4.5. DNA Extraction and WGS

DNA was extracted from an overnight culture using the QIAamp DNA Mini Kit (Qiagen, Germany) for whole-genome sequencing. The manufacturer’s protocol was followed with slight modifications. For highly mucoid K. pneumoniae isolates, a pre-lysis step was performed, which includes the preparation of a pre-lysis buffer consisting of 100 μL of Tris//EDTA (TE) buffer (ThermoFisher Scientific, Waltham, MA, USA), 1.00 μL lysozyme 10 mg/mL (final concentration 0.1 mg/mL) (Sigma-Aldrich, St. Louis, MO, USA), 0.20 μL lysostaphin 10 mg/mL (final concentration 0.02 mg/mL) (ThermoFisher Scientific, USA), and 0.10 μL RNAse A 100 mg/mL (final concentration 0.1 mg/mL) (Qiagen, Hilden, Germany). The bacterial suspension from an overnight culture was centrifuged at 8000× g for 2 min, and the pelleted bacterial cells were resuspended in 100 μL of the pre-lysis buffer by pipetting up and down several times with a P200 pipette. The suspension was then incubated for 60 min. After incubation, 1.00 μL of Proteinase K (Sigma-Aldrich, St. Louis, MO, USA) was added as a final step in the pre-lysis stage. The sample was then ready for the first step of the Qiagen DNA extraction protocol as discussed in the previous section.

After DNA quantification by NanoDrop (ThermoFisher Scientific 1000 NanoDrop Spectrophotometer), the samples were sent to microbesNG in the UKfor WGS by Illumina next-generation sequencing (https://microbesng.co.uk, Birmingham, UK, accessed on 30 June 2021) [60]. The extracted DNA was prepared following the manufacturer’s protocol as described on the company’s website as follows: ILLUMINA SEQUENCING (SGS and EGS) Genomic DNA libraries are prepared using the Nextera XT Library Prep Kit (Illumina, San Diego, CA, USA) following the manufacturer’s protocol with the following modifications: input DNA is increased 2-fold, and PCR elongation time is increased to 45 s. DNA quantification and library preparation are carried out on a Hamilton Microlab STAR automated liquid handling system (Hamilton Bonaduz AG, Bonaduz, Switzerland). Pooled libraries are quantified using the Kapa Biosystems Library Quantification Kit for Illumina. Libraries are sequenced using Illumina sequencers (HiSeq/NovaSeq) using a 250 bp paired-end protocol. Reads are adapter trimmed using Trimmomatic 0.30 with a sliding window quality cutoff of Q15 [63]. De novo assembly is performed on samples using SPAdes version 3.7 [64], and contigs are annotated using Prokka 1.11 [65].