Raman Monitoring of Staphylococcus aureus Osteomyelitis: Microbial Pathogenesis and Bone Immune Response

<i>Staphylococcus aureus</i> is the most common pathogen causing osteomyelitis, a hardly recoverable bone infection that generates significant burden to patients. Osteomyelitis mouse models have long and successfully served to provide phenomenological insights into both pathogenesis and host response. However, direct in situ monitoring of bone microbial pathogenesis and immune response at the cellular level is still conspicuously missing in the published literature. Here, we update a standard pyogenic osteomyelitis in Wistar rat model, in order to investigate bacterial localization and immune response in osteomyelitis of rat tibia upon adding in situ analyses by spectrally resolved Raman spectroscopy. Raman experiments were performed one and five weeks post infections upon increasing the initial dose of bacterial inoculation in rat tibia. Label-free in situ Raman spectroscopy clearly revealed the presence of <i>Staphylococcus aureus</i> through exploiting peculiar signals from characteristic carotenoid staphyloxanthin molecules. Data were collected as a function of both initial bacteria inoculation dose and location along the tibia. Such strong Raman signals, which relate to single and double bonds in the carbon chain backbone of carotenoids, served as efficient bacterial markers even at low levels of infection. We could also detect strong Raman signals from cytochrome <i>c</i> (and its oxidized form) from bone cells in response to infection and inflammatory paths. Although initial inoculation was restricted to a single location close to the medial condyle, bacteria spread along the entire bone down to the medial malleolus, independent of initial infection dose. Raman spectroscopic characterizations comprehensively and quantitatively revealed the metabolic state of bacteria through specific spectroscopic biomarkers linked to the length of staphyloxanthin carbon chain backbone. Moreover, the physiological response of eukaryotic cells could be quantified through monitoring the level of oxidation of mitochondrial cytochrome <i>c</i>, which featured the relative intensity of the 1644 cm^-1 signal peculiar to the oxidized molecules with respect to its pyrrole ring-breathing signal at 750 cm^-1, according to the previously published literature. In conclusion, we present here a novel Raman spectroscopic approach indexing bacterial concentration and immune response in bone tissue. This new approach enables locating and characterizing in situ bone infections, inflammatory host tissue reactions, and bacterial resistance/adaptation.