Virulence Potential of Nonclinical <i>Vibrio parahaemolyticus</i> Isolates From Vietnam: Evidence for Functional T3SS2‐Mediated Enterotoxicity

Vibrio parahaemolyticus is a leading cause of seafood-borne gastroenteritis worldwide, and its pathogenic strains typically harbor thermostable direct hemolysin (TDH) and type III secretion system 2 (T3SS2). Although these virulence factors are associated primarily with clinical isolates, their presence in nonclinical environmental and food isolates raises concerns about their potential infection risk. In this study, we investigated the pathogenic potential of nonclinical V. parahaemolyticus isolates from Vietnam, which share serotypic and genotypic characteristics with pandemic strains. Serotyping and genetic analysis of 56 isolates (35 clinical and 21 nonclinical) revealed that two nonclinical isolates from shrimp and environmental water carried the tdh gene, T3SS2α genes, and pandemic markers that clustered phylogenetically with the pandemic strains. Protein expression assays confirmed that these isolates secreted TDH and the T3SS2 translocator (VopD2) at levels similar to those in the clinical reference strain. Bile exposure induced T3SS2-related gene expression, which suggests a conserved gene regulatory mechanism. Enterotoxicity evaluated using a rabbit ileal loop assay showed that two nonclinical isolates induced significant fluid accumulation. Genetic deletion and complementation experiments confirmed that T3SS2 was essential for enterotoxicity. These findings provide the first experimental evidence that nonclinical pandemic strains of V. parahaemolyticus possess functional enteric virulence mechanisms and suggest their potential as infection sources in endemic regions.