It seems that actin cytoskeleton formation played a major role in S. aureus internalization since cytochalasin D, an inhibitor that disrupts actin filament polymerization, Stem Cells inhibitor led to up to 50% inhibition of S. aureus internalization. This finding was consistent with other studies [23,32] where actin filament was determined to play a dominant role in S. aureus internalization. One limitation of this study was that MOIs lower than 100:1 were not investigated and the higher MOI may only apply to infected tissues where numerous bacteria may exist. Future studies may need to consider lower MOIs. Note that our data confirmed that gentamicin treatment

was effective in eliminating extracellular S. aureus and the post-infection CFU was indeed from live intracellular S. aureus. Gentamicin treatment is commonly used to eliminate extracellular bacteria [21,32], but such a procedure lacks direct confirmation of live intracellular bacteria. In this study, besides culturing

the washing media collected after gentamicin treatment, the dual staining approach combined with confocal microscopy presented direct evidence that no live extracellular S. aureus was observed after the click here gentamicin treatment (Figure 3C). S. aureus has been thought to be a frequent cause for several types of chronic and recurrent infections including osteomyelitis, endovascular diseases, and chronic lung infections [33], and S.

aureus infections have been reported in clinical cases to persist asymptomatically with relapses occurring months or even years after apparent antimicrobial cure of the infections [34,35]. In these cases, S. aureus may have protected itself and escaped antibiotics and immune response of the host by “hiding” intracellularly and establishing a latent bacterial reservoir. This was supported by our observation that S. aureus could survive intracellularly for CYTH4 up to 5 and 7 days, respectively, within macrophages and osteoblasts (Figure 2). As a phagocytic cell, macrophages were obviously more effective than osteoblasts at not only phagocytizing but also destroying the intracellular bacteria. This was supported by our data showing that S. aureus infection not only significantly increased the H2O2 levels in macrophages at 1 h infection but also significantly increased the O. 2 − levels in macrophages at infection times of 0.5 and 1 h. In contrast, S. aureus infection induced significantly higher levels of H2O2 in osteoblasts at 0.5 and 1 h infection but did not induce significant changes in O. 2 − levels in osteoblasts. As a result, a significantly higher number of intracellular CFUs was found in macrophages immediately after infection while significantly less intracellular S.