2.3 Whole-genome sequencing and bioinformatics analysis
Based on drug susceptibility testing, a MDR Klebsiella pneumoniaeisolate (named as M297-1) from Red Kangaroo was identified multidrug resistant (MDR) bacteria, and subjected to WGS using Oxford Nanopore Technologies (ONT) MinION platform (Biomarker Technologies, China) (Ashton et al., 2015; Loman, Quick, & Simpson, 2015). The sequencing was carried out according to the standard protocol provided by ONT, and the high-quality genomic DNA, was extracted by Nanodrop, Qubit and 0.35% agarose gel electrophoresis for purity, concentration and integrity. Large fragments of DNA were recovered by BluePippin automatic nucleic acid recovery system. The library was constructed by ligation sequencing kit (SQK-LSK109 Ligation Sequencing Kit, Oxford Nanopore Technologies, UK), and the DNA damage repair and terminal repair, magnetic bead purification were used to connect, purify again, and the Qubit library was quantified and sequenced on the machine.
The phylogenetic tree was constructed based on 16s rRNAsKlebsiella pneumoniae isolate multilocus sequence typing (MLST) was conducted by using MLST 1.8 or PubMLST (http://pubmlst.org/)Plasmid replicon typing, plasmid multilocus sequence typing ,and identification of resistance genes were performed using Plasmid Finder (https://cge.cbs.dtu.dk/services/PlasmidFinder/) or pMLST 2.0, and Resfinder 2.0 or CARD, respectively. The comparison of the similarity between plasmids and known plasmids was conducted using PLSDB databases (https://ccb-microbe.cs.uni-saarland.de/plsdb/). Plasmid sequence was annotated with MiGAP (https://www.migap.org/), and the genomic structure was compared in EasyFig. The comparative map of plasmid genome was drawn by the Illustrator for Biological Sequences (IBS) software v1.0. (Liu et al., 2015) and modified manually. Transposons and insertion sequences were determined using ISfinder.