Uncovering the mechanisms of clinically relevant altered antibiotic responses of Staphylococcus aureus under wound infection-mimetic conditions.

Standard in vitro antimicrobial susceptibility testing (AST) using Mueller-Hinton broth (MHB) does not reflect infection-site conditions, and its results often do not correlate with therapeutic outcomes. We compared the antibiotic susceptibility of methicillin-resistant Staphylococcus aureus (MRSA), a common chronic wound pathogen, in simulated wound fluid (SWF) resembling wound exudate versus MHB, revealing discordant AST results across six of nine tested antibiotic classes. The most significant were 128-fold increased resistance to tetracyclines and 256-fold sensitization to β-lactams in SWF. Tetracycline resistance was mediated by MntC, an extracellular manganese-binding protein, whereas β-lactam sensitization was driven by cell envelope remodeling in SWF. Galleria mellonella wound infection results matched the SWF susceptibility phenotypes, suggesting SWF better predicts in vivo wound infection therapeutic outcomes. These comprehensive phenotypic and mechanistic insights into MRSA antibiotic responses under wound-infection-mimetic conditions, with direct in vivo validation, uncover a potential antibiotic adjuvant target and may guide improved MRSA wound-infection therapy.