Environmental and chemical modulation of Staphylococcus aureus Newman biofilm formation

Staphylococcus aureus biofilm formation enhances survival on host tissues and medical devices. This study tested how oxidative stress (H₂O₂), pH (5-9), NaCl (0-10%), and human serum (5-50%) affect the Newman strain biofilm and key genes (icaA, icaD, sarA). Biofilm was quantified by crystal violet assays and Lowry protein assay methods, and gene expression was measured by quantitative real-time PCR. Biofilm biomass was quantified using crystal violet staining and Lowry protein assays under various environmental conditions. Statistical significance was determined using ANOVA with post hoc analysis (p < 0.001). Hydrogen peroxide induced a dose-dependent reduction in biomass, with significant repression of icaA, icaD, and sarA expression at 3% H₂O₂ (≤ 22.8%, p < 0.001). Similarly, deviations from neutral pH markedly impaired biofilm formation, with acidic (pH 5) and alkaline (pH 9) conditions reducing biomass by 34.6% and 41.7%, respectively, accompanied by strong downregulation of biofilm-associated genes (p < 0.001). In contrast, NaCl exerted a biphasic effect: mild osmotic stress (1.25% and 5%) enhanced biofilm biomass (up to 154.2%) in the case of crystal violet assays and at 5% biomass increased to 130.8 ± 10.8*%; at 10%, it was 103.5 ± 6.1% (no significant change) in the case of protein quantification, and gene expression (icaA 160.55%, icaD 168.18%, sarA 149.8%, p < 0.001), whereas higher concentrations (≥ 10%) restored expression to near-control levels. Serum exposure produced a threshold-dependent response, with low concentrations (5-10%) slightly enhancing gene expression (~ 110%), while higher concentrations (20-50%) significantly repressed both biomass and transcription, with profound inhibition found at 50% (icaA 12.94%, icaD 10.88%, sarA 12.79%, p < 0.001). In addition, confocal laser scanning microscopy technique is used as a confirmatory step for qualitative determination of the effects of both various saline and serum concentrations on the biofilm formation, which induces similar results. Environmental stressors modulate S. aureus biofilm formation in a dose-dependent manner via regulation of the ica operon and sarA, offering molecular insights that may guide strategies for biofilm control. KEY POINTS: • Oxidative stress (H₂O₂) dose-dependently inhibits S. aureus Newman biofilms. • Mild NaCl levels enhance biofilm formation via upregulation of ica and sarA. • High serum concentrations (≥ 20%) suppress biofilm biomass and gene expression.