Abstract:The effect of ethanol on thermostable direct hemolysin (TDH) production and corresponding tdh gene expression by Vibrio parahaemolyticus was studied. Two tdh-positive V. parahaemolyticus strains, ATCC33847 and SZ32 were separately treated with 0.5%, 1.0%, 2.0%, 4.0%, and 8.0% ethanol solutions. The effects on aerobic or anaerobic growth of V. parahaemolyticus were monitored, while changes in TDH production and tdh gene expression were studied with 1.0% ethanol treatment. TDH concentration in V. parahaemolyticus culture supernatant was quantitatively analyzed using a TDH detection kit (KAP-RPLA, Denka Seiken Co., Japan), while tdh gene expression was analyzed by real-time fluorescence-based quantitative polymerase chain reaction. The results showed that V. parahaemolyticus growth was not influenced by the presence of ethanol at low concentrations (0.5%, 1.0%, and 2.0%). On the other hand, 4.0% and 8.0% ethanol caused significant and total inhibition of bacterial growth, respectively. With ethanol treatment, TDH titers of culture supernatants showed a significant increase under aerobic as well as anaerobic conditions and intracellular tdh expression was also elevated. Moreover, ATCC33847 strain showed an increase in tdh expression by 6.6- and 5.7-fold, while SZ32 strain showed an increase by 5.9- and 8.6-fold under aerobic and anaerobic conditions, respectively. Thus, ethanol treatment enhanced tdh gene expression, which may have resulted in an increase in TDH production. The effects of methanol, ethanol, and n-propanol on tdh gene expression were also compared and the results showed increased tdh gene expression by all three alcohols, however, the effects were not significantly different.
