Jpn. J. Infect. Dis., 53, 2000

Laboratory and Epidemiology Communications

The Use of Colony Hybridization in the Isolation of Thermostable
Direct Hemolysin‑Produclng Vibrio parahaemolyticus fromFoods Implicated in an Incidence of Food Poisonlng
NoboruNumata*, Makiko Ushimizu, Mayumi Ohtomo, Kyoko Chida, Hitomi Fujita,
Takuya Saito, Dai Suto, Miyako Oguro, Yasuhiko Hayakawa, Ken Sasaki,
Eiji Arakawa1, Toshio Shimadal and Haruo Watanabel
Sendai CityInstitute of Public Health,

Oroshimachi‑Higashi 2‑5‑I 0, Wakabayashi‑ku, Sendai 984‑0002 Miyagi andIDepartment ofBacteriology, National Institute ofInfectious Diseases,
Toyama I‑23‑1, Shinjuku‑ku 162‑8640 To砂oCommunicated by Kazuo Kato(Accepted May 8, 2000)

Humansare infected by Vibrio parahaemolyticus by ingest‑

ing uncooked contaminated marine fishes and shellrlShes (1).

hemolysin (TDH) and TDH‑related hemolysin (TRH) (2)In general, in optlmum temperatures, V. parahaemolyticus

Its major bacterial pathogenic factors are thermostable direct

grows more rapidly than other fわod poISOnlng‑CauSlng
bacteria, and negligence in temperature control during fわod

*Corresponding author: Fax: +8 1‑22‑236‑8601

processlng and storage is the major Cause Of poISOnlng by75

Jpn. J. Infect. Dis., 53, 2000
mantis shrimps in salt polymyxin broth∫ masticated∫ 35oC, overnight ‑ PCR

spread on TCBS agar
∫ 35oC, ovemight

the V. pwahaemolyEicus‑like colonies are collected and suspended in PBS aliquotslaliquots are titratedand tdh'is detected by PCR∫aliquots are placed on TCBS agar
∫ 35oC, Ovemightcolonies are transferred to nylonfiltersand hybridized with a I(肋IprObe∫colonies showing hybridization slgnals on X‑ray丘lms are selected and suspended in PBS

colonies are plated on TCBS agar

1 35oC, ovemightslngle colonies are selected∫

theunIque tdh'coloniesare detected by PCR
PBS : phosphate‑buffered saline
Fig. I. Flow chart of the screenlng Process Of tdh'colonies using hybridization

Nod

this pathogen.

Isolation ofTDH‑positive bacteria &omthe stool specimensof patients is relatively easy. However, isolation of thisbacteria from foods or sea water is extremely difrlCult,thoughisolation of TDH‑positive bacteria from implicated foods by

S用AlA31 2345

九1 2345using the modified Wagatsuma medium was reported (3). We
recently succeeded in an叩plication of colony hybridization

for isolation of tdh'(gene encoding TDH) bacteria from foods
implicated in a fわod poISOnlng Case. The infわrmation isdetailed in this report.
In November 1999, food poISOnlng Was Caused by Sushi

prepared by a restaurant in Sendai City. The major symptomsof the patients were dia血ea, abdominal palm, and vomiting.

From stool specimens of two patients, TDH‑positive V.
parahaemolyticus 03:K6 was isolated (isolates VPl andVP2). In order to identify foods implicated in this incident,
we tried to detect TDHIPrOducing bacteria kom various foods
stored in the restaurant. The tdh gene was detected by PCRfrom only a frozen stock of boiled mantis shrimps after culture

insalt polymyxinbrodl. V. pwahaemolytimf Ofvarious serotypes
were isolatedfrom thiosulfate‑citrate‑bilesalts‑sucrose (TCBS)
agar plates inoculated with the broth but none of the 264isolates were tdh+.
In order to screen仇ousands of colonies, colony hybridiza‑tion by uslng a tdh probe was performed. The outline of theFig. 2. Pulsed‑field gel electrophoresis (PFGE) patterns showing NotI‑

procedures is shown in Figure 1. The bacteria grown in salt

or SjiI‑ chromosomalDNA digests of Vibrio pwahaemolyticus isolates.

polymyxin broth were plated on ten TCBS agarplates. A totalof 1,369 transparent and sucrose non‑fermenting V.

LaneA3: the standard strain ofA3 subtype; Lane Al :the standard strainofAI subtype; Lane入:入DNA ladder; Lane I‑2: VP1‑2 (tdh'isolatesfrom patients); Lane 3: VP3 (tdh'isolates &om the mantis shrimps);

parahaemolylicus‑like colomies appeared. A鮎r culture over‑night, the colomies were transferred to nylonfilters (HybondTM‑

Lane 4‑5: VP4‑5 (tdh‑ isolates aomthe mantis shrimps)

N+,Amersham LIFE SCIENCE, Buckinghamshire, England)
and treated with alkaline solution for DNA denaturation andcross‑linkwith the filter. After prehybridization, the filters

3). Though of the A typeprevalent most recently,their PFGEwでre probed with the peroxidase‑labelled tdh probe (2) bypattem was different from any of the reported subtypes (4).
Two tdhl 03:KUK isolates from the same mantis shrimps

uslng ECLTM direct nucleic acid labeling and detection

systems (Amersham LIFE SCIENCE). Three plates were(VP4‑5) were examined for PFGE pattem. One (VP4) had apattem resembling that ofVP1‑3 but was not of the A type(Fig. 2, lane 4), and another (VP5) had a pattem entirelydifferent (Fig. 2, lane 5).

positive for tdh'candidate colonies. Forty colonies werechecked for the tdh gene by PCR. One colony (VP3) waspositive. Therefore, the detection rate was 0.07% (1/1 ,369).
The isolate obtained by the colony hybridization methodfrom the mands shrimp (VP3) actually produced TDH･ VP3 's

REFERENCES

serotype, which was 03 :K6, biochemlCal markers, and the

1. World Health Organization ( I 999): Vibrioparahaemoly‑
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pattem of pulsed‑field gel electrophoresis (PFGE) of DNA
digests were identical to the patient isolates'(Fig. 2, lanes 1‑76

Jpn. J. Infect. Dis., 53, 2000
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