Seqsphere software version 6

All 84 K. pneumoniae isolates were subjected to next-generation sequencing (NGS) using the Illumina HiSeq 2500 (BaseClear, Leiden, the Netherlands). The NGS data of the K. pneumoniae isolates were used for classical MLST and wgMLST analyses using the in-house wgMLST scheme in SeqSphere software version 6.0.2 (Ridom GmbH, Münster, Germany). The in-house K. pneumoniae wgMLST scheme was comprised of 4978 genes (3471 core-genome and 1507 accessory-genome targets) using K. pneumoniae MGH 78,578 (NC_009648.1) as a reference genome^{21 (link)}. For classical MLST, the existing scheme was used and cluster nomenclature were depicted in Table 1^{34 (link)}. The resulting data was imported into Bionumerics version 7.6.3 for subsequent comparative analyses (Applied Maths, Sint-Martens-Latem, Belgium). The antibiotic resistance gene profile and plasmid replicon compositions in all of the isolates were determined by interrogating the online ResFinder (version 3.1.0) and PlasmidFinder (version 2.0.2) databases available at the Center for Genomic Epidemiology website (https://cge.cbs.dtu.dk/services/)^{35 (link),36 (link)}. For ResFinder, a 90% identity threshold and a minimum length of 60% were used as criteria, whereas for PlasmidFinder, an identity of 95% was utilized.

The first VRE isolate per patient and BSI isolates were subjected to whole genome sequencing (WGS). Briefly, sequencing libraries were prepared using the Nextera XT library preparation kit (Illumina, Munich, Germany) for a 250 bp paired-end sequencing run on a MiSeq (Illumina). De novo assembly was performed using Velvet (version 1.1.04). Assembled genomes were used for core genome multilocus sequence typing (cgMLST) (1,423 alleles [18 (link)],) and traditional 7-loci MLST using SeqSphere + software version 6.0.2 (Ridom, Münster, Germany), van genes were identified using ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/). The raw sequencing reads were submitted to the European Nucleotide Archive under the study accession number PRJEB25579 [19 ].