Figure 3.

Direct evidence of reinfection using real-time confocal imaging. Overnight cultures of Salmonella Typhimurium SL1344 expressing either GFP (G) or ds-Red (R) were added to RAW264.7 macrophages. Cells were infected with R at an MOI of 50 for 15 min then, after three washes to remove any residual bacteria, an infected macrophage was identified and G was added at an MOI of 100. The infected cell was observed in multiple planes (Z-stacks) over time to confirm the presence or the absence of intracellular bacteria. In this figure, a range of confocal Z-stacks is merged together, and overlaid to the phase-contrast image obtained in transmission. The four images are from sequential timepoints: 181 s, 284 s, 321 s and 439 s. The first two images from 181 s and 284 s show the presence of the R bacterium within the macrophage. The third image at 321 s shows an extracellular G bacterium associating with the macrophage. In the final image at 439 s, the G bacterium is within the macrophage, along with the R bacterium, demonstrating a second infection event. Movies showing the Z-stacks separately are available as electronic supplementary material.