Survival in Macrophages Figure 2. S. aureus infected macrophages retained their bactericidal functions. Control hMDMs, and cells 4 days post infection with S. aureus strain Newman, were stimulated to phagocytose either live bacteria at a MOI of 1:25 (1.256107 CFU) (panel A) or latex beads (panel B). Generation of reactive oxygen species (ROS) determined as the level of the mean fluorescence intensity (MFI) was measured at various time intervals. The data shown is representative of at least three separate experiments, performed in triplicate, using hMDMs derived from different donors. doi:10.1371/journal.pone.0001409.g002 infected with strains Newman and ATCC 25923 at MOIs of 25 or 50 was only slightly above that of the control. At a MOI of 100 both S. aureus strains caused a significant increase in LDH level at 2 h which returned to basal level at 24 h. Apparently, rapid necrotic lysis of a relatively small subset of macrophages occurred, with the majority of cells surviving uptake, as confirmed by microscopic inspection of the hMDM cultures (data not shown). Indeed, the quantitative counts of total cell numbers indicated the significant loss of cells (5–10%) only after the first day postphagocytosis, at MOI = 25 and 50. The loss of cells was higher at MOI = 100, correlating well with the recorded cytotoxicity (Fig. 1D). Based on these results all further experiments were performed at a MOI of 25. The bactericidal functions of infected macrophages are preserved That the metabolic activity of the infected macrophages was maintained suggested that the bactericidal functions of these cells was also undisturbed. In order to verify this assertion we compared the respiratory burst response of control and infected hMDMs after the phagocytosis of latex beads and live bacteria. As shown in Fig. 2 the kinetics of increased mean fluorescence intensity (MFI), which is a measure of the production of reactive oxygen species (ROS) by cells, was very similar during phagocytosis of both live bacteria (Fig. 2A) and latex beads (Fig. 2B). However, at each time interval the infected hMDMs produced at least twice as much ROS as control cells that took up latex beads or live bacterial cells. Thus, it is clear that infection with S. aureus not only does not suppress antibacterial activity of macrophages, but on the contrary, infected cells were primed for a very vigorous response to phagocytic stimuli. S. aureus persists inside macrophages until the cells lyse Cultured hMDMs engulfed S. aureus strain Newman efficiently, with the majority of bacteria being internalized by 2 h. To determine the intracellular survival/killing rate of S. aureus cells the infected hMDMs were lysed at specific time points post- phagocytosis and plated onto TSA. The intracellular survival of S. aureus was dependent on the MOI. At MOI = 5 or 10 all of the intracellular bacteria were killed by 3 days postphagocytosis (data not shown). Conversely, at MOI = 25 the number of viable intracellular staphylococci decreased gradually between day 1 and day 5 by approximately five orders of magnitude compared to day 0 (Fig. 3A). Significantly, the conditioned medium of the infected cultures was sterile throughout. This suggests that hMDM effectively killed the internalized S. aureus, and the surviving bacteria did not escape intracellular confinement. Nevertheless, at higher MOI macrophages could not completely eradicate the bacteria, since an explosion of bacterial growth occurred on day 6, with similarly large numbers of S. aureus free in the medium and apparently adherent to cell corpses. At this time all macrophages that had appeared healthy on the preceding day had lost their plasma membrane integrity. Significantly, the same kinetic of S. aureus intracellular survival was observed in hMDMs cultivated for both 1 and 2 weeks, eliminating the possibility of macrophage aging triggering bacterial release (data not shown). A burst of live S. aureus in the conditioned media may indicate that the bacteria either suddenly proliferated intracellularly, with large numbers of bacterial cells being released into the media after hMDM lysis, or that a small number of surviving intracellular bacteria escaped intracellular confinement and multiplied vigor- ously in the media, killing the hMDMs in the process. To discern between these two vastly different possibilities we have simulta- neously monitored live bacteria in media, and also cell membrane damage. In four independent experiments we found that proliferation of S. aureus in the conditioned medium clearly preceded any significant cell damage as measured by the release of LDH (Fig. 3B, left panel). This result was fully corroborated by staining cells with propidium, showing that plasma membrane permeabilization was secondary to the robust growth of S. aureus in the media (Fig. 3B, right panel). Collectively these data, together with kinetics of intracellular survival (Fig. 3A), argue that S. aureus persists inside hMDM in continuously decreasing numbers until the exhausted cells allow bacteria to escape into the conditioned medium, where they proliferate. Experiments monitoring the survival of S. aureus strain Newman inside hMDMs were repeated a total of 76 times with cells PLoS ONE | www.plosone.org 4 January 2008 | Issue 1 | e1409