In-depth characterization of multidrug-resistant NDM-1 and KPC-3 co-producing Klebsiella pneumoniae bloodstream isolates from Italian hospital patients.

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-ß-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 blaNDM-1 and blaKPC-3, various AMR genes, indicative of a multidrug resistance phenotype, including colistin resistance, were found. Each cluster-representative ST512 isolate harbored a blaNDM-1 plasmid (IncC) and a blaKPC-3 plasmid [IncFIB(pQil)/IncFII(K)], while the ST11 isolate harbored a blaNDM-1 plasmid [IncFII(pKPX1)] and a blaKPC-3 plasmid [IncFIB(K)/IncFII(K)]. The blaNDM-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 blaNDM-1/blaKPC-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 blaNDM-1/blaKPC-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-ß-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.

Chip-Based Molecular Evaluation of a DNA Extraction Protocol for Candida Species from Positive Blood Cultures.

The diagnosis of Candida bloodstream infection (BSI) may rely on a PCR-based analysis of a positive blood culture (PBC) obtained from the patient at the time of BSI. In this study, a yeast DNA extraction protocol for use on PBCs was developed and evaluated with the molecular mouse (MM) yeast blood (YBL) chip-based PCR assay, which allowed us to detect nine medically relevant Candida species. We studied 125 simulated or clinical PBCs for Candida species. A positive correlation between the DNA concentration and colony-forming unit count was found for simulated (Spearman's ρ = 0.58; p < 0.0001) and clinical (Spearman's ρ = 0.23, p = 0.09) PBCs. The extracted DNA yielded positive results with the MM YBL chip assay that agreed with the Candida species-level identification results for 63 (100%) of 63 isolates from simulated PBCs and 66 (99.5%) of 67 isolates from clinical PBCs. The false-negative result was for one C. tropicalis isolate that grew together with C. albicans in PBC. None of the 30 (Candida)-negative clinical BCs included as negative controls yielded a positive result with the MM YBL chip assay. Our DNA extraction protocol for the Candida species couples efficiency and simplicity together. Nevertheless, further studies are needed before it can be adopted for use with the MM YBL chip assay.