A methodological approach to improve extracellular staphylococcal clearance in internalization assays using...

Orthopedic implant-related infections are a major cause of prosthesis failure and are associated with biofilm formation, antimicrobial tolerance, and bacterial persistence. Bacterial internalization into bone-associated host cells may also contribute to chronic or recurrent infection. However, non-standardized extracellular bacterial clearance, multiplicity-of-infection definition, and data normalization limit assay interpretation and comparability. This study aimed to establish a stepwise protocol with a structured strategy to obtain the extracellular staphylococcal clearance allowing for the unbiased quantification of host-cell internalized bacteria through identification and prevention of major methodological pitfalls. Cell-associated bacteria from host-cell lysates were a secondary endpoint. Methicillin-resistant S. aureus USA300 and S. epidermidis RP62A were used as model pathogens, whereas MG-63 osteosarcoma cells and human bone-marrow mesenchymal stromal cells served as host-cell models. Vancomycin and lysostaphin were first evaluated in the absence of host cells with bacterial inocula simulating multiplicities of infection (MOI) of 50 and 100. Clearance was more effective and consistent at the lower inoculum. Accordingly, 25 µg/mL lysostaphin for 30 min and 100 µg/mL vancomycin for 60 min were tested individually or sequentially after a 2-h infection at an MOI of 50. Vancomycin alone resulted in the highest residual extracellular bacterial recovery, precluding interpretation of CFU recovered from cell lysates. Lysostaphin-containing protocols reduced viable CFU recovered from the final treatment medium to near or below the assay detection limit. S. epidermidis recovery from cell lysates was negligible after lysostaphin-containing treatments. Phase-contrast microscopy monitored host-cell morphology, whereas confocal microscopy supported intracellular bacterial localization. These findings indicate that antimicrobial treatments should be validated within the bacteria-host-cell system rather than selected solely from cell-free assays. The framework integrates strain-specific antimicrobial testing, inoculum selection, verification of extracellular clearance, normalization to host-cell number, and microscopy-based assessment, improving the rigor and comparability of in vitro studies of staphylococcal interactions with bone-associated cells.