Staphylococcus aureus is the leading pathogen in fracture-related infection (FRI). Virulence factors vary between different strains, which may have a decisive influence on the course of infection. Previous in vitro experiments, in vivo testing in wax moth larvae, and genomic analysis of S. aureus isolates from FRI identified a low- and high-virulent strain. These findings correlated with the acute course of FRI induced by the high-virulent pathogen, whereas the low-virulent strain caused a chronic FRI in its human host. However, the role of bacterial virulence in FRI is not completely understood. Therefore, the present study aimed to compare the identified high- and low-virulent S. aureus isolates in a murine FRI model.
Skeletally mature C57Bl/6N mice received a femoral osteotomy stabilized by titanium locking plates. FRI was established by inoculation of either high-virulent S. aureus EDCC 5458 or low-virulent S. aureus EDCC 5464 in the fracture gap. Mice were euthanized 4 and 14 days after surgery, respectively. Severity and progression of infection were assessed in terms of clinical presentation, quantitative bacteriology, semiquantitative histopathologic evaluation, and serum cytokine profile.
Quantitative bacteriological results 4 days after surgery revealed a higher bacterial load in soft tissue samples in high-virulent infected animals (p =0.026). Mice infected with the high-virulent strain also displayed higher rates of organ dissemination (24/36 organs in high-virulent, versus 5/36 organs in low-virulent infected animals; p <0.0001).
In the histopathological assessment, bacterial agglomerations at the fracture ends were present to a greater extent in the high-virulent cohort and barely detectable in low-virulent infected mice. In both cohorts, no bone healing was observed after 4 days. On day 14, bone healing at the fracture site was visible in low-virulent infected animals, whereas callus formation was observed in only one animal from the high-virulent infected cohort. Furthermore, osteonecrosis and osteolysis were increased in high-virulent infected animals. Regarding serum cytokines, innate immune markers were elevated in both groups at day 4. By day 14, a more pronounced proinflammatory response indicated by increased serum cytokine levels of IFN-γ, IL-1β, and IL-6 was observed in high-virulent infected animals.
The present study demonstrated distinct bacteriological and histopathological differences between two different virulent S. aureus strains previously shown to have different courses in human patients. While host physiology is often considered to have a major impact on the course of FRI, this study highlights the critical influence of the invading pathogen and its virulence characteristics.