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Protective Effect of Japanese Green Tea Extract

on Gnotobiotic Mice Infected with an
Escherichia coli O157:H7 Strain
Article in Microbiology and Immunology February 1998
DOI: 10.1111/j.1348-0421.1998.tb02260.x Source: PubMed

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Koichi Takeshi

Tohoku University

Obihiro University of Agriculture and Veteri

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Microbiol. Immunol., 42(2), 125-128, 1998

Protective
Effect of Japanese
Green Tea Extract
on
Gnotobiotic
Mice Infected
with an Escherichia
coli
0157:H7
Strain
Emiko

Isogai*,1,

Hiroshi

Isogai2,

Koichi

Takeshi3,

and

Takeshi

Nishikawa

1Departmentof Preventive Dentistry, School of Dentistry, Health Sciences University of Hokkaido, Hokkaido 061-0212, Japan,
2Division
of Animal Experimentation, Sapporo Medical University, Sapporo, Hokkaido 060-0061, Japan, 'Hokkaido Institute
of Public Health, Sapporo, Hokkaido 060-0819, Japan, and 'Medical Science Laboratory, Hokkaido University of Education,
Ainosato, Sapporo, Hokkaido 002-8074, Japan
Received August 7, 1997; in revised form, October 23, 1997. Accepted November 7, 1997
Abstract: We examined the effect of Japanese green tea extract (JGTE) on enterohemorrhagic
Escherichia
coli (EHEC) 0157:H7 infection in a gnotobiotic mouse model. Gnotobiotic mice inoculated with an
EHEC strain developed neurologic and systemic symptoms, usually culminating in death. In contrast, none
of mice receiving dietary JGTE showed clinical signs or death. This report describes the effect of JGTE,
which includes the inhibition of bacterial growth in vivo. The Shiga-like toxin (SLT) level in the feces of the
JGTE diet group was significantly lower than that of the control group.
Key words: Escherichia

coli 0157, Japanese

green tea, Gnotobiotic mouse

Enterohemorrhagic Escherichia coli (EHEC) strains

are important causes of human hemorrhagic colitis,
hemolytic uremic syndrome (HUS) and encephalopathy
(4, 5, 9). A common histopathological finding in patients
with postdiarrheal sequelae is the destruction of endothelial cells lining small vessels in the colon, kidneys and
central nervous systems (8). Despite efforts to improve
sanitary hygiene, EHEC infection and postdiarrheal
HUS continue to cause excessive morbidity and mortality
in many developing nations.
Japanese green tea contains polyphenolic compounds,
collectively termed catechins (1, 11). These compounds
exist naturally in plants, and they possess potentially
valuable properties (1, 10-12). Japanese green tea
extracts (JGTE) or catechins showed growth inhibition of
both Gram-negative and Gram-positive bacteria when
added into bacterial culture medium (13). The mode of
bactericidal action of catechins was explained by damage
of the lipid bilayer (2). Recently, we described that
JGTE was effective in the inhibition of canine periodontal disease in vivo (3). It has been considered that
JGTE or the custom of drinking green tea may be a
candidate for preventive and therapeutic agent against
EHEC infection.
*Address

correspondence

of Preventive

Dentistry,

University

of Hokkaido,

to Dr . Emiko
School

Isogai,

of Dentistry,

Ishikari-Tobetsu

EHEC O157:H7 strain EDL 931 (9), which produces

both SLT-I and SLT-II, was used for our experiments.
The organisms were incubated in brain-heart infusion
(BHI) medium (BBL, Md., U.S.A.) for 24 hr at 37 C.
Additionally, strain #7 was also used for the experiments. After one passage (incubation for 6 hr at 37 C),
viable counts were determined by plating on BHI agar
media.
Germ-free IQI mice, which originated from ICR mice,
were obtained from Japan Clea Co., Ltd. (Tokyo).
Female and male mice were used at 4-5 weeks of age.
Catechins were extracted from the leaf of Camellia
sinensis with 95% hot ethanol. After filtration and charcoal treatment, samples were analyzed for composition
by high-performance liquid chromatography and gas
chromatography as described previously (3). The compounds contained five major catechins: (-)-epigallocatechin gallate, ( -)-epigallocatechin, (-)-epicatechin
gallate, (-)-epicatechin
and D-(+ )-catechin, 0.4%,
(48 : 32 : 11 : 8 : 1), respectively. It is noteworthy that a
cup of green tea usually contains 0.5 to 1.0 mg/ml
catechins. Therefore, a special diet with JGTE at a concentration of 1.0 mg/g catechin was prepared by Funabashi Farm Co., Ltd. (Chiba, Japan).

Department

Health

Abbreviations:

Sciences

1757, Hokkaido

orrhagic

061-

0212, Japan.

12 5

BHI, brain-heart

Escherichia

JGTE,

Japanese

green

saline;

SLT, Shiga-like

coli;

HUS,

tea extract;
toxin.

infusion;
hemolytic
PBS,

EHEC,
uremic

enterohemsyndrome;

phosphate-buffered

12 6

E. ISOGAI

Table

1. Effect of dietary

JGTE

on EHEC

ET AL

infection

EHEC strains were prepared by washing the bacterial pellet twice in phosphate-buffered saline (PBS, pH
7.4). Bacterial suspensions (0.1 ml in PBS, EHEC strain
EDL 931: 2.0 X 103/mlor 2.0 X 1010/ml,strain #7: 2.1 X
10110/m1)
were deposited intragastrically through a soft
polyethylene catheter. Immediately after inoculation, the
catheter was removed and no further manipulations were
performed. The controls received 0.1 ml of PBS.
Mice were maintained in a level 3 environment. Each
animal was fed daily 3-4 g of a pellet diet, the commercial control diet or the experimental diet containing
JGTE, during the experimental period (from 3 days
before infection). Food and drinking water for mice
were autoclaved before use. After bacterial inoculation, fecal samples were collected from each mouse.
They were suspended at the concentration of 100 mg/ml
in BHI medium, homogenized and plated on CHROMagar 0157 (chromogenic medium for the isolation and
differentiation of E. coli 0157, CHROMagar Microbiology, Paris, France) and BHI agar. In this investigation,
colonizing ability was assessed by the level at which the
strain persisted in mouse feces. The viable count of
EHEC was calculated, and the significance of difference between means of the groups was determined by
unpaired t-test. Probability values of < 0.05 were considered significant.
The SLT antigen level was determined by an ELISA
kit (Novapath EHEC, Japan Bio-Rad Laboratories,
Tokyo). This immunoassay is for the detection of both
SLT-I and -II in stool specimens and cultures. An
Escherichia coli verotoxin detection kit by reversed
passive latex agglutination (Denka Seiken Co., Ltd.,
Tokyo) was also used.
Ten days post-feeding or when signs of disease were
first evident, animals colonized with EHEC EDL 931
were sacrificed and subjected to full necropsy. Tissue
specimens were collected for histological examination.
Specimens were fixed in 10% buffered neutral formalin
and processed by standard procedures. Sections of
paraffin-embedded tissue were stained with hematoxylin
and eosin.

Fig.

1.

Pattern

0157:H7.

At

CHROMagar.
initial
the

of
the
The

colonization
mean

diet. :
109/mouse). :

colonization
time

bacterial
was

CFU

per

IQI

mice
IQI

to

indicated,

gram }

CFU/g

inoculated

limit

of feces.

S.D.

inoculated

mice

samples

detection

102

mice

germ-free

fecal

for
Each

: JGTE
with

EHEC
with

were

EHEC

by

EHEC

plated

assessment
point

diet.

on
of

represents
: Control

0157:H7
0157:H7

(2.0

(2.0 ~

102/mouse).

Mice fed the strain of EHEC became lethargic, occasionally exhibited hind-limb paralysis, stopped eating and
died within 10 days of infection (depending on the size
of the inoculum). Severe bilateral tubular necrosis of the
kidney was observed in the dead mice. Slight microhemorrhages and edematous changes of the brain were also
observed. The capillary endothelial cells were sometimes
swollen in the brains of symptomatic mice. In contrast,
mice given dietary JGTE did not die. Experimental
mice did not show clinical signs or histopathological
changes (Table 1). PBS-treated mice were healthy and
showed no histological changes.
When EHEC strain EDL 931 (inoculum size 2.0 X
109/mouse) was fed individually to IQI mice (control
diet), it colonized equally well at about 109-101()CFU/g
of feces (Fig. 1). In contrast, the JGTE diet was effective
in preventing EHEC colonization; the number of EHEC
dropped to 105 CFU/g of feces. After inoculation of

NOTES

Tat-le

2. SLT level

in the feces

12 7

of experimental

mice

after

EHEC

0157:H-;

strain EDL 931 inoculation

2.0 X 102EDL 931, the number of EHEC in feces began

to rise rapidly on day 1 and a stable level of colonization
(108-109 CFU/g of feces) was maintained in the experimental period. In these mice, the JGTE diet was effective; bacterial growth was inhibited in the intestine.
When EHEC strain #7 (inoculum size 2.1 X 109/mouse)
was fed individually to IQI mice (control diet), it colonized equally at about 1010") CFU/g of feces. In contrast, the JGTE diet was effective in preventing colonization; the number of #7 dropped to 105-106CFU/g of
feces.
The JGTE diet inhibited SLT production (Table 2).
SLT during necropsy was not detectable in either ELISA
or reversed passive latex agglutination. When mice
were fed the commercial control diet, strong SLT production was observed in the gnotobiotic mice. The SLT
level in mice inoculated with 2.0 X 109was significantly
higher than that in mice inoculated with 2.0 X 102EHEC.
The levels of SLT-I and SLT-II were equal in these mice.
Figure 2 shows the SLT level in mice which were fed the
JGTE diet or control diet. The SLT level in the JGTE
diet group was significantly lower than that in the control
diet group during the experimental period (1, 3, 5 and
7 days after infection, respectively; P<0.01).
Our results indicate that JGTE had an inhibitory effect
on bacterial growth of EHEC infection in the gnotobiotic
mouse model. The SLT level in the JGTE diet group was
significantly lower than that in the control group. No
mice with EHEC infection died when fed the JGTE
diet. Toda et al reported the bactericidal activity of
JGTE against EHEC 0157 (7, 14); JGTE at the concentration of 2.5% killed the bacteria completely within
5 hr. It has been reported that tea extracts protect against
experimental infection by Vibrio cholerae 01 (11, 12).
The custom of humans drinking green tea may relate to
the prevention of severe HUS and neurologic symptoms during EHEC infection.

Fig.
feces
Control

2.
of

Effect

of

Japanese

experimental
diet. :

(2~109/mouse). :

green

gnotobiotic
IQI

mice
IQI

mice

tea

extract

on

mice.
inoculated
inoculated

with
with

SLT

level

: JGTE

diet.

EHEC
EHEC

in

0157:

the

H7

0157:H7

(2 ~109/mouse).

JGTE could not eliminate EHEC from infected mice.

The colonization level of EHEC was 104-5/gof feces.
Microscopic changes were not observed in the kidneys or
brains of such asymptomatic mice. Therefore, it is
expected that antibiotic therapy may be effective in such
cases for humans.
The components of JGTE responsible for the observed
protective activity are probably catechins, because purified catechins, especially epigallocatechin gallate, have
bactericidal and anti-toxin activity (2, 6, 12). It has been
reported that bactericidal catechin primarily acts on and
damages bacterial membranes (2).

12 8

E. ISOGAI

References
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1992. Mitogenic activity of ( - )epigallocatechin gallate on
B-cells and investigation of its structure-functionrelationship.
Int. J. Immunopharmacol. 14: 1399-1407.
2) Ikigai, H., Nakae, T., Hara, Y., and Shimamura,T. 1993. Bactericidal catechins damage the lipid bilayer. Biochim. Biophys. Acta 1147: 132-136.
3) Isogai, E., Isogai, H., Kimura, K., Nishikawa, T., Fujii, N.,
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Immunol.39:999-1001.

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