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Staphylococcus aureus is responsible for 80% of human osteomyelitis. It can invade and persist within osteoblasts. Antibiotic resistant strains of S. aureus make successful treatment of osteomyelitis difficult. Null Hypothesis: antibiotic sensitivities of S. aureus do not change after exposure to the osteoblast intracellular environment. Human and mouse osteoblast cultures were infected and S. aureus cells were allowed to invade. Following times 0, 12, 24, and 48 h (AE the addition of erythromycin, clindamycin, and rifampin at times 0 or 12 h), the osteoblasts were lysed and intracellular bacteria enumerated. Transmission electron microscopy was performed on extracellular and intracellular S. aureus cells. In mouse osteoblasts, administration of bacteriostatic antibiotics at time 0 prevented the increase in intracellular S. aureus. If the antibiotics were delayed 12 h, this did not occur. When rifampin (bactericidal) was introduced at time 0 to human and mouse osteoblasts, there was a significant decrease in number of intracellular S. aureus within osteoblasts compared to control. If rifampin was delayed 12 h, this did not occur. Significant time-dependent S. aureus structural changes were observed after exposure to the osteoblast intracellular environment. These studies demonstrate that once S. aureus is established intracellularly for 12 h, the bacteria are less sensitive to antibiotics capable of eukaryotic cell penetration (statistically significant). These antibiotic sensitivity changes could be due in part to the observed structural changes. This leads to the rejection of our null hypotheses that the antibiotic sensitivities of S. aureus are unaltered by their location. ß

Staphylococcus aureus is the principal causative agent of the inflammatory bone disease osteomyelitis. Unfortunately, the pathogenesis of this often chronic infection is poorly understood and is complicated by the recent observation that bone-forming osteoblasts can harbor S. aureus. Such an infection presents a significant challenge for the host immune response, because osteoblasts are not known to initiate protective cell-mediated immune responses. Cultured mouse and human osteoblasts infected with S. aureus were found to express high levels of interleukin (IL)-6 and IL-12p75, on the basis of complementary investigations demonstrating both S. aureus-induced up-regulation of expression of IL-6 and IL-12p40 mRNA and secretion of IL-6 and IL-12p75 by these cells. Additionally, a quantitative bioassay demonstrated that IL-12p75 secreted after infection was biologically active. These studies are the first to demonstrate induced IL-12p75 expression by osteoblasts and suggest a previously unrecognized role for osteoblasts in initiating immune responses after S. aureus infection.

Staphylococcus aureus invades osteoblasts and is the primary cause of osteomyelitis. This study examined the ability of S. aureus to induce apoptosis in a mouse osteoblast cell line. The presence of intracellular S. aureus was demonstrated by transmission electron microscopy. Light microscopy was utilized to examine morphological changes in the osteoblasts following killing of extracellular bacteria. Cell rounding was observed, and dark centers due to condensation of chromatin were noted in cells in infected osteoblast cultures. DNA was isolated from infected osteoblast cultures, and electrophoresis revealed the laddering effect characteristic of cells undergoing apoptosis. Additionally, an in situ cell death detection assay was utilized to label apoptosis-induced DNA strand breaks. Apoptotic nuclei were present, providing further evidence that S. aureus induces apoptosis in osteoblasts.