Multi-strain probiotic suppression of Staphylococcus aureus virulence: Growth kinetics, enterotoxin production, and sea/seb gene expression under Lactobacillus casei, Lactobacillus plantarum, Bifidobacterium bifidum and consortia co-culture conditions.

BACKGROUND

Staphylococcus aureus causes staphylococcal food poisoning (SFP), mediated through heat-stable enterotoxins encoded by virulence genes sea and seb. Probiotic biocontrol represents a promising upstream intervention, yet simultaneous mechanistic evidence spanning growth inhibition, toxin suppression, and virulence gene transcriptional downregulation remains limited.

OBJECTIVES

To evaluate the capacity of Lacticaseibacillus casei (formerly Lactobacillus casei), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum), Bifidobacterium bifidum, and their defined consortia to suppress S. aureus across three analytical tiers: growth kinetics, enterotoxin production (ELISA), and sea/seb gene expression (RT-qPCR).

METHODS

Probiotic-pathogen co-cultures were established in broth and sampled at 24, 48, and 72 h. Viable counts were determined by standard plate count (Log₁₀ CFU/mL, verified by standard plate count). Enterotoxin levels were quantified by ELISA. Relative expression of sea and seb was determined by RT-qPCR using the 2⁻ᴵᴵᴺᵗ method with dual housekeeping gene normalisation (gmk and tpi).

RESULTS

All treatments reduced S. aureus viability and enterotoxin production vs. controls. The consortium was bactericidal (µ = -0.178 h⁻¹), achieving a 4.56 log₁₀ CFU/mL reduction and 58.1% enterotoxin suppression at 72 h (p < 0.01). sea was downregulated across all treatments (FC 0.0002-0.67 at 24 h). seb showed stationary-phase upregulation in individual strain treatments at 48 h, while the consortia sustained transcriptional downregulation of both genes throughout (sea: 0.0002-0.044; seb: 0.0014-0.009).

CONCLUSION

Three-tier evidence confirms that the consortia coherently suppresses S. aureus growth, enterotoxin accumulation, and virulence gene transcription. The seb stationary-phase transcriptional downregulation These results support multi-strain probiotic formulations as biocontrol agents in food safety applications.