Ion-dependent regulation of biofilm formation in Vibrio parahaemolyticus: strain-specific trade-offs between cellular proliferation and extracellular matrix production.

Biofilm formation in Vibrio parahaemolyticus is modulated by environmental factors; however, the influence of ionic composition on extracellular polymeric substance (EPS) production and biofilm architecture remains insufficiently characterized. This study examined the individual and combined effects of monovalent (K⁺) and divalent (Ca2⁺, Mg2⁺) cations on environmental (strain 5) and clinical (strains 8 and 10) biofilms under air-liquid wall (ALW) and submerged (SM) conditions. Viable cell counts (log10CFU/cm2), normalized protein concentration per viable cell (nProt), and normalized polysaccharide concentration per viable cell (nPol) were measured. ALW biofilms exhibited higher cell densities (4.40-6.49 log10CFU/cm2) than SM biofilms (4.13-6.01 log10CFU/cm2), reflecting enhanced oxygen-driven proliferation. Conversely, SM conditions yielded significantly higher nProt and nPol (p < 0.05), indicating that low oxygen promotes investment in the EPS matrix. The ion responses were strain specific. The oyster isolate Strain P5 maximized nPol/nProt with K⁺ + Ca2 + Mg2 under seawater-mimicking conditions. Clinical Strain P8 peaked under K + Ca2⁺, whereas pandemic Strain P10 favored K⁺ alone. Microscopy and three-dimensional surface plot analyses revealed relatively uniform biofilm layers at the ALW interface. In contrast, SM biofilms formed heterogeneous, tower-like structures, highlighting structural differences attributable to both ionic composition and strain. These findings demonstrate that cation composition and the interface regulate the balance between growth and matrix production in V. parahaemolyticus. Divalent cations enhance structural cohesion, whereas monovalent ions primarily support metabolic activity. This study provides a mechanistic framework linking ionic environments to biofilm architecture and emphasizes the importance of strain-specific responses in marine and food-associated environments.