pH-dependent antibacterial activity of N-acetylcysteine against Staphylococcus aureus and metabolic alterations induced at neutral pH

N-acetylcysteine (NAC) is a mucolytic and antioxidant increasingly investigated as an antibacterial agent and antibiotic adjuvant, particularly against biofilm-associated infections. Despite being a weak organic acid, solution pH is rarely reported in the literature, and reported minimum inhibitory concentrations (MIC) for S. aureus are highly inconsistent across studies, varying by more than 50-fold. We systematically assessed the pH-dependence of NAC antibacterial activity and investigated how pH-neutral NAC perturbs bacterial metabolism using LC-MS based targeted metabolomics. Without pH-adjustment, NAC at 200 mM (pH 2.8) acted bactericidal against the S. aureus strains USA300 and Mu12, while pH-adjusted NAC solutions had no effect on bacterial growth, even at concentrations approaching its solubility limit. Based on LC-MS measurement, pH adjustment did not measurably degrade NAC but significantly increased dimerization ratios, suggesting that altered ionization state rather than degradation underlies the loss of antibacterial activity at neutral pH. Despite the absence of growth inhibition, pH-neutral NAC induced concentration-dependent metabolic alterations under both planktonic and biofilm conditions. Arginine, cysteine, glycolysis, and TCA cycle were the most affected pathways, with intracellular accumulation of arginine and cystine, together with increased lactic acid production. Our findings demonstrate that the antibacterial activity of NAC against S. aureus is driven by acidification, helping reconcile contradictory MIC reports in the literature. Additionally, pH-neutral NAC alters bacterial metabolism without impairing growth, highlighting its potential to modulate bacterial physiology independently of direct antibacterial activity.