Session I - Combined Session (International Society for Fracture Repair)

Intracellular Staphylococcus Aureus: A Proposed Mechanism for the Indolence of Osteomyelitis

J. Kent Ellington, MS; Mitchel B. Harris, MD; Lawrence X. Webb, MD; Beth P. Smith, PhD; Thomas Smith, PhD; Kim Tan, PhD; Michael C. Hudson, PhD; Wake Forest University School of Medicine, Winston-Salem, North Carolina, USA

Purpose/Hypothesis: Staphylococcus aureus is the bacterial pathogen responsible for nearly 80% of all cases of human osteomyelitis. It has previously been demonstrated that this bacterium can invade and persist within the intracellular environment of the human osteoblast. To date, the fate of intracellular S. aureus after host cell (osteoblast) death has not been established. The hypothesis of this study was that intracellular S. aureus will survive the death of their host osteoblasts and retain the capacity to invade other osteoblasts.

Methods: Normal human osteoblasts were purchased and propagated in cell media. After cells reached 80% confluence (5 to 9 days), they were washed in growth media and seeded into six- well plates. The osteoblasts were then maintained in growth medium. S. aureus was grown in tryptic soy broth, harvested by centrifugation, and resuspended. The osteoblast containers were then infected at a multiplicity of infection at 250:1 with S. aureus. Following incubation (45 minutes at 37° C), the cultures were washed, and gentamicin was added to kill the extracellular S. aureus (gentamicin cannot penetrate the eukaryotic cells). Two methods were subsequently used to release the intracellular microorganisms.

First, detergent-based cell lysis techniques were used as the osteoblast cultures were washed, lysed (0.1% Triton X-100), and then transferred to tryptic soy agar (TSA) plates for overnight incubation and bacterial enumeration. The second method used 0.025% trypsin-0.01% edetic acid instead of detergent. The trypsinized cultures were then transferred to TSA plates (allowed to die because of a lack of nutrients) for overnight incubation and bacterial enumeration. Following both of these methodologies, the previously intracellular bacteria were quantified at time zero (45 minutes), 24, and 48 hours after infection.

These bacteria were then harvested, grown overnight, and used to infect other sterile osteoblast cultures. Following infection, the osteoblast cultures were incubated in gentamicin to eliminate extracellular bacteria. The new osteoblast cultures were lysed or trypsinized and plated on TSA plates for bacterial enumeration.

In an effort to determine whether the length of time the bacteria had been intracellular had an effect on their subsequent ability to re-invade osteoblasts, cultures infected with the bacteria recovered at 45 minutes and 24 hours were compared to control infected osteoblast cultures (infected with S. aureus that had not previously been intracellular).

Results: Dying and dead osteoblast cultures release viable S. aureus equivalent to those lysed by detergent. The respective colony counts demonstrated no statistically significant differences at times 0, 24, or 48 hours. Also, there was no statistical difference in the number of recovered S. aureus between the control group and the previously intracellular S. aureus cultures.

Discussion/Conclusion: Human osteoblasts whose membranes are lysed and those that undergo cell death are capable of releasing viable S. aureus. These bacteria (once released from their intracellular environment) are capable of re-infecting additional cultures of normal human osteoblasts. The results of this study suggest a potential cellular mechanism responsible for the indolent and evanescent nature of bone infection.