Jpn. J. Infect. Dis., 55, 2002

Laboratory and Epidemiology Communications

An Outbreak of Heat-Stable Enterotoxin-Producing
Escherichia coli O25: HNM Resistant to Nalidixic Acid Caused byIngestion 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 Healthand Environmental Sciences,
Arata-cho 2-1-29, Hyogo-ku, Kobe 652-0032 and1
Shiga 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 annuallyfrom 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 beenmajor causes of ETEC infections over the past decade inJapan. From September to October in 2002, Hyogo Prefecture(the end of Sept.) and a boundary area of Shiga and Fukui
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-1unch 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 ormanufacturer, were commonly used for the lunches preparedby 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, ’96E.118, isolated from a sporadic infection in 1996 in one of
our laboratories. The strain was lysine-negative, sensitive toall 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 andpositive for STh. The strains were tested for sensitivities to*Corresponding author: Tel: +81-78-511-6787, Fax: +81-78-5317080
205Jpn. J. Infect. Dis., 55, 2002

(A)

Path Typing System (Program No. 5; Bio-Rad Laboratories,Hercules, Calif., USA). Bacterial chromosomal DNAs weredigested with XbaI, BlnI, SfiI, NotI, or SpeI purchased from amanufacturer (Takara Shuzo). It was found that, of thesedigests, 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 ofthe XbaI-digests is shown in Fig. 1B. The strain from asporadic infection (’96-E.118) gave a pattern different fromthose isolates with every digest (Fig. 1A).
The occurrences of the ETEC strains resistant to NA andfluoroquinolones 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 afood poisoning outbreak caused by NA-resistant ETEC inJapan.

(B)

The present case communicated in Japanese appeared inInfectious 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 DNAanalysis. J. Clin. Microbiol., 36, 652-656.
2. Hamada, K., Tsuji, H. and Shimada, K. (1999): Outbreaksof heat stable enterotoxin-producing Escherichia coli
O169 in the Kinki district in Japan: epidemiologicalanalysis 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 electrophoresisof isolates from two outbreaks in 2000 with isolates fromfour outbreaks in 1997-1998. Jpn. J. Infect. Dis., 53, 174176.
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
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5. Matsushita, S., Kawamura, M., Takahashi, M., Yokoyama,K., Konishi, N., Hatakeyama, K., Kai, A., Morozumi,
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Fig. 1. PFGE patterns of digests with five kinds of restriction enzymesof chromosomal DNA of ETEC O25:HNM isolates. The PFGE wasperformed using a Gene Path Typing System, linear Program No. 5.
(A) XbaI-, NotI-, SfiI-, BlnI-, or SpeI-digests of four ETEC O25:HNM
isolates. The strains ’02-E.098 (one of Hyogo strains), ’02-E.142(one of Shiga strains), and ’02-E.143 (the same as ’02-E.142)
were isolated from this outbreak. The strain ’96-E.118 was froma sporadic infection in 1996.
Lanes 1-4: XbaI-, lanes 5-7: NotI-, lanes 8-10: SfiI-, lanes 11-13:BlnI-, and lanes 14-16: SpeI-digests.
Lanes 1, 5, 8, 11, and 14: ’02-E.142, lane 2: ’02-E.143, lanes 3,6, 9, 12, and 15: ’02-E.098, and lanes 4, 7, 10, 13, and 16: ’96E.118. M: λ ladder.
(B) XbaI-digests of 13 isolates from this outbreak. Lane 1: ’02-E.098
and lanes 2-13: 12 strains, all of which were isolated in Hyogo
Prefecture. M: λ ladder.

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 toNA and sensitive to the remaining drugs.
The pulsed-filed gel electrophoresis (PFGE) patterns ofDNA from ETEC O25 isolates were analyzed by using a Gene206