Aim
Discriminative animal models in which bacterial virulence factors and the impact on the host can be studied are desirable. Therefore, a porcine model of haematogenous osteomyelitis based on intraarterial inoculation of Staphylococcus aureus was developed. In the model, the pathology of osteomyelitis and the presence of bacteria and their microenvironment were elucidated.

Methods
Five 12 weeks old female pigs (BW 25 kg) were separated into two groups. Three animals were inoculated into the right femoral artery with S. aureus strain S54F9 and the control group of two animals was sham inoculated with saline. Following euthanasia, 11 days after inoculation the animals were necropsied and macroscopic bone lesions were recorded. Tissue was sampled from the lesions and following fixation in formalin and embedding in paraffin used for immunohistochemistry and peptide nuclei acid in situ hybridisation (PNA FISH) to identify S. aureus in situ.

Results
Macroscopic osteomyelitis lesions were observed in the distal metaphyseal area of the right femoral bone and in the proximal metaphyseal area of the right tibial bone of all infected animals. The lesions were made up by a purulent inflammatory material which contained sequestered trabecular bone. Sometimes the inflammation penetrated into and through the cortical bone and periosteum leading to the formation of soft tissue abscesses. Dense, soft and pink granulation tissue surrounded the lesions. S. aureus bacteria were identified in the bone abscesses by immunohistochemistry and PNA FISH as single organisms or in colonies. PNA FISH showed that the bacterial colonies were surrounded by an opaque matrix comparable to biofilm.

Conclusion
The presence of granulation tissue and biofilm formation within lesions showed that a chronic and persistent infection was established. The formation of a biofilm by S. aureus affects the pathology, since the consistent release of planktonic bacteria induces an ongoing inflammatory reaction. The presented discriminative porcine model presents an attractive model for studying the nature and role of biofilm formation in vivo and how to diagnose and treat these infections successfully.