/h2\u003e \u003cp\u003eStrain K767 was isolated in 2015 from an abdominal drainage specimen of a patient with rectal malignancy at a tertiary hospital in Zhejiang, China. The bacterial strain was preliminarily identified using matrix‑assisted laser desorption/ionization time‑of‑flight mass spectrometry (MALDI‑TOF MS). Further species‑level identification was accomplished based on average nucleotide identity (ANI) analysis of the genomic sequence [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Antimicrobial susceptibility testing (AST) was performed using the bioM\u0026eacute;rieux VITEK 2 system, and results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (2024 edition). Carbapenemase production was detected using the NG‑Test CARBA 5, a rapid immunochromatographic assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Sequencing and Sequence Assembly\u003c/h2\u003e \u003cp\u003eGenomic DNA of the strain was extracted using the Gentra Puregene Yeast/Bacteria Kit (Qiagen, Valencia, CA, USA). Libraries were constructed with the TruePrep DNA Library Prep Kit V2 and the SQK‑LSK109 Ligation Sequencing Kit, followed by sequencing on the Illumina HiSeq X Ten platform (Illumina, San Diego, CA, USA) and the GridION X5 platform (Oxford Nanopore Technologies, Oxford, UK), respectively. Raw data from the HiSeq X Ten and GridION X5 platforms were trimmed using Canu (version 1.8; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://canu.readthedocs.io/en/latest/index.html\u003c/span\u003e\u003cspan address=\"https://canu.readthedocs.io/en/latest/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to obtain high‑quality clean reads. De novo assembly was performed by combining Illumina paired‑end short reads and Nanopore long reads using Unicycler (version 0.4.5; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/rrwick/Unicycler\u003c/span\u003e\u003cspan address=\"https://github.com/rrwick/Unicycler\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Phylogenetic Tree Construction and Average Nucleotide Identity (ANI) Analysis\u003c/h2\u003e \u003cp\u003ePublicly available \u003cem\u003eK. grimontii\u003c/em\u003e genome data up to April 23, 2025, were downloaded from the GenBank database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/datasets/genome/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/datasets/genome/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). All datasets had an assembly level of contig or higher. Genome quality was assessed using Quast 5.0.2, with an N50 value of at least 20 kb required for inclusion. Subsequently, CheckM v1.2.2 was used to evaluate genome completeness and contamination, retaining only genomes with completeness\u0026thinsp;\u0026ge;\u0026thinsp;95% and contamination\u0026thinsp;\u0026le;\u0026thinsp;5%. For strains that passed quality control, average nucleotide identity analysis was performed using FastANI v1.33 to calculate the ANI values between each strain and the reference strain 2750 (GenBank accession: GCA_042137965.1). Based on the species threshold (ANI\u0026thinsp;\u0026ge;\u0026thinsp;95%), a total of 235 high-quality \u003cem\u003eK. grimontii\u003c/em\u003e genomes, including strain K767, were finally selected for subsequent analysis.\u003c/p\u003e \u003cp\u003eFurthermore, the FastANI software was employed to calculate pairwise average nucleotide identity values among the \u003cem\u003eK. grimontii\u003c/em\u003e genomes, and an ANI heatmap was generated. Additionally, using strain 2750 (GenBank accession: GCA_042137965.1) as the reference sequence and \u003cem\u003eK. oxytoca\u003c/em\u003e NCTC13727 (accession: NZ_LR134333.1) as the outgroup, the genomic sequences of all strains were aligned against the complete chromosome sequence of reference strain 2750. Core single nucleotide polymorphisms were identified using MUMmer v3.2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://mummer.sourceforge.net/\u003c/span\u003e\u003cspan address=\"https://mummer.sourceforge.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Single-nucleotide polymorphisms (SNPs) located in all repetitive DNA regions were identified and filtered out using RepeatMasker (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.repeatmasker.org/\u003c/span\u003e\u003cspan address=\"http://www.repeatmasker.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Finally, based on 86,032 filtered, non-recombinant core SNP sites derived from the chromosomal sequences of the 235 strains, a maximum likelihood phylogenetic tree was constructed using RAxML under the GTR model with 1000 bootstrap replicates, and the tree was visualized using iTOL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de\u003c/span\u003e\u003cspan address=\"https://itol.embl.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Screening for Antibiotic Resistance Genes (ARGs), Multi-Locus Sequence Typing (MLST), and Plasmid Typing\u003c/h2\u003e \u003cp\u003eA local sequence database was constructed using the software abricate (v1.2.0) based on the ResFinder database (dated 2024-03-22), with thresholds for both identity and coverage set at \u0026ge;\u0026thinsp;90%. The detected resistance genes were organized into a binary presence/absence matrix and visualized as a heatmap using the pheatmap package (v1.0.12) in R (v3.27.1; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.r-project.org\u003c/span\u003e\u003cspan address=\"https://www.r-project.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Multi-locus sequence typing (MLST) for the 235 \u003cem\u003eK. grimontii\u003c/em\u003e strains was performed via the PubMLST website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmlst.org/\u003c/span\u003e\u003cspan address=\"https://pubmlst.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). To facilitate plasmid typing, a custom plasmid sequence database was built by integrating data from the PlasmidFinder database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cge.food.dtu.dk/services/PlasmidFinder/\u003c/span\u003e\u003cspan address=\"https://cge.food.dtu.dk/services/PlasmidFinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with plasmid sequences obtained from laboratory sequencing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Genome Annotation and Comparative Analysis\u003c/h2\u003e \u003cp\u003eFunctional annotation of the sequences was performed using the RAST 2.0 online platform. Annotation of resistance genes, mobile genetic elements, and other genomic features was conducted with online databases including CARD[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], ResFinder[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], ISFinder[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], DANMEL[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Finally, Inkscape (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://inkscape.org/en/\u003c/span\u003e\u003cspan address=\"https://inkscape.org/en/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to visualize the comparative structure of plasmid mobile elements and the characteristics of resistance regions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Nucleotide sequence accession number\u003c/h2\u003e \u003cp\u003eThe complete genome sequences of the chromosome and the four plasmids (pK767-IMP, pK767-KPC, pK767-sul, and pK767-NR) of strain K767 have been deposited in the GenBank database under the accession numbers CP198962, CP198963, CP198964, CP198966, and CP198965, respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Species Identification and Antimicrobial Susceptibility Testing\u003c/h2\u003e \u003cp\u003eStrain K767 was initially identified as \u003cem\u003eK. oxytoca\u003c/em\u003e by mass spectrometry. To verify this result, ANI analysis was further performed, which revealed 99.37% identity with the reference genome of \u003cem\u003eK. grimontii\u003c/em\u003e (GenBank accession: GCA_042137965.1), confirming its identity as \u003cem\u003eK. grimontii\u003c/em\u003e at the genomic level. Antimicrobial susceptibility testing demonstrated that strain K767 was resistant to multiple antimicrobial agents, including penicillin/beta-lactamase inhibitor combinations (ticarcillin/clavulanate, piperacillin/tazobactam), cephalosporins (ceftazidime, cefoperazone/sulbactam, cefepime), monobactams (aztreonam), carbapenems (imipenem, meropenem), aminoglycosides (amikacin, tobramycin), fluoroquinolones (ciprofloxacin, levofloxacin), and trimethoprim/sulfamethoxazole. However, the strain remained susceptible to tetracyclines (doxycycline, minocycline) and tigecycline (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Furthermore, the NG Test CARBA 5 immunochromatographic assay confirmed the production of KPC type and IMP type carbapenemases by this strain (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntimicrobial drug susceptibility profiles\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAntibiotics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC (mg/L)/antimicrobial susceptibility\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eK767\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTicarcillin/Clavulanic Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;128/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePiperacilin/Tazobactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;128/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCeftazidime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;64/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCefoperazone/Sulbactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;64/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCefepime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;32/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAztreonam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;64/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImipenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;16/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMeropenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;16/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmikacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;64/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTobramycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;16/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCiprofloxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;4/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLevofloxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDoxycycline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1S\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMinocycline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;1/S\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTigecycline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.5/S\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColistin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.5/I\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrimethoprim/Sulfamethoxazole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;320/R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003eS=sensitive; R=resistant.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Overview of the Genomic Information of \u003cem\u003eK. grimontii\u003c/em\u003e K767\u003c/h2\u003e \u003cp\u003eGenomic sequencing analysis revealed that strain K767 comprises a chromosome of 6,025,577 bp in length (accession number: CP198962.1), with a GC content of 55.64%, and encodes 5,676 predicted open reading frames (ORFs) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The resistance genes \u003cem\u003edfrA1\u003c/em\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXY\u0026minus;6\u0026minus;2\u003c/sub\u003e were identified on the chromosome. Multilocus sequence typing via the PubMLST website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmlst.org/\u003c/span\u003e\u003cspan address=\"https://pubmlst.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) assigned this strain to sequence type ST172. Furthermore, K767 harbors four circular plasmids, designated pK767-IMP, pK767-KPC, pK767-sul, and pK767-NR. Among these, plasmid pK767-IMP is 339,206 bp in length, has a GC content of 47.38%, contains 369 ORFs, and carries 19 different resistance genes, including \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e; its replicon type is IncHI5. Plasmid pK767-KPC is 126,694 bp long with a GC content of 53.50%, contains 152 ORFs, carries the resistance gene \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e, and belongs to the replicon type IncFII:FIA. Plasmid pK767-sul measures 182,801 bp, has a GC content of 52.14%, comprises 223 ORFs, and carries the resistance gene \u003cem\u003esul1\u003c/em\u003e. The remaining plasmid, pK767-NR, did not contain any identified resistance genes (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eWhole genome information of \u003cem\u003eKlebsiella grimontii\u003c/em\u003e K767\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean G\u0026thinsp;+\u0026thinsp;C content (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLength (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal number of ORFs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMLST\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInc type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAccession number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eResistance genes\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecK767\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e55.64%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6,025,577\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5,676\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eST172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCP198962\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003edfrA1\u003c/em\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003eOXY\u0026minus;6\u0026minus;2\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epK767-IMP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e47.38%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e339,206\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e369\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIncHI5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCP198963\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e, \u003cem\u003efosA3\u003c/em\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u0026minus;12\u003c/sub\u003e, \u003cem\u003esul2\u003c/em\u003e, \u003cem\u003estrA\u003c/em\u003e, \u003cem\u003estrB\u003c/em\u003e, \u003cem\u003eqnrS1\u003c/em\u003e, \u003cem\u003eaacA4\u003c/em\u003e, \u003cem\u003earr3\u003c/em\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u0026minus;1\u003c/sub\u003e, \u003cem\u003edfrA12\u003c/em\u003e, \u003cem\u003eaadA2\u003c/em\u003e, \u003cem\u003eqacEDl\u003c/em\u003e, \u003cem\u003earmA\u003c/em\u003e, \u003cem\u003emsr\u003c/em\u003e(E), \u003cem\u003emph\u003c/em\u003e(E), aacC2, \u003cem\u003ecatA2\u003c/em\u003e, \u003cem\u003esul1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epK767-KPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53.50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e126,694\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e152\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIncFII:FIA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCP198964\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epK767-sul\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e52.14%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e182,801\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e223\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCP198966\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003esul1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epK767-NR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e48.12%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6,156\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCP198965\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003e-, not available\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Genetic Characterization of pK767-IMP and Comparative Analysis with Related Plasmids\u003c/h2\u003e \u003cp\u003eTo further elucidate the genetic structure of plasmid pK767-IMP, which carries the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e gene, we performed an in-depth analysis. This plasmid belongs to the IncHI5 incompatibility (Inc) group. Its structure can be divided into a backbone region and ten accessory insertion regions. The backbone region (approximately 168.9 kb) contains functional modules for replication, maintenance, and conjugative transfer: the replication region includes the replication initiation protein (Rep) and its specific binding site, regulating plasmid replication; the maintenance region includes genes such as \u003cem\u003eparA\u003c/em\u003e and \u003cem\u003eparB\u003c/em\u003e; and the conjugative transfer region contains core components of a typical type F IV secretion system (a series of \u003cem\u003etivF\u003c/em\u003e genes). The accessory modules include a Tn\u003cem\u003e6535\u003c/em\u003e-associated multidrug resistance (MDR) region of approximately 67.8 kb, which harbors a Tn\u003cem\u003e1696\u003c/em\u003e-derived resistance transposon and multiple insertion sequences (IS\u003cem\u003eEcl1\u003c/em\u003e, IS\u003cem\u003e903B\u003c/em\u003e, an IS\u003cem\u003e3\u003c/em\u003e family element, IS\u003cem\u003eEc33\u003c/em\u003e, IS\u003cem\u003eKpn28\u003c/em\u003e, IS\u003cem\u003eKpn28\u003c/em\u003e, IS\u003cem\u003e5\u003c/em\u003e, and IS\u003cem\u003eKpn37\u003c/em\u003e) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the structural relationships between pK767-IMP and similar plasmids within the same Inc group, we screened the GenBank database using the replication initiation gene \u003cem\u003erepA1\u003c/em\u003e and the carbapenemase gene \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e, identifying 30 related plasmids: p12208-IMP, p13450-IMP, p19051-IMP, among others. Analysis revealed that these 31 plasmids, including pK767-IMP from this study, are predominantly distributed in the genus \u003cem\u003eKlebsiella\u003c/em\u003e (27/31, 87.1%), followed by \u003cem\u003eRaoultella\u003c/em\u003e (4/31, 12.9%). For example, plasmids such as p12208-IMP and p13450-IMP were isolated from \u003cem\u003eKlebsiella quasipneumoniae\u003c/em\u003e, pKP18-31-IMP from \u003cem\u003eKlebsiella quasipneumoniae\u003c/em\u003e, while p208355-IMP and pMT136604 were isolated from \u003cem\u003eRaoultella\u003c/em\u003e species. Notably, with the exception of one strain for which the geographical origin was not specified, all strains were isolated from different regions of China (Table S2).\u003c/p\u003e \u003cp\u003eA comparative genomics analysis was conducted on these 31 plasmids. The results revealed that, with the exception of p19051-IMP (accession: MF344565), which lacks a conjugation transfer region, all other plasmids exhibited a highly conserved backbone in regions related to conjugation transfer and plasmid maintenance when compared to the reference plasmid pK767-IMP. However, significant variations were observed in their accessory modules.\u003c/p\u003e \u003cp\u003eSpecifically, plasmids p13450-IMP (accession: MF344564), p19051-IMP (accession: MF344565), and pRo24724 (accession: CP021328) showed a high degree of similarity to the approximately 67.8 kb multidrug resistance region found in pK767-IMP. The accessory modules in the majority of the remaining plasmids displayed varying degrees of divergence. Similarly, regions highly homologous to the Tn\u003cem\u003e1696\u003c/em\u003e-derived resistance transposon were present only in pRo24724 (accession: CP021328), p12208-IMP (accession: MF344562), p13450-IMP (accession: MF344564), and p19051-IMP (accession: MF344565). In other plasmids, this module was either partially missing or structurally incomplete. Notably, these structurally similar plasmids were all derived from strains isolated in Zhejiang Province, China (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Genetic Characterization of pK767-KPC and Comparative Analysis with Related Plasmids\u003c/h2\u003e \u003cp\u003eConcurrently, we characterized the genetic structure of plasmid pK767-KPC, which carries the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e gene. This plasmid belongs to the IncFII:IncFIA type and its structure can be divided into a backbone region and four accessory modules. The backbone region (approximately 91.64 kb) contains functional units for replication, maintenance, and conjugative transfer: the replication region is centered around the replication initiation protein; the maintenance region includes genes such as \u003cem\u003eparA\u003c/em\u003e and \u003cem\u003estbD\u003c/em\u003e/\u003cem\u003estbE\u003c/em\u003e; and the conjugative transfer region comprises core components of a typical type F IV secretion system (a series of \u003cem\u003etivF\u003c/em\u003e genes) along with the regulatory protein \u003cem\u003eFinO\u003c/em\u003e. The accessory modules include a 15.86 kb \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e resistance region and the insertion sequences IS\u003cem\u003eRor3\u003c/em\u003e, IS\u003cem\u003e903B\u003c/em\u003e, and a truncated ΔIS\u003cem\u003ePa38b\u003c/em\u003e (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSimilarly, to investigate the relationship between pK767-KPC and related plasmids within the same Inc group, we screened the GenBank database based on the replication initiation gene \u003cem\u003erepA1\u003c/em\u003e and the carbapenemase gene \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e, identifying 10 additional plasmids. Analysis showed that these 11 plasmids, including pK767-KPC from this study, are predominantly distributed in the genus \u003cem\u003eKlebsiella\u003c/em\u003e (8/11, 72.7%). However, this screening strategy does not capture related plasmid backbones that lack \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e or utilize alternative replication initiation genes. Notably, all strains harboring these plasmids were isolated from various regions within China (Table S3).\u003c/p\u003e \u003cp\u003eStructural comparison of the 11 plasmids revealed that the conjugative transfer regions of all plasmids were highly conserved compared to that of pK767-KPC. Regarding the plasmid maintenance region, plasmids pBKPC18-1 (accession: CP022275), pFAHZZU5885-2 (accession: CP135254), pK516_KPC (accession: CP022349), pK518_KPC (accession: CP023186), and pK92-KPC (OL828742) exhibited a highly similar maintenance region to pK767-KPC. The remaining plasmids showed varying degrees of insertions, deletions, or truncations in this region. Furthermore, the plasmids with highly similar maintenance regions mentioned above carried identical \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e multidrug resistance modules (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Genetic Structure of the Antimicrobial Resistance Modules in pK767-KPC and pK767-IMP\u003c/h2\u003e \u003cp\u003eThe Tn\u003cem\u003e1696\u003c/em\u003e-derived transposon carried by plasmid pK767-IMP is 49.9 kb in length and exhibits a complex structure. Tn\u003cem\u003e1696\u003c/em\u003e was originally identified in plasmid R1033, isolated from a clinical \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain in Spain in 1975 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Compared to the prototype transposon, the derivative in pK767-IMP contains an insertion of IS\u003cem\u003e5075\u003c/em\u003e within its backbone, which splits the left inverted repeat into two segments. The remainder of the backbone is identical to the prototype. Furthermore, the accessory regions of this derivative are more complex than those of the prototype, primarily consisting of four resistance modules: an integron, a truncated \u003cem\u003eaacC2-tmrB\u003c/em\u003e region, a truncated \u003cem\u003eIS26-catA2-IS26\u003c/em\u003e unit, an ars region, and ΔTn\u003cem\u003e1548\u003c/em\u003e. The integron contains a resistance gene cassette harboring the carbapenem resistance gene \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e. The truncated \u003cem\u003eaacC2\u003c/em\u003e-\u003cem\u003etmrB\u003c/em\u003e region features the gene arrangement \u003cem\u003eaacC2-tmrB-orf192-orf228-Δorf1158\u003c/em\u003e. The truncated IS\u003cem\u003e26\u003c/em\u003e-\u003cem\u003ecatA2\u003c/em\u003e-IS\u003cem\u003e26\u003c/em\u003e unit mainly comprises a truncated IS26 element. ΔTn\u003cem\u003e1548\u003c/em\u003e only includes two modules: an IS\u003cem\u003eEc29-mph(E) -\u003c/em\u003eIS\u003cem\u003e26\u003c/em\u003e unit and an IS\u003cem\u003eCR1-armA\u003c/em\u003e unit, while the right-side integron In\u003cem\u003e27\u003c/em\u003e and the IS\u003cem\u003e26\u003c/em\u003e element are absent (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe approximately 67.8-kb MDR region associated with Tn\u003cem\u003e6535\u003c/em\u003e in plasmid pK767-IMP is flanked on its left side by the transposase gene of Tn\u003cem\u003e6535\u003c/em\u003e, suggesting its likely origin from Tn\u003cem\u003e6535\u003c/em\u003e or a variant-derived structure. The 16 resistance genes it carries are located within nine distinct resistance modules: an IS\u003cem\u003e26\u003c/em\u003e-\u003cem\u003efosA3\u003c/em\u003e-IS\u003cem\u003e26\u003c/em\u003e unit, an IS\u003cem\u003e26\u003c/em\u003e-\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u0026minus;12\u003c/sub\u003e-IS\u003cem\u003e26\u003c/em\u003e unit, the unit transposon ∆Tn\u003cem\u003e5393c\u003c/em\u003e (carrying \u003cem\u003esul2\u003c/em\u003e, \u003cem\u003estrA\u003c/em\u003e, and \u003cem\u003estrB\u003c/em\u003e genes), an IS\u003cem\u003eKpn19\u003c/em\u003e-\u003cem\u003eqnrS1\u003c/em\u003e-IS\u003cem\u003e26\u003c/em\u003e unit, integron In\u003cem\u003e792\u003c/em\u003e (with the gene cassette array aacA4-arr3), unit transposon Tn6320 (carrying the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u0026minus;1\u003c/sub\u003e gene), a truncated integron In\u003cem\u003e27\u003c/em\u003e (with the gene cassette array \u003cem\u003edfrA12\u003c/em\u003e-\u003cem\u003egcuF\u003c/em\u003e-\u003cem\u003eaadA2\u003c/em\u003e), an IS\u003cem\u003eCR1\u003c/em\u003e-\u003cem\u003earmA\u003c/em\u003e unit, and an IS\u003cem\u003eEc29\u003c/em\u003e-\u003cem\u003emph(E)\u003c/em\u003e-IS\u003cem\u003e26\u003c/em\u003e unit. Collectively, these modules constitute a vast multidrug resistance region (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eThe unit transposon Tn\u003cem\u003e6296\u003c/em\u003e was originally identified in the IncFII plasmid pKP048 from \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. Its canonical structure has been described as a genetic platform harboring \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e, spanning from Tn\u003cem\u003e6376\u003c/em\u003e to Δ\u003cem\u003erepB\u003c/em\u003e, which is integrated within the Tn\u003cem\u003e1722\u003c/em\u003e transposon [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In comparison, plasmid pK767-KPC retains only a 15.9 kb segment of this \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e genetic platform. Its structure is arranged as Δ\u003cem\u003erepB\u003c/em\u003e-\u003cem\u003eorf396\u003c/em\u003e-\u003cem\u003eorf279\u003c/em\u003e-\u003cem\u003eklcA\u003c/em\u003e-\u003cem\u003ekorC\u003c/em\u003e-ΔIS\u003cem\u003eKpn6\u003c/em\u003e-\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e-Tn\u003cem\u003e6376\u003c/em\u003e. Notably, the backbone structure of Tn\u003cem\u003e6296\u003c/em\u003e (\u003cem\u003etnpA\u003c/em\u003e-\u003cem\u003etnpR\u003c/em\u003e-\u003cem\u003eres\u003c/em\u003e) as well as a truncated \u003cem\u003emcp\u003c/em\u003e gene are absent in this plasmid (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Global Spatiotemporal Distribution Characteristics of \u003cem\u003eK. grimontii\u003c/em\u003e Strains\u003c/h2\u003e \u003cp\u003eWe retrieved and downloaded all available \u003cem\u003eK. grimontii\u003c/em\u003e genome data from the GenBank database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/datasets/genome/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/datasets/genome/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) up to April 23, 2025. After quality control screening, high-quality genomes were selected. Combined with one newly sequenced strain from this study, a total of 235 genomes were included in the analysis. The isolation years of these strains spanned from 1997 to 2024, and they were distributed across six continents and 22 countries/regions worldwide. Regarding sample sources, isolates from clinical origins were relatively dominant (60.85%, 143/235), environmental isolates accounted for 24.68% (58/235), and the source information for 34 strains (14.47%) was unknown (Table S4).\u003c/p\u003e \u003cp\u003eSpatially, European strains constituted the highest proportion (156/235, 66.38%), primarily collected from the United Kingdom (92/235, 39.15%) and Switzerland (34/235, 14.47%) in Europe. The remaining strains were mostly distributed across Asia and the Americas. Temporal dynamic analysis revealed that the prevalence of \u003cem\u003eK. grimontii\u003c/em\u003e displayed distinct phase characteristics. The highest number of isolate data uploaded to NCBI occurred between 2017 and 2018, totaling 133 strains, which accounted for 56.60% of all strains. In contrast, the prevalence intensity weakened before 2017 and after 2018 but persisted (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, TableS5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Spatiotemporal Distribution and Phylogenetic Analysis of Global \u003cem\u003eK. grimontii\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eUsing MLST, a key tool in bacterial epidemiology, we identified 76 distinct STs among all 235 strains. MLST typing revealed that ST408 was the predominant type (15/235, 6.38%), followed by ST186 (14 strains) and ST517 (10 strains).\u003c/p\u003e \u003cp\u003eTo gain deeper insight into the genetic background of \u003cem\u003eK. grimontii\u003c/em\u003e, a maximum likelihood phylogenetic tree was constructed based on filtered, non-recombinant core SNP sites from the chromosomal sequences of the 235 strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Phylogenetic analysis showed that all strains could be clearly divided into three evolutionary clusters (Cluster I\u0026ndash;III). The ANI heatmap (Fig S2, Table S5) and the SNP heatmap (Fig S3, Table S6) further validated the reliability of this clustering structure. Strains formed three distinct, well-separated genomic clusters along the diagonal with high homology (ANI\u0026thinsp;\u0026gt;\u0026thinsp;98.5%), which was highly consistent with the topological structure of the phylogenetic tree branches. In terms of cluster size, Cluster III was the largest, containing 221 strains (94.04%), followed by Cluster I with 12 strains (5.1%). The strain sequenced in this study is located within Cluster III. Strains in Cluster I were isolated in 2017, 2018, 2019, and 2022 and included various STs such as ST351 and ST386. Cluster II contained only two strains, both from European clinical samples. Cluster III had a broad temporal span (1997\u0026ndash;2024) and exhibited a prevalence pattern characterized by coexistence across multiple countries and diverse STs. Strains within this cluster originated from several countries including the United Kingdom, the United States, and Australia. The STs in this cluster were highly diverse, comprising 66 types including ST186, ST168, and ST215. The dominant ST in this cluster was ST186 (6.33%, 14/221). Notably, potential clonal expansion was observed among European strains; for example, isolates from Europe in 2018 were predominantly of specific STs (ST408, ST577). Overall, this bacterial population exhibited high genetic diversity (Fig S4, Table S4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Plasmid and Antimicrobial Resistance Gene Distribution in Global \u003cem\u003eK. grimontii\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eScreening of plasmid replicons in the strain genomes identified 17 replicon types, including IncHI2, IncQ1, IncFII, IncX3, and IncFIB. Among these, the IncFIB type was the most prevalent (150/235, 63.83%), followed by IncFII (146/235, 62.13%) and IncFIA (72/235, 30.64%), while types such as IncN1 and IncHI5 occurred at lower frequencies (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis of antimicrobial resistance genes in the 235 \u003cem\u003eK. grimontii\u003c/em\u003e genomes revealed that these strains harbor a substantial number of acquired resistance genes. A total of 75 distinct resistance genes were identified, covering 10 major classes of antimicrobial agents: β‑lactams, polymyxins, tetracyclines, aminoglycosides, macrolides, phenicols, quinolones, rifampicin, sulfonamides, and trimethoprim. β‑Lactam and aminoglycoside resistance genes showed the highest diversity, with 31 and 13 different genes detected, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Table S7).\u003c/p\u003e \u003cp\u003eRegarding detection frequency, the quinolone resistance gene \u003cem\u003eoqxB\u003c/em\u003e was detected in the majority of strains, showing the highest prevalence (231/235, 97.88%), suggesting it is likely located on the chromosome. This was followed by the β-lactamase genes \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXY\u0026minus;6\u0026minus;4\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXY\u0026minus;6\u0026minus;2\u003c/sub\u003e, detected in 32.63% (77/235) and 31.78% (75/235) of strains, respectively. A total of eight carbapenemase genes were identified, including two \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e variants (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;3\u003c/sub\u003e), one \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e variant (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e), four \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u003c/sub\u003e variants (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;1\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;22\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;38\u003c/sub\u003e), and one \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM\u003c/sub\u003e variant (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM\u0026minus;1\u003c/sub\u003e). Strains carrying these carbapenemase genes predominantly belonged to STs 186 and 131 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e; Table S8). Within Cluster III, six ST371 strains isolated in 2019 all carried an identical set of multiple resistance genes, including \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXY\u0026minus;6\u003c/sub\u003e, \u003cem\u003eoqxB\u003c/em\u003e, and \u003cem\u003eaac(3)-IIa\u003c/em\u003e, and all harbored IncFIB and IncFII type plasmids, indicating a clear clonal spread event (Table S4). Additionally, multiple ST431 strains isolated in Portugal in 2023 carried resistance genes such as \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;22\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;3\u003c/sub\u003e, and all contained IncFIB, IncFII, and IncFIA type plasmids (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDue to current technical limitations, we cannot directly confirm that resistance genes and plasmid replicons reside on the same DNA fragment. Our statistical analysis, based on Spearman correlation, revealed that certain resistance genes and plasmid replicons exhibit similar prevalence patterns across genomes (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). While this indicates a trend of co-occurrence at the population level, it does not provide direct evidence of physical co-localization on the same plasmid. For instance, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;22\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;3\u003c/sub\u003e, \u003cem\u003eaph(3'')-Ib\u003c/em\u003e, and \u003cem\u003etet(A)\u003c/em\u003e show correlated prevalence patterns with each other and with the plasmid replicon IncHI3; IncHI2 is correlated with genes such as \u003cem\u003emcr-9.1\u003c/em\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eLAP\u0026minus;2\u003c/sub\u003e, and IncC correlates with \u003cem\u003edfrA19\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e; Table S9). These results reflect statistical co-occurrence rather than physical co-location, and plasmids may serve as potential vectors for these resistance genes.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eIn this study, we performed genomic sequencing and in-depth characterization of \u003cem\u003eK. grimontii\u003c/em\u003e K767, a clinical isolate co-harboring the carbapenemase genes \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e. By further integrating resources from the GenBank public database, we conducted a systematic epidemiological analysis of 235 \u003cem\u003eK. grimontii\u003c/em\u003e genomes from a global collection. This comprehensive approach elucidated the resistome profile, transmission mechanisms, and global epidemiological characteristics of this species.\u003c/p\u003e \u003cp\u003eStrain K767 demonstrated resistance to multiple classes of critical antimicrobial agents, including carbapenems and cephalosporins, but remained susceptible to tetracyclines and tigecycline. This resistance phenotype closely aligns with its carriage of corresponding resistance genes. Of particular significance is the concurrent production of both KPC and IMP type carbapenemases by K767. The co-production of two carbapenemases belonging to different Ambler classes (Class A and Class B) within a single strain may lead to the failure of various therapeutic regimens, including those involving novel β-lactam/β-lactamase inhibitor combinations. This severely limits treatment options and substantially increases the difficulty of infection control [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs crucial mobile genetic elements, plasmids serve as the primary vectors mediating the spread of carbapenemase genes [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Among these, IncFII-type plasmids are common and significant carriers facilitating the dissemination of carbapenemase genes such as \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e in Enterobacteriaceae [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. When IncFII and IncFIA replicons coexist, they often form hybrid plasmids, which can enhance plasmid stability within the host and broaden the host range [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].In this study, pK767-KPC, which carries \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e, belongs to the IncFII:IncFIA plasmid type. Hybrid IncFII:IncFIA plasmids are frequently reported to mediate the spread of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e genes in Enterobacteriaceae [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Further analysis revealed that all strains included in this study carrying structurally similar IncF-KPC-2 plasmids were isolated from various regions of China. Notably, some of these plasmids (e.g., pBKPC18-1) exhibited a highly consistent backbone and resistance module with pK767-KPC, further substantiating the significant role of this plasmid type in mediating the cross-regional transmission of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eIncHI5-type plasmids constitute a class of large, broad-host-range conjugative plasmids in \u003cem\u003eEnterobacteriaceae\u003c/em\u003e, typically exceeding 200 kb in size [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Studies indicate that bacteria harboring IncHI5 plasmids are primarily isolated from human specimens, and these plasmids possess a wide dissemination capability among \u003cem\u003eEnterobacteriaceae\u003c/em\u003e [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Notably, most IncHI5 plasmids are closely associated with multidrug resistance, with nearly all carrying various types of antibiotic resistance genes [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These resistance genes are often linked to transposons such as Tn\u003cem\u003e1696\u003c/em\u003e or Tn\u003cem\u003e6535\u003c/em\u003e [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The plasmid pK767-IMP identified in this study belongs to the IncHI5 type. Its structure comprises a conserved backbone and two highly variable multidrug resistance regions: a 49.9 kb Tn\u003cem\u003e1696\u003c/em\u003e-derived transposon and a 67.8 kb Tn\u003cem\u003e6535\u003c/em\u003e-associated multidrug resistance region. These two large resistance regions function like dual \"resistance gene toolkits,\" significantly enhancing the strain's ability to acquire and accumulate diverse resistance determinants. Comparative genomic analysis revealed that strains carrying structurally highly similar IncHI5-IMP-4 plasmids (e.g., p13450-IMP, p19051-IMP) were almost exclusively from the genus \u003cem\u003eKlebsiella\u003c/em\u003e (27/31, 87.1%) and were all isolated in China, suggesting this plasmid type mediates the horizontal transfer and dissemination of the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e gene among \u003cem\u003eKlebsiella\u003c/em\u003e species within China.\u003c/p\u003e \u003cp\u003eWhole-genome screening for resistance genes confirmed that \u003cem\u003eK. grimontii\u003c/em\u003e constitutes a significant reservoir of resistance determinants, harboring a wide variety of acquired resistance genes. Statistical analysis revealed that the IncFIB (63.83%) and IncFII (62.13%) plasmid replicon types were the most prevalent, underscoring their important role in mediating the dissemination of resistance genes among these strains. Furthermore, statistical analysis identified significant correlations between specific resistance genes and particular plasmid replicon types (e.g., \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;22\u003c/sub\u003e with IncHI3, and \u003cem\u003emcr-9.1\u003c/em\u003e with IncHI2). Although physical linkage cannot be directly confirmed from the assembled data, this co-occurrence strongly suggests that plasmids serve as key vectors driving the co-dissemination of these resistance genes across different strains.\u003c/p\u003e \u003cp\u003eThe isolation of strains showed distinct spatiotemporal clustering. Temporally, 2017 to 2018 marked a peak period for genome data submissions, which may reflect heightened clinical attention or outbreak events during that time. Spatially, Europe, particularly the United Kingdom and Switzerland, is currently the primary source of genome data. This likely relates to robust microbial surveillance and genome sequencing initiatives in these regions and does not necessarily indicate the true epicenter of prevalence. Notably, within specific timeframes and locations (e.g., Europe in 2018), clonal expansion dominated by STs such as ST408 and ST577 was observed, suggesting potential local transmission chains within healthcare or community settings. Furthermore, the ST371 and ST431 clonal groups identified within Cluster III provide compelling evidence for clonal spread co-occurring with the dissemination of plasmids and resistance genes. Members of these groups not only shared identical STs but also carried nearly identical resistance gene and plasmid profiles.\u003c/p\u003e \u003cp\u003eThis study has several main limitations. First, the genomic data were primarily sourced from public databases, which introduces potential geographical and temporal sampling biases. Therefore, the data may not fully represent the true global distribution of this bacterial species. Second, the analysis was largely based on short-read sequencing data. While bioinformatics methods allowed for the accurate identification of resistance genes and plasmid replicons and confirmed their statistical associations, the limitations of short read lengths hindered the complete assembly of plasmids and the precise localization of resistance genes. Consequently, direct physical evidence confirming that resistance genes and plasmids reside on the same DNA molecule could not be obtained, which may affect the precision of inferences regarding plasmid transmission dynamics. Future research should involve more prospective, standardized surveillance. This should be combined with long-read sequencing technologies to accurately resolve the complete structure of resistance plasmids, alongside in vitro and in vivo conjugation experiments to empirically validate plasmid transferability. Such approaches will provide a stronger scientific foundation for developing more effective infection prevention and control strategies.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eThis study reports a clinical strain of \u003cem\u003eK. grimontii\u003c/em\u003e, K767, co-harboring the carbapenemase genes \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e and blaIMP-4. It exhibits resistance to multiple classes of antimicrobial agents, including carbapenems and cephalosporins, representing a clinically high-risk case of accumulated resistance. Our analysis confirmed that the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e gene is carried by an IncFII:IncFIA-type hybrid plasmid (pK767-KPC). This plasmid type is widely disseminated across different regions in China and serves as a primary vector driving the cross-regional spread of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e. The \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e gene is carried by a large IncHI5-type conjugative plasmid (pK767-IMP). This plasmid contains two large and variable \"resistance gene toolkits,\" including a Tn\u003cem\u003e1696\u003c/em\u003e-derived transposon and a Tn\u003cem\u003e6535\u003c/em\u003e-associated element, which substantially enhance the multidrug resistance capacity of the host bacterium. Strains carrying similar IncHI5-IMP-4 plasmids are predominantly \u003cem\u003eKlebsiella\u003c/em\u003e species of Chinese origin, indicating this plasmid type mediates the dissemination of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e within this genus in China. Whole-genome analysis confirmed that this bacterial species harbors a wide array of acquired resistance genes, highlighting the key role of plasmids in driving their co-dissemination. Phylogenetic analysis revealed high genetic diversity within the global \u003cem\u003eK. grimontii\u003c/em\u003e population, with its prevalence demonstrating spatiotemporal heterogeneity. The observed clonal spread events underscore the complexity of its transmission mechanisms.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e \u003cp\u003e \u003cem\u003eK. oxytoca Klebsiella oxytoca\u003c/em\u003e \u003c/p\u003e \u003cp\u003eANI Average Nucleotide Identity\u003c/p\u003e \u003cp\u003eAST Antimicrobial susceptibility testing\u003c/p\u003e \u003cp\u003eCLSI Clinical and Laboratory Standards Institute\u003c/p\u003e \u003cp\u003eSNPs Single-nucleotide polymorphisms\u003c/p\u003e \u003cp\u003eARGs Antibiotic Resistance Genes\u003c/p\u003e \u003cp\u003eMLST Multi-Locus Sequence Types\u003c/p\u003e \u003cp\u003eSTs Sequence types\u003c/p\u003e \u003cp\u003eORFs Open reading frames\u003c/p\u003e \u003cp\u003eRep Replicon\u003c/p\u003e \u003cp\u003eInc Incompatibility\u003c/p\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eThis study was conducted in accordance with the Declaration of Helsinki. The use of human specimens and all related experimental protocols was reviewed and approved by the Ethics Committee of Taizhou Municipal Hospital, Zhejiang, China, in accordance with the medical research regulations of the Ministry of Health, China. Research and all related procedures involving biohazardous materials were approved by the Biosafety Committee of Taizhou Municipal Hospital. The Ethics Committee of Taizhou Municipal Hospital, Zhejiang, China granted a waiver of the requirement for informed consent for this study. This research was conducted in China.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Medical and Health Science and Technology Project of Zhejiang Province (2025KY461), and the Natural Science Foundation of Zhejiang Province (LTGY23H190003).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, X.H.L., Y.L.Z., and Z.G.J.; methodology, Y.L.Z., Y.Z., J.B.Y., H.M.C., Z.G.J., and J.L.; data analysis, X.H.L., Y.L.Z., X.L., J.L., Y.Z., and H.M.C.; resources, X.L., J.B.Y. and H.M.C.; writing-original draft, Y.L.Z. and Z.G.J.; writing-review and editing, X.H.L. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and analyzed during the current study are available in the public NCBI GenBank database, under the following accession numbers: CP198962, CP198963, CP198964, CP198966, and CP198965.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDong N, et al. Klebsiella species: Taxonomy, hypervirulence and multidrug resistance. EBioMedicine. 2022;79:103998.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLei TY, et al. Hypervirulent and carbapenem-resistant Klebsiella pneumoniae: A global public health threat. Microbiol Res. 2024;288:127839.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKocsis B. 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Front Microbiol. 2021;12:636396.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Klebsiella grimontii, carbapenemase genes, bla KPC−2, bla IMP−4, sequence types, Global population analysis","lastPublishedDoi":"10.21203/rs.3.rs-9296935/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9296935/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eKlebsiella grimontii\u003c/em\u003e is a recently confirmed member of the \u003cem\u003eKlebsiella oxytoca\u003c/em\u003e complex with significant clinical importance. As an opportunistic pathogen, reports of infections it causes are increasing, and the associated threats warrant attention. However, its phylogeny and epidemiological patterns remain poorly studied.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn this study, we identified a strain of \u003cem\u003eKlebsiella grimontii\u003c/em\u003e, designated K767, simultaneously carrying the carbapenemase genes \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e through phenotypic assays and sequencing techniques from a tertiary hospital in Zhejiang Province, China. After performing whole-genome analysis of this strain, we integrated data from the NCBI public database, incorporating genomic information from a total of 235 \u003cem\u003eKlebsiella grimontii\u003c/em\u003e strains for in-depth bioinformatics analysis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eGenomic analysis revealed that in strain K767, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e is located on an IncHI5-type plasmid. This plasmid harbors multiple resistance modules and is highly conserved among similar plasmids originating from China. \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e is located on an IncFII:IncFIA-type hybrid plasmid, a type predominantly found in \u003cem\u003eKlebsiella\u003c/em\u003e species within China. Analysis of the 235 global strains indicated spatiotemporal heterogeneity in their prevalence, with Europe being the primary region. A total of 76 sequence types were identified, with Cluster III being the predominant evolutionary clade. The strains carried 75 acquired antimicrobial resistance genes, including various carbapenemase genes, and a significant co-occurrence was observed between certain resistance genes and specific plasmid replicons.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study reports a strain of \u003cem\u003eKlebsiella grimontii\u003c/em\u003e simultaneously carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;4\u003c/sub\u003e. These genes are carried by an IncFII:IncFIA-type hybrid plasmid and an IncHI5-type conjugative plasmid, respectively, both of which are widely disseminated in China. Global population analysis suggests that this species exhibits high genetic diversity, with spatiotemporal heterogeneity in its prevalence, and that plasmids play a key role in the transmission of multidrug resistance. The findings provide an important basis for the clinical prevention and control of such resistant pathogens.\u003c/p\u003e","manuscriptTitle":"Co-occurrence of blaIMP-4 and blaKPC-2 in a Clinical Isolate and Global Evolutionary Genomics of Klebsiella grimontii","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-22 20:51:34","doi":"10.21203/rs.3.rs-9296935/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-19T06:13:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-18T06:39:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-22T08:10:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-20T08:18:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4390663414488697659769137294705383765","date":"2026-04-18T09:24:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"260582887782539993550957140854671228471","date":"2026-04-16T09:48:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"193412539973495629442386633559011029964","date":"2026-04-14T00:53:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-13T15:01:46+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-06T13:13:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-04T14:44:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-04T14:43:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2026-04-02T01:53:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"27b7695b-182f-4510-9836-7ac8ab411304","owner":[],"postedDate":"April 22nd, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-19T06:13:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-18T06:39:41+00:00","index":19,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-19T06:25:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-22 20:51:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9296935","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9296935","identity":"rs-9296935","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}