Comparative Genomics of Bacterial Isolates

We performed whole genome sequencing (WGS) on a representative subset of bacterial isolates, stratified by bacterial species and month of isolation. All bacterial isolates were routinely grown on Heart Infusion agar (Oxoid) for 16 h at 37°C, inoculated into 3 mL Heart Infusion broth (Oxoid), and grown for a further 16 h at 37°C with orbital shaking at 150 rpm. Genomic DNA was extracted from 300 µL of liquid bacterial culture using the GenFind V3 Reagent Kit (Beckman Coulter) as per manufacturer's instructions. Libraries were prepared using the Nextera Flex DNA Library Prep Kit (Illumina), and 150 bp paired-end sequencing was performed on the NovaSeq 6000 system (Illumina). Genomes were assembled using Unicycler (v.0.4.9) [14] (link). Multi-locus sequence types (STs) were identified using mlst (https://github.com/tseemann/mlst).
For the genomes of the most prevalent ST, we determined relatedness by calculating pairwise single nucleotide variation (SNV) distances between all isolates. Genomes were assembled using SKESA v.2.3 [15] (link) using default parameters. Each pair of genome assemblies was compared using Catpac (https://github.com/rrwick/Catpac) to determine the number of SNVs between pairs. Pairs with ≤5 SNVs per mbp between them were considered likely candidates for strain transmission, as defined by Gorrie et al. [16] (link).