Isolation of Vibrio parahaemolyticus O3:K6 from incriminated foodstuffs, September 1999 - Oita, Japan
(IASR 2000; 21:34-35)

September 26, 1999, a medical facility in O County, Oita Prefecture, reported to the health center in jurisdiction that a patient visited with gastrointestinal symptoms such as vomiting, diarrhea, abdominal pain. Investigation revealed that common meal was served at an open-house party on September 25, and suspected foods included lunch box, nigiri-sushi, which were prepared at the same restaurant, and sekihan-rice, boiled vegetable, and maki-sushi, which were cooked at home. Forty-seven persons ate the meal, and 17 developed illness during 1-21 O'clock of September 26. Major symptoms included 15 cases of diarrhea (88%), 11 abdominal pain (65%), 6 fever (35%), 6 vomiting (35%). Average time period to onset was 18.5 hours.

Six specimens from the patients were collected (3 on September 26, and 3 on September 28). Twenty-three specimens were selected from the remained foods. These specimens were inoculated directly on isolation agar plates including TCBS. Colonies grown were inoculated on Salt-polymixin broth and cultured for 16 hours at 37C. These colonies were separately cultured using TCBS and Vibrio agar plates. Polymerase Chain Reaction (PCR) was applied with the colonies to detect tdh and trh gene fragments. Food specimens were inoculated on Salt-polymixin broth and other isolation agar plates, and densely grown colonies were scratched and inoculated to the Trypto-soy broth (hereafter, this method is described as the colony scratching method). The broth was cultured at 37C for 7 hours, and then, the tdh and trh genes were screened by PCR. Isolates from the stool and the food were classified by serotype, and TDH production by reversed passive latex agglutination (RPLA) reaction. In addition, these isolates were examined for DNA fragment pattern by pulsed-field gel electrophoresis (PFGE) using Not I restriction enzyme.

Vibrio parahaemolyticus O3:K6 (TDH production positive) was isolated from the 3 of 6 stool specimens, which were collected on September 26. Regarding with 23 food specimens, specimens from the Salt-polymixin broth examination was all negative in PCR. A specimen by the colony scratching method was positive by PCR tdh gene screening (Table). Isolation of bacteria was attempted using the isolation agar plate, which was screening positive. Eitghy-one colonies on the plate were examined for the tdh gene by PCR. One strain had the tdh gene. This strain was identified by a routine method, as well as its serotype, detection of TDH by RPLA, and it was confirmed V. parahaemolyticus O3:K6 (TDH +). PFGE pattern using Not I restriction enzyme was identical between the stool and the food specimens (Figure).

Most of V. parahaemolyticus isolates from patients' stool have both or either of the tdh and trh genes. Whereas, environmental specimens such as seawater and food specimens using marine products hardly have these genes. Thus, it is believed difficult to detect TDH+ V. parahaemolyticus from food specimen. We have reported that combination of the colony scratching method and PCR methods can efficiently detect pathogenic Escherichia coli. We could detect TDH+ V. parahaemolyticus from suspected food items by these methods, as well as conventional ones.

Reported by Kikuyo Ogata, Yoshiaki Abe, Yuichi Fuchi, Kikuo Hoashi, Oita Prefectural Institute of Health and Environment, Oita Prefectural Mie Health Center.

Correspondence: Kikuyo Ogata;
E-mail: kiogata@eiken.oita-ri.go.jp

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