Physiological Characteristics of Viable-but-nonculturable Vibrio parahaemolyticus upon Prolonged Exposure to the Refrigerator Temperature

Abstract

Although it has been reported that viable-but-nonculturable (VBNC) cells of Vibrio parahaemolyticus can be developed by a prolonged duration of cold-starvation there are restricted cellular characteristics available on understanding the exact mechanisms governing the entry of pathogens into the VBNC state. Therefore, this research was aimed at determining the cellular profile of VBNC cells of V. parahaemolyticus upon exposure to the refrigerator temperature. Strains of V. parahaemolyticus were incubated in artificial sea water (ASW) microcosms (pH 6) added with different amounts of NaCl at 4{degrees}C until these pathogens entered into such a dormant state. At a regular time-interval, both culturability and viability of these bacteria were enumerated, and then cellular profiling were carried out in terms of cellular membrane permeability, enzymatic activity, hydrophobicity, fatty acid composition, and morphological changes after cells of V. parahaemolyticus became the VBNC state. Three strains of V. parahaemolyticus used in this study showed that VBNC cells retained the strong virulent properties to Vero and CACO-2 cell lines, re-gained the cytotoxicity even after resuscitation, became permeabilized in terms of the outer membrane, showed lower levels of enzymatic (catalase and glutathione-S-transferase) activities, exerted the increasing hydrophobicity, and then exhibited increasing amounts of saturated fatty acids.\n\nIMPORTANCETo the current best knowledge, there are restricted information available on understanding the physiological characterization of viable-but-nonculturable cells. Most previous studies are still making a degree of efforts in discovering the causative effector causing microorganisms to be induced into the VBNC state. Herein, the present study showed that pathogenic V. parahaemolyticus can enter into the VBNC state when challenged by a certain environmental stress where higher amounts of NaCl combined with acidic pHs was artificially controlled. Importantly, it was indicated that VBNC V. parahaemolyticus maintained peculiarly different physiological characteristics. Furthermore, this study proposed a novel approach on the transient/stepwise conversion of the bacteria into the VBNC state. Specific alternative tools for measuring and controlling the incidence of VBNC pathogens on food are not established until now. In this aspect, results obtained from this study will used to provide an effective insight in determining physiological properties of viable-but-nonculturable V. parahaemolyticus.