2.4. Whole genome sequence (WGS) analysis
For selected ESBL-producing E. coli strain, genomic DNA was
extracted using a PureLinkTM Quick Gel Extraction Kit (Life
Technologies, Carlsbad, CA), and a genomic paired-end library (75 x 2
bp) was prepared using a Nextera XT DNA Library Preparation Kit
(Illumina Inc., Cambridge, UK) according to the manufacturer’s
instructions. The whole genome was sequenced on the NextSeq platform
(Illumina). De novo genome assembly and contig annotation was
carried out using CLC Genomics Workbench 12.0.3. Multilocus sequence
type (MLST), plasmid replicons, resistome and serotype were identified
using MLST v2.0 (Larsen et al. , 2012), PlasmidFinder v2.1
(Carattoli et al. , 2014), ResFinder v3.2 (Zankari et al. ,
2012), and SerotypeFinder v2.0 (Jenkins, 2015) tools, respectively, from
Center for Genomic Epidemiology (http://www.genomicepidemiology.org/).
Clinically important virulence factors were detected and compared by
ABRicate v0.9.8 (https://github.com/tseemann/abricate) using data from
the Escherichia coli Virulence Factors
(https://github.com/phac-nml/ecoli_vf) and the Virulence Factor
Database (VFDB) (http://www.mgc.ac.cn/VFs/). Heavy metal (HM) and
biocides genes were detected using the BacMet2 experimentally confirmed
database (http://bacmet.biomedicine.gu.se). For whole genome of
selected ESBL-producing E. coli identified in this study, a
minimum spanning tree was constructed in Enterobase using the MSTree V2
algorithm and the wgMLST scheme
(https://enterobase.warwick.ac.uk/species/index/ecoli). This scheme
consists of 25,002 pan-genome genes present in E. coli genomes,
which represented most of the diversity in Enterobase at the time (March
2020)
(https://bitbucket.org/enterobase/enterobase-web/wiki/Escherichia%20Statistics).
All images were generated with iTOL v.5.5 (https://itol.embl.de).