RESUMEN
Vibrio parahaemolyticus is a common foodborne pathogen frequently causing outbreaks in summer. Maintenance of virulence by the viable but nonculturable (VBNC) state of this pathogen would allow its threat to human health to persist. This study reports on the change in virulence and concomitant changes in activity of two enzymes and fatty acid profiles when V. parahaemolyticus ST550 entered the VBNC state in the modified Morita mineral salt-0.5% NaCl medium incubated at 4 degrees C. The major change in fatty acid composition occurred in the first week, with a rapid increase in C15:0 fatty acid and saturated/unsaturated ratio while a rapid decrease in C16:1 was observed. The activity level of the inducible protective enzyme superoxide dismutase became undetectable in the VBNC state, whereas that of constitutive glucose-6-phosphate dehydrogenase did not change in either the exponential phase or the VBNC state. Cytotoxicity against HEp-2 cells and a suckling mouse assay showed that virulence was lowered in the VBNC state compared with exponential-phase cells. Longer incubation times were required by the VBNC cells to achieve the same level of virulence as seen in exponential-phase cells. Culturable cells were recovered on selective agar medium from the VBNC cultures injected into suckling mice, probably as the result of in vivo resuscitation. Results of this study add to our understanding of the biochemical and physiological changes that have not been reported when V. parahaemolyticus enters into the VBNC state.
Asunto(s)
Medios de Cultivo/metabolismo , Ácidos Grasos/análisis , Microbiología de Alimentos , Vibrio parahaemolyticus , Animales , Animales Lactantes , Recuento de Colonia Microbiana , Glucosafosfato Deshidrogenasa/metabolismo , Ratones , Superóxido Dismutasa/metabolismo , Factores de Tiempo , Vibrio parahaemolyticus/enzimología , Vibrio parahaemolyticus/crecimiento & desarrollo , Vibrio parahaemolyticus/patogenicidad , VirulenciaRESUMEN
Vibrio parahaemolyticus, an important seafood-associated enteropathogen, usually encounters different adverse conditions in its native or food-processing environment, and the stresses resulting from these conditions may affect the survival of this pathogen and thus change its risk with regard to food hygiene. In this study, we investigated the thermotolerance of V. parahaemolyticus under sublethal heat shock and characterized this response by examining the changes in protein profiles and toxin production. Logarithmically grown cells heat shocked at 42 degrees C for 30 min were more resistant to thermal inactivation at 47 degrees C than were unshocked cells. After the 25 degrees C culture was heat shocked, 24 species of proteins were induced, while 13 species were inhibited, as indicated by polyacrylamide gel electrophoresis. DnaJ-, GroEL-, and GroES-like proteins with molecular sizes of 47, 62, and 12 kDa, respectively, were detected by immunoblotting with antibodies raised against the Escherichia coli proteins. During 1 to 8 h of heat shock, GroEL-like protein was produced in substantial amounts and was present in the periplasmic and extracellular fractions, while DnaJ- and GroES-like proteins were present mainly in the total cellular fraction. DnaK-like protein was not detected; nevertheless, the presence of the dnaK-like genetic element was revealed by Southern blotting. Production of thermostable direct hemolysin, the major virulence factor in V. parahaemolyticus, was enhanced in the cells heat shocked at 42 degrees C but not in those heat shocked at 37 degrees C.