Abstract

Background/Objectives: Klebsiella pneumoniae complex, Escherichia coli, and Enterobacter cloacae complex are considered the most prevalent carbapenemase-producing (CP) Enterobacterales in Spain. However, temporal changes in carbapenemase distribution, clonal diversity, and the mobile genetic elements (MGEs) involved in their dissemination have not been systematically characterised in our region. To address this knowledge gap, this study investigated the epidemiology of these organisms over a seven-year period. Methods: CP Enterobacterales isolates recovered between 2019 and 2025 from rectal screening swabs and clinical specimens (blood, urine, respiratory, wound, and sterile fluid samples) were included. All 297 isolates underwent phenotypic characterisation. Carbapenemase detection and sequence type (ST) assignment were performed for the 282 isolates available for molecular analysis. Following the gradual implementation of long-read whole-genome sequencing (WGS) beginning in 2022, MGEs associated with carbapenemase dissemination were characterised, and selected high-risk clones were analysed by SNP-based phylogenetic analysis. Results: Fifty isolates were recovered from ICU admission rectal screening, and 247 from clinical samples. Although the proportion of CP Enterobacterales among Enterobacterales isolates remained stable throughout the study period, significant changes were observed between 2019 and 2022 and between 2023 and 2025 in carbapenemase types, with a statistically significant increase in metallo-β-lactamases (MBLs) (p < 0.001), as well as in species distribution. Furthermore, predominant plasmid replicons associated with the dissemination of MBL genes were characterised. A K. pneumoniae ST147 clone co-harbouring blaOXA-48 and blaNDM-1 emerged and expanded significantly during the study period (p < 0.05). In addition, an ST225 NDM-1 clone was identified. SNP-based phylogenetic analysis revealed distinct genetic clusters within both lineages. Conclusions: Temporal shifts in the epidemiology of CP Enterobacterales, including the emergence and expansion of high-risk clones, were observed. High-resolution genomic analyses provided additional insight into the genetic relationships among circulating lineages.

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