Session I - Basic Science

Antibiotic Resistance in Staphylococcal Osteomyelitis

J. Kent Ellington, MS; Mitchel B. Harris, MD; Lawrence X. Webb, MD; Beth P. Smith, PhD; Tom Smith, PhD; Michael D. Hudson, PhD; Wake Forest University Health Sciences Center, Winston-Salem, North Carolina, USA

Purpose: Staphylococcus aureus is capable of invading and persisting within osteoblasts. Antibiotic resistant strains of S. aureus are now commonplace and make successful treatment of osteomyelitis difficult. Hypothesis: Antibiotic sensitivity of S. aureus does not change after exposure to the osteoblast intracellular environment.

Methods: Human osteoblast cultures were grown to maturation. Prior to infection and invasion of the osteoblasts, the minimum inhibitory concentration of both bacteriostatic (clindamycin, erythromycin) and bacteriocidal (rifampin) antibiotics capable of penetrating and accumulating within osteoblasts was calculated for a human osteomyelitic S. aureus isolate. Osteoblast cultures were then infected and the bacteria were allowed to invade. At times 0, 12, 24, and 48 hours after invasion, the osteoblasts were lysed and the intracellular organisms enumerated. At times 0 or 12 hours after invasion, antibiotics capable of penetrating the osteoblast cell membrane were added. At times 12, 24, and 48 hours after invasion, the osteoblasts were lysed and the intracellular S. aureus enumerated. In addition, both extracellular and previously intracellular S. aureus cells were examined with transmission electron microscopy.

Results: Control assays demonstrated no significant increase in the number of intracellular S. aureus recovered from human osteoblasts 12, 24, and 48 hours after invasion. Thus, the introduction of bacteriostatic drugs capable of penetrating the human osteoblasts had no apparent effect on the numbers of intracellular S. aureus. Rifampin (bacteriocidal) was effective in killing intracellular S. aureus when administered at time zero. However, when administered to the osteoblast cultures 12 hours after invasion, the bacteriocidal capacity was greatly reduced, as the numbers of recovered intracellular S. aureus closely approximated the control assays. Analysis of the transmission electron microscope images revealed the presence of structural changes to S. aureus when the bacteria were exposed to the osteoblast intracellular environment. A new bacterial "capsular material" was observed on the surface of S. aureus that was previously intracellular.

Conclusion: The results of these studies demonstrate that once S. aureus are established intracellularly within the human osteoblasts for 12 hours, they become resistant to bacteriocidal antibiotics capable of eukaryotic cell penetration. The effect of the introduction of bacteriostatic drugs that are capable of cell membrane penetration was negligible, because we did not observe an increase in the intracellular numbers of S. aureus. The increased resistance to bacteriocidal drugs could be related in part to the structural changes observed, such as the formation of a capsule. This leads to the rejection of our null hypothesis that the antibiotic sensitivities of S. aureus are unaltered by their intracellular location. Additionally, there appears to be no beneficial effect of bacteriostatic drugs in the eradication of intracellular bacteria.

Significance: Antibiotic resistance occurs through several proposed pathways. By better understanding the pathogenesis of S. aureus infection, particularly its capacity to invade and persist within human osteoblasts and its increased resistance to antibiotics once in the intracellular milieu, our ability to treat patients with osteomyelitis should improve significantly. The ability of S. aureus to invade osteoblasts protects it from the host's humoral immune response and conventional antibiotic therapy. This may help explain the chronic and unpredictable nature of osteomyelitis.