Effective Detection of Staphylococcal Infections in Human Bone Tissue using Combined Raman Microscopy and Micro-Computed Tomography

Richard Andreas Lindtner¹Lukas Kampik¹Larissa Noack¹David Putzer¹Rohit Arora¹Débora Coraça-Huber¹Michael Schirmer¹Gerald Degenhart¹Michaela Lackner¹Jürgen Popp²Jovan Badzoka³Christoph Kappacher³Christian Wolfgang Huck³Johannes Dominikus Pallua^1*

• ¹Innsbruck Medical University, Innsbruck, Tyrol, Austria
• ²Leibniz Institute of Photonic Technology (IPHT), Jena, Thuringia, Germany
• ³University of Innsbruck, Innsbruck, Tyrol, Austria

Bone infections caused by Staphylococcus aureus and Staphylococcus epidermidis are serious complications in orthopedic surgery. These infections commonly occur in joint replacements, fracture management, and bone grafting procedures. Rapid and accurate pathogen-specific diagnostic methods are urgently needed to support early clinical decisions. Current culture-based methods are slow and delay effective treatment. This study evaluated the diagnostic value of combining Raman microscopy with high-resolution micro-computed tomography (micro-CT). Human bone samples, either uninfected or inoculated with S. aureus or S. epidermidis, were analyzed. Raman spectroscopy detected distinct spectral changes in inoculated bones, including reduced intensity of phosphate (v₁PO₄³⁻), Amide III, and CH₂ deformation bands. A single principal component explained 96-98% of the variance in these infection-related markers. Specifically, the v₁PO₄³⁻ and CH₂ deformation bands effectively differentiated between S. aureus and S. epidermidis infections, capturing 99-100% variance. Micro-CT analysis showed significant structural changes in inoculated bones. Trabecular volume, number, and spacing were particularly affected. Among these, VOX-BV/TV and Mean1 best differentiated between S. aureus and S. epidermidis infections (both p < 0.0001). Support vector machine (SVM) classification repeated stratified k-folg cross-validation accurately detected inoculation status. Combining Raman and micro-CT features yielded moderately improved classification performance in pathogen-specific discrimination. These findings demonstrate that combining molecular (Raman spectroscopy) and structural (micro-CT) methods allows rapid, nondestructive diagnosis of bone infections. This multimodal approach may improve diagnostic precision, supports timely clinical decisions, and ultimately improves patient outcomes in orthopedic and trauma surgery.