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PubMed2026

Genomic and functional characterization of ST11-KL64 hypervirulence-associated carbapenem-resistant Klebsiella pneumoniae co-harboring bla KPC-2 and bla NDM-13.

BACKGROUND: Hypervirulence-associated carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is a major clinical and public health threat. However, ST11-KL64 hv-CRKP co-harboring bla KPC-2 and bla NDM-13 remains poorly characterized, particularly regarding genomic relatedness, plasmid dynamics, and attenuated virulence-associated phenotypes. METHODS: We retrospectively investigated clinical K. pneumoniae isolates collected at a tertiary hospital in Chengdu, China, between January and December 2024. Hypervirulence-associated markers were screened by PCR, followed by antimicrobial susceptibility testing and carbapenemase inhibitor enhancement assay to identify genotype-defined hv-CRKP. All isolates were subjected to molecular typing. ST11-KL64 isolates co-harboring bla KPC-2 and bla NDM-13 were subjected to Illumina sequencing, with the representative isolate K3 undergoing hybrid whole-genome sequencing and functional characterization. RESULTS: Among the 46 hvKP isolates recovered from 43 patients, 35 were identified as hv-CRKP, predominantly ST11-KL64. Three ST11-KL64 hv-CRKP isolates co-harbored bla KPC-2/bla NDM-13, and Illumina sequencing coupled with core-genome SNP (cgSNP) typing revealed minimal genetic variation. The expanded cgSNP analysis supported close relatedness between K3 and Beijing isolate K56649. K3 carried a pLVPK-like virulence plasmid, a bla KPC-2-bearing IncFII/IncR plasmid, and a bla NDM-13-bearing IncI1 plasmid. Relative to pK2044, K3 exhibited an rmpA-proximal ISKpn26-associated insertion and a complex alteration of the 5'-terminal coding region of rmpA. The bla NDM-13 plasmid was conjugatively transferred to Escherichia coli C600 with a mean conjugation frequency of 5.213 × 10-3 transconjugants per recipient cell and bla NDM-13 maintained high stability following approximately 100 generations of antibiotic-free passage, whereas bla KPC-2 was not detected under the tested conditions. Phenotypically, K3 showed a negative string test, low mucoviscosity, and attenuated virulence-associated phenotypes. CONCLUSION: Our results reveal that the three isolates formed a closely related local genomic cluster, among which K3 was closely related to the K56649 clone. In addition, K3 exhibited conjugative transfer capacity of the bla NDM-13-bearing IncI1 plasmid, and alterations at the rmpA locus accompanied by reduced rmpA transcript abundance were associated with low mucoviscosity.

IncI1 plasmid↗

PubMed2025

Multiplex PCR assay for the rapid detection of Klebsiella pneumoniae pathotypes.

Introduction. Klebsiella pneumoniae (Kp) is a major cause of nosocomial infections, with its evolving pathotypes including multidrug-resistant, hypervirulent (hvKp) and convergent strains posing significant diagnostic and treatment challenges due to combined antimicrobial resistance and virulence.Gap Statement. While there is a pressing requirement for thorough detection of Kp pathotypes, current assays in resource-limited environments are unable to effectively focus on essential carbapenemase and hypervirulence genes with the necessary reliability and precision.Aim. To develop and validate a multiplex PCR (m-PCR) assay capable of simultaneously detecting Kp isolates including those carrying partial or full virulence markers, alongside antimicrobial resistance.Methodology. In this study, an m-PCR assay was designed and optimized for the simultaneous detection of key biomarkers associated with hypervirulent (rmpA, rmpA2, iucA, peg344 and iroB), carbapenem-resistant (bla NDM, bla OXA-48-like and bla KPC) and convergent Kp pathotypes in clinical isolates. The assay was evaluated on clinical isolates and validated against whole-genome sequencing (WGS) data for accuracy, specificity and sensitivity.Results. The developed m-PCR assay exhibited 100% specificity when compared to WGS data, successfully detecting all target genes without cross-amplification in ATCC control strains. The assay demonstrated high sensitivity, efficiently amplifying bacterial genomes from minimal DNA input as low as 1 ng µl-1. Additionally, validation through sequencing confirmed the accuracy of detected amplicons.Conclusion. This m-PCR assay offers a rapid, sensitive and specific diagnostic tool for differentiating Kp pathotypes in clinical settings, aiding in timely intervention and improved infection control measures.

Klebsiella pneumoniae↗

PubMed2026

Deciphering the genomic landscape of novel Acinetobacter non-baumannii lineages causing neonatal septicemia: carbapenem resistance and virulence.

BACKGROUND: Acinetobacter non-baumannii (Anb) species are reported worldwide to cause infections in both adults and neonates, although less frequently than Acinetobacter baumannii. However, limited information is available on their genomic diversity, resistance mechanisms, and virulence potential. This study investigates novel Anb isolates causing neonatal septicemia in India to characterize their resistance and pathogenic traits. METHODS: Anb isolates from neonatal blood cultures (2007-2025) were identified by VITEK2 Compact system, MALDI-TOF MS, and Whole-genome sequencing (WGS). Antimicrobial susceptibility was tested by VITEK2. Genomic analysis included MLST, resistome, virulome, plasmid typing, integrons, and core-genome phylogeny analysis. In vitro and in vivo studies assessed pathogenic potential of Anb species. RESULTS: Anb infections were low (11%) among the neonates during the study period. WGS revealed 11 novel Sequence Types (STs) which include A. indicus, A. variabilis, A. schindleri, and A. bereziniae. Six out of these eleven Anbs harbored carbapenemases such as bla NDM-1 and/or bla OXA-58-like genes (bla OXA-58, bla OXA-420). bla NDM-1 was acquired via Tn125 transposon. ISAba125 was located upstream of bla NDM-1, and a conserved structure extending to IS91 family transposase was detected in bla NDM-1-harboring genomes. bla OXA-58-like genes were found to be associated with ISAba3. Most carbapenemases were likely located on chromosome. Class 1 integrons carrying multiple antimicrobial resistance genes (ARGs) and diverse plasmid replicase families were detected in Anbs. Core genome phylogeny showed that the study Anbs were not closely related to the global Anbs. In vitro virulence-associated assays (biofilm formation, surface motility, adherence/invasion, apoptosis) and in vivo lethality in murine infection model showed reduced pathogenicity, reinforcing earlier observations that Anb species are generally less virulent than A. baumannii. Several virulence factors (VFs) were detected; however, no clear correlation was observed between virulence genes, in vitro pathogenicity, and in vivo lethality. CONCLUSION: These results indicate the multifactorial nature of Anb pathogenicity and the current limitations of knowledge of its VFs. However, the presence of numerous VFs suggests a capacity to cause disease, particularly in vulnerable host populations such as neonates. Furthermore, the presence of multiple ARGs indicates a strong potential for persistence and dissemination in hospital environments with high antibiotic pressure. Overall, these findings underscore the importance of continued AMR surveillance, genome characterization and further investigations into Anb pathogenicity.

Acinetobacter non-baumannii↗

PubMed2026

Genetic characterization of carbapenem-resistant Klebsiella pneumoniae bloodstream isolates with reduced susceptibility to cefiderocol.

OBJECTIVES: To assess cefiderocol activity against carbapenem-resistant Klebsiella pneumoniae (CRKP) bloodstream isolates collected before local clinical introduction and to characterize the distribution of borderline MIC elevation across major genomic backgrounds. METHODS: We retrospectively analyzed 389 episodes of K. pneumoniae bloodstream infection at a tertiary hospital in China during 2018-2024. All 83 carbapenem-resistant isolates underwent cefiderocol broth microdilution testing and whole-genome sequencing. For epidemiological analysis, reduced susceptibility was prespecified as an MIC of 4-16 mg/L and was not intended to replace clinical breakpoint interpretation. RESULTS: CRKP accounted for 21.3% of K. pneumoniae bloodstream infections and remained associated with in-hospital mortality after adjustment for infection severity and source. By CLSI criteria, 83.1% of isolates were cefiderocol susceptible; the MIC50 and MIC90 were 4 and 8 mg/L, respectively, and 41.0% met the reduced-susceptibility definition. ST11 predominated, with KL47 and KL64 as the main capsular loci. Cefiderocol MICs were higher among KL47/KL64 and virulence-plasmid-associated isolates than among comparator backgrounds. In multivariable analysis, bla NDM-1, bla SHV-12, and the aerobactin locus remained associated with reduced susceptibility, although the findings require cautious interpretation because of limited sample size and possible effects of clonal background. No inactivating mutations were identified in cirA, fepA, or fiu. CONCLUSIONS: Borderline cefiderocol MIC elevation was present before local drug exposure and was more frequent in locally prevalent ST11-KL47/KL64 and virulence-plasmid-associated CRKP. These findings provide a bloodstream-specific pre-introduction baseline and support prospective surveillance of numerical MIC distributions and associated genomic backgrounds.

Cefiderocol↗

PubMed2026

Coexistence of carbapenemase and hypervirulence-associated genes among Klebsiella pneumoniae high-risk clones in Hungary.

INTRODUCTION: Strains of Klebsiella pneumoniae carrying hypervirulence and carbapenemase genes represent a rapidly emerging global public health threat. Our study aimed to comprehensively characterise the genomics of hypervirulence-associated and carbapenemase genes carrying K. pneumoniae (hv(a)CpKp) isolates in Hungary. MATERIALS AND METHODS: Between January 2022 and April 2024, 89 aerobactin (iucA-D/iutA)-positive non-duplicate carbapenemase-producing K. pneumoniae isolates from 15 Hungarian healthcare institutes underwent short-read (Illumina, MiSeq, NextSeq) whole-genome sequencing, followed by detailed plasmid analysis using long-read sequencing (Nanopore, MinION) in a representative subset of 32 strains. RESULTS: Most isolates (79/89) belonged to the high-risk clone ST147. Hypervirulence-associated (hva) genes-including rmpA/rmpA2, peg344, shiF, iucA-D, and iutA-were universally present, and 59 isolates possessed chromosomally integrated yersiniabactin loci. Most isolates (87/89) carried the bla NDM-1 carbapenemase gene. Hypervirulence-associated genes were most frequently (29/32) associated with IncHI1B/IncFIB(Mar) plasmids. Notably, we identified plasmids carrying both hva and carbapenemase genes-designated as hybrid plasmids-in 13 of 32 strains. The bla NDM-1 was linked to the IS26 transposase and was present in conserved, identical cassettes on all bla NDM-1-carrying plasmids. DISCUSSION/CONCLUSION: Our study identified hv(a)CpKp strains, particularly the ST147 clone, circulating in Hungary. Our findings highlight the need for routine virulence gene monitoring and continuous genomic and plasmid-based surveillance to mitigate the clinical and epidemiological impact of emerging hv(a)CpKp lineages.

Klebsiella pneumoniae↗

PubMed2026

Carbapenemase-producing Acinetobacter baumannii from Spanish hospitals, 2016-2020: Interregional spread of ST2PAS isolates co-harbouring blaOXA-23, blaOXA-66 and armA genes.

INTRODUCTION AND AIMS: Carbapenem-resistant Acinetobacter baumannii (CRAB) poses a serious global threat, prioritized by the World Health Organization for new antibiotic development. The European Centre for Disease Prevention and Control proposed the integration of genomic sequencing into surveillance. This study characterizes phenotypic and genotypic features of nationwide-collected CRAB isolates from Spanish hospitals (2016-2020). MATERIALS AND METHODS: A total of 822 CRAB isolates sent to the Spanish reference laboratory were analysed, and 108 representative isolates were selected from 48 hospitals in 25 Spanish provinces. Antibiotic susceptibility was determined according to European Committee on Antimicrobial Susceptibility Testing guidelines, and WGS was performed by Illumina. Resistome, virulome, phylogeny (core-genome Multi-Locus Sequence-Typing; 2390 genes), insertion sequences and plasmids were analysed. RESULTS: Of the 108 representative isolates, 68.5% produced OXA-23, 10.2% OXA-24, 7.4% OXA-58, 1.9% NDM-1 and 2.7% others. 3.7% co-produced two acquired carbapenemases, and six that did not have them showed the ISAba1 upstream of the blaOXA-51 like. The predominant sequence types were ST2pas (82.4%) and ST218Oxf (61.1%). blaOXA-66 chromosomal carbapenemase allele was present in 75% of the 108 isolates, armA 16 s rRNA methyltransferase gene in 61.1%, and the kL-7/OCL-18 was present in 59.3% of the isolates. The most active antibiotic was colistin. Overall, 65.4% of invasive cases were caused by isolates harbouring all 38 virulence genes tested. In 64.8% of isolates a plasmid type was detected, mainly pS32-1-like (54.6%). CONCLUSIONS: In Spain, CRAB dissemination is driven by interregional spread of isolates belonging to the ST218Oxf/ST2Pas clones, characterized by the blaOXA-23/blaOXA-66 genotype, the presence of armA, the KL7/OCL18 capsular profile and the presence of pS32-1 plasmid.

K Locus↗

PubMed2026

Characteristics of an NDM-1-producing Klebsiella pneumoniae strain belonging to ST105.

Uncommon multilocus sequence types (MLSTs) of NDM-producing Klebsiella pneumoniae may pose a significant threat to patients, although they are often overlooked in surveillance studies. Characterizing these isolates is therefore important for infection control. In this study, the antimicrobial susceptibility and pathogenicity of K. pneumoniae strain KP_WXD, pertaining to the atypical sequence type ST105, were evaluated, including capsular polysaccharide (CPS) production, biofilm formation, and resistance to serum killing. Whole-genome sequencing (WGS) was performed to analyze its genomic features. K. pneumoniae KP_WXD strain was resistant to all tested β-lactam agents. Its virulence was lower than that of K. pneumoniae strains ST11-KL64 and NTUH-k2044 used as references, while its biofilm formation ability was stronger than that of both strains. WGS analysis revealed carriage of IncF and IncN plasmids carrying multiple antibiotic resistance genes, alongside blaNDM-1 and blaCTX-M, integrated into well-characterized mobile genetic elements. Moreover, both blaNDM-1 and blaCTX-M-harboring plasmids were transferable to E. coli J53 by conjugation without significant fitness cost on the recipient strain.

Klebsiella pneumoniae↗

PubMed2026

Silent carriage of tigecycline- and carbapenem-resistant Klebsiella quasipneumoniae co-harbouring tetX(4) and blaNDM-1 in healthy individuals in China.

OBJECTIVES: To investigate the occurrence and genomic characteristics of tigecycline- and carbapenem-resistant Klebsiella quasipneumoniae isolated from healthy individuals in China. METHODS: During a nationwide screening programme, faecal samples from healthy community individuals were cultured for carbapenem-resistant Enterobacterales. Antimicrobial susceptibility testing, whole-genome sequencing and conjugation assays were performed for two K. quasipneumoniae isolates co-harbouring tet(X4) and blaNDM-1. RESULTS: Two K. quasipneumoniae strains carrying tet(X4) and blaNDM-1 were recovered from healthy individuals without recent hospitalisation or antibiotic exposure. Both isolates showed resistance to tigecycline (>8 μg/mL) and carbapenems (≥4 μg/mL). Genomic analysis demonstrated close clonal relatedness between the isolates (ST6460-1LV, KL151). The blaNDM-1 gene was located on an IncX3 plasmid associated with mobile genetic elements, whereas tet(X4) was carried by an IncX1 plasmid. Conjugation assays confirmed successful transfer of both resistance genes, which frequently co-transferred into recipient strains. CONCLUSIONS: To our knowledge, this is the first report of K. quasipneumoniae co-harbouring tet(X4) and blaNDM-1 in healthy individuals in China. The findings indicate that healthy community populations may serve as a hidden reservoir for last-resort antimicrobial resistance genes and underscore the importance of community-based surveillance within a One Health framework.

Klebsiella quasipneumoniae↗

PubMed2025

Genomic and phenotypic insights into ST164 blaNDM-1-positive Acinetobacter baumannii from intestinal colonization in China.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CRAB) poses a critical global threat, especially in ICUs. Yet, reports on ST164 CRAB harboring blaNDM-1 remain scarce. This study investigates two clinical CRAB isolates, L4773hy and L4796hy, derived from intestinal colonization in Hangzhou, China, focusing on their phenotypic and genomic characteristics as well as the broader transmission of ST164 A. baumannii. METHODS: Bacterial identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) mass spectrometry. Antimicrobial susceptibility was assessed via agar and broth microdilution. Whole-genome sequencing employed Illumina NovaSeq 6000 and Oxford Nanopore platforms. Resistance genes, insertion elements, transposons, and integrons were detected using ResFinder, PlasmidFinder, VFDB, ISFinder, pdifFinder, and IntegronFinder. Strains were typed by MLST, and a phylogenetic tree was constructed with kSNP3.0. Genetic environment diagrams were generated using Easyfig 2.2.5. RESULTS: Two blaNDM-1-carrying A. baumannii isolates exhibiting extensive resistance to carbapenems, cephalosporins, and fluoroquinolones. Whole-genome sequencing and genetic environment analysis revealed the presence of a conserved structural sequence (ISAba14-ISAba14-aphA-ISAba125-blaNDM-1-bleMBL) on their chromosomes. Phylogenetic and clonal dissemination analysis showed that ST164 CRAB is primarily distributed in China and exhibits clonal spread. Pathogenicity studies indicated that blaNDM-1-positive ST164 strains have enhanced survival under immune pressure but do not display increased virulence in infection models. CONCLUSION: This study provides the genomic and phenotypic characterization of intestinally colonized ST164 blaNDM-1 positive CRAB in Hangzhou, China. The elucidation of the genetic environment of blaNDM-1 further confirms the clonal dissemination of ST164 isolates, highlighting the importance of enhanced surveillance and infection control measures to mitigate the spread of these multidrug-resistant pathogens.

Acinetobacter baumannii↗