Pandora Tsolakidou

Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a growing threat in Greek hospitals, with increasing reports of multidrug- and pandrug-resistant strains; however, molecular data from regional centers remain limited. This study aimed to investigate the molecular epidemiology, resistance mechanisms, and transmission dynamics of CRKP isolates collected at the General Hospital of Volos, Central Greece, between 2022 and 2024. Thirty-seven non-duplicate CRKP isolates were analyzed. Identification and antibiotic susceptibility testing were performed using VITEK® 2, disk diffusion, Etest®, and broth microdilution. Carbapenemase production was assessed using the NG-Test® Carba-5. Eight isolates underwent multilocus sequence typing (MLST). All isolates were resistant to carbapenems, cephalosporins, and fluoroquinolones; furthermore, 40% were colistin-resistant. The dominant carbapenemase genes were bla_NDM-1 (45.9%), bla_KPC-2 (18.9%), and bla_VIM-1 (27.0%), with co-expression of multiple carbapenemases in 30% of the isolates. MLST revealed the high-risk clones ST11, ST15, and ST323, and three intra-intensive care unit (ICU) transmission clusters. The emergence of dual-carbapenemase and colistin-resistant clones underscores the need for local genomic surveillance, improved infection control, and access to newer antimicrobials in non-tertiary settings.

Background/Objectives: Carbapenem-resistant Klebsiella pneumoniae (CRKP) threatens Intensive Care Units (ICU), particularly in settings where serine (KPC) and metallo-β-lactamases (NDM) co-circulate. The aim of this study was to assess CRKP susceptibility especially to novel β-lactam/β-lactamase inhibitor combinations, characterize the genetic determinants of resistance, and contribute to the understanding of local epidemiology in the ICU of our hospital. Methods: We studied 32 non-duplicate CRKP isolates (30 ICU, 2 wards) collected at Hippokration General Hospital, Thessaloniki (May–Oct 2023). Bacterial identification and Antimicrobial susceptibility testing (AST) were performed by VITEK-2; Minimum inhibitory concentrations (MICs) for ceftazidime/avibactam (CAZ/AVI), meropenem/vaborbactam (MER/VAB), and imipenem/relebactam (IMI/REL) were determined by E-tests. Colistin MICs were performed by broth microdilution. Carbapenemases were screened phenotypically and by immunochromatography and confirmed by multiplex PCR. One bronchial isolate co-harboring bla_NDM and bla_KPC genes underwent WGS. Results: All isolates were carbapenem-resistant and showed extensive resistance to β-lactams and fluoroquinolones. By PCR, 8/32 (25%) carried bla_KPC alone, 8/32 (25.0%) bla_NDM alone, and 16/32 (50%) co-harbored bla_KPC and bla_NDM. KPC-only isolates were generally susceptible in vitro to CAZ/AVI, MER/VAB, and IMI/REL, whereas dual KPC-NDM producers were resistant to all three combinations. Tigecycline showed the highest retained activity; colistin remained active in a minority. WGS of one ST512 (CG258) isolate revealed co-harboring bla_NDM-1 and bla_KPC-3 with additional resistance determinants and plasmid replicons, consistent with high-risk spread. Conclusions: Half of CRKP isolates in this ICU-predominant series co-produced KPC and NDM, severely limiting β-lactam/β-lactamase inhibitor options. These data support routine screening for carbapenemases, strict infection prevention, antimicrobial stewardship, and access to agents active against MBLs.

Carbapenemase-producing Klebsiella pneumoniae (CRKP) is a critical public health threat, particularly in Greece, where high prevalence limits therapeutic options. This retrospective study analyzed 26 CRKP isolates recovered at the General Hospital of Volos between July 2024 and January 2025, aiming to correlate carbapenemase phenotypes with clinical and epidemiological parameters. Demographic, clinical, and microbiological data were extracted from patient records, and isolates underwent phenotypic carbapenemase detection, antimicrobial susceptibility testing, and molecular characterization using real-time PCR; four isolates were further analyzed using whole-genome sequencing. CRKP was detected across multiple hospital departments, notably in the Emergency Department (n = 5) and Intensive Care Unit (n = 6). KPC producers predominated (n = 9), followed by NDM (n = 6), VIM (n = 1), and OXA-48 (n = 6). All VIM- or NDM + VIM-positive cases were associated with mortality. High-risk clones, including ST15, ST11, and ST307, were identified, with one ST15 isolate harboring bla_NDM-1, bla_VIM-1, and chromosomal colistin resistance; this is the first such report in Greece. Colistin and gentamicin were the most active agents in vitro; three isolates were pan-drug-resistant. The findings highlight significant CRKP circulation outside ICUs, the role of horizontal gene transfer in resistance dissemination, and the need to expand screening and rapid diagnostics to non-ICU settings. Enhanced molecular surveillance targeted at infection control and strengthened antimicrobial stewardship programs are essential for limiting the spread of CRKP.

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 bla_NDM-1 and bla_VIM-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 bla_NDM-1 and bla_VIM-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.

Background/Objectives: Klebsiella pneumoniae ST307 is emerging as a significant global high-risk antimicrobial-resistant (AMR) clone with a notable capacity to acquire and disseminate resistance genes. However, there is limited research on the pathogenicity, virulence, and adaptation of ST307 strains and on the clinical characteristics of infected patients. Methods: In this study, a carbapenem-resistant K. pneumoniae (CRKP) ST307 strain named U989 was isolated from a urine culture of a hospitalized patient in Volos, Greece, in July 2024. Whole-genome sequencing was performed to identify resistance genes to β-lactams bla_KPC-2, bla_CTX-M-15, bla_TEM-1B, bla_OXA-1, bla_OXA-10, bla_SHV-106, and bla_VEB-1 and resistance genes to other antibiotics. Results: A genomic analysis also revealed the presence of virulence factors such as iutA, clpK1, fyuA, fimH, mrkA, Irp2, and TraT and an IncFiB(pQil)/IncFII(K) replicon, which harbors the bla_KPC-2 gene. Additionally, the transposable element Tn4401 was identified as a key vehicle for the mobilization of the bla_KPC-2 resistance gene. Finally, this is the report of a high-risk CRKP ST307 clone expressing KPC-2, SHV-106, CTX-M-15, and VEB-1 bla genes in Greece. Conclusions: The coexistence of these resistance genes in addition to aminoglycoside, quinolone, and other resistance genes results in difficult-to-treat infections caused by respective carrier strains, often requiring the use of last-resort antibiotics and contributing to the global challenge of antimicrobial resistance.

The spread of NDM-1-harboring Klebsiella pneumoniae is a worldwide concern. In this study the whole-genome sequence (WGS) of a carbapenem- and colistin-resistant K. pneumoniae 838Gr strain is presented. This strain was isolated from a urine sample of a patient in the Intensive Care Unit (ICU) at Volos Hospital, Greece. The initial assembly produced 224 contigs with a combined genome size of 5,561,803 bp and a GC content of 57.21%. The K. pneumoniae strain carried IncR, IncFIA, IncC, and repB (R1701) replicons. Multilocus sequence typing (MLST) analysis revealed that the isolate belonged to the sequence type 11 (ST11) and serogroup KL24 and O2a. The WGS analysis identified several beta-lactamase genes (bla_TEM-1B, bla_CTX-M-15, bla_NDM-1, bla_OXA-1, bla_VEB-1, bla_OXA-10, and bla_SHV-11) alongside resistance genes for other antibiotic classes, including floR2, cmlA1, cmlA5, catB3, arr-3, aph(6)-Id, aadA2. Colistin resistance was attributed to specific point mutations in pmrB (R256G, T140P). This is the first report of a carbapenem- and colistin-resistant K. pneumoniae ST11 strain in Greece. The findings of this study highlight the urgent need for increased surveillance and stringent infection control.

Klebsiella pneumoniae is a major pathogen associated with hospital-acquired infections, particularly those involving multidrug-resistant strains. Carbapenem resistance, often driven by carbapenemases such as KPC, VIM, OXA-48, and NDM, poses a significant challenge in clinical settings. This study reports on K. pneumoniae strain A165, isolated from a blood culture of a 51-year-old female patient hospitalized for respiratory distress post-SARS-CoV-2 infection. This K. pneumoniae strain exhibited resistance to several antibiotics, including carbapenems, cephalosporins, aminoglycosides, and fluoroquinolones, but remained susceptible to gentamicin, colistin, and trimethoprim-sulfamethoxazole. Next-generation sequencing was performed on Ion torrent platform, that revealed a genome size of 5,676,404 bp, including a chromosome and six plasmids. The strain was classified as sequence type 11 (ST11), a high-risk lineage associated with carbapenem resistance. The resistome of A165 included multiple β-lactamase genes, such as bla_NDM-1 and bla_OXA-48, as well as genes conferring resistance to other antibiotic classes. The virulome analysis identified genes involved in iron acquisition (yersiniabactin operon genes: ybtE, ybtT, irp1, irp2; aerobactin receptor: iutA), adhesion (mrkA-J, fimA-K), capsule and biofilm formation (rcsA, rcsB, ompA) and resistance to complement (traT) contributing to its pathogenic potential. The mobilome analysis revealed nine insertion sequences, including ISKpn1, ISKpn18, ISKpn43, ISKpn28, ISKpn14, ISEcp1, and IS6100. The strain also harbored six replicons: Col440II, ColRNAI, IncFIA(HI1), IncFIB(K), IncFII(K), and IncR, which are associated with the horizontal transfer of resistance and virulence genes. Comparative analysis with global isolates demonstrated the widespread dissemination of carbapenemase-producing K. pneumoniae, with notable occurrences in Europe, Asia, and the Americas. This study highlights the growing concern of multidrug-resistant K. pneumoniae in hospital settings and emphasizes the need for robust surveillance and infection control measures.

This study investigated a strain of Klebsiella pneumoniae, identified as GRTHES, which exhibited extensive antibiotic resistance. The strain was resistant to all beta-lactams, including combinations with newer agents such as meropenem/vaborbactam and imipenem/relebactam, as well as to aminoglycosides, fluoroquinolones, fosfomycin, trimethoprim-sulfamethoxazole and colistin. It remained susceptible to tigecycline. Whole-genome sequencing was performed by Ion Torrent platform on the K. pneumoniae strain. Genomic analysis revealed a genome length of 5,808,650 bp and a GC content of 56.9%. Advanced sequencing techniques and bioinformatic tools were used to assess resistance genes and plasmid replicons, highlighting the emergence of multidrug resistance and virulence traits. The strain carried bla_NDM-1 and bla_KPC-3 genes and was designated to KL107 O2afg type. Colistin resistance-associated mgrB/pmrB gene mutations were present, and the strain also harbored yersiniabactin-encoding ybt gene. Our findings provide insights into the genomic context of bla_NDM-1 and bla_KPC-3 carbapenemase-producing K. pneumoniae and emphasize the importance of continuous surveillance and novel therapeutic strategies to combat multidrug-resistant bacterial infections. It is the first time that an NDM-1 and KPC-3 co-producing strain of K. pneumoniae ST512 is identified in Greece. This study highlights the essential role of genomic surveillance as a proactive strategy to control the spread of carbapenemase-producing K. pneumoniae isolates, particularly when key antimicrobial resistance genes, such as bla_NDM-1 and bla_KPC-3, are plasmid-mediated. Detailed characterization of these isolates could reveal plasmid similarities that facilitate adaptation and transmission within and between hospitals. Although data on patient movements are limited, it is plausible that carbapenem-resistant isolate was selected to co-produce KPC and NDM through plasmid acquisition.

Escherichia coli A382 was isolated in July 2024 from a positive blood culture obtained from the central venous catheter of a male patient undergoing chemotherapy at the Hospital of Volos, Thessaly, Greece. Whole-genome sequencing analysis revealed that the isolate A382 is E. coli belonging to the ST410 high-risk clone, which co-harbors the bla_KPC-3 and bla_SHV-182 genes on an IncX3 plasmid. It also harbors bla_TEM-1 and has five replicons, as follows: IncX3, IncQ1, CoIRNAI, IncF1A, and IncFIB. Complete genome analysis revealed that E. coli A382 isolate carries a range of virulence factors (iutA, iucC, fimH, fdeC, yehA, yehD, yehC, yehB, cgs, ahha, ccI, hlyE, papC, irp2, fyuA, lpfA, and nlpl) and many other non-beta-lactam resistance determinants, including dfrA14 and sul2, but it is susceptible to aminoglycosides, nitrofurantoin, tigecycline, colistin and ceftazidime-avibactam. In conclusion in this study, we describe the phenotypic and genome characteristics of an extensively drug-resistant E. coli ST410.

Klebsiella pneumoniae is a major human pathogen, because it causes both community- and hospital-acquired infections. Several multidrug-resistant high-risk clones of K. pneumoniae have been reported worldwide, and these are responsible for high numbers of difficult-to-treat infections. In Greece, a K. pneumoniae ST39 high-risk clone was detected in 2019 in a survey of carbapenem- and/or colistin-resistant Enterobacteriacae. The present study included nine carbapenem-resistant K. pneumoniae (CRKP) isolates collected during a retrospective analysis from October 2020 to December 2020. They were isolated from nine different patients hospitalized in the intensive care unit (ICU) of a hospital in Volos, Greece, and they were selected for analysis due to their phenotypic profile. In this study, we analyzed A165 strain K. pneumoniae ST39 isolated from a blood culture in November 2020. Whole-genome sequencing (WGS) was performed using Ion Torrent Platform, and resistance genes, virulence determinants, capsular types, insertion sequences, phage regions, and clustered regularly interspaced palindromic repeats (CRISPR) regions were detected by bioinformatic analysis. The molecular characterization revealed antimicrobial resistance genes, including sul2 for sulfamethoxazole; dfrA1 for trimethoprim; bla_VIM-1 and bla_KPC-2 for carbapenems; aac(6′)-II for aminoglycosides; fosA for fosfomycin and aad1 for streptomycin, bla_SHV-40, bla_SHV-85, bla_SHV-79, bla_SHV-56, and bla_SHV-89 for beta-lactams. Point mutations were identified in ompK36, and ompK37 and in acrR, gyrA, parC. Several replicons were found, including CoIRNA, IncC, IncFIB(K), IncFIB(pQiL), and IncFII(K). The capsular typing revealed that the strain was KL23, O2afg. The genome sequence of A165 was submitted to NCBI under PRJNA1074377 and have been assigned to Genbank accession number JAZIBV000000000.