Environmental triggers of enterotoxin production in foodborne Staphylococcus aureus: A focus on growth-dependent risk

Staphylococcus aureus is a major foodborne pathogen whose virulence is mediated by heat-stable staphylococcal enterotoxins (SEs). Understanding environmental and metabolic drivers of SE production is critical for risk assessment in ready-to-eat (RTE) foods. This study investigated the impact of cell density, environmental stressors, and metabolic profiles on SEs expression. Additionally, metabolic signatures were investigated as potential predictive biomarkers. A total of 138 isolates from diverse food sources were characterized. Enterotoxin production was assessed under varying conditions, and LC-MS-based metabolomic profiling was conducted on reference strains harboring sea, seb, sec, and sed. The sea gene was detected in 14.5 % of isolates, with serotype Ⅴ predominating among enterotoxigenic strains. Penicillin resistance was nearly universal (95.7 %), and 29.7 % of isolates exhibited multidrug resistance. SEA was distributed across multiple clonal lineages, whereas SEB was restricted to ST2176. Enterotoxin detection consistently occurred at ∼6 log CFU/mL, with earlier detection at higher inocula, indicating quorum-sensing-based regulation. Under low temperatures or high salinity levels, SEs were detected at lower apparent thresholds, attributable to the induction of viable but non-culturable (VBNC) states and the presence of biofilm-associated microenvironments. Metabolomic profiling revealed dynamic strain- and time-dependent changes. However, robust toxin-type-specific biomarkers were not identified. Collectively, these results demonstrate that SEs production is governed primarily by population thresholds rather than environmental stress, while VBNC and biofilm states pose hidden risks beyond CFU-based monitoring. The lack of reproducible metabolic markers underscores the potential value of integrative multi-omics methods in supporting early-warning biomarker identification and improving food safety surveillance.