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

Laboratory and Epidemiology Communications

Outbreaks of Heat Stable Enterotoxin‑Produclng Escherichia coli 0 1 69

in the Kinki District in Japan: Genotypic Comparison by Pulsed‑Field
Gel Electrophoresis of Isolates from Two Outbreaks in 2000

With Isolates from Four Outbreaks in 1997‑1998
Kokichi Hamada*, Tomohiro Oshibe, Hidetaka Tsuji, Satoshi Yoshidal and Yoshinari Aoki.
Division ofMicy10biology, Hyogo Prefectural Institute of Public Health,Arata‑cho 2‑1‑29, Hyogo‑ku, Kobe 652‑0032 andlDepartment of Preventive Health, Nara Prefectural Institute for Public Health,Omori‑cho 57‑6, Nara 630‑8131
Communicated by Shinsaku lmashuku(Accepted September 1 1 , 2000)

An outbreak of enterotoxigenic Escherichia colt serotype

Hyogo Prefecture in May (case E). Two hundred and sixty‑six

0169:H41 infection was flrSt reported in Japan in 1991 (1,2).
From June 1997 to August 1998, we investigated four
outbreaks (cases A‑D) due to the same organism in Hyogo

persons among three package‑tour groups from neighboring

Prefectures ate a Japanese‑Style box lunch prepared by a hotelnear the site of the exposition. About half of the members

and neighboring Prefectures (3). In 2000, two more outbreaks(1 2 1 persons) experienced watery dia汀hea an〟or abdominal

pain. We identified E. coli 01 69:H4 1 in fecal specimens fromoccu汀ed.
One outbreak occu汀ed at the lntemational Gardening and22 patients and from one of 19 cooks at the hotel. The otherLandscaplng Exhibition Japan Flora 2000 at Awaji Island inoutbreak occurred in April 2000 in a reformatory in Nara
Prefecture (case F)･ Seventy‑three of88 boys aged 16‑20 ye?rs*Co汀eSpOnding author: Fax: +8 I ‑78‑53 1 ‑7080

became ill wi也symptoms Including dia汀hea, abdominal palm,

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

Fig.2. PFGE pattems ofXbal digests ofchromosomal DNAsofE. colt

Fig. 1. PFGE pattems of.粗aI digests ofchromosomal DNAsofE. colt'0169:H41 isolated from case E outbreak (lanes 2‑12). The PFGE

O1 69:H4 I isolates from various outbreaks. The PFGE was performed

was perf♭med with non‑linear program No.23. Lanes 1 and 1 3 arewith linearprogram No.5. Lanes 1, 8, and 15:入DNA ladders; lanes入DNA ladders.

2‑3: isolates &om patients in case A; lane 4: an isolate hm patientsin case D; lane 5: an isolate from patients in case B; lane 6: an isolate

kom the causative food, wakame, ln Case B; lane 7: an isolate kom

patients in case C; lanes 9‑10: isolatesfrom patients in case E; lanes
I 1‑12: isolates from patients in case F; lane 13:anisolate from thetraveler to India in 1999 as a reference unrelated to cases A‑F; laneand vomiting. They had eaten lunch served by the institution.
Twelve non‑motile E. co/i 01 69 isolates were obtained斤om

fecal specimens.

14: the same bacteria as that in lane 7 with no treatment by XbaI.
Chr: chromosome.

The strains were examined fわr genes encoding ul STh,and STD by polymerase chainreaction uslng pnmerS Purchasedfrom Takara Shuzo Co. Ltd. (Kyoto). All of the isolates werestrains. The two STp negatives probably had lost the toxin‑

fosfomycin (FOM), chloramphenicol (CP), sulfamethoxazole‑
trimethoprim (ST), and nalidixic acid (NA).

encoding Ent plasmid (4) sometime before the test. All of theisolates were tested by Sensi Disc (Nippon Becton Dickinson

case F (Fig･ 2) were examined by pulsed‑field gel electro‑

negative for LT and STh, and positive for STp except for two

The 23 isolates from case E (Fig. 1 and 2) and two fromCo., Ltd., Tokyo), 23 isolates from case E and ll isolates

fTrom case F, were resistant to tetracyline (TC);intermediately

phoresis (PFGE) uslng a Gene Path Typing System (NipponBio‑Rad, Tokyo). The PFGE pattern of XbaI‑digested

sensitive to streptomycin (SM); and sensitive to ampicillinchromosomal DNAsfor the isolates from case E (Fig. 1 and(ABPC), cefotaxime (CTX), kanamycin (KM), gentamicinlanes 9 and 10 in Fig. 2) was identical to也at f♭r case F (lanes(GM), trimethoprim (TMP), ciplonoxacin (CPFX),ll and 12 in Fig. 2); i.e., the two outbreaks were probably

昌昌蔓昌書芸…蔓昌蔓墨…

･‑1･II I I･1‑‑‑･‑‑‑‑･･1‑･ ー ･‑ ‑■ 1 ‑‑̲･IlT･‑】7 ‑ ･ ‑ ‑‑ー…

昌Kb

1,･･ll一tl

Case B

Case B*
Case C

Case D‑ ‑ ‑ ‑1‑1‑

Case A‑1
Case A‑2

1 ‑ ‑■ ‑ ‑ ■‑ ‑‑1 ‑ JI L ｢̲. 1､=‑1.

̲.

･ ‑ 1 ‑ ･1‑. ‑Case E‑1
Case E‑2

Case F‑1
Case F‑2
TFig.3. Cluster analysis of PFGE patterns ofE. colt O169:H41 isolates shown in Fig. 2. The strains were all from patients,except in case B* (an isolate &om wakame). T indicates an isolate from a traveler to India in 1999. Two isolates from thesame outbreak are shown f♭r cases A, E, and F.

175

Jpn. J. Infect. Dis., 53, 2000
caused by closely related strains. The pattem was compared

REFERENCES

with those of the strains from the four previous outbreaks inthe Kinki district (case A, Takasago City and neighboring

1. Ando, K., Itaya, T.,Aoki,A., Saito,A., Masaki, H. andTowns in Hyogo Prefecture in June 1997; case B, Sakai City

Tokumara, Y. (1993): An outbreak of food poisoning
caused by enterotoxigenic Escherichia coli 0 1 69:H4 1.

in Osaka Prefecture and Sumoto City in Hyogo Prefecture inJpn. J. Food Microbio1.,10, 77‑81 (in Japanese).
2. Nishikawa, Y., Hanaoka, M., Ogasawara, J., Moyer, N. P.

April 1998; case C, Ohtsu City in Shiga Prefecture in June
1998; and case D, Seidan Town in Hyogo Prefecture inAugust 1998) (3) and that of an E. coli O169:H41 isolateand Kimura, T. ( I 995): Heat‑stable Enterotoxin‑produc‑from a traveler (T) infected in India in 1999 (Fig. 2). Isolates]ng Escherichia coli 0 169:H4 1 in Japan. Emerg. Infect.

incasesA‑F (A, lanes2 and3; B, lanes 5 and6; C, lane 7; D,Dis.,I, 61.

lane4; E, lanes 9 and 10; andF, lanes ll and 12) andanisolate in case T (lane 1 3) showed similar but slightly different
PFGE pattems.
A dendrogram obtained by molecular ana)ysIS software

3. Hamada, K., Tsuji, H. and Shimada, K. (1999): Outbreaksof heat stable enterotoxin‑producing Escherichia colt
･0169 in the Kinki district in Japan: epidemiologlCalanaIysis by pulsed‑field gel electrophoresis. Jpn. J. Infect.

Dis., 52, 165‑167.

(Finger Printing PLUS, Bio‑Rad, Hercules, Calif., USA) (Fig.
3) indicated the presence of three distinct groups, designated

4･ Macrina, F.し. (1984): Molecular cloning or bacterial

as Group 1 (cases B, C, and D), Group 2 (caseA), and Group

antIgens and virulence determinants.Am. Rev. Microbio1.,

3 (cases E, F, and T). Thus, the outbreaks of STp‑producing

38, 193‑219.

E･ colt 0169:1141 in the Kinki district over the last 3 ‑ 4 years

were caused by more or less genotypically different bacteria.

176