Whole genome sequencing and sequence analysis was done as previously described [19 (link)]. Briefly, DNA was extracted from bacterial cultures using a DNeasy UltraClean 96 Kit (Qiagen, Venlo, Netherlands). Library preparation and sequencing was performed by a commercial service provider (Novogene, Cambridge, United Kingdom) using Illumina chemistry. Sequencing was carried out on a NovaSeq 6000 flow cell using a paired-end sequencing strategy of 2 × 150 bp. Analysis of phylogenetic relatedness was carried out using the pan-genome pipeline Roary [20 (link)]. In silico screening for antimicrobial resistance genes was performed with ABRicate (accessible at https://github.com/tseemann/abricate ) using the Comprehensive Antibiotic Resistance Database (CARD) [21 (link)] as reference. We also used ABRicate to screen the genomes of K. pneumoniae isolates for hypervirulence genes by searching against gene entries of hypervirulence plasmid pLVPK (NC_005249.1) as the reference. Plasmid-related sequences were analysed with RFplasmid (accessible at http://klif.uu.nl/rfplasmid ) and plasmidMLST (accessible at https://pubmlst.org/bacteria/plasmid-mlst ). All sequence data generated for this study were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra ) and are available under BioProject accession number PRJNA891092.
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