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Bloodstream infection (BSI) caused by carbapenem-resistant Klebsiella pneumoniae (KP) poses significant challenges, particularly when the infecting isolate carries multiple antimicrobial resistance (AMR) genes/determinants. This study, employing short- and long-read whole-genome sequencing, characterizes six New Delhi metallo-β-lactamase (NDM) 1 and KP carbapenemase (KPC) 3 co-producing KP isolates, the largest cohort investigated in Europe to date. Five [sequence type (ST) 512] and one (ST11) isolates were recovered from patients who developed BSI from February to August 2022 or February 2023 at two different hospitals in Rome, Italy. Phylogenetic analysis revealed two distinct clusters among ST512 isolates and a separate cluster for the ST11 isolate. Beyond bla NDM-1 and bla KPC-3 , various AMR genes, indicative of a multidrug resistance phenotype, including colistin resistance, were found. Each cluster-representative ST512 isolate harbored a bla NDM-1 plasmid (IncC) and a bla KPC-3 plasmid [IncFIB(pQil)/IncFII(K)], while the ST11 isolate harbored a bla NDM-1 plasmid [IncFII(pKPX1)] and a bla KPC-3 plasmid [IncFIB(K)/IncFII(K)]. The bla NDM-1 plasmids carried genes conferring resistance to clinically relevant antimicrobial agents, and the aminoglycoside resistance gene aac ( 6 ')- Ib was found on different plasmids. Colistin resistance-associated mgrB / pmrB gene mutations were present in all isolates, and the yersiniabactin-encoding ybt gene was unique to the ST11 isolate. In conclusion, our findings provide insights into the genomic context of bla NDM-1 / bla KPC-3 carbapenemase-producing KP isolates.IMPORTANCEThis study underscores the critical role of genomic surveillance as a proactive measure to restrict the spread of carbapenemase-producing KP isolates, especially when key antimicrobial resistance genes, such as bla NDM-1 / bla KPC-3 , are plasmid borne. In-depth characterization of these isolates may help identify plasmid similarities contributing to their intra-hospital/inter-hospital adaptation and transmission. Despite the lack of data on patient movements, it is possible that carbapenem-resistant isolates were selected to co-produce KP carbapenemase and New Delhi metallo-β-lactamase via plasmid acquisition. Studies employing long-read whole-genome sequencing should be encouraged to address the emergence of KP clones with converging phenotypes of virulence and resistance to last-resort antimicrobial agents., Competing Interests: The authors declare no conflict of interest.

We aimed to report a 32-month laboratory experience with the eazyplex ® CSF direct panel assay for the rapid diagnosis of meningitis due to six most common bacterial species (Escherichia coli, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus agalactiae, and Streptococcus pneumoniae). We included all cerebrospinal fluid (CSF) samples from patients admitted with a clinical suspicion of meningitis/encephalitis between May 2016 and December 2018 at our hospital. In addition to the eazyplex ® assay, both Gram stain microscopy and culture were performed, and results were confirmed with 16S rRNA PCR/sequencing. Patients' demographics and relevant clinical information were collected. Of 135 studied patients, 44 (32.6%) had a microbiologically documented diagnosis of meningitis. Overall, we identified 21 S. pneumoniae, 10 N. meningitidis, 6 L. monocytogenes, 3 E. coli, 2 Streptococcus pyogenes, 1 S. agalactiae, and 1 Citrobacter koseri as aetiological agents. The eazyplex ® assay allowed identification in 40 (90.9%) cases, with four not identified cases due to microorganisms not included in the panel at the time of testing. Thirty-two (72.7%) cases had positive culture results, whereas 28 (63.6%) cases had positive Gram stain results. Notably, combining Gram stain and eazyplex ® assay allowed identification in 100% of cases. After notification of rapid results, physicians modified the empiric antibiotic therapy, which became appropriate in three patients (all with L. monocytogenes meningitis). The eazyplex ® CSF panel assay worked better than culture in detecting the most common agents of bacterial meningitis and accelerated the diagnosis leading to timely initiation or continuation of appropriate antibiotic therapy.