Table 1_A methodological approach to improve extracellular staphylococcal clearance in internalization assays using bone-associated host-cell models of orthopedic infections.docx

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.</p>