Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/14/2010
Publication Date: 7/15/2010
Citation: Whitaker, B.W., Michelle, P.A., Naughton, L.M., Richards, G.P., Blumerman, S.L., Boyd, F.E. 2010. Growth of Vibrio parahaemolyticus O3:K6 at Different Salt Concentrations Modulates Responses to pH and Temperature Stresses. Applied and Environmental Microbiology. 76:720-729. Interpretive Summary: Vibrio parahaemolyticus is a naturally occurring bacteria found in salt water environments. It is the major cause of shellfish-related bacterial illness in the United States. Vibrio growth in the marine environment requires the presence of 1 to 9% salt (sodium chloride), but low salt conditions place stress on the bacteria. This study identified whether salt concentration played a role in the ability of V. parahaemolyticus O3:K6, a pandemic strain that was isolated in Japan in 1996, to survive acid and temperature stress. Salt concentrations of 1% and 3%, near the minimum and maximum concentrations found in oyster harvesting areas, were examined. We found that bacterial growth in 1% salt had a negative effect when compounded with high or low temperatures. In 3% salt, bacteria survived freezing and thawing better than those grown in 1% salt. We generated two mutant strains of V. parahaemolyticus which were lacking genes known to enhance bacterial invasiveness and response to stress. Both of these mutants showed reduced survival under acid stress conditions. Additionally, human cell cultures were used to evaluate whether the vibrios were more toxic when grown under 1% or 3% salt and we learned that 1% NaCl produced cells which were more toxic. Overall, this information may be beneficial in: a) predicting shellfish harvesting areas which are at increased risk of V. parahaemolyticus O3:K6 presence, b) the development of harvesting and processing interventions to reduce or eliminate vibrios from shellfish, and c) providing information on factors which influence V. parahaemolyticus infectivity in humans.
Technical Abstract: Vibrio parahaemolyticus inhabits marine, brackish, and estuarine waters worldwide, where fluctuations in salinity pose a constant challenge to the osmotic stress response of the organism. Vibrio parahaemolyticus is a moderate halophile, having an absolute requirement for salt for survival, and is capable of growth at 1 to 9% NaCl. It is the leading cause of seafood-related bacterial gastroenteritis in the United States and much of Asia. We determined whether growth in differing NaCl concentrations alters the susceptibility of V. parahaemolyticus O3:K6 to other environmental stresses. Vibrio parahaemolyticus was grown at 1% or 3% NaCl concentrations, and the growth and survival of the organism was examined under acid or temperature stress conditions. Growth of V. parahaemolyticus in 3% NaCl versus 1% NaCl increased survival in both inorganic (HCl) and organic (acetic acid) acid conditions. In addition, at 42°C and -20°C, 1% NaCl had a detrimental effect on growth. The expression of lysine decarboxylase (cadA), the organism’s main acid stress response system, was induced by both NaCl and acid conditions. To begin to address the mechanism of regulation of stress response mechanisms, we constructed a knockout mutation in rpoS, which encodes the alternative stress sigma factor, and in toxRS, a two component regulator common to Vibrio species. Both mutant strains had significantly reduced survival under acid stress conditions. The effect of V. parahaemolyticus growth in 1% or 3% NaCl in vivo was examined using a cytotoxicity assay. Vibrio parahaemolyticus grown in 1% NaCl were significantly more toxic that those grown in 3% NaCl.