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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 (blaKPC-3 and blaOXA-48) along with additional extended-spectrum β-lactamase coding loci (blaCTX-M-15, blaSHV-12, blaSHV-27, and blaSHV-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 blaKPC-3 carbapenemase gene, bore the chromosomal broad spectrum β-lactamase blaLEN-2 coding gene, oqxAB and fosA resistance loci. The blaKPC-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 blaKPC-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.

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Klebsiella pneumoniae is an important nosocomial infectious agent with a high antimicrobial resistance (AMR) burden. The application of long read sequencing technologies is providing insights into bacterial chromosomal and putative extra-chromosomal genetic elements (PEGEs) associated with AMR, but also epigenetic DNA methylation, which is thought to play a role in cleavage of foreign DNA and expression regulation. Here, we apply the PacBio sequencing platform to eight Portuguese hospital isolates, including one carbapenemase producing isolate, to identify methylation motifs. The resulting assembled chromosomes were between 5.2 and 5.5Mbp in length, and twenty-six PEGEs were found. Four of our eight samples carry blaCTX-M-15, a dominant Extended Spectrum Beta Lactamase in Europe. We identified methylation motifs that control Restriction–Modification systems, including GATC of the DNA adenine methylase (Dam), which methylates N6-methyladenine (m6A) across all our K. pneumoniae assemblies. There was a consistent lack of methylation by Dam of the GATC motif downstream of two genes: fosA, a locus associated with low level fosfomycin resistance, and tnpB transposase on IncFIB(K) plasmids. Overall, we have constructed eight high quality reference genomes of K. pneumoniae, with insights into horizontal gene transfer and methylation m6A motifs.