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

## TL;DR

This study examines how Staphylococcus aureus bacteria from joint infections resist antibiotics, especially in biofilms, and how this resistance affects treatment outcomes.

## Contribution

The study identifies specific genes linked to antibiotic tolerance in S. aureus biofilms and shows that rifampin tolerance correlates with treatment failure.

## Key findings

- Biofilm formation significantly increases antibiotic tolerance, especially to rifampin.
- Rifampin tolerance in biofilms is strongly associated with poor clinical outcomes.
- Genes like sspA, leuS, prs, and pgm are linked to antibiotic tolerance in S. aureus isolates.

## Abstract

Background: Antibiotic tolerance in Staphylococcus aureus biofilms poses a major clinical challenge in prosthetic joint infections (PJIs). This study aimed to characterize the antibiotic tolerance of clinical S. aureus 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. Methods: Twelve clinical S. aureus 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. Results: 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 (p = 0.01). Rifampin tolerance in biofilm-embedded bacteria was significantly higher in isolates from patients with treatment failure (p < 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 sspA and leuS in biofilm isolates, and between rifampin tolerance and prs and pgm. Conclusions: In this study, clinical S. aureus 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.