Jpn. J. Infect. Dis., 53 (2), 75-77, 2000

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Laboratory and Epidemiology Communications

The Use of Colony Hybridization in the Isolation of Thermostable Direct Hemolysin-Producing Vibrio parahaemolyticus from Foods Implicated
in an Incidence of Food Poisoning

Noboru Numata*, Makiko Ushimizu, Mayumi Ohtomo, Kyoko Chida, Hitomi Fujita, Takuya Saito, Dai Suto, Miyako Oguro, Yasuhiko Hayakawa, Ken Sasaki, Eiji Arakawa¹, Toshio Shimada¹ and Haruo Watanabe¹

Sendai City Institute of Public Health, Oroshimachi-Higashi 2-5-10, Wakabayashi-ku, Sendai 984-0002 Miyagi and ¹Department of Bacteriology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku 162-8640 Tokyo

Communicated by Kazuo Kato

(Accepted May 8, 2000)

Humans are infected by Vibrio parahaemolyticus by ingesting uncooked contaminated marine fishes and shellfishes (1). Its major bacterial pathogenic factors are thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) (2).

In general, in optimum temperatures, V. parahaemolyticus grows more rapidly than other food poisoning-causing bacteria, and negligence in temperature control during food processing and storage is the major cause of poisoning by this pathogen.

Isolation of TDH-positive bacteria from the stool specimens of patients is relatively easy. However, isolation of this bacteria from foods or sea water is extremely difficult, though isolation of TDH-positive bacteria from implicated foods by using the modified Wagatsuma medium was reported (3). We recently succeeded in an application of colony hybridization for isolation of tdh⁺ (gene encoding TDH) bacteria from foods implicated in a food poisoning case. The information is detailed in this report.

In November 1999, food poisoning was caused by Sushi prepared by a restaurant in Sendai City. The major symptoms of the patients were diarrhea, abdominal pain, and vomiting. From stool specimens of two patients, TDH-positive V. parahaemolyticus O3:K6 was isolated (isolates VP1 and VP2). In order to identify foods implicated in this incident, we tried to detect TDH-producing bacteria from various foods stored in the restaurant. The tdh gene was detected by PCR from only a frozen stock of boiled mantis shrimps after culture in salt polymyxin broth. V. parahaemolyticus of various serotypes were isolated from thiosulfate-citrate-bilesalts-sucrose (TCBS) agar plates inoculated with the broth but none of the 264 isolates were tdh⁺.

In order to screen thousands of colonies, colony hybridization by using a tdh probe was performed. The outline of the procedures is shown in Figure 1. The bacteria grown in salt polymyxin broth were plated on ten TCBS agar plates. A total of 1,369 transparent and sucrose non-fermenting V. parahaemolyticus-like colonies appeared. After culture overnight, the colonies were transferred to nylon filters (Hybond^TM-N+, Amersham LIFE SCIENCE, Buckinghamshire, England) and treated with alkaline solution for DNA denaturation and cross-link with the filter. After prehybridization, the filters were probed with the peroxidase-labelled tdh probe (2) by using ECL^TM direct nucleic acid labeling and detection systems (Amersham LIFE SCIENCE). Three plates were positive for tdh⁺ candidate colonies. Forty colonies were checked for the tdh gene by PCR. One colony (VP3) was positive. Therefore, the detection rate was 0.07% (1/1,369).

The isolate obtained by the colony hybridization method from the mantis shrimp (VP3) actually produced TDH. VP3's serotype, which was O3:K6, biochemical markers, and the pattern of pulsed-field gel electrophoresis (PFGE) of DNA digests were identical to the patient isolates' (Fig. 2, lanes 1-3). Though of the A type prevalent most recently, their PFGE pattern was different from any of the reported subtypes (4). Two tdh^- O3:KUK isolates from the same mantis shrimps (VP4-5) were examined for PFGE pattern. One (VP4) had a pattern resembling that of VP1-3 but was not of the A type (Fig. 2, lane 4), and another (VP5) had a pattern entirely different (Fig. 2, lane 5).

REFERENCES

1. World Health Organization (1999): Vibrio parahaemolyticus, Japan, 1996-1998. Wkly. Epidemiol. Rec., 74, 361-363.
2. Tada, J., Ohashi, T., Nishimura, N., Shirasaki, Y., Ozaki, H., Fukushima, S., Takano, J., Nishibuchi, M. and Takeda, Y. (1992): Detection of the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction. Mol. Cell. Probes, 6,
   477-487.
3. Sugiyama, A., Nakano, Y., Iwade, Y., Yamauchi, A., Nakayama, O., Matsumoto, T., Kou, Y., Ito, T., Nishinaka, T. and Shoji T. (1999): Isolation of thermostable direct hemolysin-producing Vibrio parahaemolyticus from food implicated in food poisoning in Mie Prefecture. Infect. Agents Surveillance Rep., 20, 189-192 (in Japanese).
4. Arakawa, E., Murase, T., Shimada, T., Okitsu, T., Yamai, S. and Watanabe, H.(1999): Emergence and prevalence of a novel Vibrio parahaemolyticus O3:K6 clone in Japan. Jpn. J. Infect. Dis., 52, 246-247.

*Corresponding author: Fax: +81-22-236-8601

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