SNP-calling method

For WGS, total DNA of K. pneumoniae isolates DT1 and DT12 was extracted on a QiaSymphony SP instrument using QiaSymphony mericon extraction kits (Qiagen). WGS libraries were constructed with NEB NextUltra DNA library Prep Kit for Illumina (NEB) and sequenced with 2 × 250 cycles on an Illumina MiSeq instrument. A total of 3 583 958  paired-end, 251 nt Illumina MiSeq reads were generated from the Illumina MiSeq sequencer for the isolate K. pneumoniae DT12. Bases less than Q20, as well as adapter sequences of the reads, were trimmed and any reads shorter than 126 nt were removed using Trimmomatic v0.33.7 Retained high-quality MiSeq reads were used as input data for the SPAdes assembler (version 3.7.1),8 resulting in 144 high-coverage and long contigs (coverage >10 and length >1000). Contigs were used as input for PLACNET,9 resulting in several clusters. The cluster carrying blaCTX-M-14 consists of four contigs and several reference plasmids (NC_021502.1, NC_023027.1, NZ_CP015075.2, NZ_CP015071.1, NC_021488.1, NZ_CP016927.1 and NC_021078.1). Based on these reference plasmids, the four contigs were oriented to a putative plasmid using CONTIGuator v2.7.10 All sequence gaps in the plasmid were successfully closed using GapFiller v1.1011 and the paired-end MiSeq reads. Rapid annotations using subsystems technology12 analysis identified 99 putative ORFs from the putative plasmid. The plasmid harbouring blaCTX-M-14 from the isolate K. pneumoniae DT1 (with a total of 1 628 935 paired-end, 251 nt Illumina MiSeq reads) was assembled and annotated using the same methods described above. Sequences of blaCTX-M-14 in K. pneumoniae DT1, DT12 and additional isolates from the outbreak were verified by Sanger sequencing using primers CTX-M_seq_for and CTX-M_seq_rev (Table S1, available as Supplementary data at JAC Online).

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