| Summary: | Persistent <i>Vibrio-parahaemolyticus</i>-associated vibriosis cases, attributed, in part, to the inefficient techniques for detecting viable-but-non-culturable (VBNC) <i>Vibrio</i> pathogens and the ingestion of undercooked seafood, is the leading cause of bacterial seafood-borne outbreaks, hospitalizations, and deaths in the United States. The effect of extreme heat processing on <i>Vibrio</i> biology and its potential food safety implication has been underexplored. In the present work, environmental samples from the wet market, lagoon, and estuarine environments were analyzed for <i>V. parahaemolyticus</i> recovery using a modified, temperature-dependent, two-step enrichment method followed by culture-based isolation, phenotype, and genotype characterizations. The work recovered novel strains (30% of 12 isolates) of <i>V. parahaemolyticus</i> from prolonged-heat-processing conditions (80 °C, 20 min), as confirmed by 16S rDNA bacterial identification. Select strains, VHT1 and VHT2, were determined to be hemolysis- and urease-positive pathogens. PCR analyses of chromosomal DNA implicated the <i>tdh</i>-independent, <i>tlh</i>-associated hemolysis in these strains. Both strains exhibited significant, diverse antibiotic profiles (<i>p</i> < 0.05). Turbidimetric and viable count assays revealed the pasteurization-resistant <i>V. parahaemolyticus</i> VHT1/VHT2 (62 °C, 8 h). These findings disclose the efficiency of <i>Vibrio</i> extremist recovery by the modified, two-step enrichment technique and improve knowledge of <i>Vibrio</i> biology essential to food safety reformation.
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