Clinical Spectroscopic Diagnostics

Introduction: One possible long-term consequence of sepsis is hematogenous osteomyelitis, i.e. the spread of bacteria from the blood stream to the bone. The clinical picture of this disease is swelling and deformation of the bone, pain, redness, fever and weakness. The most common underlying pathogen is the Gram positive Staphylococcus aureus, which is one of the most frequently found pathogen in hospital. These bacteria can invade cells, adopt an intracellular life cycle and persist in the bone for years before causing symptoms. Their intracellular appearance is especially difficult to treat with antibiotics because of a reduced metabolic rate and the challenge to target the drugs into the host cells. Objectives: In this study we aim to define the localization of bacteria within the bone in an established mouse model [1] during the acute and chronic state of hematogenous osteomyelitis. Methods: Mice were infected via a lateral tail vein with S. aureus and pathological changes in behavior and by X-ray were recorded. After 1 week (acute phase) or 6 weeks (chronic phase) of infection mice were sacrificed, bones were isolated and fixed in 4% paraformaldehyde. After decalcification and cryosectioning, the slices were immunolabelled using fluorescent secondary antibodies to visualize S. aureus and osteocalcin, a marker for bone formation. Cellular structures were counterstained using DAPI (nuclei) and I555-phalloidin (actin fibres). For histopathological comparison hematoxilin & eosin (HE) staining was applied to some slices. Images were taken using confocal or two photon microscopy (Zeiss CLSM 780 Meta). Results: An activation of the bone marrow was detected by HE staining and residing pathogens could be identified in the immunolabelled slices from both the acute and the chronic phase of hematogenous osteomyelitis. Pathogen load was high but not restricted to sites of bone deformation identified by X-ray in the chronic osteomyelitis model. In the acute phase no deformations were detectable by X-ray, still we found numerous bacteria in these bones. Remarkably, sites of bacteria detection were quite diverse ranging from the hard bone, bone marrow to associated connective tissue and attached muscle fibres in both the acute and the chronic phase. Intracellular localization could be proven for both the acute and the chronic phase. Colocalization with osteocalcin, a marker of bone formation, which is produced by osteoblasts and secreted into the extracellular matrix of bone, was detected for some of the bacteria, especially in the acute phase of osteomyelitis. Conclusions: S. aureus can be found intracellularly and residing at multiple different locations in bone, both in the acute and in the chronic phase of osteomyelitis. The infection leads to an activation of the bone marrow and S. aureus can be found partly in areas of bone formation. Treatment strategies should therefore have a broad drug distribution and cover the different sites of bacterial residence. Reference: [1] S.A. Horst et al. A novel mouse model of Staphylococcus aureus chronic osteomyelitis that closely mimics the human infection: an integrated view of disease pathogenesis. Am J Pathol, 181, 1206–1124 (2012). Acknowledgement: Financial support from the BMBF via the CSCC (FKZ01EO1502) and DGF (JBIL, FKZ PO 633/29-1, BA 1601/10-1) and Leibniz ScienceCampus InfectoOptics (SAS-2015-HKI-LWC) is highly acknowledged.