Genomic characterization of the emerging multidrug-resistant Klebsiella pneumoniae ST6668 clone in northern Italy, 2020-2025.

Carbapenem-resistant Klebsiella pneumoniae carrying New Delhi metallo-β-lactamase (NDM) enzymes poses a major clinical and public health challenge due to their ability to spread rapidly in hospitals, and their resistance to most β-lactam antibiotics. Following the first report of the emerging NDM-producing K. pneumoniae lineage ST6668 in Pavia, Italy, we conducted a retrospective analysis at San Raffaele Hospital (Milan). We identified 87 ST6668 isolates, several of which were collected prior to the initial report from Pavia. Whole-genome sequencing data were used to compare the Milan isolates with the 52 genomes from Pavia, in order to characterize a total of 139 ST6668 isolates collected between 2020 and 2025 from three hospitals in northern Italy. Phylogenetic and comparative genomic analyses were performed based on single-nucleotide polymorphisms (core SNPs), including 310 ST147 genomes for comparison. Accessory genome profiling was further validated by hybrid assemblies of three representative strains. A phylogenetic cluster containing isolates from different hospitals suggested possible interhospital transmission. Although the origin of ST6668 remains unknown, it clustered closely with KL64-ST147. The hybrid assemblies revealed that blaNDM-1 is carried by the IncFIB(pQil) or IncHI1BIncFIB(Mar) plasmids. ST6668 lacks virulence markers such as iuc and rmpADC. All ST6668 isolates analyzed here carry a prophage identical to vB_Kpn_147Tu, which has been previously reported in ST147 isolates from the Tuscany outbreak. Together, these findings suggest either recent divergence from a common ancestor or convergence via mobile genetic elements.IMPORTANCECarbapenem-resistant Klebsiella pneumoniae producing New Delhi metallo-β-lactamase (NDM)-type enzymes represent a serious threat to infection control and patient safety. Understanding how novel resistant lineages emerge and spread is essential to limit hospital outbreaks. Our study traced the early presence of the recently identified ST6668 lineage in Milan, showing that it was circulating before its first description in Pavia. By linking this clone to the well-known high-risk lineage ST147, we demonstrate how resistance and genetic elements move between successful bacterial populations. These insights highlight how genomic surveillance can uncover hidden transmission pathways and guide interventions to prevent further dissemination of multidrug-resistant K. pneumoniae in healthcare settings.