Figure 3.

Evaluation of the resistance to phagocytosis of K. pneumoniae using D. discoideum as host cell model. (A) Extracellular viable bacteria count after different times of Klebsiella-amoeba co-incubation. At each indicated time, the mixed cultures were centrifuged at low rpm in order to sediment D. discoideum cells and intracellular bacteria, and the CFU remaining in the supernatant were titrated. The symbols represent the mean of three independent experiments and the error bars correspond to the standard deviation. At times 3 and 24 h, Kp RYC492 and Kp 700603 showed a significantly higher number of extracellular bacteria, compared with K. aerogenes (Two-way ANOVA, ^*p < 0.05, ^****p < 0.0001). (B) Confocal microscopy images representative of the experiment showed in (A). At each indicated time, an aliquot of the mixed cultures was directly mounted for microscopic observation (without centrifuging). D. discoideum cells are green and bacterial cells are red. Bright field and fluorescence images were blended in order to better appreciate the boundaries of the amoeba cells. Scale bar, 10 μm.