A Study of Antibiotic Tolerance to Levofloxacin and Rifampin in Staphylococcus aureus Isolates Causing Prosthetic Joint Infections: Clinical Relevance and Treatment Challenges

<b>Background</b>: Antibiotic tolerance in <i>Staphylococcus aureus</i> biofilms poses a major clinical challenge in prosthetic joint infections (PJIs). This study aimed to characterize the antibiotic tolerance of clinical <i>S. aureus</i> isolates recovered from cases of PJI under different stress conditions, including biofilm formation and antibiotic exposure. The correlation between tolerance level, the presence of specific tolerance-related genes, and clinical outcome was also evaluated. <b>Methods</b>: Twelve clinical <i>S. aureus</i> isolates were analyzed. To assess tolerance, the TDtest was used on exponentially growing bacteria, 48 h biofilms, and biofilms treated with levofloxacin and/or rifampin. Whole-genome sequencing was performed to identify tolerance-associated genes. <b>Results</b>: All isolates were phenotypically susceptible to rifampin and levofloxacin. Although all strains exhibited basal tolerance levels, biofilm formation led to heightened antibiotic tolerance, particularly those treated with rifampin as compared to levofloxacin: 29.5 vs. 17 (<i>p</i> = 0.01). Rifampin tolerance in biofilm-embedded bacteria was significantly higher in isolates from patients with treatment failure (<i>p</i> < 0.0001). Levofloxacin tolerance showed no significant association with clinical outcomes. There was no correlation between reduction in biofilm bacterial burden after treatment and tolerance levels. Genomic analysis identified associations between higher levofloxacin tolerance and the presence of <i>sspA</i> and <i>leuS</i> in biofilm isolates, and between rifampin tolerance and <i>prs</i> and <i>pgm</i>. <b>Conclusions</b>: In this study, clinical <i>S. aureus</i> strains isolated from prosthetic joint infections exhibited considerable inter-strain variability in antibiotic tolerance, particularly under biofilm conditions. Elevated rifampin tolerance in biofilm-embedded bacteria was associated with poor clinical outcomes, underscoring the need for tolerance assessment beyond standard susceptibility testing.