Abstract
The emergence of carbapenemase-producing Klebsiella pneumoniae poses a significant global health threat, particularly in hospital settings. This study reports on the first detection of a pandrug-resistant (PDR) high-risk ST15 K. pneumoniae strain co-producing NDM-1 and VIM-1 in Greece. The isolate was recovered from a blood culture of a male patient admitted to the Intensive Care Unit (ICU) of Volos Hospital in July 2024. Next generation Sequencing (NGS) confirmed the presence of blaNDM-1 and blaVIM-1 genes. Other beta-lactamase type (CTX-M-15) was detected in association with NDM and VIM enzymes. Furthermore, this isolate was resistant to other antimicrobial agents, including aminoglycosides [aac(3)-II, aac(3)-IIe, aac(6′)-Ib, aadA1, aph(3″)-Ib, aph(6)-Id, aph(3′)-Ia), chloramphenicol (catB3), fluoroquinolones (qnrS1) and sulfonamides (sul1 and sul2). The Multilocus Sequence Typing revealed that the strain belonged to ST15. According to Kaptive the strain belonged to KL48. Our study provides new data about MBL producing K. pneumoniae in Greece. Thus, we report for the first time the co-expression of blaNDM-1 and blaVIM-1 in our country in ST15 K. pneumoniae. This study provides crucial epidemiological data on MBL-producing K. pneumoniae in Greece and highlights the urgent need for enhanced surveillance, infection control strategies, and access to last-resort antibiotics such as aztreonam-avibactam.
Introduction
A significant challenge in treating infections caused by Klebsiella pneumoniae is the production of carbapenemase enzymes, which confer resistance to beta-lactam antibiotics [1]. The availability of effective treatment options is increasingly limited. Newer beta-lactamase inhibitors, such as avibactam and vaborbactam, are used for infections caused by KPC-producing K. pneumoniae [2]. However, for metallo-beta-lactamase (MBL)-producing strains, treatment relies on aztreonam-avibactam and cefiderocol, which are available in certain countries but not in Greece [3, 4]. Additionally, aztreonam—potentially effective against such infections—is unavailable in many Greek hospitals due to financial constraints. Despite its relatively low cost, pharmaceutical companies in Greece are reluctant to distribute aztreonam nationwide, further limiting treatment options for patients with MBL-producing infections.
In addition, the co-existence of NDM-producing K. pneumoniae with other carbapenemases has been widely reported across the globe, highlighting the increasing complexity of antimicrobial resistance and the challenges in treatment strategies [5–10]. According to this phenomenon many studies from Greece have documented the double carbapenemase producers [11, 12]. An NDM-1 and VIM-1 co-producing K. pneumoniae strain belonging to sequence type (ST) 11 was first reported in Greece by Papagiannitsis et al. in 2017 [7].
The ST15 lineage has been increasingly associated with multidrug resistance and nosocomial outbreaks worldwide. An ST15 KPC-2-producing K. pneumoniae outbreak was reported in Bulgaria in 2015 by Markovska R. et al. [9]. An NDM-1-producing ST15 K. pneumoniae strain was reported in Bulgaria in 2017 by Savov et al. [10]. In Greece, previous studies have reported ST15 strains carrying NDM-1 (Politi et al., 2019) but this is the first report of an ST15 strain harboring both NDM-1 and VIM-1. To the best of our knowledge, this is the first report of a pandrug-resistant (PDR) K. pneumoniae strain co-producing NDM and VIM belonging to the high-risk ST15 clone in Greece. The presence of such highly resistant strains in the Greek healthcare system is concerning, given the limited access to novel combination therapies such as aztreonam-avibactam. Additionally, the economic barriers limiting the availability of aztreonam in many Greek hospitals further exacerbate the challenges in treating such infections.
Materials and methods
The K. pneumoniae A436 strain was isolated from a positive blood culture of a male patient hospitalized in the Intensive Care Unit (ICU) of Volos Hospital, Greece, in July 2024.
The identification and antimicrobial susceptibility testing were conducted using the Vitek-2 automated system (Biomerieux, Marcy-l'Étoile, France). The susceptibility testing for newer beta-lactam/beta-lactamase inhibitor combinations was performed using gradient E-tests (Liofilchem). The determination of minimal inhibition concentration was performed according to Eucast guidelines (Eucast 2024, assessed on July 2024).
The detection of carbapenemase enzymes was carried out using immunochromatographic assay (NG Biotech).
Next Generation Sequencing was performed in a private laboratory in Greece. Libraries were prepared using Ion Torrent technology and Ion Chef workflows (Thermo Scientific). Sequencing was performed in the S5XLS system and analysis of primary data was conducted with Ion Torrent Suite v.5.10.0.
Genome assembly was performed with Spades (Galaxy Version 3.15.5+galaxy2). Quast Genome assembly Quality (Galaxy Version 5.3.0+galaxy0) was used in order to assess the quality of the assembly. Resistance profiling was performed with AMR Finder plus (Galaxy Version 3.12.8+galaxy0). Mlst via (Galaxy Version 2.22.0). Replicons were detected via PlasmidFinder (Galaxy Version 2.1.6+galaxy1). Integrons were detected via Integron Finder (Galaxy Version 2.0.5+galaxy0). Kaptive was used in order to find the K locus group of the strain (https://kaptive-web.erc.monash.edu/).
Results
Antimicrobial susceptibility testing
The isolate demonstrated pandrug-resistance (PDR), exhibiting high-level resistance to all tested beta-lactams, aminoglycosides, fluoroquinolones, and colistin. The minimum inhibitory concentration (MIC) values and their respective interpretations are summarized in Table 1.
Susceptibility testing of K. pneumoniae A436 strain
| Antimicrobial | MIC (µg mL−1) | Interpretation |
| Ertapenem | ≥8 | R |
| Ampicillin | ≥32 | R |
| Amoxicillin/Clavulanic Acid | ≥32 | R |
| Ampicillin/Sulbactam | ≥32 | R |
| Ticarcillin/Clavulanic Acid | ≥128 | R |
| Piperacillin | ≥128 | R |
| Piperacillin/Tazobactam | ≥128 | R |
| Cefalotin | ≥64 | IE |
| Cefuroxime | ≥64 | R |
| Cefuroxime Axetil | ≥64 | R |
| Cefoxitin | ≥64 | IE |
| Cefixime | ≥4 | R |
| Cefotaxime | ≥64 | R |
| Ceftazidime | ≥64 | R |
| Ceftazidime-avibactam | ≥64 | R |
| Ceftriaxone | ≥64 | R |
| Cefepime | ≥64 | R |
| Aztreonam | ≥64 | R |
| Imipenem | ≥16 | R |
| Meropenem | ≥16 | R |
| Amikacin | 32 | R |
| Gentamicin | ≥16 | R |
| Tobramycin | ≥16 | R |
| Nalidixic Acid | (−) | R |
| Ciprofloxacin | ≥4 | R |
| Levofloxacin | ≥8 | R |
| Moxifloxacin | ≥8 | R |
| Ofloxacin | ≥8 | R |
| Tigecycline | 1 | R |
| Chloramphenicol | ≤2 | IE |
| Colistin | ≥16 | R |
| Trimethoprim/Sulfamethoxazole | ≥320 | R |
The isolate was resistant to all beta-lactams, including carbapenems and cephalosporins. It also showed high-level resistance to aminoglycosides, fluoroquinolones, colistin, and trimethoprim/sulfamethoxazole. The resistance to tigecycline (MIC = 1 μg mL−1) was noted, despite its activity against certain carbapenem-resistant strains.
Genotypic characterization
Whole-genome sequencing (WGS) analysis revealed that the isolate belonged to the ST15 lineage, a high-risk clone associated with multidrug resistance and nosocomial outbreaks. The strain was found to harbor both blaVIM-1 and blaNDM-1 genes, encoding metallo-beta-lactamases responsible for carbapenem resistance. Additional beta-lactamase genes detected included blaCTX-M-15, blaTEM-1, blaOXA-1, and blaSHV-28 (Table 2).
Resistance genes of K. pneumoniae A436 strain
| Resistance gene | Function | Targeted antibiotics |
| blaVIM-1, blaNDM-1 | metallo-beta-lactamases (class B) | Carbapenems |
| oqxA, oqxB | Multidrug efflux RND transporter periplasmic adaptor subunit OqxA, OqxB | Multiple drug classes |
| blaSHV-28, blaTEM-1, blaOXA-1 | Broad-spectrum beta-lactamase SHV-28, TEM-1 (class A); oxacillin-hydrolyzing beta-lactamase OXA-1 (class D) | beta-lactams |
| blaCTX-M-15 | Extended-spectrum beta-lactamase CTX-M-15 (class A) | Third generation Cephalosporins, beta-lactams |
| parC_S80I, gyrA_D87A, gyrA_S83F | K. pneumoniae quinolone-resistant ParC, GyrA | Fluoroquinolones |
| qnrS1 | Quinolone resistance pentapeptide repeat protein QnrS1 | Fluoroquinolones |
| fosA | FosA5 family fosfomycin resistance glutathione transferase | Fosfomycin |
| sul1, sul2 | Sulfonamide-resistant dihydropteroate synthase Sul1, Sul2 | Sulfonamides |
| dfrA1, dfrA14 | Trimethoprim-resistant dihydrofolate reductase DfrA1, DfrA14 | Trimethoprim |
| aph(3″)-Ib, aph(6)-Id, aph(3′)-Ia | Aminoglycoside O-phosphotransferase APH(3″)-Ib, APH(6)-Id, APH(3′)-Ia | Aminoglycosides |
| aadA1 | ANT(3″)-Ia family aminoglycoside nucleotidyltransferase AadA1 | Aminoglycosides |
| aac(6′)-Il, aac(3)-IIe, aac(6′)-Ib | Aminoglycoside N-acetyltransferase AAC(6′)-Il, AAC(3)-IIe, AAC(6′)-Ib | Aminoglycosides |
| qacEdelta1 | Quaternary ammonium compound efflux SMR transporter QacE delta 1 | Disinfectants, biocides |
| mph(A) | Mph(A) family macrolide 2′-phosphotransferase | Macrolides |
| ble | Bleomycin binding protein Ble-MBL | Bleomycin |
| catB3 | Type B-3 chloramphenicol O-acetyltransferase CatB3 | Chloramphenicol |
Whole-genome sequencing identified mutations possibly associated with colistin resistance in the K. pneumoniae A436 isolate. Specifically, mutations in the lpxM (S253G) and arnC (S30T). The lpxM_S253G, a mutation in the lpxM gene, which is involved in the lipid A biosynthesis pathway, potentially altering the lipopolysaccharide (LPS) structure, leading to reduced colistin binding and resistance.
ArnC_S30T a mutation in the arnC gene, which plays a role in the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to lipid A, a modification known to reduce colistin susceptibility. These findings suggest that chromosomal modifications rather than plasmid-mediated mcr genes are responsible for the high-level colistin resistance observed in this isolate.
The integron of K. pneumoniae ST15, harbors multiple resistance genes, contributing to its multidrug-resistant phenotype. The integron includes:
Efflux pump gene (qacEΔ1), which may play a role in resistance to disinfectants and antiseptics.
Aminoglycoside resistance genes (aadA1, aac(6′)-II), encoding enzymes that modify aminoglycosides and confer resistance.
Trimethoprim resistance gene (dfrA1), which affects susceptibility to trimethoprim by encoding an alternative dihydrofolate reductase.
Carbapenemase gene (blaVIM-1), encoding a Verona Integron-encoded Metallo-beta-Lactamase (VIM-1), a key enzyme conferring resistance to carbapenems.
This integron-mediated accumulation of resistance genes enhances K. pneumoniae ST15's ability to survive under antimicrobial pressure, making it a high-risk multidrug-resistant (MDR) clone with limited treatment options.
Virulence and plasmid analysis
Virulence factor analysis identified the presence of Type 1 and Type 3 fimbriae genes (mrk and fim clusters), the yersiniabactin siderophore, and genes associated with capsular polysaccharide production (Table 3).
Virulence of the study strain K. pneumoniae A436
| Category | Genes | Locus |
| Type 3 fimbriae | mrk (B, C, D, F, H, I, J) | orf03846, orf03845, orf03844, orf03843, orf03840, orf03841, orf03842 |
| Type I fimbriae | fimA, fimB, fim(C, D, E, F, G, H, I, K) | orf04753, orf04756, orf02699; orf04751, orf02698; orf03632; orf04750, orf04754, orf04749; orf05254, orf04748, orf04747; orf05253, orf04752, orf04746; orf05252 |
| Capsule | – | orf00410 - orf00416; orf00427 - orf00433; orf04379 |
| AcrAB | acrA, acrB | orf01193, orf01192; orf02899 |
| Aerobactin | iutA | orf02105 |
| Ent siderophore | entA, entB, entC, entD, entE, entF, entS, fep(A, B, C, D, G), fes | orf02519, orf02518, orf02516, orf02505, orf02517, orf02509, orf02514, orf01535; orf02506, orf02515, orf02510, orf02512, orf02511, orf02507 |
| Salmochelin | iroE, iroN | orf03382, orf01702 |
| Yersiniabactin | fyuA, irp1, irp2, ybt(A, E, P, Q, S, T, U, X) | orf00515, orf00519, orf00520, orf00521, orf00516, orf00522, orf00523, orf00525, orf00517, orf00518, orf00524 |
| RcsAB | rcsA, rcsB | orf00546, orf00273 |
| T6SS-I | clpV/tssH, dotU/tssL, hcp/tssD, icmF/tssM, impA/tssA, ompA, sciN/tssJ, tss(F, G), vasE/tssK, vgrG/tssI, vip(A, B) | orf04830, orf04833, orf04831, orf03111, orf03112, orf04832, orf03117, orf03115; orf03736, orf03116, orf04834, orf05465, orf04836, orf04835 |
| T6SS-II | clpV, dotU, icmF, imp(F, H, J), ompA, sciN, vgrG | orf02902, orf03729, orf03735, orf03739, orf03737, orf03728, orf03730, orf03738, orf03731 |
| T6SS-III | dotU, icmF, imp(A, F, G, H, J), ompA, sciN | orf04605, orf02295, orf02300, orf02299, orf02296, orf02297, orf04606, orf04604, orf02298 |
Bold: Virulence of K. pneumoniae A436 strain.
Plasmid replicon typing identified the presence of IncA/C2, IncFIA(HI1), IncFIB(K), and IncFII(K) plasmids, which are commonly associated with carbapenemase-encoding genes and multidrug resistance.
Genome assembly and quality metrics
The genome assembly analysis yielded a total of 209 contigs, with a total genome length of 5,719,458 bp. The largest contig measured 689,525 bp, and the N50 value was 198,607 bp, indicating a well-assembled genome (Table 4).
Genome Assembly Metrics for K. pneumoniae A436 strain
| Statistic | Value |
| # Contigs | 103 |
| # Contigs (≥0 bp) | 209 |
| # Contigs (≥1,000 bp) | 83 |
| Largest Contig | 689,525 |
| Total Length | 5,685,559 |
| Total Length (≥0 bp) | 5,719,458 |
| Total Length (≥1,000 bp) | 5,672,811 |
| N50 | 198,607 |
| N90 | 62,718 |
| auN | 242,994 |
| L50 | 10 |
| L90 | 29 |
| GC (%) | 56.96 |
| Per Base Quality | – |
| #N's per 100 kbp | 0 |
| #N's | 0 |
Discussion
ST15 K. pneumoniae is a high-risk lineage that has expanded across different regions. It has been associated with blaKPC-2 gene in the past, but later on it has been associated in the Balkan region with blaNDM-1, as lineage ST11. ST15 is recognized as a high-risk clone associated with multidrug resistance and has been implicated in various outbreaks globally. The KL48 capsular type, while less commonly reported in association with ST15, has been identified in certain studies. For instance, a population genomic analysis of clinical ST15 K. pneumoniae strains in China identified four clades, with one clade (C3) associated with the KL48 capsular type, although this clade represented a small proportion (0.7%) of the studied strains [13].
The study isolate A436 exhibited resistance to all tested beta-lactams, aminoglycosides, fluoroquinolones, and colistin, underscoring the critical limitation of available treatment options. The detection of both blaNDM-1 and blaVIM-1 indicates a dual metallo-beta-lactamase (MBL) production, a rare but increasingly reported mechanism in multidrug-resistant K. pneumoniae strains [5–7]. This co-production of MBLs renders all carbapenems ineffective, with aztreonam-avibactam remaining as the only potential treatment option, which, however, is not currently available in Greece. The absence of mcr genes suggests that colistin resistance is likely due to chromosomal mutations in the lpxM and arnC genes, leading to modifications in lipid A and reducing colistin binding.
Given the rapid evolution and dissemination of carbapenemase-producing K. pneumoniae, enhanced infection control measures are urgently needed. Routine surveillance, strict antimicrobial stewardship programs, and rapid molecular diagnostics should be prioritized to limit the spread of PDR pathogens. Furthermore, the monitoring of integron-carrying strains is essential, as they represent a major reservoir for the horizontal gene transfer of resistance determinants. Whole-genome sequencing (WGS) and epidemiological tracking of high-risk clones like ST15 can provide valuable insights into their transmission dynamics and guide targeted containment strategies.
A major limitation of the study is the absence of phylogenic correlation with other strains ST15 of the Hospital of Volos. Such comparative analyses are crucial for understanding the genetic relationships, potential transmission pathways, and evolutionary dynamics of this high-risk lineage within the hospital setting. Thus, incorporating phylogenetic comparisons with local ST15 strains would enhance the study's findings by providing a clearer picture of the genetic landscape and transmission dynamics of K. pneumoniae within the Hospital of Volos.
Conclusion
This study highlights the first detection of a PDR K. pneumoniae ST15 strain co-producing NDM-1 and VIM-1 in Greece, emphasizing the growing threat of metallo-beta-lactamase-producing K. pneumoniae in hospital settings. The integron-mediated accumulation of resistance genes in this strain further complicates treatment options and underscores the need for urgent infection control measures, antimicrobial stewardship programs, and access to last-resort antibiotics. Given the global dissemination of ST15 high-risk clones, continuous surveillance is critical to prevent the establishment and spread of such extensively resistant pathogens.
Author contributions
CM: conceptualization methodology and design of the study, resources, data curation, writing—original draft preparation, writing—review and editing. TP: writing- original draft preparation. MAK: review and editing. SV: laboratory testing. FC: laboratory testing. IF: laboratory testing and analysis. KAK: software, validation, formal analysis. KK: editing. MS: writing—review and editing. PS: editing. All authors have read and agreed to the published version of the manuscript.
Funding
This study received no external funding.
Institutional review board statement
Not applicable.
Informed consent statement
Not applicable.
Data availability statement
The whole genome of K. pneumoniae has been deposited at DDBJ/ENA/GenBank under the accession Number PRJNA1222132.
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