Search Results (60)

Background/Objectives: Cefiderocol is a novel siderophore cephalosporin with potent in vitro activity against a broad spectrum of Gram-negative bacteria, including carbapenemase-producing Enterobacterales (CPE). However, the recent emergence of resistance in clinical settings raises important concerns regarding its long-term effectiveness. This study aims to investigate the genomic determinants associated with cefiderocol resistance in CPE isolates of human origin. Methods: Comparative genomic analyses were conducted between cefiderocol-susceptible and -resistant CPE isolates recovered from human clinical and epidemiological samples at a tertiary care hospital. Whole-genome sequencing, variant annotation, structural modelling, and pangenome analysis were performed to characterize resistance mechanisms. Results: A total of 59 isolates (29 resistant and 30 susceptible) were analyzed, predominantly comprising Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae. The most frequent carbapenemase gene among the resistant isolates was bla_NDM, which was also present in a subset of susceptible strains. The resistant isolates exhibited a significantly higher burden of non-synonymous mutations in their siderophore receptor genes, notably within fecR, fecA, fiu, and cirA. Structural modelling predicted deleterious effects for mutations such as fecR:G104S and fecA:A190T. Additionally, porin loss and loop 3 insertions (e.g., GD/TD) in OmpK36, as well as OmpK35 truncations, were more frequent in the resistant isolates, particularly in high-risk clones such as ST395 and ST512. Genes associated with toxin–antitoxin systems (chpB2, pemI) and a hypothetical metalloprotease (group_2577) were uniquely found in the resistant group. Conclusions: Cefiderocol resistance in CPE appears to be multifactorial. NDM-type metallo-β-lactamases and missense mutations in siderophore uptake systems—especially in those encoded by fec, fhu, and cir operons—play a central role. These may be further potentiated by alterations in membrane permeability, such as porin disruption and efflux deregulation. The integration of genomic and structural approaches provides valuable insights into emerging resistance mechanisms and may support the development of diagnostic tools and therapeutic strategies. Full article

Klebsiella pneumoniae is a major public health concern due to its role in Gram-negative bacteremia, which leads to high mortality and increased healthcare costs. This study characterizes phenotypic and genomic features of K. pneumoniae isolates from clinical samples in Karachi, Pakistan. Among 507 isolates, 213 (42%) were carbapenem-resistant based on disk diffusion and MIC testing. Urine (29.7%) and blood (28.3%) were the most common sources, with infections predominantly affecting males (64.7%) and individuals aged 50–70 years. Colistin was the only antibiotic showing consistent activity against these isolates. The whole-genome sequencing of 24 carbapenem-resistant K. pneumoniae (CR-KP) isolates revealed bla_NDM-5 (45.8%) as the dominant carbapenemase gene, followed by bla_NDM-1 (12.5%) and bla_OXA-232 (54.2%). Other detected bla_OXA variants included bla_OXA-1, bla_OXA-4, bla_OXA-10, and bla_OXA-18. The predominant beta-lactamase gene was bla_CTX-M-15 (91.6%), followed by bla_CTX-M-163, bla_CTX-M-186, and bla_CTX-M-194. Sequence types ST147, ST231, ST29, and ST11 were associated with resistance. Plasmid profiling revealed IncR (61.5%), IncL (15.4%), and IncC (7.7%) as common plasmid types. Importantly, resistance was driven not only by acquired genes but also by chromosomal mutations. Porin mutations in OmpK36 and OmpK37 (e.g., P170M, I128M, N230G, A217S) reduced drug influx, while acrR and ramR mutations (e.g., P161R, G164A, P157*) led to efflux pump overexpression, enhancing resistance to fluoroquinolones and tigecycline. These findings highlight a complex resistance landscape driven by diverse carbapenemases and ESBLs, underlining the urgent need for robust antimicrobial stewardship and surveillance strategies. Full article

Background: The qacE gene and its variant, qacE∆1, have been associated with resistance to antimicrobials and biocides. This poses a threat to infection prevention, control and treatment. Several studies investigated this relationship with conflicting results. The aim of this research was to determine the prevalence of qacE and qacE∆1 in clinical extended spectrum β-lactamase- (ESBL) and carbapenemase-producing Klebsiella spp. and Enterobacter spp. and elucidate the association of these genes with resistance to sodium hypochlorite. Methods: This study included 216 isolates of ESBL- and carbapenemase-producing multidrug-resistant (MDR) Klebsiella spp. and Enterobacter spp. These isolates were identified by VITEK-2 analyser. The MIC and MBC of sodium hypochlorite were determined using the microbroth serial-dilution method. PCR was used to detect gene variants. A regression analysis investigated any association between qacE genotypes, MIC and MBC, as well as antimicrobial drug resistance profiles. Results: Overall, there was a high prevalence of qacE and qacE∆1 variants (84.7%; 95% CI, 79.2–89.2). There was a high prevalence of qacE∆1 (80.6%; 95% CI, 74.6–85.6) as compared to qacE (15.3%, 95% CI, 10.8–20.8). The MIC₅₀ and MIC₉₀ of the isolates ranged between 7031 mg/L and 9375 mg/L and 14,060 mg/L and 18,750 mg/L, respectively, while the MBC ranged from 48,750 mg/L to 18,750 mg/L. There was no association between qacE genotypes and high MIC and MBC as well as antimicrobial drug resistance. Conclusions: The MIC and MBC of sodium hypochlorite are higher than what is currently used for disinfection in Botswana. There is a high prevalence of qacE and qacE∆1; however, these genes do not seem to be associated with resistance to sodium hypochlorite. Full article

Background/Objectives. Carbapenemase production is the most diffused carbapenem-resistance mechanism among Enterobacterales, with Klebsiella pneumoniae carbapenemase (KPC), Verona-imipenemase (VIM), New-Delhi metallo-β-lactamase (NDM), imipenemase (IMP), and oxacillinase (OXA-48) being reported as the main types within Europe. Particularly, Southern Italy holds a concerningly high percentage of carbapenemases-producing Enterobacterales diffused among different hospital settings. These strains may colonize critical patients’ gastrointestinal tracts, often causing disseminations and severe complications. Scientific data recently reported carbapenemase variants’ worldwide diffusion and several double-carbapenemases reports. The diagnostic routine needs devices whose detection rates are extended to similar epidemiological conditions, avoiding a lack of specificity and potential negative results. Methods. We planned a retrospective study including carbapenem- and/or ceftazidime/avibactam-resistant Enterobacterales (62) which were tested with the KPC/IMP/NDM/VIM/OXA-48 Combo Test Kit (KINVO, Medomics Medical Technology, Nanjing, Jiangsu, China) based on the lateral flow assay (LFA) method. Results. We compared its results to the phenotypic antimicrobial susceptibility testing (AST) MIC results, obtaining a 100% agreement rate. The LFA kit reported carbapenemases in all the tested strains, also identifying cases of KPC variants and double-carbapenemases production. Conclusions. Our data demonstrated how LFAs may represent a reliable alternative requiring minimum economic and personnel resources along with simple result interpretations. Future studies will be necessary to further investigate the system effectiveness on a larger isolates’ number and a broad carbapenemase variant spectrum. Full article

Background/Objectives: Proteus mirabilis is a frequent causative agent of urinary tract and wound infections in community and hospital settings. It develops resistance to expanded-spectrum cephalosporins (ESC) due to the production of extended-spectrum β-lactamases (ESBLs) or plasmid-mediated AmpC β-lactamases (p-AmpC). Here, we report the characteristics of ESBLs and p-AmpC β-lactamases encountered among hospital and community isolates of P. mirabilis in two hospitals and the community settings in Zagreb, Croatia. Methods: Antibiotic susceptibility testing was performed using disk-diffusion and broth dilution methods. The double-disk-synergy test (DDST) and inhibitor-based test with clavulanic and cloxacillin were applied to screen for ESBLs and p-AmpC, respectively. PCR investigated the nature of ESBL, carbapenemases, and fluoroquinolone resistance determinants. Selected strains were subjected to molecular analysis of resistance traits by the Inter-Array CarbaResist Kit and whole-genome sequencing (WGS). Results: In total, 39 isolates were analyzed. Twenty-two isolates phenotypically tested positive for p-AmpC and seventeen for ESBLs. AmpC-producing organisms exhibited uniform resistance to amoxicillin-clavulanate, ESC, ciprofloxacin, and sulphamethoxazole-trimethoprim, and uniform susceptibility to carbapenems and piperacillin-tazobactam and all harbored bla_CMY-16 genes. ESBL-positive isolates demonstrated resistance to amoxicillin-clavulanate, cefuroxime, cefotaxime, ceftriaxone, and ciprofloxacin but variable susceptibility to cefepime and aminoglycosides. They possessed bla_CTX-M genes that belong to cluster 1 (n = 5) or 9 (n = 12), with CTX-M-14 and CTX-M-65 as the dominant allelic variants. Conclusions: The study demonstrated the presence of CTX-M ESBL and CMY-16 p-AmpC among hospital and community-acquired isolates. AmpC-producing isolates showed uniform resistance patterns, whereas ESBL-positive strains had variable degrees of susceptibility/resistance to non-β-lactam antibiotics, resulting in more diverse susceptibility patterns. The study found an accumulation of various resistance determinants among hospital and outpatient isolates, mandating improvement in detecting β-lactamases during routine laboratory work. Full article

Background: The emergence of carbapenem-resistant Enterobacterales is prevalent and poses a significant threat to health systems worldwide. This study aimed to conduct a molecular analysis of tigecycline resistance in 100 CRE isolates from Mthatha Hospital and surrounding hospitals. Methods: A retrospective study among patients who attended Nelson Mandela Academic Hospital (NMAH) and Mthatha Regional Hospital (MRH), Eastern Cape, South Africa. Enterobacterales isolates were identified using the Vitek2^® system (bioMérieux); an E-test was performed on 100 CRE isolates according to the manufacturer’s instructions. PCR assays for rapid detection of tet(X) and its variants, including tet(X1) and tet(X2), and high-level tigecycline resistance genes tet(X3), tet(X4), and tet(X5) were developed. Results: The results show a notably high prevalence of CRE infections in neonatal, male surgical, and maternal and pediatric wards, predominantly driven by Klebsiella species (53.4%), followed by Enterobacter species (20.5%) and then Escherichia coli (6.7%), and 7.2% of CRE isolates were resistant to tigecycline (E-test). In this study, tet(X) genes were not identified as the primary mechanism of tigecycline resistance. The risk factors associated with tigecycline resistance in CRE include age, pre-exposure to antibiotics, prolonged hospitalization, and undergoing invasive procedures, indicated by strong r = 0.9501. Conclusions: CRE gradually evolves, posing a significant threat to patients of all ages; early detection of carbapenemase production in clinical infections, carriage states, or both is essential to prevent hospital-based outbreaks. Full article

The management of infectious diseases has proven to be a daunting task for clinicians worldwide, and the rapid development of antibiotic resistance among Gram-negative bacteria is making it even more challenging. The first-line therapy is empirical, and it most often comprises β-lactam antibiotics. Among Gram-negative bacteria, Proteus mirabilis, an important community and hospital pathogen associated primarily with urinary tract and wound infection, holds a special place. This review’s aim was to collate and examine recent studies investigating β-lactam resistance phenotypes and mechanisms of Proteus species and the global significance of its β-lactam resistance evolution. Moreover, the genetic background of resistance traits and the role of mobile genetic elements in the dissemination of resistance genes were evaluated. P. mirabilis as the dominant pathogen develops resistance to expanded-spectrum cephalosporins (ESC) by producing extended-spectrum β-lactamases (ESBL) and plasmid-mediated AmpC β-lactamases (p-AmpC). β-lactamase-mediated resistance to carbapenems in Enterobacterales, including Proteus spp., is mostly due to expression of carbapenemases of class A (KPC); class B (metallo-β-lactamases or MBLs of IMP, VIM, or NDM series); or class D or carbapenem-hydrolyzing oxacillinases (CHDL). Previously, a dominant ESBL type in P. mirabilis was TEM-52; yet, lately, it has been replaced by CTX-M variants, particularly CTX-M-14. ESC resistance can also be mediated by p-AmpC, with CMY-16 as the dominant variant. Carbapenem resistance in Proteus spp. is a challenge due to its intrinsic resistance to colistin and tigecyclin. The first carbapenemases reported belonged to class B, most frequently VIM-1 and NDM-5. In Europe, predominantly France and Belgium, a clonal lineage positive for OXA-23 CHDL spreads rapidly undetected, due to its low-level resistance to carbapenems. The amazing capacity of Proteus spp. to accumulate a plethora of various resistance traits is leading to multidrug or extensively drug-resistant phenotypes. Full article

Ceftazidime–avibactam (CZA) is a potent broad-spectrum drug combination covering extended-spectrum β-lactamases, AmpC, and carbapenemases of class A and D, OXA-48-type producers. Rapid antimicrobial susceptibility testing is crucial for the timely de-escalation/escalation of therapy. We evaluate CZA susceptibility using the CE-IVD FASTgramneg kit (FASTinov^®), a ground-breaking 2 h assay, based on flow cytometry technology for antimicrobial susceptibility testing. The assay involved rapid bacterial extraction and purification from positive blood cultures (PBCs), followed by a 1 h 37 °C incubation and flow cytometry analysis (Cytoflex, Beckman-Coulter). The susceptibility report was generated using a proprietary software and interpreted using EUCAST and CLSI 2024 criteria. Sensitivity and specificity were calculated against a reference standardized method (disk diffusion) according to ISO20776-2:2021. Overall, 135 Enterobacterales and 73 Pseudomonas aeruginosa isolates were studied. Thirty-four isolates were resistant to CZA, including six P. aeruginosa and 28 Enterobacterales (24 metallo-beta-lactamase producers, three KPC variants, and one co-producing KPC+NDM). Sensitivity and specificity reached 100% when using EUCAST and CLSI criteria compared with the reference method. The FASTinov ultra-rapid susceptibility assay for CZA demonstrated excellent results, potentially enabling de-escalation/escalation even before the second dose. Combining the speed of a molecular assay with the comprehensive information of a phenotypic test offers valuable insights for treatment decisions. Full article

Antibiotic resistance, in particular the dissemination of carbapenemase-producing organisms, poses a significant threat to global healthcare. This study introduces the qPCR CarbaScan LyoBead assay, a robust, accurate, and efficient tool for detecting key carbapenemase genes, including blaKPC, blaNDM, blaOXA-48, and blaVIM. The assay utilizes lyophilized beads, a technological advancement that enhances stability, simplifies handling, and eliminates the need for refrigeration. This feature renders it particularly well-suited for point-of-care diagnostics and resource-limited settings. The assay’s capacity to detect carbapenemase genes directly from bacterial colonies without the need for extensive sample preparation has been demonstrated to streamline workflows and enable rapid diagnostic results. The assay demonstrated 100% specificity and sensitivity across a diverse range of bacterial strains, including multiple allelic variants of target genes, facilitating precise identification of resistance mechanisms. Bacterial strains of the species Acinetobacter baumannii, Citrobacter freundii, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae and Pseudomonas aeruginosa were utilized as reference material for assay development (n = 9) and validation (n = 28). It is notable that the assay’s long shelf life and minimal operational complexity further enhance its utility for large-scale implementation in healthcare, food safety, and environmental monitoring. The findings emphasize the necessity of continuous surveillance and the implementation of rapid diagnostic methods for the effective detection of resistance genes. Furthermore, the assay’s potential applications in other fields, such as toxin-antitoxin system research and monitoring of resistant bacteria in the community, highlight its versatility. In conclusion, the qPCR CarbaScan LyoBead assay is a valuable tool that can contribute to the urgent need to combat antibiotic resistance and improve global public health outcomes. Full article

Background/Objectives: Carbapenem-resistant Klebsiella pneumoniae has become endemic in Europe, including in Italy, where its prevalence has risen dramatically, primarily due to epidemic clones harboring metallo-enzymes. This study aims to investigate the dissemination of K. pneumoniae strains co-producing OXA-48 and NDM-1 between two hospitals in southern Italy using molecular analyses. Methods: A total of 49 K. pneumoniae strains, predominantly co-producing OXA-48 and NDM-1, were collected between March and December 2023. Antibiotic susceptibility testing was conducted following EUCAST guidelines. Whole-genome sequencing (Illumina MiSeq) and bioinformatics tools (CARD, CLC Genomics Workbench) were used to identify resistance and virulence genes, capsule loci, and phylogenetic relationships. Results: All isolates exhibited multidrug-resistant or extensively drug-resistant profiles, including resistance to ceftazidime/avibactam and meropenem/vaborbactam. Genomic analysis revealed diverse resistance genes such as bla_OXA-48, bla_NDM-1, bla_CTX-M-15, and bla_SHV variants. Virulence genes associated with capsules, fimbriae, and siderophores were widespread. Most strains were classified as ST147 by MLST and contained various plasmids known to carry antimicrobial resistance. Phylogenetic analysis confirmed their clonal relatedness, highlighting the intra-hospital dissemination of high-risk clones. Conclusions: High-risk K. pneumoniae clones, particularly ST147, pose significant challenges in healthcare settings due to the extensive antimicrobial resistance driven by plasmid-borne resistance genes, including those that co-produce carbapenemases, like bla_NDM-1 and bla_OXA-48. Molecular monitoring of these clones is essential for improving targeted infection control strategies, mitigating the spread of multidrug-resistant pathogens, and managing their clinical impact effectively. Full article

Background/Objectives: Carbapenem resistance poses a significant health threat. This study reports the first detection and characterization of a novel variant of New Delhi metallo-β-lactamase (bla_NDM-60) in Escherichia coli from the United Arab Emirates (UAE), including its genetic context and relationship to global strains. Methods: NDM-60-producing E. coli was isolated from a rectal swab during routine screening. Characterization involved whole-genome sequencing, antimicrobial susceptibility testing, and comparative genomic analysis with 66 known NDM variants. Core genome analysis was performed against 42 global E. coli strains, including the single other reported NDM-60-positive isolate. Results: The strain demonstrated extensive drug resistance, including resistance to novel β-lactam/β-lactamase inhibitor combinations, notably taniborbactam. NDM-60 differs from the closely related NDM-5 by a single amino acid substitution (Asp202Asn) and two amino acid substitutions (Val88Leu and Met154Leu) compared to NDM-1. NDM-60 is located on a nonconjugative IncX3 plasmid. The strain belongs to sequence type 940 (ST940). Phylogenetic analysis revealed high diversity among the global ST940 strains, which carry a plethora of resistance genes and originated from humans, animals, and the environment from diverse geographic locations. Conclusions: NDM-60 emergence in the UAE represents a significant evolution in carbapenemase diversity. Its presence on a nonconjugative plasmid may limit spread; however, its extensive resistance profile is concerning. Further studies are needed to determine the prevalence, dissemination, and clinical impact of NDM-60. NDM evolution underscores the ongoing challenge in managing antimicrobial resistance and the critical importance of vigilant molecular surveillance. It also highlights the pressing demand to discover new antibiotics to fight resistant bacteria. Full article

The COVID-19 pandemic has exacerbated the global antimicrobial resistance (AMR) crisis. Consequently, it is more urgent than ever to prioritize AMR containment and support countries in improving the detection, characterization, and rapid response to emerging AMR threats. We conducted a prospective, multicenter study to assess the prevalence of carbapenemase-producing Enterobacterales in infectious processes in Argentina during the post-COVID-19 pandemic period and explore therapeutic alternatives for their treatment (RECAPT-AR study). Methods: A total of 182 hospitals participated by submitting Enterobacterales clinical isolates to the National Reference Laboratory (NRL) during the first three weeks of November 2021. Inclusion criteria were defined as an ertapenem MIC ≥ 0.5 mg/L, a zone diameter ≤ 22 mm. Carbapenemase genes and those coding for major extended-spectrum β-lactamases were molecularly characterized using multiplex PCR at the NRL. Antibiotic susceptibility testing followed international standards (CLSI and EUCAST). Results: The NRL analyzed 821 Enterobacterales isolates. Metallo-β-lactamase (MBL, 42.0%) and KPC (39.8%) accounted for 81.8% of carbapenemases, followed by OXA-163 (7.4%), a variant of OXA-48 with additional activity against extended-spectrum cephalosporins, and enzyme combinations (8.3%). These combinations included NDM plus KPC (3.4%), OXA-163 plus KPC (2.4%), and OXA-163 plus NDM (2.1%). Klebsiella pneumoniae was the main species recovered, representing 76% of the isolates. According to the carbapenemase classes or combinations, tigecycline exhibited a susceptibility range of 33–83%, fosfomycin 59–81%, colistin 27–78%, and amikacin 17–81%. Ceftazidime-avibactam (CZA) and imipenem-relebactam (IMR) showed 92% and 98% susceptibility against serine carbapenemases, respectively. Meanwhile, aztreonam-avibactam (AZA) exhibited 96–98% susceptibility against all carbapenemase classes. Conclusions: A new epidemiological landscape has emerged, characterized by the equivalent circulation of NDM and KPC. K. pneumoniae remains the primary species responsible for their dissemination. The co-production of carbapenemase combinations, particularly KPC plus NDM, was confirmed, mainly in K. pneumoniae. High activity was observed for AZA against MBLs and for CZA and IMR against KPC and OXA-163 carbapenemases. Full article

Background/Objectives: Antimicrobial resistance represents a challenge to public health systems because of the array of resistance and virulence mechanisms that lead to treatment failure and increased mortality rates. Although for years the main driver of carbapenem resistance in Italy has been the Klebsiella pneumoniae KPC carbapenemase, recent years have seen an increase in VIM and NDM metallo-beta-lactamases (MBLs). We conducted a five-year survey of New Delhi Metallo-beta-Lactamase (NDM)-producing Klebsiella pneumoniae (NDM-Kpn) clinical isolates from the Lazio region, Italy; the study aimed to elucidate the molecular mechanisms underpinning their resistant and virulent phenotype. Methods: Antimicrobial susceptibility was evaluated by automated systems and broth microdilution. In silico analysis of acquired resistance and virulence genes was performed using whole-genome sequencing (WGS), molecular typing through MLST, and core genome multi-locus sequence typing (cgMLST). Conclusions: A total of 126 clinical NDM-Kpn isolates were collected from 19 distinct hospitals in the Lazio region. Molecular analysis highlighted the existence of NDM-1 (108/126) and NDM-5 (18/126) variants, 18 Sequence Types (STs), and 15 Cluster Types (CTs). Notably, 31/126 isolates displayed a virulence score of 4, carrying ybt, ICEKp, iuc, and rmp genes. This study identified a variety of NDM-Kpn STs, mainly carrying the bla_NDM-1 gene, with a significant number linked to high-risk clones. Of these isolates, 24.6% showed high-level resistance and virulence, emphasizing the risk of the spread of strains that combine multi-drug-resistance (MDR) and virulence. Proactive surveillance and international collaborations are needed to prevent the spread of high-risk clones, as well as further research into new antimicrobial agents to fight antibiotic resistance. Full article

Background: Extended-spectrum β-lactamases (ESBL) in Escherichia coli are a serious concern due to their role in developing multidrug resistance (MDR) and difficult-to-treat infections. Objective: This study aimed to identify ESBL-carrying E. coli strains from both clinical and environmental sources in Lusaka District, Zambia. Methods: This cross-sectional study included 58 ESBL-producing E. coli strains from hospital inpatients, outpatients, and non-hospital environments. Antimicrobial susceptibility was assessed using the Kirby–Bauer disk diffusion method and the VITEK^® 2 Compact System, while genotypic analyses utilised the Illumina NextSeq 2000 sequencing platform. Results: Among the strains isolated strains, phylogroup B2 was the most common, with resistant MLST sequence types including ST131, ST167, ST156, and ST69. ESBL genes such as bla_TEM-1B, bla_CTX-M,bla_OXA-1, bla_NDM-5, and bla_CMY were identified, with ST131 and ST410 being the most common. ST131 exhibited a high prevalence of bla_CTX-M-15 and resistance to fluoroquinolones. Clinical and environmental isolates carried bla_NDM-5 (3.4%), with clinical isolates showing a higher risk of carbapenemase resistance genes and the frequent occurrence of bla_CTX-M and bla_TEM variants, especially bla_CTX-M-15 in ST131. Conclusions: This study underscores the public health risks of bla_CTX-M-15- and bla_NDM-5-carrying E. coli. The strengthening antimicrobial stewardship programmes and the continuous surveillance of AMR in clinical and environmental settings are recommended to mitigate the spread of resistant pathogens. Full article

Background. In the context of increasing antimicrobial resistance (AMR), whole-genome sequencing (WGS) of bacteria is considered a highly accurate and comprehensive surveillance method for detecting and tracking the spread of resistant pathogens. Two primary sequencing technologies exist: short-read sequencing (50–300 base pairs) and long-read sequencing (thousands of base pairs). The former, based on Illumina sequencing platforms (ISPs), provides extensive coverage and high accuracy for detecting single nucleotide polymorphisms (SNPs) and small insertions/deletions, but is limited by its read length. The latter, based on platforms such as Oxford Nanopore Technologies (ONT), enables the assembly of genomes, particularly those with repetitive regions and structural variants, although its accuracy has historically been lower. Results. We performed a head-to-head comparison of these techniques to sequence the K. pneumoniae VS17 isolate, focusing on bla_NDM resistance gene alleles in the context of a surveillance program. Discrepancies between the ISP (bla_NDM-4 allele identified) and ONT (bla_NDM-1 and bla_NDM-5 alleles identified) were observed. Conjugation assays and Sanger sequencing, used as the gold standard, confirmed the validity of ONT results. This study demonstrates the importance of long-read or hybrid assemblies for accurate carbapenemase resistance gene identification and highlights the limitations of short reads in the context of gene duplications or multiple alleles. Conclusions. In this proof-of-concept study, we conclude that recent long-read sequencing technology may outperform standard short-read sequencing for the accurate identification of carbapenemase alleles. Such information is crucial given the rising prevalence of strains producing multiple carbapenemases, especially as WGS is increasingly used for epidemiological surveillance and infection control. Full article