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Appl Environ Microbiol, April 1998, p. 1532-1535, Vol. 64, No. 4
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Enterohemorrhagic Escherichia coli O157:H7 Present
in Radish Sprouts
Yoshinori
Itoh,1
Yoshiko
Sugita-Konishi,1
Fumiko
Kasuga,1
Masaaki
Iwaki,1
Yukiko
Hara-Kudo,1
Noriko
Saito,2
Yoko
Noguchi,3
Hirotaka
Konuma,4 and
Susumu
Kumagai1,*
Department of Biomedical Food
Research,1
Laboratory of Electron
Microscopy,2 and
Department of
Veterinary Science,3 National Institute of
Infectious Diseases, Shinjuku-ku, and
Division of
Microbiology, National Institute of Health Sciences,
Setagaya-ku,4 Tokyo 162, Japan
Received 8 December 1997/Accepted 9 January 1998
 |
ABSTRACT |
Using cultivation, immunofluorescence microscopy, and scanning
electron microscopy, we demonstrated the presence of viable enterohemorrhagic Escherichia coli O157:H7 not only on the
outer surfaces but also in the inner tissues and stomata of cotyledons of radish sprouts grown from seeds experimentally contaminated with the
bacterium. HgCl2 treatment of the outer surface of the hypocotyl did not kill the contaminating bacteria, which emphasized the
importance of either using seeds free from E. coli O157:H7 in the production of radish sprouts or heating the sprouts before they
are eaten.
| |
TEXT |
Hydroponically grown radish
(Raphanus sativus) sprouts were suspected to be the vehicle
of a large outbreak of Escherichia coli O157:H7 infections
in Sakai City, Japan, in 1996 (8) and in small outbreaks in
Yokohama and Gamagori City, Japan, in 1997. To determine whether radish
sprouts could serve as a vehicle for transmission of human-pathogenic
microorganisms, we investigated the proliferation of E. coli
O157:H7 during growth of radish sprouts and demonstrated
previously (4) that in experimentally contaminated radish
seeds, E. coli O157:H7 proliferated 103-
to 105-fold at an early stage of plant growth, including
germination, and that the bacteria were located in edible parts of
radish sprouts. Also, we demonstrated that the edible parts (hypocotyls
and cotyledons) of radish sprouts could be contaminated when the roots
were immersed in a bacterial suspension. These findings suggested that
like alfalfa (2, 3, 5, 7, 9) and mung beans (1), radish sprouts could pose a health risk if seeds or hydroponic water
were contaminated with bacteria which cause food-borne diseases. In
this study we tried to determine whether E. coli O157:H7,
which was considered a nonphytopathogenic bacterium, could exist in a
viable and culturable form in radish sprouts.
Detection of E. coli O157:H7 by immunofluorescence
microscopy.
Based on our previous study (4), we
developed an experimental model for radish sprouts highly contaminated
with E. coli O157:H7. An isolate of E. coli
O157:H7 from a patient from the outbreak in Sakai City (strain 212) was
grown in Trypticase soy broth (Becton Dickinson, Cockeysville, Md.)
overnight at 37°C and then diluted with sterile water to a
concentration of 103 CFU/ml. Radish seeds purchased from a
retail store in Kanagawa Prefecture, Japan, were soaked in the
water containing E. coli O157:H7, and then sprouts
were grown at room temperature (18 to 25°C) for 7 days.
The localization of E. coli O157:H7 in the edible parts of
contaminated radish sprouts was studied by immunofluorescence microscopy and immunoelectron microscopy. For immunofluorescence microscopy, the edible parts were fixed in formalin-acetic acid-70% ethanol (1:1:18), dehydrated in a graded ethanol series, and used to
prepare conventional paraffin sections. The sections were dewaxed with
xylene and then hydrated in a graded ethanol series. After nonspecific
binding was blocked with 2% normal goat serum in phosphate-buffered saline (PBS) for 1 h and the preparations were washed with PBS six
times (5 min each), the sections were incubated for 90 min with primary
rabbit anti-E. coli O157:H7 polyclonal antibody (a kind gift
from K. Tamura, Department of Bacteriology, National Institute of
Infectious Diseases), which has been found not to react to bacteria
other than E. coli. The sections were then washed with PBS.
They were then incubated with the fluorescein-conjugated goat
anti-rabbit immunoglobulin G Fab fragment (Cappel Research Products, Durham, N.C.) for 30 min, washed six times with PBS and three
times with distilled water, and then immersed in 90% glycerol in PBS
for fluorescence microscopy. Rabbit antibody against Salmonella
enteritidis was used as a negative control.
There was positive fluorescence in and on the walls of vessels
and just beneath the epidermis of the hypocotyls of contaminated sprouts (Fig. 1a). This fluorescence was
not detected in noncontaminated sprouts (Fig. 1b), suggesting that
E. coli O157:H7 was present in the inner tissues of
contaminated sprouts.
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FIG. 1.
Contaminated radish sprouts as shown by fluorescence
microscopy. Horizontal sections were prepared and stained with the
antibody against E. coli O157:H7. (a) Contaminated sprouts.
The fluorescence was caused by antibody against E. coli
O157:H7 binding. (b) Noncontaminated control sprouts. (a and b)
Magnification, ×100. (Insets) Magnification, ×400.
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Detection of E. coli O157:H7 by scanning immunoelectron
microscopy.
For scanning electron microscopy, the tissue
specimens were further fixed with 4% paraformaldehyde and 0.25%
glutaraldehyde for 2 h, washed with PBS, and incubated with
1% bovine serum albumin in PBS for 30 min. They were again washed with
PBS and incubated for 1 h with the same primary antibody
solution containing 0.1% bovine serum albumin. After being
washed with PBS, they were dipped in a 15-nm-diameter colloidal
gold-labeled anti-rabbit immunoglobulin (electron microscopy grade;
British Biocell International, Cardiff, United Kingdom) solution,
washed again with PBS, and then postfixed with 2% paraformaldehyde and
2.5% glutaraldehyde for 30 min. After dehydration in a graded acetone
series, they were dried by critical point drying with liquid
CO2 dryer (HCP-2 type; Hitachi), coated with osmium with a
model NL-OPC 80A osmium plasma coater (Nippon Laser & Electronic
Laboratory), and examined by scanning electron microscopy with a
Hitachi model S-5000 microscope.
Numerous bacteria were present on the outer surfaces, including the
stomata, of contaminated sprouts (Fig.
2a). As determined
by the immunostaining
studies, almost all of the bacterial cells
on the outer surfaces of the
radish sprouts which were grown from
contaminated seeds appeared to be
E. coli O157:H7 cells (Fig.
2a, inset). Several kinds of
bacteria were present inside the
hypocotyls of radish sprouts (Fig.
2b). Although
E. coli O157:H7
was not present inside the
hypocotyls as frequently as other species
of bacteria, there certainly
were bacterial cells bound to anti-
E. coli O157:H7 (Fig.
2b,
inset).
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FIG. 2.
Contaminated radish sprouts as shown by scanning
electron microscopy. (a) Outer surface. The arrow indicates a stomate,
and the arrowheads indicate bacteria. (b) Inner surface. The arrow
indicates the xylem, and the arrowheads indicate bacteria. Insets show
sections stained with E. coli O157:H7 antibody. (a and b)
Bar = 5 µm. (Insets) Bar = 50 nm.
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Detection of E. coli O157:H7 by cultivation.
In
order to ascertain whether the E. coli O157:H7 in
the inner tissues of cotyledons was culturable, 4-cm sections of the edible parts (cotyledons and hypocotyls) of the sprouts grown from
E. coli O157:H7-contaminated seeds were subjected to a
common plant surface disinfection procedure involving
HgCl2 (6), and then the sections were examined
for the presence of the bacteria. For surface disinfection, the
sections were immersed in 80% ethanol for 4 to 5 s and in 0.1%
HgCl2 for various periods of time and then washed in
sterile water three times to remove the HgCl2. The excess
water on the surface was wiped off with sterilized filter paper. During
this procedure, each section was held upside down to prevent the cut
edge from absorbing the HgCl2 into the hypocotyl. For 20 sections, the 1.5-cm disinfected part of the hypocotyl was cut out, and
the specimens were then placed on rainbow agar O157 plates (Biolog
Inc., Hayward, Calif.). Another 20 pieces were sliced in half with a
surgical knife from the base of the cotyledon parallel to the vascular
bundle of the hypocotyl, and the sliced surface of each hypocotyl was
placed on an agar plate. After being kept on the agar for 10 min, all
of the sections were removed from the plates, which were then incubated
overnight at 37°C and examined for colony formation. As shown in
Table 1, E. coli O157:H7 was
recovered from the outer surface of 1 of the 20 sections treated with
HgCl2 for 1 or 2 min, but not from any of the sections
treated for 4 or 10 min. On the other hand, when the sliced surfaces
were used, bacteria were detected with two and seven of the sections
treated for 4 and 10 min, respectively. The colonies were confirmed to
be E. coli O157:H7 colonies with an E. coli
O157 latex agglutination assay kit (UNI kit; Unipath Ltd., Hampshire, United Kingdom) and by a PCR performed with DNA probes
for verotoxins (O157 PCR screening set; Takara Biomedicals, Tokyo,
Japan).
Ten unsliced hypocotyl sections which had been placed on an agar plate
were crushed by hand to push out their contents into
10 ml of
Trypticase soy broth before stomaching with a model 400
Stomacher
(A. J. Seward, London, United Kingdom), and the number
of
E. coli O157:H7 cells in the homogenate was determined by using
rainbow agar O157 plates (Table
1).
E. coli O157:H7 was
detected
in the homogenate of the sections treated with
HgCl
2 for 4 or
10 min. These results indicated that
E. coli O157:H7 was present
in a viable form in the
hypocotyl at a location that the surface
disinfectant HgCl
2
could not reach in 10 min.
Thus, all of the results demonstrated that
E. coli O157:H7
was present not only on outer surfaces but also in inner tissues
of
cotyledons and in stomata when radish sprouts were raised from
E. coli O157:H7-contaminated seeds; furthermore, HgCl
2
treatment
of the outer surfaces of cotyledons did not kill the
bacteria.
The viable
E. coli O157:H7 found after
HgCl
2 treatment might have
been bacteria that were present
around the vessels and in the
stomata of cotyledons. The
ineffectiveness of disinfecting the
outer surfaces of cotyledons
indicates that using seeds free from
E. coli O157:H7 in the
production of radish sprouts or heating
the sprouts to kill the
bacteria is critical for preventing
E. coli O157:H7
infections caused by eating raw radish sprouts.
| |
ACKNOWLEDGMENTS |
We thank Yuichi Takikawa (Shizuoka University) for valuable and
helpful comments.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biomedical Food Research, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162, Japan. Phone: 81-3-5285-1111, ext. 2300. Fax: 81-3-5285-1176. E-mail:
skumagai{at}nih.go.jp.
| |
REFERENCES |
| 1.
|
Andrews, W. H.,
P. B. Mislivec,
C. R. Wilson,
V. R. Bruce,
P. L. Poelma,
R. Gibson,
M. W. Trucksess, and K. Young.
1982.
Microbial hazards associated with bean sprouting.
J. Assoc. Off. Anal. Chem.
65:241-248[Medline].
|
| 2.
|
Beuchat, L. R.
1997.
Comparison of chemical treatment to kill salmonella on alfalfa seeds destined for sprout production.
Int. J. Food Microbiol.
34:329-333[Medline].
|
| 3.
|
Como-Sabetti, K.,
S. Reagan,
S. Allaire,
K. Parrott,
C. M. Simonds,
S. Hrabowy,
B. Ritter,
W. Hall,
J. Altamirano,
R. Martin,
F. Downes,
G. Jennings,
R. Barrie,
M. F. Dorman,
N. Keon,
M. Kucab,
A. Al Shab,
B. Robinson-Dunn,
S. Dietrich,
L. Moshur,
L. Reese,
J. Smith,
K. Wilcox,
J. Tilden,
G. Wojtala,
J. D. Park,
M. Winnett,
L. Petrilack,
L. Vasquez,
S. Jenkins,
E. Barrett,
M. Linn,
D. Woolard,
R. Hackler,
H. Martin,
D. McWilliams,
B. Rouse,
S. Willis,
J. Rullan,
G. Miller, Jr.,
S. Henderson,
J. Pearson,
J. Beers,
R. Davis,
D. Saunders, and Food-Borne and Diarrheal Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention.
1997.
Outbreaks of Escherichia coli O157:H7 infection associated with eating alfalfa sprouts Michigan and Virginia, June-July 1997.
Morbid. Mortal. Weekly Rep.
46:741-744.
|
| 4.
|
Hara-Kudo, Y.,
H. Konuma,
M. Iwaki,
F. Kasuga,
Y. Sugita-Konishi,
Y. Ito, and S. Kumagai.
1997.
Potential hazard of radish sprouts as a vehicle of Escherichia coli O157:H7.
J. Food Prot.
60:1125-1127.
|
| 5.
|
Jaquette, C. B.,
L. R. Beuchat, and B. E. Mahon.
1996.
Efficacy of chlorine and heat treatment in killing Salmonella stanley inoculated onto alfalfa seeds and growth and survival of the pathogen during sprouting and storage.
Appl. Environ. Microbiol.
62:2212-2215[Abstract].
|
| 6.
|
Johnston, A., and C. Booth (ed.).
1983.
, p. 30-45.
Plant pathologist's pocketbook, 2nd ed.
Commonwealth Agricultural Bureaux, Slough, United Kingdom.
|
| 7.
|
Mahon, B. E.,
A. Pönkä,
W. N. Hall,
K. Komatsu,
S. E. Dietrich,
A. Siitonen,
G. Cage,
P. S. Hayes,
M. A. Lambert-Fair,
N. H. Bean,
P. M. Griffin, and L. Slutsker.
1997.
An international outbreak of salmonella infections caused by alfalfa sprouts grown from contaminated seeds.
J. Infect. Dis.
175:876-882[Medline].
|
| 8.
|
National Institute of Infectious Diseases and Infectious Diseases Control Division, Ministry of Health and Welfare of Japan.
1997.
Verocytotoxin-producing Escherichia coli (enterohemorrhagic E. coli) infection, Japan, 1996-June 1997.
Infect. Agents Surveillance Rep.
18:153'-154'.
|
| 9.
|
Pönkä, A.,
Y. Andersson,
A. Siitonen,
B. de Jong,
M. Jahkola,
O. Haikala,
A. Kuhmonen, and P. Pakksala.
1995.
Salmonella in alfalfa sprouts.
Lancet
345:462-463[Medline].
|
Appl Environ Microbiol, April 1998, p. 1532-1535, Vol. 64, No. 4
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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