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Infection and Immunity, February 2001, p. 1101-1108, Vol. 69, No. 2
Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186-86501
Research Institute, International Medical Center of Japan,
1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655,2
School of Medicine, Tsukuba University, Tsukuba
305-8575,3 and National Institute of
Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo
162-8640,4 Japan
Received 24 July 2000/Returned for modification 8 September
2000/Accepted 17 November 2000
We examined colonization patterns of Shiga toxin-producing
Escherichia coli (STEC), concentrations of Shiga toxins
(Stxs) and specific immunoglobulin A (lgA) against Stxs and STEC
bacterial cell surface antigen in various portions of the
gastrointestinal tract in an infant rabbit infection model. After
inoculation of 3-day-old infant rabbits with STEC strain
89020087 at low doses (~103 CFU/body), numbers of
colonizing STEC bacteria and concentrations of Stxs in the intestine
increased dramatically and the animals developed diarrhea within a
couple of days after infection. Daily administration of
Lactobacillus casei from the day of birth dramatically decreased the severity of diarrhea and lowered STEC colonization levels
in the gastrointestinal tract 100-fold day 7 after infection. Both Stx1
and Stx2 concentrations in the intestines and histological damage to
the intestinal mucus induced by STEC infection were decreased by the
administration of L. casei. Examination of the concentrations of volatile fatty acids and pH of the intestinal contents revealed that the protective effect of L. casei
administration against STEC infection was not due to fermented products
such as lactic acid in the gastrointestinal tract. Administration of L. casei increased levels of lgAs against Stx1, Stx2, and
formalin-killed STEC cells in the colon approximately two-, four-, and
threefold, respectively, compared with those of the untreated controls
by day 7 after infection. These results suggest that administration of
L. casei strain Shirota enhances the local immune responses to STEC cells and Stxs and leads to elimination of STEC and thus decreases Stx concentrations in the intestines.
Shiga toxin-producing
Escherichia coli (STEC) O157:H7 is characterized by
production of two kinds of Shiga toxins (Stxs), Stx1 and Stx2, which
cause hemorrhagic colitis followed, in some cases, by hemolytic-uremic
syndrome and central nervous system complications (4, 25, 45, 51,
57). The mechanism of infection, however, remains unclear; thus,
preventive and therapeutic measures have not been established.
A complex intestinal microflora provides protection against
colonization by many pathogenic infectious agents (for reviews, see
references 8 and 60). Increased susceptibility to STEC infection due to treatment of mice with antibiotics can be explained by
disruption of the normal indigenous intestinal microflora (15, 27, 32, 59, 61). A hypothesis that foods fermented by lactobacilli help maintain a balance between lactobacilli and the
indigenous intestinal flora, originally proposed by Metchnikoff (33) as early as 1908, has been supported by many
investigators (3, 12, 41, 54), and probiotic
Lactobacillus strains have been shown to protect against
infection by pathogens such as Shigella sonnei (2,
35), Listeria monocytogenes (39, 52,
53), E. coli (40), Salmonella
enterica serovar Typhimurium (13, 16) and rotavirus
(20, 29).
In this study, by using newborn rabbits as an experimental infection
model, the protective effects of oral administration of probiotic
Lactobacillus casei strain Shirota (24) against STEC infection were investigated.
Bacterial strain and growth conditions.
Clinically isolated
STEC O157:H7 strain 89020087, which produces both Stx1 and Stx2, was
used throughout. Cells were grown overnight in Casamino Acids-yeast
extract (CA-YE) broth (11) at 37°C. L. casei
strain Shirota is a stock culture of the Yakult Central Institute for
Microbiological Research and was grown overnight anaerobically at
37°C in De Man-Rogosa-Sharpe (MRS) broth (Difco Laboratories,
Detroit, Mich.) which had been purged of oxygen with nitrogen.
Animals.
Specific-pathogen-free Japanese White rabbits
(pregnant for 24 days) were purchased from Kitayama Rabesu Co. Ltd.,
Ina, Japan. After parturition, neonatal rabbits 1 day old were isolated
from their dams and housed in a clean isolator system with automatic control of temperature (25 ± 0.5°C), humidity (55% ± 5%),
and light conditions (14 h of light and 10 h of darkness) and were kept individually in polypropylene cages (CLEA Japan, Inc., Tokyo, Japan) with stainless steel lids and sterilized paper bedding (CLEA).
They were fed 20% pasteurized (65°C, 30 min) artificial milk for pet
(PetAg, Inc., Hampshire, Ill.) intragastrically by a soft
polyethylene catheter tube (Fuchigami Co., Kyoto, Japan) attached
to a 5-ml syringe twice a day throughout the experiment (1, 34,
36).
Preparation of bacterial suspension and inoculation of infant
rabbits.
After two passages in CA-YE broth, STEC was grown
overnight in CA-YE broth at 37°C on a shaker for 18 h and the
bacterial cells were washed once with saline by centrifugation. The
precipitating bacterial cells were then suspended and diluted with
saline to an optical density at 600nm (OD600) of 0.1 as
determined with a spectrophotometer (U-2001; Hitachi Co. Ltd., Tokyo,
Japan). This bacterial suspension was then diluted 104-fold
with saline to make a suspension of ~2 × 103
CFU/ml. An aliquot of 500 µl of the suspension was given orally to
infant rabbits by using the catheter tube described above. For exact
enumeration of an inoculum, this bacterial suspension was serially
diluted with phosphate-buffered saline (PBS) and plated on Trypticase
soybean agar plates (BBL Microbiology Systems, Cockeysville, Md.) and
incubated overnight at 37°C.
Preparation of milk supplemented with L. casei.
After
two passages in MRS broth at 37°C anaerobically, L. casei
strain Shirota was grown in MRS broth overnight and the culture was
centrifuged at 1,700 × g for 20 min (Kubota Co. Ltd.,
Tokyo, Japan). The bacterial pellet was resuspended in sterile
artificial milk to a concentration of 108 CFU/ml. For exact
enumeration of an inoculum, L. casei-supplemented milk was
serially diluted with PBS, plated on lactitol-LBS-vancomycin (LLV) agar
plates (66), and incubated aerobically for 48 h at 37°C. LLV agar contains the following (per liter): tryptic peptone (BBL), 10 g; yeast extract (Difco), 5 g;
KH2PO4, 6 g; triammonium citrate, 2 g; sodium acetate · 3H2O, 25 g;
MgSO4 · 7H2O, 0.58 g;
MnSO4 · 2H2O, 0.12 g;
FeSO4 · 7H2O, 0.034 g; Tween 80, 1 g; lactitol, 20 g; Bacto Agar (Difco), 15 g; vancomycin
hydrochloride (Sigma Chemical Co., St. Louis, Mo.), 10 mg. The pH of
the medium was adjusted to 6.1.
Experimental design.
Animals were divided into two groups.
One was fed sterilized artificial milk supplemented with L. casei strain Shirota at a concentration of 108 CFU/ml
(total number, 18), and the other was fed only sterilized artificial
milk (total number, 14). Feeding was carried out twice a day (10:00 am
and 6:00 pm), from day 1 after birth until the morning of the day the
animals turned 10 days old. Exactly the same amounts of the milk
preparations were given to the animals of both groups. The amounts were
as follows: 3.0 ml twice a day on days 1 and 2, 5 ml twice a day on
days 3 and 4, and 7.0 ml twice a day on days 5 and 6 and once in the
morning of day 7. When the animals became 3 days old, they were
individually inoculated with 0.5 ml of STEC suspension with a sterile
flexible polyethylene catheter tube. After infection, they were weighed
daily and checked for diarrhea until day 7 after infection. Diarrhea
was classified into the following severity groups: I, no diarrhea; II,
slight diarrhea (mixed soft and hard stools); III, mild diarrhea (feces stuck to perineum and hind legs); IV, severe diarrhea (feces stuck to
hind legs, wet tail, and prolapse of rectum).
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.2.1101-1108.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Protective Effect of Lactobacillus casei Strain
Shirota on Shiga Toxin-Producing Escherichia coli
O157:H7 Infection in Infant Rabbits
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Bacteriological examination. The removed gastrointestinal tracts were segmented into stomach, small intestine, cecum, and colon, and each section was weighed and homogenized in 5 ml of ice-cold saline. The suspension was centrifuged at 17,000 × g for 15 min (Microfuge R; Beckman Instruments Inc., Palo Alto, Calif.), and the pellet was washed once with PBS by centrifugation, resuspended in PBS, and diluted 10-fold serially with PBS for evaluation of bacterial CFU; 100-µl samples of the suspension were spread onto sorbitol MacConkey agar plates (Eiken Chem. Co. Ltd., Tokyo, Japan) and LLV agar plates for counting of STEC and L. casei strain Shirota colonies, respectively. Sorbitol MacConkey agar plates and LLV agar plates were incubated at 37°C overnight and for 48 h, respectively.
Gastrointestinal pH and concentrations of VFAs.
To examine
pH values and concentrations of VFAs in the gastrointestinal contents
of test animals, sections of gastrointestinal tracts were treated as
described above. After homogenization, samples were centrifuged at
1,700 × g for 30 min and supernatants were filtered
(pore size, 0.8 µm). The pH of filtrates was measured with a
hand-held pH meter (B-212; Horiba Ltd., Kyoto, Japan). For VFA
analysis, supernatants were mixed with 10% (vol/vol ratio, 9:1)
trichloroacetic acid, incubated overnight at 4°C, and centrifuged at
20,000 × g for 10 min. After filtration (pore size,
0.22 µm), samples were analyzed by high-pressure liquid
chromatography (TOA Electronics Ltd., Tokyo, Japan). The millimolar
concentration of lactic acid in an undissociated form was calculated by
the following formula: undissociated lactic acid = millimolar
total lactic acid/(1 + 10pH
pKa).
Stx assay. Concentrations of Stx1 and Stx2 in intestinal contents were assayed by a bead enzyme-linked immunosorbent assay (ELISA) method described previously (42, 67). Briefly, sections of gastrointestinal tracts were treated as described above. After homogenization, samples were centrifuged at 1,700 × g for 30 min and the supernatants were filtered (pore size, 0.8 µm). The filtrates were diluted twofold with PBS supplemented with 2% bovine serum albumin (BSA; Nakalai Tesque Inc., Kyoto, Japan) and 0.02% NaN3. Solid-phase beads coated with rabbit polyclonal lgG against purified Stx1 or Stx2 were added, and the mixtures were incubated at 37°C for 1 h. After being washed twice with PBS, the beads were incubated with goat anti-rabbit lgG-Fab'-horseradish peroxidase (HRP) conjugate diluted in PBS containing 2% BSA at 37°C for 1 h. After the beads had been washed twice with PBS, the enzymatic activity of the HRP bound to the beads was assayed by addition of 3, 3', 5, 5'-tetramethylbenzidine as a substrate and the OD450 was measured with a spectrophotometer. Stx concentrations in intestinal contents were calculated relative to a standard curve of purified Stx1 or Stx2 and expressed as nanograms per gram of tissue.
lgA assay. Samples were prepared as described above. Determination of lgA levels was performed as described by Keren et al. (22) and MacQueen et al. (31). Briefly, 96-well polystyrene microtiter plates (Maxisorp; Nalge Nunc International, Roskilde, Denmark) were coated with 100-µl portions of purified Stx1 or Stx2 at a concentration of 2.5 µg/ml in carbonate buffer (coating buffer), pH 9.6, or with a formalin-killed STEC whole-cell suspension diluted with coating buffer to an OD600 of 0.01. Purified Stx1 and Stx2 were prepared as described previously (38, 43, 65). Intact STEC cells were prepared as follows. STEC was cultured in CA-YE broth at 37°C for 18 h, washed twice with saline by centrifugation, suspended in saline containing 0.5% neutralized formalin, stored at room temperature for 3 days, and then washed three times with saline to remove free Stxs. Plates were incubated overnight at 4°C in a moist chamber to prevent evaporation. After incubation, wells were washed three times with PBS containing 0.02% Tween 20 (PBST) to remove the free Stx solution or STEC suspension. Wells were sequentially incubated with samples, 1/500 goat anti-rabbit lgA (Nordic Immunological Laboratories, Tilburg, The Netherlands) coupled to HRP, and finally a 2, 2'-azino+bis[3-ethylbenzthiazoline sulfonate (6)] (ABTS) substrate (Sigma). Wells were washed with PBST three times between incubations and reacted with PBST supplemented with 1% BSA at 37°C for 1 h before addition of the primary samples and before addition of the secondary antibody conjugate to inhibit nonspecific adherence. The OD405 of wells was measured in a microplate reader (Inter Med Immunomini NJ-2300; Nalge Nunc International), and results were expressed as OD405 units per gram of tissue. The background OD405 for this ELISA was 0.2/g of tissue.
Histopathological examinations. Segments of the ileum, cecum, and distal colon were surgically removed, washed, and then fixed in 4% buffered formalin, blocked in paraffin, and sectioned. Sections were stained with hematoxylin and eosin, and examined under a microscope for pathological changes such as exfoliation, necrosis, edema, STEC cells attached to epithelial cells, and pseudoeosinophil infiltration. In addition, immunohistochemical staining of STEC cells was performed using goat anti-E. coli O157:H7 antibody (Kirkegaard & Perry Laboratories Inc., Gaithersburg, Md.), biotinylated rabbit anti-goat immunoglobulin, and 3, 3-diaminobenzidine in a chromogen solution (DAKO Japan, Kyoto, Japan).
Statistical examinations. Statistical differences between the control group and the L. casei-treated group were evaluated with the cumulative chi-square test for the incidence of diarrhea and with Student's t test for other benchmarks. P < 0.05 was considered significant.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Clinical symptoms.
Most of the infected rabbits began to show
diarrhea within 3 days. No animal in either group developed bloody
diarrhea. By day 7 after infection, 77.3% of the rabbits in the
control group suffered from severe diarrhea whereas only 16.0% of the
rabbits in the L. casei-treated group showed severe diarrhea
(Table 1). On the whole, the severity of
diarrhea was less pronounced in the L. casei-treated group
than in the control group. L. casei did not delay the onset
of diarrhea. No clinical symptoms were observed in the group fed
L. casei alone (data not shown).
|
Colonization and distribution of STEC in the gastrointestinal
tract.
Viable STEC counts in the gastrointestinal tracts of mice
in the control group increased dramatically to
107~109 CFU/g of organ homogenate by
day 7 after infection (Fig.
1a). Although STEC
colonization levels in the L. casei-treated group were
similar to those in the control group by day 4 after infection, they
were approximately 100-fold lower than in the control group by day 7 after infection. Viable counts of L. casei in the
gastrointestinal tract were sustained at higher than 108
CFU/g of organ throughout the experimental period (Fig. 1b).
|
pH and lactic acid concentration in the gastrointestinal
tract.
No differences in pH of the gastrointestinal contents on
day 7 after infection was observed between the control and
L. casei-treated groups (Table
2). In both groups, the pH of the stomach
contents was approximately 5.1 and was higher than 6.5 in other parts
of the intestine. Concentrations of lactic acid in the gastrointestinal tract were slightly higher in the L. casei-treated group
than in the control group (Table 2). In both groups, concentrations of
undissociated lactic acid in the gastrointestinal tract were less than
1.0 mM (data not shown).
|
Stx concentrations in the intestinal contents.
Concentrations
of both Stx1 and Stx2 in the cecum and colon in the control group
increased at day 4 after infection, and further significant increases
were observed at day 7. In contrast, Stx concentrations in the L. casei-treated group increased by day 4 after infection but
showed no further increase thereafter (Fig. 2). Stx concentrations in the small
intestine were quite low compared with those in the cecum and
colon in both groups.
|
Specific IgA concentrations in the intestinal contents.
In the
small intestine, concentrations of IgA against Stx and STEC cells
remained low and no differences were observed between the control and
L. casei-treated groups (Fig.
3). In the colon, in contrast,
concentrations of IgAs against Stx1, Stx2, and intact STEC cells in the
L. casei-treated group increased approximately two-, four-,
and threefold, respectively, over those in the control group.
|
Histopathology.
In the control group, STEC infection led to
vacuolation of epithelial cells (arrows) with attached STEC cells
(arrowhead) on top of the villi and necrosis due to massive growth of
STEC cells (asterisk) in the small intestine (Fig.
4A), exfoliation of epithelial cells
(large arrows), pseudoeosinophil infiltration (arrowheads),
and mitotic activity (small arrow) in the cecum (Fig. 4B), and
exfoliation and necrosis (arrow) in the colon due to STEC cells
attached to epithelial cells (arrowheads) (Fig. 4C). In contrast, no
notable pathological changes except for low mitotic activity in the
cecum and slight exfoliation of the epithelium in the colon were
observed in the L. casei-treated group (Fig. 4D to F).
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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A number of experimental animal models have been proposed to study the pathogenicity of STEC (63), including gnotobiotic mice (17, 18, 64), streptomycin-treated mice (15, 27, 32, 61, 62), gnotobiotic piglets (14, 59), newborn chickens (5), and infant rabbits (26, 36, 44, 50). While mouse models have been used most frequently to date, most are not appropriate because high doses of inoculum, more than 107 CFU/body, are usually required for establishment of STEC infection, and often mice do not develop diarrhea even when they are inoculated with such high doses of STEC (21, 23). On the contrary, the rabbit model has the major advantages of the reproducibility of diarrhea and susceptibility to STEC infection and Stx toxicity (7, 26, 36, 44, 50, 55). In this study, we used the infant rabbit model with administration of a lower number of bacteria (~103 CFU/body) than reported previously (36, 44, 50).
STEC infection in the infant rabbit model occurs reproducibly with an inoculum as small as 103 CFU/body (Table 1). Concentrations of Stxs in the gastrointestinal tract also increased in proportion to the time (days) after infection (Fig. 2). Both the number of colonizing bacteria and the concentration of Stxs were higher in the large intestine than in the small intestine. In particular, the concentration of Stx2 was significantly lower in the small intestine than in the cecum and the colon. Histological studies also showed that damage to the intestinal epithelium was more pronounced in the cecum and the colon than in the small intestine (Fig. 4). These findings are in agreement with the previously reported results that showed that the major colonization site of STEC in rabbit is the large intestine rather than the small intestine (36, 44, 50).
In rabbits administered L. casei strain Shirota, although levels of STEC colonization increased to levels similar to those of the control group by day 4 after infection, thereafter they remained unchanged or even decreased 100-fold by day 7 (Fig. 1). Concentrations of Stx1 and Stx2 were also lower in the L. casei-treated group, in parallel with decreased STEC colonization, compared with the controls (Fig. 2). Stx2 levels, in particular, were significantly lower in rabbits given L. casei in addition to STEC. The differences in diarrhea severity (Table 1) and mucosal damage in the intestines (Fig. 4) between the two groups was closely related to the differences in STEC colonization and Stx levels in the gastrointestinal tract.
Probiotics, including lactobacilli, are known to produce short-chain
VFAs such as lactic acid (3, 12, 58). It has been reported
that VFAs possess potent bactericidal activity and that the
bactericidal activity of the organic acids depends mainly on their
undissociated form (6, 10). Undissociated organic acids
can permeate the cell membrane by diffusion and release protons in the
cell. The influx of protons is thought to induce acidification of the
cytoplasm and dissipate the membrane proton potential (
pH) (6,
9, 10). In a recent report, we demonstrated that L. casei strain Shirota exerts a bactericidal effect on STEC strains
during coculture and that this effect is dependent on the lactic acid
produced by L. casei during culture (M. Ogawa, K. Shimizu,
K. Nomoto, R. Tanaka, T. Hamabata, S. Yamasaki, T. Takeda, and Y. Takeda, submitted for publication). In this study, there were no
differences in the pH of the gastrointestinal contents between the
control and L. casei-treated groups (Table 2).
Concentrations of undissociated lactic acid in the gastrointestinal
tract were less than 1.0 mM in both groups (data not shown). We
previously showed that undissociated lactic acid at a concentration of
more than 3.2 mM is required to inhibit the growth of STEC strain
89020087 (Ogawa et al., submitted). These results suggest that lactic
acid produced by L. casei strain Shirota in the intestines
may not contribute to its inhibitory effect on STEC in the
gastrointestinal tract in infant rabbits.
It has been shown that probiotics, such as lactobacilli or bifidobacteria, can enhance specific and total IgA secretion when used as an oral adjuvant (19, 28, 30, 41, 49). Perdigon et al. reported that L. casei had a protective effect on intestinal infections with S. enterica serovar Typhimurium and E. coli by enhancing the secretion of specific IgA (47, 48). In this study, secretion of specific IgA antibodies against Stx1, Stx2, and STEC cells in the colon was enhanced locally by L. casei administration by 4 days after infection (Fig. 3). The most marked increase in the level of IgA was observed in the colon, where high levels of Stxs were detected. It has been reported that when antigens such as RDEC-1 (rabbit diarrheal E. coli) or Shiga toxin are used to inoculate rabbits by the oral route, mucosal IgA levels in the intestines increase within 7 days (22, 31, 56). The levels of IgA against Stxs and STEC cells in the colon in the L. casei-treated rabbits increased over the same period when STEC colonization and Stxs levels were reduced (days 4 to 7 after infection). It was also observed that levels of total IgA in the colon were increased by the administration of L. casei strain Shirota during STEC infection (data not shown). Paton et al. reported that antibody specific to STEC cells, especially lipopolysaccharide, inhibited the adherence of STEC to a human intestinal epithelial cell line (46). Therefore, a local increase in secretion of IgA, including specific IgA against Stxs and STEC bacterial cell surface antigen, by administration of L. casei strain Shirota may lead to the elimination of STEC and thus decrease the Stx concentration in the intestines.
In conclusion, the results obtained in this study suggest that preventive administration of probiotic lactobacilli to infants may lead to enhanced resistance to acute STEC infection due to acceleration of a specific humoral immune response to STEC, as well as Stxs.
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FOOTNOTES |
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* Corresponding author. Mailing address: Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186-8650, Japan. Phone: 81 (42) 577 8962. Fax: 81 (42) 577 3020. E-mail: koji-nomoto{at}yakult.co.jp.
Editor: A. D. O'Brien
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Abstract
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Materials and Methods
Results
Discussion
References
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Infection and Immunity, February 2001, p. 1101-1108, Vol. 69, No. 2
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.2.1101-1108.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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