Outbreak of High-Risk Clone ST323 Klebsiella pneumoniae Resistant to Ceftazidime–Avibactam Due to Acquisition of blaVEB-25 and to Cefiderocol Due to Mutated fiu Gene
by
, , , , and
Irene Galani
1,*
,
Ilias Karaiskos
2,
Maria Souli
3,
Vassiliki Papoutsaki
4,
Aikaterini Gkoufa
2,5,
Anastasia Antoniadou
1,6 and
Helen Giamarellou
2 1
Infectious Diseases Laboratory, 4th Department of Internal Medicine, National and Kapodistrian University of Athens, 12462 Athens, Greece
2
1st Internal Medicine & Infectious Diseases Department, Hygeia General Hospital, 15123 Athens, Greece
3
Duke Clinical Research Institute, Duke University Medical Center, Durham, NC 27710, USA
4
Infectious Diseases Laboratory, Hygeia General Hospital, 15123 Athens, Greece
5
Department of Infectious Diseases, First Department of Internal Medicine, National and Kapodistrian University of Athens, Laikon General Hospital, 11527 Athens, Greece
6
Medical School, University of Cyprus, 2029 Nicosia, Cyprus
*
Author to whom correspondence should be addressed.
Antibiotics 2025, 14(3), 223; https://doi.org/10.3390/antibiotics14030223
Submission received: 22 January 2025
/
Revised: 16 February 2025
/
Accepted: 19 February 2025
/
Published: 21 February 2025
(This article belongs to the Special Issue A Themed Issue in Honor of Professor Helen Giamarellou—Outstanding Contributions in the Fields of Antimicrobial Resistance and Difficult-to-Treat Resistance Pathogens)
Abstract
:Background/Objectives: The incidence of Ceftazidime/Avibactam (CZA)-resistant Klebsiella pneumoniae isolate co-producing Klebsiella pneumoniae carbapenemase 2 (KPC-2) and Vietnamese extended-spectrum β-lactamase 25 (VEB-25) has been on the rise in Greece over the past five years. This study investigates the isolation of ST323 K. pneumoniae isolates co-resistant to CZA and cefiderocol (FDC) from colonized and infected patients in a single hospital in Athens. Methods: CZA-resistant K. pneumoniae strains were isolated from 5 ICU patients from 27 December 2023 to 22 January 2024. Antimicrobial susceptibility was tested against a panel of agents. Whole-genome sequencing of the isolates was carried out to identify the acquired resistance genes and mutations that were associated with CZA and FDC resistance. Results: The K. pneumoniae isolates belonged to ST323 and harbored blaKPC-2 and blaVEB-25. The isolates had a minimum inhibitory concentration (MIC) of >256 mg/L for CZA and 32 mg/L for FDC, due to the disrupted catecholate siderophore receptor Fiu. blaVEB-25 was located on an IncC non-conjugative plasmid and on a ~14 kb multidrug resistance (MDR) region comprising 15 further acquired resistance genes. Transformation studies showed that the blaVEB-25-carrying plasmid provided resistance to most of the β-lactams tested, including CZA. The isolates remained susceptible to carbapenems, imipenem/relebactam, and meropenem/vaborbactam. The plasmid harbored the citrate-dependent iron (III) uptake system (fecIRABCDE), which increased the MIC of FDC from ≤0.08 mg/L to 2 mg/L. Conclusions: The blaVEB-25 gene was associated with IncC plasmids which are important contributors to the spread of key antibiotic resistance genes. Strict infection control measures must be elaborated upon to prevent the spread of extensively drug-resistant organisms such as those described here.
1. Introduction
Antibiotic resistance is a dynamic and evolving process among bacteria, primarily driven by prolonged exposure to antimicrobial agents. This underscores the necessity for continuous surveillance to enable early detection of resistance mechanisms, leveraging advanced genetic assays and molecular technologies [1]. Such efforts are particularly critical following the commercial approval of novel β-lactam–β-lactamase inhibitor (BLBLI) combinations. These agents have demonstrated high clinical efficacy, and a more favorable adverse event profile compared to older antibiotics, thereby enhancing therapeutic outcomes of infections caused by carbapenem-resistant pathogens [2].
Klebsiella pneumoniae is a frequent cause of healthcare-associated infections including pneumonia, urinary tract, wound, and bloodstream infections [3]. Hospital infections caused by carbapenemase-producing K. pneumoniae (CPKP) are a worldwide problem with high treatment failure and mortality rates.
The worldwide spread of CPKP is driven by the transmission of international high-risk clones in healthcare facilities [4,5]. In Greece, Tryfinopoulou et al. reported the dissemination of high-risk clones of CPKP and showed the persistent spread of previously established (ST258, ST11, and ST147) as well as newly emerging high-risk clones (ST39 and ST323) in Greek hospitals over a 10-year period [6]. ST323 has been mainly associated with the blaCTX-M-15 extended-spectrum β-lactamase gene and has been linked to healthcare-associated infections in Australia and Poland [7,8] or community-acquired urinary tract infections in Tunisia [9]. Sporadic cases of ST323 CPKP carrying blaVIM or both blaKPC-2 and blaVIM have been reported in Greece in 2005 and 2011 [10,11], and Zarras et al. reported the isolation of such isolates from three ICU patients in Thessaloniki in 2019 [12]. ST323 was not detected in the carbapenem- and/or colistin-resistant Enterobacterales (CCRE) survey conducted in 2019 in 15 Greek hospitals. However, this high-risk clone had rapidly spread across six Greek hospitals in Attica, Peloponnese, and Crete by 2022. Interestingly, this clone was not found in the six participating hospitals in northern and central Greece [6].
VEB-25 (Vietnamese extended-spectrum β-lactamase) was first reported in 2019 in two KPC-KP isolates from patients in two Greek hospitals [13] and caused an outbreak in two ICUs of a hospital in Athens. This outbreak affected seven patients over five weeks and was linked to ceftazidime/avibactam (CZA) resistance [14]. The isolates belonged to ST147 and ST258. Later, Zarras et al. reported a case of neonatal sepsis due to CZA-resistant KPC-KP carrying blaVEB-25 belonging to ST35 that was successfully treated with non-conventional “off-label” antimicrobial agents [15]. Furthermore, in 2023, Findlay et al. reported the isolation of an ST323 KPC-producing Klebsiella pneumoniae (KPC-KP) resistant to CZA due to the acquisition of VEB-25. The source was a patient in Switzerland who had previously been hospitalized in Greece [16]. VEB-type β-lactamases are a group of Ambler class A enzymes inhibited by avibactam, although VEB-25 differs from VEB-1 by a substitution of lysine by an arginine at position 237 (K234R, per Ambler numbering scheme), due to a nt A710G substitution [13], which compromises the inhibitory efficiency of avibactam [17]. To our knowledge, this variant has only been reported in Greece and in one patient in Switzerland who was epidemiologically linked with Greece.
Cefiderocol (FDC) is a new siderophore cephalosporin that penetrates the outer membrane of Gram-negative bacteria using their iron transport system, binds to penicillin-binding proteins (PBPs), and inhibits the bacterial wall synthesis [18]. The activity of FDC against aerobic Gram-negative bacilli is equal to or superior to that of CZA and meropenem due to its high stability against β-lactamases including ESBLs, AmpC, and carbapenemases. In a recent Greek study, FDC inhibited 80% of the CPKP isolates with minimum inhibitory concentrations (MICs) ranging from 0.006 to 32 mg/L, and the concentration of FDC inhibiting 90% (MIC90) of the isolates was 4 mg/L [19]. FDC was approved by the US Food and Drug Administration and the European Medicines Agency in 2020 to treat Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa invasive infections caused by carbapenem- and CZA-resistant strains [20,21]. However, despite the initial promising in vitro and in vivo results, the emergence of resistance has been observed worldwide. The mechanisms underlying FDC resistance remain poorly described. Combined factors such as modification of the target (PBP-3), mutations in iron transporters (mainly cirA and fiu), expression of β-lactamases, porin loss, or efflux pump overexpression by some isolates have been reported [22]. Additionally, the acquisition of a transferable extrachromosomal fec operon has been associated with a cefiderocol MIC increase in Enterobacterales [23,24]. Unfortunately, this fec gene cluster is present in many K. pneumoniae strains, including those isolated before the introduction of FDC in clinical therapy [24]. According to Polani et al., in the future, FDC may act as a positive selector for plasmids carrying the fec genes, for which prevalence can be expected to increase [24].
We report the isolation of ST323 carbapenem-resistant K. pneumoniae isolates co-resistant to CZA, due to VEB-25 production, and FDC from colonized and/or infected patients in a single hospital in Athens. FDC was introduced in Greece in 2024 and is supplied by the Institute of Pharmaceutical Research & Technology (IFET), which imports and distributes essential pharmaceutical products to the Greek market. Since no pharmaceutical company has officially launched FDC in the country, IFET ensures its availability for clinical use. The increasing use of CZA and FDC is expected to further elevate the clinical significance of VEB-25- and fec gene cluster-mediated resistance. Notably, these resistance determinants are located on the same plasmid, which has already been acquired by the high-risk CPKP clone. This raises significant concerns about the potential emergence of pan-drug resistance, posing a serious threat to antimicrobial treatment options.
2. Results
2.1. Antibiotic Susceptibility Testing and Molecular Investigation of the Isolates
Susceptibility testing evaluated according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints [25] revealed that K. pneumoniae isolates were resistant to all β-lactams tested, including carbapenems, FDC, and the BLBLI combinations CZA and aztreonam/avibactam (AZA). The isolates remained susceptible to imipenem/relebactam (IMR) (MIC 0.5–1 mg/L) and meropenem/vaborbactam (MEV) (MIC 0.125–0.25 mg/L) (Table 1). All harbored blaKPC-2 as the sole carbapenemase gene, as initially detected with NG Test CARBA 5 and confirmed by PCR and sequencing (Table 1). Additionally, all isolates harbored the blaVEB-25 variant, which explained the CZA resistance.
All isolates exhibited an identical pulsed-field gel electrophoresis pulsotype using enzyme SpeI and conditions as previously described [26]. Representative isolates were subjected to whole-genome sequencing (WGS), which identified that the isolates belonged to clonal lineage ST323 and harbored blaKPC-2, blaVEB-25, and 15 further acquired resistance genes encoding resistance to multiple antibiotic classes: tet(G) (tetracycline efflux pump), qnrS1 (quinolones), cmlA5 (phenicols), arr-2 (rifiampicin), dfrA23 (trimethoprim), sul1 and sul2 (sulphonamides), and seven aminoglycoside resistance genes (rmtB1, aph(3″)-Ia, ant(2″)-Ia, ant(3″)-Ia, aph(6)-Id and aph(3″)-Ib) (Table 2).
The isolates had an intact OmpK35 and an OmpK36_v6 variant [27]. OmpK36_v6 exhibited a substitution of amino acids, Ala267-Gly268-Ser269-Leu270 to Phe267-Ser268-Gly269-Asn270, and an insertion of amino acids, Glu272-Ser273-Asp274-Ser275-Ile276-Ser277-Gly278, in loop L6, an external loop, facing the cell exterior.
A frameshift mutation in the fiu gene (insertion of an adenine after 652 nucleotides) and a subsequent disruption of the coding catecholate siderophore receptor Fiu protein, due to a stop codon after amino acid 250, were also found, contributing to FDC resistance [22,28].
No mutations were found in ramA or acrR genes (tigecycline resistance determinant genes); however, the amino acid replacement of N87D in GyrA occurred in both isolates, contributing to quinolone resistance.
Plasmid replicon type analyses revealed a number of replicon types present, comprising Col440II, ColRNAI, IncC, IncFIB(pQIL), IncFII(K), IncFII(pKP91), IncR, and repB(R1701) (Table 2).
Conjugation experiments were unsuccessful; however, E. coli TOP10 transformants harboring the blaVEB-25-carrying plasmid were confirmed by susceptibility testing and subsequent amplification and sequencing of the blaVEB-25 gene.
E. coli TOP10 transformants harboring blaVEB-25 were resistant to most of the β-lactams tested, including the BLBLI combinations CZA and AZA. The isolates remained susceptible to FDC, carbapenems, IMR, and MEV (Table 1). The blaVEB-25 gene was located on an IncC plasmid in a ~14 kb multidrug resistance region comprising IS10A, blaVEB-25, aadB, arr-2, cmlA5, blaOXA-10, aadA1, qacEΔ1, Δsul1, floR2, tetR, tet(G), lysR, ISCR3-like, groEL, ISL3, rmtB1, and blaTEM-1. This region was similar to the ~14 kb MDR region previously described by Voulgari et al., while the plasmid was similar to N3418 plasmid pVEB-25_IncC (GenBank: OQ362291.1) described by Findlay et al. [13,16], with most of the differences found in this MDR region, due to the lack of a ~5 kb region that includes rmtB1 and blaTEM-1. Similar to N3418 plasmid pVEB-25_IncC, the plasmid in this study harbored the mercury resistance operon (merACPTR), the HigAB type II toxin–antitoxin system (typical of IncC plasmids), and the citrate-dependent iron (III) uptake system (fecIRABCDE), which has recently been linked to reduced susceptibility to FDC [23]. FDC MICs increased from the initial ≤ 0.08 mg/L (MIC of E. coli TOP10) to 2 mg/L in both transformants (Table 1).
2.2. Epidemiologic Investigation of the Outbreak
In four of the patients in this study, colonization by the CZA-resistant KPC-KP isolate followed prior exposure to carbapenem therapy. Three of the five colonized patients presented with a septic episode caused by the same isolate (Patients 1, 3, and 4 in Table 3) and the fourth patient by a KPC-KP isolate susceptible to CZA and to all aminoglycosides (Patient 2 in Table 3).
Novel BLBLIs were utilized for the treatment of the infectious episodes in these patients. Specifically, three patients received meropenem–vaborbactam, while one patient was treated with imipenem–cilastatin–relebactam. These therapies were administered for conditions including ventilator-associated pneumonia (VAP) and bloodstream infection (BSI) associated with central venous catheter (CVC) use. Source control strategies played a pivotal role in infection management for two patients. All patients were treated promptly with novel BLBLIs; however, 28-day mortality was 50% in the infected patients. Given the small sample size, these findings should be interpreted with caution. The observed 50% mortality rate is likely influenced by severe underlying conditions, prolonged hospitalization, and critical illness, aligning with previous reports of up to 60% mortality for CZA–AVI-resistant infections [29]. The baseline characteristics of these patients are outlined in Table 3.
A thorough investigation was conducted to trace the source and transmission pathways of the outbreak. Epidemiologic data were gathered, including patient histories, antimicrobial usage, and infection timelines. The spatial layout and patient flow within the ICUs were reviewed to identify potential points of environmental contamination or breaches in infection control practices. The outbreak was successfully contained by a combination of intensified infection control measures and rigorous adherence to infection prevention protocols. These included isolating colonized patients in single rooms, enhancing implementation of personal protective equipment protocols, and dedicating resources to environmental cleaning and surveillance of hand hygiene compliance. The absence of new cases after January 2024 indicates the effectiveness of these interventions.
3. Discussion
CZA demonstrates high in vitro activity against non-metallo-β-lactamase-producing K. pneumoniae strains [26], although VEB-mediated resistance has been well documented in Greece [13,14,15]. Hopefully, only rare cases have been reported so far, as rare cases of CZA resistance among KPC-producing isolates, endowed with other resistance mechanisms, have also been reported.
In most cases reported so far, and in the cases reported in this study, blaVEB-25 is located in a ~14 kb multidrug resistance (MDR) region comprising IS10A, blaVEB-25, aadB, arr-2, cmlA5, blaOXA-10, aadA1, qacEΔ1, Δsul1, floR2, tetR, tet(G), lysR, ISCR3-like, groEL, ISL3, rmtB1, and blaTEM-1 [13]. In only one case reported in Switzerland, from a patient who had previously been hospitalized in Greece, this MDR region was smaller (~9 kb), lacking ~5 kb that include rmtB1 and blaTEM-1 [16]. The MDR region is always located on IncC plasmids, conjugative [13,14] or not [16], in different sequence types of K. pneumoniae isolates (ST147, ST258, ST35, and ST323) that always produce KPC (mostly KPC-2).
Previous epidemiologic analyses indicated that a conjugative plasmid co-harboring blaVEB-1, blaOXA-10, blaTEM-1B, and rmtB is present in 8% of all bla KPC-positive isolates in Greece [30], while among aminoglycoside-resistant K. pneumoniae isolates, 37.8% harbored a 16S rRNA methylase-coding gene, mainly rmtB, located on an Inc A/C plasmid along with blaVEB, blaOXA-10, and blaTEM [31].
The backbone of the IncC plasmids in this study, carrying the MDR region, was similar to that of N3418 plasmid pVEB-25_IncC (GenBank: OQ362291.1) described by Findlay et al. [16], harboring the mercury resistance operon (merACPTR), typical of IncC plasmids’ HigAB type II toxin–antitoxin system and the citrate-dependent iron (III) uptake system (fecIRABCDE).
Additionally, the K. pneumoniae isolates in this study, similar to the N3418 isolate reported in Switzerland [16], belong to ST323, a sequence type that was rapidly spread in Greek hospitals in 2022 [6].
The MDR region carrying the blaVEB-25 gene is an already-established vehicle that enhances dissemination of VEB-mediated CZA resistance in KPC-KP of different sequence types in Greek hospitals. The emergence of CZA resistance dramatically limits treatment options against carbapenemase-producing Enterobacterales. In this report, infected patients were treated with novel BLBLIs; however, 28-day mortality was 50% among infected patients.
Finally, ST323 isolates in this study and the N3418 isolate from Switzerland were resistant to the recently approved cefiderocol with MICs ranging from 32 to 64 mg/L due to fiu chromosomal gene disruption and to the presence of fecIRABCDE gene cluster in the IncC plasmid. The deletion of cirA or fiu in an E. coli isolate increased the MIC of FDC within 2-fold of that required for the parent strain, while double knockout of cirA and fiu was needed to increase the MIC of cefiderocol 16-fold [28]. In the isolates presented in this study, cirA was intact and only fiu had a frameshift mutation resulting in a smaller protein. Furthermore, the isolates had an intact OmpK35 and an OmpK36_v6 variant which was previously associated with the unrelated ST323 and ST383 K. pneumoniae isolates from Greece, affecting the entry of β-lactams [27,32]. This variant of OmpK36 must be further investigated for a possible contribution to FDC resistance. Additionally, the presence of the plasmidic fecIRABCDE gene cluster, which increased the MIC of FDC in the E. coli transformants by at least 5-fold (from ≤0.08 mg/L to 2 mg/L), may contribute synergistically to the increased FDC MICs (32–64 mg/L) observed in the K. pneumoniae isolates.
The acquisition of the transferable extrachromosomal fec operon has been associated with a cefiderocol MIC increase in Enterobacterales [23,24], and recently, Polani et al. reported that the fec gene cluster is present in many K. pneumoniae strains, including those isolated before the introduction of FDC in clinical therapy [24]. Although Poirel et al. have reported that VEB-1 does not hydrolyze FDC [33], the hydrolytic activity of VEB-25 against FDC must be investigated. Previous studies that reported VEB-25-producing K. pneumoniae isolates have not determined the MIC for FDC [13,14,15].
Here, we report the co-emergence of resistance to CZA due to VEB-25 production and to FDC due to mutated fiu and co-existence of the fec gene cluster in the successful high-risk clone ST323. This is a public health problem that needs to be monitored and also poses a challenge for clinical microbiology laboratories. Neither of the patients was previously treated with CZA or FDC, indicating that this clone is likely circulating in Greece.
In conclusion, the dissemination of VEB-25 among multidrug-resistant organisms is an unwelcome event. The blaVEB-25 gene was associated with IncC plasmids which are important contributors to the spread of key antibiotic resistance genes. The co-presence of the fec cluster in the same plasmid is even more worrisome. As already reported, the use of FDC may also act as a positive selector for plasmids carrying the fec genes, for which prevalence can be expected to increase [24]. Our findings highlight the importance of genomic data surveillance to detect onward pathogen and plasmid transmission and persistence. Strict infection control measures must be elaborated upon to prevent the spread of extensively drug-resistant organisms such as those described here that co-produced KPC-2 and VEB-25 and were also resistant to cefiderocol.
4. Materials and Methods
4.1. Detection of the Outbreak
This study was conducted at a hospital comprising two mixed ICUs located in separate areas. ICU-1 includes a 14-bed open space and four single-patient rooms, while ICU-2 consists of an 11-bed open space and five single-patient rooms. In this case, routine rectal surveillance cultures were instrumental in identifying carbapenemase-producing bacteria among patients in the ICUs. These cultures were performed biweekly on all ICU patients. The outbreak was identified when five patients tested positive for colonization with KPC-KP which were resistant to CZA.
4.2. Bacterial Isolates
Eight KPC-KP isolates, resistant to CZA, were sent to the Infectious Diseases Laboratory of the 4th Dept of Internal Medicine, Faculty of Medicine, National and Kapodistrian University of Athens, for further testing. The strains were isolated from five ICU patients during the period between 27 December 2023 and 22 January 2024. All five patients were colonized by a CZA-resistant KPC-KP strain and three of them developed an infection from this isolate.
4.3. Susceptibility Testing
Species identification and primary determination of MICs were performed by Vitek®2 (bioMérieux, Marcy-l’Etoile, France). Carbapenemase production was defined by the multiplex immunochromatographic assay NG Test CARBA 5 (NG Biotech, Guipry, France). Susceptibility testing of FDC and AZA was evaluated by disk diffusion, while MICs of FDC and colistin were determined by the commercially available broth microdilution method ComASP according to the manufacturer’s instructions. Ceftazidime, CZA, meropenem, MEV, IMR, amikacin, gentamicin, tigecycline, fosfomycin, and chloramphenicol MICs were also determined by Liofilchem® MIC Test Strips (Liofilchem srl, Roseto degli Abruzzi, Italy) according to the manufacturer’s instructions. Escherichia coli ATCC 25922, E. coli NCTC 13846, and K. pneumoniae ATCC 700603 were used as quality control strains. Antimicrobial susceptibility results were interpreted according to the EUCAST recommendations [25].
4.4. DNA Extraction, Genome Sequencing, and Data Analysis
DNA was extracted from K. pneumoniae isolates using the PureLink® Genomic DNA mini kit (Life Technologies, InvitrogenTM, Carlsbad, CA, USA) following the manufacturer’s Gram-Negative Bacterial Cell DNA extraction protocol. Libraries were prepared using Ion Torrent technology and Ion Chef workflows (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing was performed in the S5XLS system and analysis of primary data was conducted with Ion Torrent Suite v.5.10.0. The raw reads were assembled using the SPAdes assembler available from the Bacterial and Viral Bioinformatics Resource Center (BV-BRC), accessed on 12 April 2024, using default settings [34]. Automatic annotation of the bacterial genome was performed with RASTtk available from the BV-BRC [34]. Assembled contigs were also submitted to the Center for Genomic Epidemiology platform for species identification (KmerFinder 3.2), Multilocus Sequence Typing (MLST 2.0), detection of antimicrobial resistance genes (ResFinder 4.6.0), identification of plasmid incompatibility groups (PlasmidFinder 2.1), and plasmid MLST (pMLST 2.0), accessed on 13 September 2024 [35]. Capsule and lipopolysaccharide serotype were predicted by the Kaptive Web tool, accessed on 13 September 2024 [36]. Selected genes were also aligned to reference genes with the NCBI BLAST algorithm version BLAST+ 2.16.0 [37].
4.5. Transferability of blaVEB-25 Genes
Conjugation of the blaVEB-25-harboring plasmids was attempted using a rifampicin-resistant E. coli RC85 R K12 as a recipient strain, as previously described [14], with selection on rifampicin at 512 mg/L and ceftazidimeat at 256 mg/L or gentamicin at 50 mg/L, with no success.
Transformation of competent E. coli TOP10 cells (Invitrogen, Milan, Italy) was performed by plasmid DNA that was extracted (PureLink HiPure plasmid filter midiprep kit; Invitrogen, Milan, Italy) from all eight VEB-25-producing isolates. Transformants were selected on Luria Bertani agar (Sigma-Aldrich Inc., St. Louis, MO, USA) plates containing 256 mg/L ceftazidime. Transformants were subjected to antibiotic susceptibility testing and PCR with specific primers to determine the presence of bla genes.
Author Contributions
Conceptualization, I.G. and I.K.; Data curation, I.G. and I.K.; Investigation, I.G., I.K., V.P. and A.G.; Methodology, I.G. and V.P.; Project administration, A.A. and H.G.; Resources, A.A.; Supervision, A.A. and H.G.; Visualization, I.G., I.K. and M.S.; Writing—original draft, I.G. and I.K.; Writing—review and editing, M.S. and A.A. All authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding.
Institutional Review Board Statement
This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Hygeia General Hospital (registration no. 627/26-09-2018).
Informed Consent Statement
Informed consent was obtained from all subjects involved in this study.
Data Availability Statement
The whole genome sequencing data for isolates 4-BS.3 and 3-BL.6 have been deposited in the NCBI under BioProject accession number PRJNA1215006 and BioSample accession numbers SAMN46391955 and SAMN46391956.
Acknowledgments
The authors would like to acknowledge Irene Karantani from the Infectious Diseases Laboratory of Hygeia Hospital and Anastasia Molla from the Infectious Diseases Laboratory of the 4th Department of Internal Medicine for outstanding technical work.
Conflicts of Interest
The authors declare no conflicts of interest.
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Table 1.
Antimicrobial susceptibility and genotypic characteristics of the Klebsiella pneumoniae isolates and their transformants.
Table 1.
Antimicrobial susceptibility and genotypic characteristics of the Klebsiella pneumoniae isolates and their transformants.
| Patient (No., Sex, Age) | 1 | 2 | 3 | 4 | 5 | Laboratory Strain | ||||
|---|---|---|---|---|---|---|---|---|---|---|
| Male | Female | Male | Male | Male | ||||||
| 75 | 71 | 46 | 79 | 77 | ||||||
| Isolate | 1-R.2 | 1-S.7 | 2-R.1 | 3-R.5 | 3-BL.6 | 4-R.8 | 4-BS.3 | 5-R.4 | TOP10/pl-VEB-25 * | TOP10 |
| Source | Rectal | Sputum | Rectal | Rectal | Blood | Rectal | Bronchial secretions | Rectal | Transformant | Laboratory strain |
| Day of isolation | 27 December 2023 | 9 January 2024 | 2 January 2024 | 2 January 2024 | 22 January 2024 | 22 January 2024 | 10 January 2024 | 22 January 2024 | - | - |
| MLST-type | ST323 | ST323 | ST323 | ST323 | ST323 | ST323 | ST323 | ST323 | ND | ND |
| KPC-type | KPC-2 | KPC-2 | KPC-2 | KPC-2 | KPC-2 | KPC-2 | KPC-2 | KPC-2 | None | None |
| VEB-type | VEB-25 | VEB-25 | VEB-25 | VEB-25 | VEB-25 | VEB-25 | VEB-25 | VEB-25 | VEB-25 | None |
| Antibiotics tested | in mg/L | |||||||||
| Penicillins | ||||||||||
| Amoxicillin/ Clavulanate | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | ≤4 |
| Ampicillin/ Sulbactam | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | 4 |
| Piperacillin/ Tazobactam | >64 | >64 | >64 | >64 | >64 | >64 | >64 | >64 | 64 | ≤4 |
| Cephalosporins | ||||||||||
| Cefuroxime | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | 4 |
| Cefoxitin | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | 8 | 8 |
| Cefotaxime | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | 32 | ≤0.25 |
| Ceftazidime | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | 0.25 |
| Ceft/avi | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | 0.25 |
| Ceftriaxone | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | ≤0.25 |
| Ceft/taz | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | ≤0.25 |
| Cefepime | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | 8 | ≤0.12 |
| Cefiderocol | 32 | 32 | 32 | 32 | 32 | 32 | 32 | 32 | 2 | 0.016 |
| Monobactams | ||||||||||
| Aztreonam | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | ≤1 |
| Aztreonam/Avibactam ** | R | R | R | R | R | R | R | R | R | S |
| Carbapenems | ||||||||||
| Ertapenem | >4 | >4 | >4 | >4 | >4 | >4 | >4 | >4 | ≤0.12 | ≤0.12 |
| Imipenem | >8 | >8 | >8 | >8 | >8 | >8 | >8 | >8 | ≤0.25 | ≤0.25 |
| Imipenem/ Relebactam | 0.5 | 0.5 | 0.5 | 0.5 | 1 | 0.5 | 1 | 0.5 | 0.5 | 0.25 |
| Meropenem | >8 | >8 | >8 | 16 | 16 | >32 | >32 | >8 | 0.032 | 0.032 |
| Meropenem/ Vaborbactam | 0.25 | 0.25 | 0.25 | 0.125 | 0.125 | 0.25 | 0.25 | 0.25 | 0.032 | 0.032 |
| Aminoglycosides | ||||||||||
| Amikacin | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | 2 |
| Gentamicin | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | 0.5 |
| Tobramycin | >8 | >8 | >8 | >8 | >8 | >8 | >8 | >8 | >8 | ≤1 |
| Tetracyclines | ||||||||||
| Tigecycline | >4 | >4 | >4 | >4 | >4 | >4 | >4 | >4 | 0.25 | 0.25 |
| Miscellaneous agents | ||||||||||
| Fosfomycin | 64 | 64 | 64 | 64 | 64 | 64 | 64 | 64 | 16 | 0.25 |
| Colistin | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | >4/2 | >4/2 | 0.5 | ≤0.25 | ≤0.25 |
| Trimethoprim/ Sulphamethoxazole | >160 | >160 | >160 | >160 | >160 | >160 | >160 | >160 | >160 | ≤20 |
| Chloramphenicol | 128 | 128 | 64 | 128 | 128 | 64 | 64 | 64 | 16 | 2 |
ND, not defined; R, resistant; S, susceptible. * All transformants had identical susceptibilities. ** Susceptibility to Aztreonam/Avibactam was determined by disk diffusion method. All resistant isolates exhibited disk diameters of 17–18 mm, while the susceptible laboratory strain TOP10 exhibited a zone diameter of 34 mm.
Table 2.
Acquired antibiotic resistance genes and plasmid replicons of Klebsiella pneumoniae clinical isolates and their transformants.
Table 2.
Acquired antibiotic resistance genes and plasmid replicons of Klebsiella pneumoniae clinical isolates and their transformants.
| Isolate | 4-BS.3 | TOP10/pl3 | 3-BL.6 | TOP10/pl6 |
|---|---|---|---|---|
| Source | Bronchial secretions | Transformant | Blood | Transformant |
| Day of isolation | 10 January 2024 | - | 22 January 2024 | - |
| Acquired resistance genes | ||||
| β-lactamase genes | blaSHV-11, blaKPC-2, blaVEB-25, blaOXA-10, blaTEM-1B | blaKPC-2, blaVEB-25, blaOXA-10, blaTEM-1B | blaSHV-11, blaKPC-2, blaVEB-25, blaOXA-10, blaTEM-1B | blaKPC-2, blaVEB-25, blaOXA-10, blaTEM-1B |
| Aminoglycoside-modifying enzyme-coding genes | aph(6)-Id, aph(3″)-Ib, aadA1, ant(2″)-Ia, rmtB | aph(6)-Id, aph(3″)-Ib, aadA1, ant(2″)-Ia, rmtB | aph(6)-Id, aph(3″)-Ib, aadA1, ant(2″)-Ia, aph(3′)-Ia, rmtB | aph(6)-Id, aph(3″)-Ib, aadA1, ant(2″)-Ia, aph(3′)-Ia, rmtB |
| Quinolones | qnrS1 | qnrS1 | qnrS1 | qnrS1 |
| Tetracycline | tet(A), tet(G) | tet(A), tet(G) | tet(A), tet(G) | tet(A), tet(G) |
| Fosfomycin | fosA7, fosA6 | fosA7, fosA6 | ||
| Trimethoprim | dfrA23 | dfrA23 | dfrA23 | dfrA23 |
| Phenicol | cmlA5 | cmlA5 | cmlA5 | cmlA5 |
| Sulfonamide | sul1, sul2 | sul1, sul2 | sul1, sul2 | sul1, sul2 |
| Plasmid replicons | ||||
| Plasmid replicons | Col440II, ColRNAI, IncC, IncFIB(pQil), IncFII(K), IncFII(pKP91), IncR, repB(R1701) | IncC | Col440II, ColRNAI, IncC, IncFIB(pQil), IncFII(K), IncFII(pKP91), IncR, repB(R1701) | IncC |
Table 3.
Baseline characteristics of the patients colonized by a CZA-R KPC-K. pneumoniae isolate.
| A/A Patient | Gender, Age | Charlson Comorbidity Index | Cause of ICU Admission | Date of Colonization by CZA-R KPC-KP Isolate | Antibiotics Prescribed Prior to Colonization | Prior Carbapenem Use (Days) | Type/Date of Infection for BLBLI Administration | Antimicrobial Treatment | Previous Treatment with BLBLI | Treatment Outcome | 28-Day Outcome |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | Male, 75 | 9 | Respiratory failure | 27 December 2024 | CRO, TZP, MFX | No | VAP by CZA-R KPC-KP/9 January 2024 | MER/VAB | No | Reduction of ventilation support, transfer to rehabilitation center | Death on 14th day after infection |
| 2 | Female, 71 | 7 | Brain injury/Subdural hematoma | 2 January 2024 | VAN, TZP, CRO | Yes (7) | CRBSI by KPC-KP/9 January 2024 | MER/VAB | No | Sterile blood cultures, removal of central line | Alive |
| 3 | Male, 46 | 1 | Ischemic stroke | 2 January 2024 | CRO, TZP, VAN, MEM | Yes (3) | CRBSI by CZA-R KPC-KP/22 January 2024 | IMI/REL | No | Sterile blood cultures, removal of central line | Alive |
| 4 | Male, 79 | 8 | Respiratory failure | 8 January 2024 | TZP, VAN, MEM | Yes (13) | VAP by CZA-R KPC-KP/10 January 2024 | MER/VAB | No | Complicated with super infection with CRAB | Death on 10th day after infection |
| 5 | Male, 77 | 9 | Chest injury | 22 January 2024 | LZD, PZT, MEM | Yes (3) | - | - | No | - | Death occurred within 24 h after BSI with KPC-K. pneumoniae susceptible to CZA |
Abbreviations: BLBLI: β-lactam/β-lactamase inhibitor; BSI: bloodstream infection; CRBSI: catheter-related bloodstream infection; CRO: ceftriaxone; IMI/REL: imipenem–cilastatin–relebactam; ICU: intensive care unit; LZD: linezolid; MEM: meropenem; MER/VAB: meropenem–vaborbactam; MFX: moxifloxacin; TZP: piperacillin–tazobactam; VAN: vancomycin; VAP: ventilator-associated pneumonia.
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Galani, I.; Karaiskos, I.; Souli, M.; Papoutsaki, V.; Gkoufa, A.; Antoniadou, A.; Giamarellou, H. Outbreak of High-Risk Clone ST323 Klebsiella pneumoniae Resistant to Ceftazidime–Avibactam Due to Acquisition of blaVEB-25 and to Cefiderocol Due to Mutated fiu Gene. Antibiotics 2025, 14, 223. https://doi.org/10.3390/antibiotics14030223
AMA Style
Galani I, Karaiskos I, Souli M, Papoutsaki V, Gkoufa A, Antoniadou A, Giamarellou H. Outbreak of High-Risk Clone ST323 Klebsiella pneumoniae Resistant to Ceftazidime–Avibactam Due to Acquisition of blaVEB-25 and to Cefiderocol Due to Mutated fiu Gene. Antibiotics. 2025; 14(3):223. https://doi.org/10.3390/antibiotics14030223
Chicago/Turabian StyleGalani, Irene, Ilias Karaiskos, Maria Souli, Vassiliki Papoutsaki, Aikaterini Gkoufa, Anastasia Antoniadou, and Helen Giamarellou. 2025. "Outbreak of High-Risk Clone ST323 Klebsiella pneumoniae Resistant to Ceftazidime–Avibactam Due to Acquisition of blaVEB-25 and to Cefiderocol Due to Mutated fiu Gene" Antibiotics 14, no. 3: 223. https://doi.org/10.3390/antibiotics14030223
APA StyleGalani, I., Karaiskos, I., Souli, M., Papoutsaki, V., Gkoufa, A., Antoniadou, A., & Giamarellou, H. (2025). Outbreak of High-Risk Clone ST323 Klebsiella pneumoniae Resistant to Ceftazidime–Avibactam Due to Acquisition of blaVEB-25 and to Cefiderocol Due to Mutated fiu Gene. Antibiotics, 14(3), 223. https://doi.org/10.3390/antibiotics14030223
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