Shaking Angles Determine Staphylococcus aureus Virulence Via Oxygen-Glucose Metabolic Crosstalk in Vitro

Culture tubes positioned at 45° or 90° angles on shakers are routinely utilized for in vitro bacterial growth, but their influence on bacterial virulence, particularly in Staphylococcus aureus, remains poorly characterized. This study systematically quantified how these shaking angles modulate S. aureus virulence through cytotoxicity assays, virulence factor quantification, and oxidative stress/neutrophil function analyses. This work demonstrated that high-oxygen conditions (shaking at 45° angle) drastically suppressed S. aureus cytotoxicity against human erythrocytes and neutrophils, correlating with the downregulation of multiple core virulence factors. Glucose addition reversed the suppressed hemolytic activity under high-oxygen conditions, which was inhibited by glycolytic inhibitors 2-deoxy-D-glucose (targeting hexokinase) and oxalate (targeting pyruvate kinase), confirming glycolysis-dependent virulence regulation. Conversely, low-oxygen conditions (shaking at 90° angle) supplemented with glucose increased lactate production while reducing hemolysis. In glucose-rich medium, inhibition of lactate dehydrogenase by oxamate reduced lactate levels but enhanced hemolysis under low-oxygen conditions, directly linking lactate metabolism to S. aureus cytotoxicity. Additionally, low-oxygen conditions enhanced S. aureus oxidative stress resistance and neutrophil migration, and impaired neutrophil antimicrobial ability. Collectively, these findings reveal that glucose-oxygen crosstalk determines S. aureus virulence and neutrophil responses, highlighting the critical importance of selecting appropriate bacterial culture methods when studying in vitro virulence.