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

Laboratory and Epidemiology Communications

A Case of Hemolytic-Uremic Syndrome Associated withShiga Toxin 2-Producing Escherichia coli O121 Infection Caused
by Drinking Water Contaminated with Bovine Feces
Jun Yatsuyanagi*, Shioko Saito and Isao Ito
Akita Prefectural Institute of Public Health,6-6 Sensyu kubota-machi, Akita 010-0874
Communicated by Kazuo Kato
(Accepted November 11, 2002)

Shiga toxin (stx)-producing Escherichia coli (STEC) is an
important cause of hemorrhagic colitis (HC) and hemolyticuremic syndrome (HUS) in humans (1). STEC O157:H7 isthe serotype most commonly isolated from HUS patients, butnon-O157 STEC have also been implicated in HC and HUS
cases (2,3). Cattle are known as carriers of both O157 andnon-O157 STEC (4,5). STEC infection is primarily a food-borne
disease. Foods, such as unpasteurized milk, undercooked
hamburgers, salad, and white radish sprouts, have been
identified as vehicles in STEC infection outbreaks (6).
However, vehicles have rarely been identified in sporadic
cases of either O157 or non-O157 STEC infections. We report
here an HUS case in Akita Prefecture that was caused by anstx 2 gene (stx2)-positive STEC O121. The case involved
family members and the vehicle was traced to contaminateddrinking well water.
Mackonkey agar supplemented with Cefixime Tellurite
supplement (Oxoid, Basingstoke, England) (CT-Mac) wasused to select STEC O121, which formed distinctive clear
and slightly brown-colored colonies on the agar. CT-Mac andEC broth were inoculated with stool specimens and incubatedovernight at 37°C. Three liters of drinking water samples were
filtered through 0.45 μm pore-size membrane filters, whichwere then placed in trypticase soy broth and incubatedovernight at 35°C. Environmental swabs were cultured andincubated in the same manner as the membrane filters.
Manure and bovine feces were cultured and incubated in thesame manner as the stool specimens. Each culture broth wasanalyzed by PCR using VTcom primers in order to screen forstx-positive strains (7). The stx and E. coli attaching andeffacing (eaeA) gene were typed and detected, respectively,by PCR as described previously (7). Pulsed-field gel electrophoresis (PFGE) analysis of the STEC isolates was performed
using XbaI digests of the chromosomal DNAs (7).
A 2-year-old girl developed watery diarrhea on October 3,
2000, which progressed to bloody diarrhea on October 5. She
was hospitalized on October 6, 2000, and developed HUS onOctober 7. The hospital laboratory sent us an E. coli isolate
from the patient’s stool for stx evaluation on October 7. The
strain was identified as stx2-positive STEC O121. A hospital
doctor notified the local public health center of the STECO121 HUS case on October 7. Stool specimens collected fromthe family members by the officials were sent to us on October

7 for STEC examination.
As shown in Table 1, the examination revealed that thepatient’s father and brother were infected with stx2-positive
STEC O121, while the patient’s mother and aunt wereinfected with stx1-positive STEC O91, indicating that the case
was an intra-family infection involving two STEC serotypes.
None of the individuals infected with STEC O121 or O91
showed clinical symptoms. All of the STEC O121 isolates
were eaeA positive, while neither of the two STEC O91
isolates were positive for the gene.
The local public health center officials’ epidemiologicalinvestigation revealed that the family utilized un-chlorinatedwell water that was pumped up at a well and sent through 80
m long piping to their house. Seven cattle were raised in acattle shed near the well. The well was impaired so that
surface water could flow into the well. The local public healthcenter officials collected water samples, environmental
samples, and bovine feces and sent them to us for STEC
examination. Table 2 summarizes the results of the STEC
evaluation of these samples. The drinking water samples
collected on October 7 and the water of the well and the tap
water in the patient’s house collected on October 11 werepositive for stx2-harboring STEC O121. The tap water sample
collected on October 10 was STEC negative, indicating thatthe water was intermittently contaminated with STEC O121.
Cows No. 5 and No. 7 were, respectively, positive for stx2positive STEC O121 and stx1-positive STEC O111. No othercattle were positive for STEC, including STEC O91.
The stx2-positive STEC O121 strains isolated from thepatient, the family members, water samples, and bovine
feces were subjected to XbaI PFGE pattern analysis. The
STEC O121 strains had identical XbaI PFGE patterns,
indicating that they were of a common origin. From these

Table 1. Results of evaluation for STEC in stool specimens
obtained from patient’s family on October 7, 2000

*Corresponding author: Tel: +81-18-832-5005, Fax: +81-18-8325938, E-mail: jyatsu@spica.freemail.ne.jp
174

Relationship to patientResults

FatherMother
BrotherGrandmother
Great-grandmother
AuntCousin

STEC O121 stx2+ positive
STEC O91 stx1+ positive
STEC O121 stx2+ positive
STEC negative
STEC negative
STEC O91 stx1+ positive
STEC negativeJpn. J. Infect. Dis., 55, 2002
Table 2. Results of evaluation for STEC in drinking water, environmental samples, and bovine feces
SamplesCollection date

Results

Drinking water
Drinking water1)
Drinking water2)
Drinking water1)
Drinking water1)
Drinking water2)
Manure No.1
Manure No.2
Manure No.3
Environmental swabs3)
Tap water of cow house
Ditch water No.14)
Ditch water No.24)
Ditch water No.34)
Bovine No.1 feces
Bovine No.2 feces
Bovine No.3 feces
Bovine No.4 feces
Bovine No.5 feces
Bovine No.6 feces
Bovine No.7 feces

Oct. 7, 2000
Oct. 10, 2000
Oct. 11, 2000
Oct. 11, 2000
Oct. 31, 2000
Oct. 31, 2000
Oct. 7, 2000
Oct. 31, 2000
Oct. 31, 2000
Oct. 7, 2000
Oct. 31, 2000
Oct. 10, 2000
Oct. 31, 2000
Oct. 31, 2000
Oct. 31, 2000
Oct. 31, 2000
Oct. 31, 2000
Oct. 31, 2000
Oct. 31, 2000
Oct. 31, 2000
Oct. 31, 2000

STEC O121 stx2+ positive
STEC Negative
STEC O121 stx2+ positive
STEC O121 stx2+ positive
STEC Negative
STEC Negative
STEC Negative
STEC Negative
STEC Negative
STEC Negative
STEC Negative
STEC Negative
STEC Negative
STEC Negative
STEC Negative
STEC Negative
STEC Negative
STEC Negative
STEC O121 stx2+, STEC O111 stx1+ positive
STEC Negative
STEC O121 stx2+, STEC O111 stx1+ positive

1)

Collected at the well.
Tap water collected at the patient’s house.
3)
Obtained in the patient’s house: 12 swab samples were collected and subjected to STEC examination.
4)
Collected downstream of the cow house drainage influx point.
2)

response to non-O157 vero toxin-producing Escherichia
coli in patients with hemolytic uremic syndrome. J.
Infect. Dis., 174, 1028-1039.
4. Wells, J. G., Shipman, L. D., Greene, K. D., Sowers, E.
G., Green, J. H., Cameron, D. N., Downes, F. P., Martin,M. L., Griffin, P. M., Ostroff, S. M., Potter, M. E., Tauxe, R.
V. and Wachsmuth, I. K. (1991): Isolation of Escherichia
coli serotype O157:H7 and other shiga-like-toxin-producing E. coli from dairy cattle. J. Clin. Microbiol., 29, 985989.
5. Montenegro, M. A., Bülte, M., Trumpf, T., Aleksic, S.,
Reuter, G., Bulling, E. and Helmuth, R. (1990): Detection and characterization of fecal verotoxin-producing
Escherichia coli from healthy cattle. J. Clin. Microbiol.,28, 1417-1421.
6. Michino, H., Araki, K., Minami, S., Nakayama, T., Ejima,Y., Hiroe, K., Tanaka, H., Fujita, N., Usami, S.,
Yonekawa, M., Sadamoto, K., Takaya, S. and Sakai, N.
(1998): Recent outbreaks of infections caused by Escherichia coli O157:H7 in Japan. In Kaper, J. B. and O’Brien,
A. D. (ed), Escherichia coli O157:H7 and other shiga
toxin-producing E. coli strains. American Society for
Microbiology, Washington, D.C.
7. Yatsuyanagi, J., Kinouchi, Y., Saito, S., Suzuki, Y., Sato, H.
and Miyajima, Y. (1999): Epidemiological characteristics
and virulence factor of verotoxin-producing Escherichia
coli O121:H19 isolated in Akita prefecture in July 1997.
J. Jpn. Assoc. Infect. Dis., 73, 218-224 (in Japanese).
8. McCarty, T. A., Barrett, N. L., Hadler, J. L., Salsbury,B., Howard, R. T., Dingman, D. W., Brinkman, C. D.,Bibb, W. F. and Cartter, M. L. (2001): Hemolyticuremic syndrome and Escherichia coli O121 at a lake inConnecticut, 1999. Pediatrics, 108, E59.
9. Proesmans, C. G., Dediste, A., Jacobs, F., Van de Walle,

and the epidemiological investigation results, we concluded
that the well water contaminated with stx2-positive STECO121 was the vehicle in this infectious case. The well water
was possibly contaminated by the influx of surface watercontaining the contaminated bovine feces.
O121 is a rare STEC serotype associated with humandiseases in Japan, but a few infectious cases have been
identified yearly in this prefecture since 1997 (7). This is thefirst HUS case associated with STEC O121 and none of theother non-O157 STEC strains has been associated with thedevelopment of HUS in this prefecture. HUS outbreakassociated with STEC O121 infection was also reported inConnecticut, U.S. in 1999 (8) and a few other cases of HUS
associated with STEC O121 have also been reported (9,10).
O121 should be considered an important STEC serotype thatcauses HUS, a serious public health problem.
We previously reported a familial infectious case associatedwith stx1-positive STEC O103:H2, and a cow was identified
as an infectious source as in this case (11). The significanceof cattle as infectious sources of human STEC infections
should be further investigated.
This article appeared in the Infectious Agents Surveillance
Report, vol. 22, no. 6, p. 141-142, 2001 in Japanese.
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Jpn. J. Infect. Dis., 55, 2002
J., Mertens, A., Ramet, J. and Lauwers, S. (1999):
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