Trypcase soy agar plates

AchroS and AchroR (Table 1) were cultured on trypcase soy agar plates (BioMérieux, Marcy l’Etoile, France), inoculated into liquid medium brain-heart-infusion (BHI) broth (Oxoid, Basingstoke, UK) and grown overnight (~16-hrs) at 37 °C. Cells were harvested, washed with phosphate-buffered saline (PBS, pH 7.4), and genomic DNA extraction was performed using a phenol-chloroform extraction as previously described^{49 (link)}.

K. pneumoniae O1 (Kp4, Kp16, Kp24, Kp69, Kp71, Kp75, Kp76, Kp88, Kp111) and O2 (Kp30) isolates used in this study were obtained from clinical specimens. Prototype strains PCM-27 (O2 gml+) and PCM-11 (O3:K11) were purchased from the Polish Collection of Microorganisms (PCM, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland). Bacteria were grown on Trypcase Soy Agar plates (BioMérieux) and Luria-Bertani (LB) broth. For large scale LPS preparation, strains were cultured in LB broth in a 10 L fermenter (37°C, 200 rpm, aeration of 5 L/min), killed with 0.5% phenol for 2 h at 60°C, washed with water and freeze-dried. For molecular cloning and cultivation of trans-complemented strains (Kp4/pKP100 and Kp4/pSU2718), selective medium supplemented with chloramphenicol (20 μg/ml) was used.