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

Laboratory and Epidemiology Communications

An Outbreak of Enterohemorrhagic Escherichia coli O157 Caused byIngestion of Contaminated Beef at Grilled Meat-Restaurant Chain Storesin the Kinki District in Japan: Epidemiological Analysis byPulsed-Field Gel Electrophoresis
Hidetaka Tsuji, Tomohiro Oshibe, Kokichi Hamada*, Shinya Kawanishi1,Akifumi Nakayama2 and Hiroshi Nakajima3
Infectious Disease Research Division, Hyogo Prefectural Institute of
Public Health and Environmental Sciences,Arata-cho 2-1-29, Hyogo-ku, Kobe 652-0032,
lHimeji City Institute of Environment and Health,
Sakata-machi 3, Himeji 670-0931,
2
Division of Virology and Bacteriology,
Nara Prefectural Institute for Hygiene and Environment,
Ohmori-cho 57-6, Nara 630-8131 and3
Department of Microbiology,
Okayama Prefectural Institute for Environmental Science and Public Health,
Uchio 739-1, Okayama 701-0298
Communicated by Takashi Kawamura(Accepted July 9, 2002)
revealed all the isolates were sensitive to all the 12 antibiotics:
ampicillin, cefotaxime, kanamycin, gentamicin, streptomycin,
tetracycline, trimethoprim, ciplofloxacin, fosfomycin,
chloramphenicol, sulfamethoxazole-trimethoprim, and nalidixicacid.
Pulsed-field gel electrophoretic (PFGE) patterns of the 27
isolates were analyzed by using a gene path typing system(Program No. 5 or No. 27; Nippon Bio-Rad, Tokyo) in amanner reported previously (9). The typical PFGE patternsof XbaI-digested chromosomal DNAs are shown in Fig. 1.
Five PFGE patterns were observed among human isolates
and three patterns among beef isolates. Nineteen out of the23 isolates from humans (lanes 1-7 in Fig. 1A and lanes 1-4,5-6, 9-10, and 12 in Fig. 1B) and one out of four from food(lane 12 in Fig. 1A) gave an identical pattern, whereas fourothers from humans (’02-E.019 in lane 8 in Fig. 1A; ’02E.012 in lane 7, ’02-E.013 in lane 8, and ’02-E.015 in lane 11in Fig. 1B) and three others from food (lanes 9-11 in Fig. 1A;
PFGE patterns in lanes 10 and 11 were identical) weredifferent from the predominant isolates from humans, e.g.,’02-E.004 (lane 1 in Figs. 1A and 1B).
Three kinds of genetically different strains were isolated
from four samples of beef, suggesting that beef contained avariety of EHECs. We speculate that patients were infectedby strains predominant among the EHEC population present
in the food. Probably 19 patients ate food contaminated by
the dominant type ’02-E.004, and at least two of the remaining
four ate beef containing EHEC other than ’02-E.004, such as’02-E.012 or ’02-E.013. The origins of ’02-E.012 and ’02E.013 were probably different from that of ’02-E.004 because
the PFGE patterns were largely different between the twogroups. Meanwhile, we speculate that ’02-E.015 and ’02E.019 were of the same origin as ’02-E.004 and the change
took place in a short period in infected humans owing to thehigh mutability of EHEC (10). This is because ’02-E.015 andEnterohemorrhagic Escherichia coli (EHEC) is a highly
virulent enteric pathogen. Since 1982, it has been foundworldwide including the United States, Europe, and Japan(1). E. coli O157: H7 has been the most prevalent serotype
among them (1). A large-scale outbreak occurred in 1996 inSakai City in Osaka Prefecture (2). The EHEC epidemic has
continued ever since (3-5).
Cattle are considered to be an important reservoir for EHEC,and so beef is one of the major causative foods for EHEC
infection (1,6). From the end of April to the middle of May in
2002, an outbreak of the EHEC O157: H7 appearing first in
Hyogo Prefecture and its boundary areas extended to Osaka,Kyoto, Nara, Wakayama, and Okayama Prefectures. Theoutbreak was due to contaminated raw beef materials procured
by a restaurant center (in Himeji City in Hyogo) for its 63branch restaurants which served grilled beef.
Investigations revealed 43 persons (1 to 77 years of age)
who consumed beef at 19 such branch restaurants dispersed
in Hyogo, Kyoto, Nara, Wakayama, and Okayama within ashort range of time between April 26 and May 1 (except one
person who ate beef on May 9). Twenty-eight among themdeveloped enterocolitic symptoms several days later. The
remaining 15 had no symptoms. We obtained a total of 23
isolates from their fecal specimens. In addition, we obtained
four strains from suspected beef stocked in the central andthree branch restaurants in Hyogo.
The strains were examined for genes encoding verotoxin(VT) using polymerase chain reaction employing EVT- (forVT1) and EVS- (for VT2) primers purchased from Takara
Shuzo Co., Ltd., (Kyoto) (7). A11 the isolates were positive
for both VT1 and VT2, except one isolate (’02-E.013; seelane 8 in Fig. 1B) which was positive for only VT1. Sensi
Disk (Nippon Becton Dickinson Co., Ltd., Tokyo) assay (8)*Corresponding author: Fax: +81-78-531-7080
91Jpn. J. Infect. Dis., 55, 2002
(A)

We thank the authorities concerned of Kyoto Prefecture,
Wakayama Prefecture, Kobe City, Nara City, Okayama City,and Kurashiki City, for the E. coli O157 isolates.
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(B)Fig. 1. PFGE patterns of XbaI-digests of chromosomal DNA ofEscherichia coli O157 isolates. PFGEs were performed using Program
No. 27. Otherwise the isolates from beef are noted in parentheses,
and all the lane wells contain the isolates from human feces.
(A) M:λDNA ladder. Lane 1: ’02-E.004. Lane 2: ’02-E.032.
Lane 3: ’02-E.005. Lane 4: ’02-E.031. Lane 5: ’02-E.027.
Lane 6: ’02-E.028. Lane 7: ’02-E.029. Lane 8: ’02-E.019.
Lane 9: ’02-E.035 (from heart). Lane 10: ’02-E.039 (from liver).
Lane 11: ’02-E.048 (from stringy beef).
Lane 12: ’02-E.044 (from rib).
(B) M: λDNA ladder. Lane 1: ’02-E.004. Lane 2: ’02-E.007.
Lane 3: ’02-E.008. Lane 4: ’02-E.018. Lane 5: ’02-E.010.
Lane 6: ’02-E.011. Lane 7: ’02-E.012. Lane 8: ’02-E.013.
Lane 9: ’02-E.014. Lane 10: ’02-E.016. Lane 11: ’02-E.015.
Lane 12: ’02-E.017.

’02-E.019 were only slightly different from ’02-E.004 inPFGE, and especially because the patient from whom ’02E.015 was isolated and his brother from whom ’02-E.014,
identical to ’02-E.004 in PFGE pattern (compare lanes 9 and 1in Fig. 1B), was isolated ate beef together at the same restaurant.
The outbreak reported here was caused by a large amountof beef procured by a distribution center and supplied to branchrestaurants. This epidemic is a typical diffuse outbreak. The
epidemic demonstrates an inherent risk of this food delivery
system of spreading food poisoning in a large geographicalarea.

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