Effective detection of staphylococcal infections in human bone tissue using combined raman microscopy and micro-computed tomography

Bone infections caused by <i>Staphylococcus aureus</i> and <i>Staphylococcus epidermidis</i> 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 <i>S. aureus</i> or <i>S. epidermidis</i>, were analyzed. Raman spectroscopy detected distinct spectral changes in inoculated bones, including reduced intensity of phosphate (v₁PO₄ ^3-), Amide III, and CH₂ deformation bands. A single principal component explained 96%-98% of the variance in these infection-related markers. Specifically, the v₁PO₄ ^3- and CH₂ deformation bands effectively differentiated between <i>S. aureus</i> and <i>S. epidermidis</i> 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 <i>S. aureus</i> and <i>S. epidermidis</i> 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, non-destructive 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.