Klebsiella pneumoniae has emerged as an important nosocomial pathogen, with whole-genome sequencing (WGS) significantly improving our ability to characterize associated outbreaks. Our study sought to perform a genome-wide analysis of multiclonal K. pneumoniae isolates (n=39; 23 patients) producing extended spectrum beta-lactamases and/or carbapenemases sourced between 2011 and 2016 in a Portuguese tertiary-care hospital. All isolates showed resistance to third-generation cephalosporins and six isolates (five patients) were also carbapenem resistant. Genome-wide-based phylogenetic analysis revealed a topology representing ongoing dissemination of three main sequence-type (ST) clades (ST15, ST147 and ST307) and transmission across different wards, compatible with missing links that can take the form of undetected colonized patients. Two carbapenemase-coding genes were detected: bla_KPC-3 , located on a Tn4401d transposon, and bla_GES-5 on a novel class 3 integron. Additionally, four genes coding for ESBLs (bla_BEL-1 , bla_CTX-M-8 , bla_CTX-M-15 and bla_CTX-M-32 ) were also detected. ESBL horizontal dissemination across five clades is highlighted by the similar genetic environments of bla_CTX-M-15 gene upstream of ISEcp1 on a Tn3-like transposon. Overall, this study provides a high-resolution genome-wide perspective on the epidemiology of ESBL and carbapenemase-producing K. pneumoniae in a healthcare setting while contributing for the adoption of appropriate intervention and prevention strategies.

The evolutionary epidemiology, resistome, virulome and mobilome of thirty-one multidrug resistant Klebsiella pneumoniae clinical isolates from the northern Vila Real region of Portugal were characterized using whole-genome sequencing and bioinformatic analysis. The genomic population structure was dominated by two main sequence types (STs): ST147 (n = 17; 54.8%) and ST15 (n = 6; 19.4%) comprising four distinct genomic clusters. Two main carbapenemase coding genes were detected (bla_KPC-3 and bla_OXA-48) along with additional extended-spectrum β-lactamase coding loci (bla_CTX-M-15, bla_SHV-12, bla_SHV-27, and bla_SHV-187). Moreover, whole genome sequencing enabled the identification of one Klebsiella variicola KPC-3 producer isolate previously misidentified as K. pneumoniae, which in addition to the bla_KPC-3 carbapenemase gene, bore the chromosomal broad spectrum β-lactamase bla_LEN-2 coding gene, oqxAB and fosA resistance loci. The bla_KPC-3 genes were located in a Tn4401b transposon (K. variicolan = 1; K. pneumoniaen = 2) and Tn4401d isoform (K. pneumoniaen = 28). Overall, our work describes the first report of a bla_KPC-3 producing K. variicola, as well as the detection of this species during infection control measures in surveillance cultures from infected patients. It also highlights the importance of additional control measures to overcome the clonal dissemination of carbapenemase producing clones.