Phagocytosis Assay of K. pneumoniae

The phagocytosis assay of K. pneumoniae strains was performed by an established method (23 (link)). An inoculum of 10⁷ CFU was mixed with 10⁶ freshly isolated human neutrophils or mouse macrophage cell line RAW-264.7 in RPMI 1640 media (23 (link)). After 15 min of incubation, extracellular bacteria were washed out, and 100 μg/ml gentamicin was added to kill any remaining extracellular bacteria. Cell-associated bacteria were quantified at zero and 30 min after the end of incubation by plating on LB agar plates after lysis of the cells with 0.1% Triton X-100. Initial experiments established that the magA knockout mutants were no more sensitive to the detergent than the wild type. To determine whether cell-associated bacteria counted by the aforementioned method were indeed ingested by human neutrophils, fluorescence microscopy using confocal microscope was performed. Before fluorescence microscopy, plasmid GFPuv (CLONTECH Laboratories Inc.), which carries a gene that encodes green fluorescent protein, was electroporated into selected K. pneumoniae strains.