K. pneumoniae strain 52145 (MOI 500:1, 5 h) triggered 30.2 ± 0.28% cytotoxicity, which was approximately 1.5 times higher than that induced by strain 52K10 (20.2 ± 2.19%). Formazan is produced by reduction of MTS tetrazolium by metabolically active cells and thus serves as an indicator of cell viability. Formazan production (% viability) was lower
in strain 52145-infected cells (32.9 ± 6.5%) than in non-infected (100%) or 52K10-infected cells (134 ± 4.9%). DNA fragmentation is taken as a sign of cell death by apoptosis. A prominent DNA laddering/degradation could be seen after 6 h of infection with K. pneumoniae strains 52145, 43816 and 1850 (Fig. 3A). However, DNA extracted from cells infected with strain 52K10 was intact, similar to DNA obtained from non-infected cells (Fig. 3A). Finally, we analysed the uptake of ethidium bromide P505-15 by infected cells. Ethidium bromide is taken up by the cells only when integrity of the plasma membrane
is lost. Red fluorescence staining of nuclei is therefore an indicator of plasma membrane integrity loss. The percentage of cells which had taken up the dye was higher in 52145-infected cells (21.2 ± 2.2%) than in 52K10-infected cells (1.74 ± 0.9%) or in non-infected cells (0%). Representative pictures are shown in Fig. 3B. Figure 3 Klebsiella induced cytotoxicity is observed by disintegration of host genomic DNA and loss of host plasma membrane integrity. A. Ethidium bromide staining after agarose gel-electrophoresis of genomic DNA isolated from A549 epithelial cells infected with K. pneumoniae strains Calpain 52145, 43816, 1850 or 52K10. B. A549 lung epithelial cells were not infected 3-MA purchase (left), infected with K. pneumoniae strain 52K10 (middle), or strain 52145 (right). The cells were stained with ethidium bromide and analysed by fluorescence microscopy. Necrotic or apoptotic cells had normal/condensed nuclei that were brightly stained with ethidium bromide and appeared red (white Selleckchem Lonafarnib arrows). In summary, these findings indicate that K. pneumoniae alters host cell viability in a process dependent on the presence of CPS. Correlation between K. pneumoniae-induced cell cytotoxicity
and virulence It is well known that CPS is essential for K. pneumoniae-induced pneumonia  and we have established here that Klebsiella-induced cytotoxicity depends on the presence of CPS. We sought then to determine whether induction of cytotoxicity is sufficient for K. pneumoniae virulence using an intranasal model of infection. As an infection marker, we determined the bacterial loads in lung, liver and spleen for K. pneumoniae strains 52145, 43816, 1850. Strain 52145 successfully infected mouse lungs (Fig. 4A and 4B, left) and disseminated to liver (Fig. 4A and 4B, middle) and spleen (Fig. 4A and 4B, right). No decrease in the bacterial load, which was higher in lung than in liver and spleen, was observed in any organ at 72 h post-infection.