HYPOXIA-DRIVEN SDHB-MEDIATED ANTIBIOTIC TOLERANCE IMPAIRS PROPHYLACTIC ANTIBIOTICS AGAINST MRSA IN A MOUSE PROSTHETIC JOINT INFECTION MODEL

Aim To determine if antibiotic tolerance impairs prophylactic antibiotics against Methicillin Resistant Staphylococcus aureus (MRSA) in orthopedic surgery Method We evaluated MRSA strains for resistance to vancomycin and ceftobiprole, using broth microdilution (BMD). We evaluated antibiotic tolerance using the Tolerance Disk Test (TD-test). Whole genome sequencing was used to identify mutations underlying tolerance. Tolerant and non-tolerant strains were subjected to a mouse prosthetic joint infection (PJI) model following a single dose of vancomycin given 60 minutes prior to surgery and infection. Bacterial load was evaluated by in vivo bioluminescent imaging for 30 days and by ex vivo enumeration of tissue bacterial colony forming units (CFUs) after completion of the experiment. Tolerance was further compared between wild type, mutant and deletion strains using TD-test and kill curve analysis in normal and low oxygen tension (normoxia and hypoxia, respectively). Results We tested 17 osteogenic MRSA strains of which none were resistant to vancomycin. Two strains, however, were highly tolerant to vancomycin (Figure 1A-B). Whole genome sequencing revealed a mutation in the sdhB gene, which is part of the succinate dehydrogenase (SDH) enzyme complex and essential for energy production and respiration. In a mouse prosthetic joint infection model, the mutant ( sdhB N51S ) led to a ~100-fold higher bacterial load post-antibiotic treatment compared to wild-type (p=0.0286; Figure 1C-E). Deletion of sdhB significantly reduced antibiotic tolerance for both vancomycin and ceftobiprole (Figure 1F-H). Hypoxia increased antibiotic tolerance in wild-type and sdhB N51S mutant strain but did not impact the D sdhB deletion strain (Figure 1I-K). Conclusions A mutation in sdhB , a key regulator of metabolism and energy production in S. aureus , is associated with hypoxia-driven antibiotic tolerance and reduces the efficacy of prophylactic antibiotics. Interestingly, deletion of the gene renders the bacteria highly susceptible to antibiotics, highlighting this mechanism as a potential therapeutic target. For any figures or tables, please contact the authors directly.