Jpn. J. Infect. Dis., 55, 205-206, 2002

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Laboratory and Epidemiology Communications

An Outbreak of Heat-Stable Enterotoxin-Producing Escherichia coli O25: HNM Resistant to Nalidixic Acid Caused by Ingestion of Contaminated Box-Lunch in September-October 2002 in Hyogo and Shiga Prefectures

Hidetaka Tsuji, Kahori Oshima, Kokichi Hamada*, Kazuhiro Ishikawa¹ and Ken-ichi Hayashi¹

Infectious Disease Research Division, Hyogo Prefectural Institute of Public Health and Environmental Sciences, Arata-cho 2-1-29, Hyogo-ku, Kobe 652-0032 and ¹Shiga Prefectural Institute of Public Health and Environmental Science, Gotenhama 13-45, Ohtsu 520-0834

Communicated by Takashi Kawamura

(Accepted December 16, 2002)

It has been considered in the late 1960s that enterotoxigenic Escherichia coli (ETEC) infections in Japan were primarily infections of overseas travelers. However, these infections have been increasingly identified among patients with diarrhea who had not traveled abroad. For the past two decades, approximately 800 ETEC strains have been isolated annually from Japanese patients, and about half of these patients had not traveled to foreign countries prior to their illness (1). E. coli serotypes O25: HNM (1) and O169: H41 (2,3) have been major causes of ETEC infections over the past decade in Japan. From September to October in 2002, Hyogo Prefecture (the end of Sept.) and a boundary area of Shiga and Fnkui Prefectures (early Oct.) experienced an outbreak due to such ETECs showing nalidixic acid (NA)-resistance.

Among 1,838 persons belonging to 117 groups who ingested the suspected box-lunch as supplied by two delivery companies in Cities A and B in Hyogo and Shiga Prefectures, respectively, 328 persons of 20-67 years of age developed symptoms such as diarrhea, fever, and abdominal pain. Two food materials, each produced by the same producer or manufacturer, were commonly used for the lunches prepared by the two companies. Of 119 patients and 34 employees working for the companies investigated, lysine-negative E. coli O25:HNM were isolated in stool specimens of 50 customers showing severe symptoms and in one employee. As a reference, we used one E. coli O25:HNM strain, '96-E.118, isolated from a sporadic infection in 1996 in one of our laboratories. The strain was lysine-negative, sensitive to all the drugs tested (see the below), and an STh enterotoxin producer.

The 51 strains from the outbreak were examined for genes encoding LT, STh, and STp by polymerase chain reaction using primers purchased from Takara Shuzo Co. Ltd. (Kyoto). All of the isolates were negative for LT and STp and positive for STh. The strains were tested for sensitivities to ampicillin, cefotaxime, kanamycin, gentamicin, streptomycin, tetracycline, trimethoprim, ciplofloxacin, fosfomycin, chloramphenicol, sulphamethoxazole-trimethoprim, and NA by using antibiotic disks (Becton Dickinson Microbiology Systems, Cockeysville, Md., USA)(4). All were resistant to NA and sensitive to the remaining drugs.

The pulsed-filed gel electrophoresis (PFGE) patterns of DNA from ETEC O25 isolates were analyzed by using a Gene Path Typing System (Program No. 5; Bio-Rad Laboratories, Hercules, Calif., USA). Bacterial chromosomal DNAs were digested with XbaI, BlnI, SfiI, NotI, or SpeI purchased from a manufacturer (Takara Shuzo). It was found that, of these digests, XbaI-digests gave the most suitable PFGE patterns for classifying the isolates (Fig. 1A). A pattern of 13 isolates from this food poisoning outbreak identical to the pattern of the XbaI-digests is shown in Fig. 1B. The strain from a sporadic infection ('96-E.118) gave a pattern different from those isolates with every digest (Fig. 1A).

The occurrences of the ETEC strains resistant to NA and fluoroquinolones have been rare up to the middle of the 1990s in Japan. Such strains started to increase abruptly from 1996 (5). The present case is probably the first reported case of a food poisoning outbreak caused by NA-resistant ETEC in Japan.

The present case communicated in Japanese appeared in Infectious Agents Surveillance Report (IASR), vol. 23, p. 317, 2002.

REFERENCES

1. Mitsuda, T., Muto, T., Yamada, M., Kobayashi, N., Toba, M., Aihara, Y., Ito, A. and Yokota, S. (1998): Epidemiological study of a food-borne outbreak of enterotoxigenic Escherichia coli O25:NM by pulsed-field gel electrophoresis and randomly amplified polymorphic DNA analysis. J. Clin. Microbiol., 36, 652-656.
2. Hamada, K., Tsuji, H. and Shimada, K. (1999): Outbreaks of heat stable enterotoxin-producing Escherichia coli O169 in the Kinki district in Japan: epidemiological analysis by pulsed-field gel electrophoresis. Jpn. J. Infect. Dis. 52, 165-167.
3. Hamada, K., Oshibe, T., Tsuji, H., Yoshida, S. and Aoki, Y. (2000): Outbreaks of heat stable enterotoxin-producing Escherichia coli O169 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. Jpn. J. Infect. Dis., 53, 174-176.
4. Jorgensen, J. H., Turnidge, J.D. and Washington, J. A. (1999): Antibacterial susceptibility tests: dilution and disk diffusion methods. p. 1526-1543. In P.R. Murray, E.J. Baron, M.A. Pfaller, F.C. Tenover and R.H. Yolken (ed.), Manual of Clinical Microbiology. 7th ed. American Society for Microbiology, Washington, D.C.
5. Matsushita, S., Kawamura, M., Takahashi, M., Yokoyama, K., Konishi, N., Hatakeyama, K., Kai, A., Morozumi, S., Morita, K., Watanabe, N., Kanamori, M. and Kudoh, Y. (2001): Increasing fluoroquinolone low-sensitivity in entrotoxigenic Escherichia coli isolated from diarrhea of overseas travelers in Tokyo. J. Jpn. Assoc. Infect. Dis., 75, 785-791 (in Japanese).

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