Jpn. J. Infect. Dis., 52, 1999

Laboratory and Epidemiology Communications

Outbreaks of Heat Stable Enterotoxin-Producing Escherichia coli 0169 in the Kinki District in Japan: Epidemiological Analysis by Pulsed-Field Gel Electrophoresis

Kokichi Hamada*, Hidetaka Tsuji and Kunio Shimada

Division of Microbiology, Hyogo Prefectural Institute of Public Health
Arata-Cho 2-1-29, Hyogo-Ku, Kobe 652-0032, Japan

Communicated by Shunsuke Imai

(Accepted August 16, 1999)

An outbreak of enterotoxigenic Escherichia coli serotype O169:H41 infection was first reported in Japan in 1991 (1,2). From June 1997 to August 1998, four outbreaks due to the same organism occurred in Hyogo and neighboring Prefectures. Case A was identified in Takasago City in Hyogo Prefecture in June 1997. In April 1998, case B occurred in Sakai City in Osaka Prefecture and Sumoto City in Hyogo Prefecture. Case C was in Ohtsu City in Shiga Prefecture in June 1998, and case D was in Seidan Town in Hyogo Prefecture in August 1998 (Table 1 and Fig. 1). We obtained 50 isolates from the stool of symptomatic (diarrhea and/or abdominal pain) and asymptomatic patients. We isolated 3 strains from wakame seaweed implicated in outbreak B. In order to isolate the bacteria from wakame, the seaweed was first incubated in tetracycline-containing nutrient broth and then the broth was streaked on teracycline-containing Lactose-MacConkey agar plates.

The isolates were examined for genes encoding LT, STh, STp, VT1, VT2, and InvE by polymerase chain reaction using primers purchased from Takara Shuzo Co. Ltd. (Kyoto). All 53 strains were negative for LT, STh, VT1, VT2, and InvE. Forty-nine strains were positive for the toxin gene STp but four were negative. The negative results might be explained by the curing of the Ent plasmid, which carries the toxin gene (3). All of the isolates were Tet resistant and NFLX sensitive; sensitivity to KM, SM, NA, and FOM was variable.

The isolates were examined by pulsed-field gel electrophoresis (PFGE) using the Gene Path Typing System (Nippon Bio-Rad, Tokyo). PFGE of XbaI digests gave two patterns (Fig. 2); samples from cases B and C had a heavy extra band (97-145.5 kb), which was absent in the samples derived from cases A and D. This observation indicated that cases B and C were caused by one strain and that cases A and D were caused by another. However, as the overall PFGE pattern was similar in both strains, they probably arose by mutation from a common strain. In large outbreaks of Shiga-toxin producing E. coli (STEC) in 1996 involving many locations in Japan (4,5), six types were detected, as distinguished by PFGE (4). In the STEC O157 outbreak in 1998 caused by contaminated ikura (salmon roe)-sushi , two different strains were isolated by PFGE (6).

We thank Dr. K. Hayashi, Shiga Prefectural Institute of Public Health and Environmental Science and Dr. T. Ohnaka, Sakai City Institute of Public Health, for the E. coli O169 isolates.

REFERENCES

  1. Ando, K., Itaya, T., Aoki, A., Saito, A., Masaki, H. and Tokumura, Y (1993): An outbreak of food poisoning caused by enterotoxigenic Escherichia coli O169:H41. Jpn. J. Food Microbiol.,10, 77-81 (in Japanese).
  2. Nishikawa, Y., Hanaoka, M., Ogasawara, J., Moyer, N.P. and Kimura, T. (1995): Heat-stable enterotoxin-producing-Escherichia coli O169:H41 in Japan. Emerging Infect. Dis., 1, 61.
  3. Macrina, F.L. (1984): Molecular cloning of bacterial antigens and virulence determinants. Ann. Rev. Microbiol., 38, 193-219.
  4. Izumiya, H., Terajima, J., Wada, A., Inagaki, Y., Itoh, K., Tamura, K. and Watanabe, H. (1997): Molecular typing of Escherichia coli O157:H7 isolates in Japan by using pulsed-field gel electrophoresis. J. Clin. Microbiol., 35, 1675-1680.
  5. Hamada, K., Shimada, K., Tsuji, H., and Ono, K. (1997): Isolation and characterization of enterohemorrhagic Escherichia coli in Hyogo Prefecture during outbreaks of the bacteria throughout Japan. 1996. Bull. Hyogo Prefect. Inst. Public Health, 32, 107-110 (in Japanese).
  6. Terajima, J., Izumiya, H., Iyoda, S., Tamura, K. and Watanabe, H. (1999): Detection of a multi-prefectural E. coli O157:H7 outbreak caused by contaminated ikura-sushi ingestion. Jpn. J. Infect. Dis., 52, 52-53.

*Corresponding author: Fax: +81-78-531-7080

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