Genomic Epidemiology of Carbapenem-Resistant Enterobacterales in a Bulgarian University Hospital (2022–2025): Emergence and Persistence of Klebsiella pneumoniae ST6260 Carrying blaNDM-5

Carbapenem-resistant Enterobacterales (CRE) are a major public health concern, yet genomic data from Bulgaria remain limited. We investigated the genomic epidemiology and genomic characteristics of clinically significant CRE recovered at University Hospital “Lozenetz”, Sofia, between 2022 and 2025. Antimicrobial susceptibility testing, phenotypic carbapenemase characterization, and PCR were combined with whole-genome sequencing of 23 purposively selected isolates chosen to capture temporal, species, carbapenemase, specimen, ward, and resistance-phenotype diversity. The proportion of CRE among all Enterobacterales remained broadly stable over the study period, and Klebsiella pneumoniae was the predominant species throughout. NDM-type carbapenemases were the most frequent overall, although VIM-, KPC-, and OXA-48-like enzymes were also detected. Among the sequenced isolates K. pneumoniae ST6260 (n = 16) was predominant. The remaining isolates included single K. pneumoniae isolates belonging to ST11, ST101, and ST258; two Enterobacter hormaechei ST114; one Proteus mirabilis ST93; and one Providencia stuartii ST46. All ST6260 isolates carried chromosomal blaNDM-5 within a conserved multidrug-resistance module with variable downstream regions, whereas blaOXA-232 was situated on a small ColKP3 plasmid within a defined subcluster. Overall, the data indicate that ST6260 is an emerging hospital-associated multidrug-resistant lineage.