The DNA of each CRKP isolate was purified and recovered by a silica gel column (D3146, HiPure Bacterial DNA kit) after incubation. Paired-end libraries with insert sizes of ~300 bp were prepared following Illumina’s standard genomic DNA library preparation procedure (VAHTS Universal DNA Library Prep kit for Illumina V3) and sequenced on an Illumina NovaSeq 6000 using the S4 reagent kits (v1.5) according to the manufacturer’s instructions. The sequencing reads of each isolate were quality trimmed and assembled into contigs using EToKi (45 (link)). The ST, antibiotic resistance genes, and virulence determinants of each isolate were predicted based on its genomic assemblies using kleborate (46 (link)). All the isolates from ST11 were aligned using the EToKi align module onto a reference genome (GCF_011066505.1 ; sample from blood, Hong Kong, China, 2016) to obtain a multiple sequence alignment of the nonrepetitive, core genomic regions that were shared by ≥95% of genomes. The resulting alignments were subjected to a maximum-likelihood phylogeny by the EToKi phylo module, had the recombinant regions removed using RecHMM (47 (link)), and were visualized using GrapeTree online software (48 (link)). Similarly, we also aligned all the ST14 isolates onto a reference genome (GCF_001521895.1 , sample from urine, Jiangxi, China, 2014) and estimated a maximum-likelihood phylogeny of ST14.
Free full text: Click here
