Bacterial dna kit

Genomic DNA was extracted by using a Bacterial DNA Kit (QIAGEN, Hilden, Germany). The harvested DNA was detected by the agarose gel electrophoresis and quantified by Qubit^® 2.0 Fluorometer (Thermo Scientific). Then the DNA was sequenced both on the Illumina NovaSeq 6000 (Illumina, San Diego, CA, United States) and Oxford Nanopore platforms (Oxford Nanopore Technologies, Oxford, United Kingdom) to obtain short-read data and long-read data, respectively. The raw llumina reads were assembled using SPAdes3.10.0, and then the sequencing results were hybrid assembled with Unicycler v0.4.7 to get the complete genome sequence (Wick et al., 2017 (link)). The bacterial genomes were annotated using Prokka. Additionally, the acquired antimicrobial resistance genes and replicon type of plasmid were determined using online tools (http://www.genomicepidemiology.org/), while the transposon and IS elements were identified using the ISFinder database (http://www-is.biotoul.fr/). The virulence genes of isolates were identified using VFDB. Finally, the circular image of multiple plasmids comparisons was plotted by the BLAST Ring Image Generator (BRIG) (Alikhan et al., 2011 (link)). The comparison figure of the genetic environment surrounding the mcr-1 gene was generated by Easyfig 2.2.3 (Sullivan et al., 2011 (link)).

The genome of K. pneumoniae 5589 was extracted using a specific bacterial DNA Kit (QIAGEN, Hilden, Germany). To better understand the genetic features, DNA sequencing was performed on the Illumina NovaSeq 6000 (Illumina, San Diego, CA, United States) and the Oxford Nanopore (Oxford Nanopore Technologies, Oxford, United Kingdom) platform (Bao et al., 2022 (link)). Then, the whole genome was annotated with Prokka. Additionally, the acquired ARGs were detected by ResFinder 4.1¹, and the plasmid replicon type was identified by PlasmidFinder 2.1.² The transposon and insertion sequence were detected using the ISFinder database.³ Finally, the circular comparison images of multiplex plasmids were generated by BLAST Ring Image Generator (BRIG). The linear comparison figures of multiple genomic loci surrounding the bla_OXA-181 and mcr-8 were generated by Easyfig 2.0 software (Sullivan et al., 2011 (link)).