2162/2301 (94.0%) samples submitted as K. pneumoniae during the EuSCAPE survey [14 (link)] were prepared for whole genome sequencing. The remaining 139/2301 (6.0%) were discarded due to a lack of growth, contamination of the culture plates, or having multiple phenotypes suggestive of contamination with different species.
470/2162 samples were plated on MacConkey agar (Sigma Aldrich) overnight at 37°C and a single colony representing each sample was picked into phosphate buffered saline (PBS) solution. For the remaining 1692/2162 samples, a storage bead was picked into 1.5mL of low salt Luria-Bertani (LB) broth in a well of a 96-well S-block (Qiagen), and incubated at 37°C overnight with shaking. The bacteria were pelleted and re-suspended in 1xPBS solution. Lysozyme (Sigma Aldrich), RNase A (Invitrogen) and Proteinase K (Qiagen) were added to all samples, which were incubated for 1h at 37°C. DNA was extracted using the QIAamp 96-well kit on the QIAcube HT system (Qiagen). Isolates were sequenced using the Illumina HiSeq platform with 125bp paired-end reads.
470/2162 samples were plated on MacConkey agar (Sigma Aldrich) overnight at 37°C and a single colony representing each sample was picked into phosphate buffered saline (PBS) solution. For the remaining 1692/2162 samples, a storage bead was picked into 1.5mL of low salt Luria-Bertani (LB) broth in a well of a 96-well S-block (Qiagen), and incubated at 37°C overnight with shaking. The bacteria were pelleted and re-suspended in 1xPBS solution. Lysozyme (Sigma Aldrich), RNase A (Invitrogen) and Proteinase K (Qiagen) were added to all samples, which were incubated for 1h at 37°C. DNA was extracted using the QIAamp 96-well kit on the QIAcube HT system (Qiagen). Isolates were sequenced using the Illumina HiSeq platform with 125bp paired-end reads.