Although thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus has caused many infections in Asian countries, the United States, and other countries, it has been difficult to detect the same pathogen in seafoods and other environmental samples. In this study, we detected and enumerated tdh gene-positive V. parahaemolyticus in Japanese seafoods with a tdh-specific PCR method, a chromogenic agar medium, and a most-probable-number method. The tdh gene was detected in 33 of 329 seafood samples (10.0%). The number of tdh-positive V. parahaemolyticus ranged from <3 to 93/10 g. The incidence of tdhpositive V. parahaemolyticus tended to be high in samples contaminated with relatively high levels of total V. parahaemolyticus. TDH-producing strains of V. parahaemolyticus were isolated from 11 of 33 tdh-positive samples (short-necked clam, hen clam, and rock oyster). TDH-producing strains of V. parahaemolyticus were also isolated from the sediments of rivers near the coast in Japan. Representative strains of the seafood and sediment isolates were examined for the O:K serovar and by the PCR method specific to the pandemic clone and arbitrarily primed PCR and pulsed-field gel electrophoresis techniques. The results indicated that most O3:K6 tdh-positive strains belonged to the pandemic O3:K6 clone and suggested that serovariation took place in the Japanese environment.

We have developed a new, effective procedure for detecting Vibrio parahaemolyticus in seafoods using enrichment and plating onto a chromogenic agar medium. Samples were cultured in salt Trypticase soy broth, which is a nonselective medium, and then a portion of the culture was cultured with salt polymyxin broth, which is a selective medium for V. parahaemolyticus. This two-step enrichment was more effective than the one-step enrichment in salt polymyxin broth alone. The enrichment cultures were then plated onto a new chromogenic agar containing substrates for beta-galactosidase. The V. parahaemolyticus colonies developed a purple color on this growth medium that distinguished them from other related bacterial strains. V. parahaemolyticus was isolated more frequently from naturally contaminated seafood samples using the chromogenic agar than thiosulfate citrate bile salts sucrose agar medium, which is currently used for the isolation of V. parahaemolyticus. Our findings suggest that this new enrichment and isolation scheme is more sensitive and accurate for identifying V. parahaemolyticus in seafood samples than previously used methods.Vibrio parahaemolyticus is a major food-borne pathogen that causes worldwide health problems. Prevention of V. parahaemolyticus contamination of foods, especially contamination with serotype O3:K6, which has been associated with recent outbreaks (3,5,6,7), is an important public health concern. It has been observed that the new O3:K6 clone of V. parahaemolyticus spread to Taiwan, Laos, Japan, Thailand, Korea, and the United States between 1997 and 1998 (10).To establish effective control measures to reduce the risk of V. parahaemolyticus infection and to ensure the safety of foods, efficient analytical methods for the detection of V. parahaemolyticus in foods and the environment must be available. Selective enrichment with alkaline peptone water (APW) or salt polymyxin broth (SPB) and plating of the enrichment culture onto thiosulfate citrate bile salts sucrose (TCBS) agar have been widely used for selective isolation of V. parahaemolyticus from foods. Although this existing method for detecting this organism in foods is inadequate, V. parahaemolyticus isolation schemes have not been carefully studied. It has been observed for other bacteria that nonselective enrichment prior to selective enrichment is effective for the detection of bacteria injured by various environmental stresses (4, 13). Therefore, we studied the effectiveness of nonselective enrichment prior to selective enrichment for V. parahaemolyticus. Furthermore, we have noted in a survey of seafoods that V. parahaemolyticus colonies on TCBS agar are difficult to distinguish visually from other bacterial colonies, since they can be covered by a yellow color produced by sucrose-fermenting bacteria. To develop a more efficient method, we studied the effectiveness of a method involving a two-step enrichment with nonselective and selective media and plating onto a chromogenic agar medium. This agar medium containing substrat...

Using cultivation, immunofluorescence microscopy, and scanning electron microscopy, we demonstrated the presence of viable enterohemorrhagic Escherichia coli O157:H7 not only on the outer surfaces but also in the inner tissues and stomata of cotyledons of radish sprouts grown from seeds experimentally contaminated with the bacterium. HgCl2 treatment of the outer surface of the hypocotyl did not kill the contaminating bacteria, which emphasized the importance of either using seeds free from E. coli O157:H7 in the production of radish sprouts or heating the sprouts before they are eaten.

To study the correlation between emetic toxin and HEp-2 vacuole activity produced by Bacillus cereus isolated from an outbreak of vomiting-type food poisoning, some properties and emetic activities of both purified HEp-2 factor (cereulide) and partially purified factor to rhesus monkeys were determined. The results indicate that both cereulide and partially purified factor were very stable to digestion with proteolytic enzymes, different pH, and heating. Vomiting was induced in the rhesus monkeys orally administered with both substances. From these findings, cereulide (or HEp-2 vacuole factor) is strongly suggested to be an emetic toxin itself.

We studied the contamination of radish sprouts after exposure to Escherichia coli O157:H7-inoculated water in the laboratory. The edible parts, the cotyledons and hypocotyl, became heavily contaminated with E. coli O157:H7 when they were grown from seeds soaked in E. coli O157:H7-inoculated water. These same parts became contaminated with E. coli O157:H7 when their roots were dipped into E. coli O157:H7-inoculated water. These findings suggest that E. coli O157:H7 contamination in the edible parts of radish sprouts could pose a serious hazard if the seeds or hydroponic water are contaminated with the bacterium.