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FEMS Immunology and Medical Microbiology 30 (2001) 43^47
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Comparison of mucosal adhesion and species identi¢cation of
bi¢dobacteria isolated from healthy and allergic infants
Fang He
a;b
, Arthur C. Ouwehand a; *, Erika Isolauri c , Hideo Hashimoto b ,
Yoshimi Benno d , Seppo Salminen a
a
Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland
Technical Research Laboratory, Takanashi Milk Products Co. Ltd., Yokohama, Japan
c
Department of Paediatrics, Turku University Central Hospital, Turku, Finland
Japan Collection of Microorganisms (JCM), Institute of Physical and Chemical Research (RIKEN), Wako-Shi, Japan
b
d
Received 11 October 2000; accepted 27 October 2000
Abstract
Fifty bifidobacteria strains were isolated from fecal samples of allergic and age matched healthy infants. Allergic infants were found to
have an adult type Bifidobacterium flora with high levels of Bifidobacterium adolescentis. Healthy infants had a typical infant
Bifidobacterium flora with high levels of Bifidobacterium bifidum. These isolates were tested for their adhesive properties to human intestinal
mucus. The adhesion of the fecal bifidobacteria from healthy infants was significantly higher (P 6 0.0001) than for allergic infants. This
suggests a correlation between allergic disease and the composition of the intestinal bifidobacteria flora which has reduced adhesive abilities
to the intestinal mucus. Therefore, dietary supplementation of bifidobacteria typical for healthy infants, may be beneficial in the treatment
of allergic disorders. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Mucus; Adhesion ; Bi¢dobacterium; Lactic acid bacterium ; Probiotics; Allergy
1. Introduction
Bi¢dobacteria are among the predominant bacteria in
the human intestinal micro£ora, they colonize the neonatal intestine from the ¢rst week after birth and inhabit the
gastrointestinal tract throughout life [1,2]. However, it has
been shown that the mode of delivery has a signi¢cant
in£uence on the rate of colonization and the composition
of the micro£ora colonizing the gastrointestinal tract of an
infant [3]. The same researchers have shown that microbial
colonization also in£uences the immune development of
the infant. The presence of bi¢dobacteria in the human
intestine is believed to contribute to human health and
well being, and many health bene¢ts of these microorganisms have been documented [4^8]. However, the available
information on the adhesion properties of bi¢dobacteria is
still limited.
The presence of allergic diseases is characterized by en-
* Corresponding author. Tel. : +358 (2) 3336894;
Fax: +358 (2) 3336860; E-mail: arthur.ouwehand@utu.¢
hanced immunoglobulin (Ig) E responses to common environmental antigens, is increasing world wide, especially
in Western industrialized countries [9]. Atopic dermatitis
and food allergy represent the earliest manifestations of
allergic disease, occurring in infancy and early childhood
[10]. Although the etiology of such allergic disease is still
not completely understood, a disturbance of the intestinal
micro£ora, especially of endogenous lactic acid bacteria,
has been reported as one of the important events related
to allergic diseases [11^13]. The immune development may
be related to the development and composition of the intestinal micro£ora. Since bi¢dobacteria are a major member of the intestinal micro£ora, it is important to understand their role in allergic and healthy infants.
Adhesion to intestinal mucus is regarded as a prerequisite for colonization by microorganisms. Therefore, mucosal adhesion has been proposed as one of the main
selection criteria for probiotic strains [7,14]. Adhesion of
probiotics to the intestinal mucosa is also considered important for modulation of the immune system [15]. The
di¡erent capacities to adhere to human mucus have been
reported for some probiotic, dairy and clinical lactic bacteria strains [16^19]. However, no studies have been per-
0928-8244 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 8 - 8 2 4 4 ( 0 0 ) 0 0 2 3 2 - 7
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F. He et al. / FEMS Immunology and Medical Microbiology 30 (2001) 43^47
formed to compare the adhesive capacity of human endogenous bi¢dobacteria from healthy and allergic infants to
human intestinal mucus.
The present study was conducted to investigate the differences between Bi¢dobacterium strains in the feces of
allergic and healthy, age matched, infants. The main
goal was to establish if di¡erences exist in the composition
and the adhesion properties of bi¢dobacterial strains isolated from healthy and allergic infants. In the present
study, 50 strains of bi¢dobacteria were isolated from the
feces of healthy and allergic infants. The strains were identi¢ed to species level and the adhesion to human intestinal
mucus was examined. During the adhesion studies, the
probiotic strains Lactobacillus rhamnosus GG (ATCC
53103) and Bi¢dobacterium lactis Bb12 were included for
comparison.
2. Material and methods
2.1. Bacteria
Fecal samples were collected from four infants diagnosed with food allergy and from six age matched healthy
infants (2^7 months of age), all infants were breast fed. All
allergic infants manifested atopic eczema according to the
Hani¢n criteria [20]. The collected fecal samples were kept
at 4³C and analyzed within 6 h. Fecal bi¢dobacteria were
isolated and identi¢ed using the methods reported by Mitsuoka [5] and Hosoda and co-workers [21]. In brief, the
fecal samples were homogenized, and a 10-fold serial dilution was prepared with an anaerobic bu¡er solution.
Fifty microliters of appropriate dilutions were plated on
the Blood Liver agar (BL agar; Nissui Seiyaku Co., Tokyo, Japan). The inoculum was incubated under anaerobic
conditions at 37³C for 72 h. The characteristic brown colonies formed on BL agar were randomly picked as potential bi¢dobacteria and further con¢rmed by Gram stain,
cell morphology, absence of growth under aerobic conditions and spore formation. The strains were identi¢ed to
species level based on their sugar fermentation pattern, as
described by Mitsuoka [5]. Isolated bi¢dobacteria were
stored at 375³C in 50% glycerol until use.
B. lactis Bb12 and L. rhamnosus GG (ATCC 53103)
were obtained from Chr. Hansen Ltd. (HÖrsholm, Denmark) and Valio Ltd. (Helsinki, Finland) respectively.
2.2. Mucus preparation
Mucus was isolated from the feces by extraction and
dual ethanol precipitation [16,22]. In short, feces from
healthy adult volunteers were suspended in ice cold PBS
containing protease inhibitors and sodium azide. Fecal
extracts were prepared by centrifuging the suspensions at
15 000Ug at 4³C. Mucus was isolated from the fecal extract by dual ethanol precipitation. The pooled mucus
was dissolved in HEPES (N-(2-hydroxyethyl)piperazine
N-2(ethane sulfonic acid)) bu¡ered Hanks' balanced salt
solution (HH; 10 mM HEPES; pH 7.4) at a concentration
of 1 mg ml31 . Any particulate material was removed from
the suspension by centrifugation (2000Ug for 10 min).
2.3. In vitro adhesion assay
The tested bacteria were grown in 1 ml GAM broth
(Nissui Seiyaku Co., Tokyo, Japan) from frozen stocks.
To metabolically radiolabel the bacteria, 20 Wl (methyl1,2-3 H)thymidine (117 Ci mmol31 ) was added to the medium. After approximately 36 h of anaerobic growth at
37³C, the bacteria were harvested by centrifugation
(2000Ug, 7 min), washed twice and resuspended in HH
bu¡er. The absorbance at 600 nm was adjusted to
0.25 þ 0.01 in order to standardize the number of bacteria
(approximately 107 CFU ml31 ).
The adhesion assay was performed essentially as reported earlier [16,18]. In short, the intestinal mucus was
passively immobilized on polystyrene microtiter plate wells
by overnight incubation at 4³C. The wells were washed
with HH bu¡er and the radioactively labelled bacteria
were added. After 1.5 h incubation at 37³C, the wells
were washed and the adhered bacteria were lysed with
1% SDS in 0.1 M NaOH at 60³C for 1 h. The radioactivity
of the lysed bacteria was assessed by liquid scintillation.
Adhesion is expressed as the percentage of radioactivity
recovered from the wells compared to radioactivity added
to the wells.
2.4. Statistical analysis
The results from the adhesion experiments are expressed
as the average of three or four independent experiments.
Each experiment was performed with four parallels to
correct for intra-assay variation. The Mann^Whitney
U-test was used to evaluate the statistical signi¢cance
(P 6 0.05) of the di¡erences in the ability to adhere to
intestinal mucus of di¡erent Bi¢dobacterium species and
between bi¢dobacteria from the two di¡erent infant
groups.
3. Results
A total of 50 strains of the predominant bi¢dobacteria
in the fecal samples from healthy and allergic infants were
isolated. The strains were identi¢ed to species level (Tables
1 and 2) and a distinct pro¢le of the bi¢dobacterial £ora
was detected. Among the isolates from the allergic infants,
Bi¢dobacterium adolescentis was the most predominant
species (20 out of 24 isolates), while in the healthy infants
Bi¢dobacterium bi¢dum was found to be the most common
species (15 out of 26 isolates).
The isolated bi¢dobacteria were tested for their ability
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F. He et al. / FEMS Immunology and Medical Microbiology 30 (2001) 43^47
Table 1
Bi¢dobacterium strains isolated from healthy infants and the adhesion of
these strains to human intestinal mucus
Species identi¢cation
Adhesion (%) (mean þ S.D.)
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
5.3 þ 5.1
10.4 þ 6.1
12.8 þ 1.4
8.7 þ 5.6
11.3 þ 4.5
5.6 þ 7.5
8.0 þ 4.1
14.3 þ 6.0
7.0 þ 8.4
13.6 þ 3.9
9.2 þ 4.9
13.3 þ 8.4
8.1 þ 5.1
14.6 þ 4.8
9.4 þ 5.2
11.0 þ 3.0
9.6 þ 6.3
11.7 þ 14.1
5.6 þ 4.6
2.6 þ 2.2
5.6 þ 2.6
9.1 þ 3.0
8.1 þ 5.8
10.7 þ 9.9
9.7 þ 3.3
5.6 þ 4.8
infantis H-1(1)
infantis H-1(2)
breve H-1(3)
breve H-1(4)
breve H-1(5)
breve H-1(6)
infantis H-1(7)
breve H-1(8)
infantis H-1(9)
breve H-1(10)
bi¢dum H-2-1
bi¢dum H-2-2
bi¢dum H-2-3
bi¢dum H-2-4
bi¢dum H-2-5
bi¢dum H-2-6
bi¢dum H-3-1
bi¢dum H-3-4
bi¢dum H-3-5
bi¢dum H-3-6
bi¢dum H-3-7
bi¢dum H-3-8
bi¢dum H-3-9
bi¢dum H-3-10
bi¢dum H-3-11
breve H-3-12
to adhere to human fecal mucus. Each of the tested fecal
bacteria exhibited their characteristic adhesion to fecal
mucus ranging from 0.9 to 14.6% (Tables 1 and 2). The
adhesion of the fecal bi¢dobacteria from healthy infants
(9.3%; S.D. 3.1%) was signi¢cantly higher (P 6 0.0001)
than the adhesion of the isolates from allergic infants
(3.9%; S.D. 2.3%). Bi¢dobacterium infantis was the only
Bi¢dobacterium species allergic and healthy infants had in
common. No signi¢cant di¡erence in adhesion was observed for this species between the two groups. B. adolescentis was found to be the least adhesive species, 3.5%
(S.D. 1.9%); P = 0.0044. None of the other isolated species
adhered signi¢cantly di¡erent from each other. The probiotic strains, Lactobacillus GG and B. lactis Bb12 which
were included for comparison, adhered 38.5% (S.D. 7.8%)
and 19.0% (S.D. 7.7%) respectively.
4. Discussion
Several studies have indicated that physiological disorders and infectious diseases are associated with a disturbance of human intestinal micro£ora characterized by low
numbers of bi¢dobacteria and lactobacilli. For example, in
infective and antibiotic associated diarrhea, constipation,
Crohn's disease, gastritis and in infants with biliary atresia, the intestinal bi¢dobacteria were found to be signi¢cantly decreased [5,6,23,24]. A similar tendency has also
45
been observed in some cases of allergic disease. The micro£ora of children su¡ering from food allergy and atopic
dermatitis were described by low numbers of bi¢dobacteria and lactobacilli [11,12]. However, no direct evidence on
the role of intestinal microbes in the mechanism of food
allergy exists [25]. In the present study, the composition of
the fecal bi¢dobacteria £ora from healthy and allergic infants was determined. Also, the adhesion of these bi¢dobacteria and two probiotic bacteria, Lactobacillus GG and
B. lactis Bb12, to human intestinal mucus, was examined.
Bi¢dobacteria have been reported to ¢rst appear in the
neonatal intestine between days 2 and 5 after birth, and
reaching a level of up to 99% of the fecal £ora within one
week [1,2]. The implantation and development of bi¢dobacteria in the neonate are greatly in£uenced by prematurity, method of birth, early feeding regime, endogenous
substrate availability from breast milk or formula and the
environment [3,26,27]. Upon weaning, the numbers of bi¢dobacteria decrease and stay at a more constant level in
the adult, then decreasing further among the elderly
[2,28,29].
In the current study, the composition of the fecal bi¢dobacteria £ora was found to be very di¡erent between
the healthy and allergic infants. Healthy infants had a
typical infant bi¢dobacteria £ora with B. bi¢dum, B. breve
and B. infantis. Allergic infants, in contrast, were mainly
colonized with B. adolescentis and some B. infantis, resembling a more adult-like Bi¢dobacterium £ora [1,30].
The probiotic strains, Lactobacillus GG and B. lactis
Bb12, exhibited the highest adhesion to human mucus.
Table 2
Bi¢dobacterium strains isolated from allergic infants and the adhesion of
these strains to human intestinal mucus
Species identi¢cation
Adhesion (%) (mean þ S.D.)
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
9.2 þ 1.3
3.6 þ 4.1
9.3 þ 6.3
2.9 þ 1.3
6.6 þ 2.2
2.6 þ 1.9
2.9 þ 3.7
6.1 þ 1.4
1.9 þ 1.4
4.7 þ 2.5
0.9 þ 0.2
2.8 þ 1.9
6.6 þ 4.9
5.4 þ 3.8
3.7 þ 3.4
4.5 þ 3.9
4.2 þ 2.3
2.3 þ 0.6
2.1 þ 1.2
2.0 þ 0.8
0.3 þ 0.2
5.5 þ 8.7
3.5 þ 2.4
1.3 þ 0.7
infantis 12(1)
infantis 12(2)
infantis 12(3)
infantis 12(4)
adolescentis b
adolescentis b
adolescentis b
adolescentis a
adolescentis a
adolescentis a
adolescentis a
adolescentis a
adolescentis a
adolescentis a
adolescentis c
adolescentis c
adolescentis c
adolescentis c
adolescentis c
adolescentis c
adolescentis c
adolescentis c
adolescentis c
adolescentis c
FEMSIM 1285 2-2-01
12(5)
28(1)
28(2)
IS-8(1)
IS-8(2)
IS-8(4)
IS-8(5)
IS-8(6)
IS-8(7)
IS-8(8)
66(1)
66(5)
66(6)
66(7)
66(8)
66(9)
66(10)
66(12)
66(13)
66(17)
46
F. He et al. / FEMS Immunology and Medical Microbiology 30 (2001) 43^47
The adhesion of these probiotic strains is in agreement
with previous reports, in which Lactobacillus GG and B.
lactis Bb12 showed high adhesive capacities to human intestinal mucus and Caco-2 tissue culture cell line [16^18].
Conversely, most of the fecal bi¢dobacteria exhibited a
relatively low a¤nity for human intestinal mucus. These
results suggest that for persistence of the endogenous bi¢dobacteria, factors other than adhesion are likely to be
involved. The colon is the main habitat of intestinal bi¢dobacteria, the relatively low £ow in this part of the gastrointestinal tract may explain the persistence of low adhering strains.
The present work is the ¢rst to determine the adhesive
abilities of endogenous bi¢dobacteria from allergic and
healthy infants. In the current study, the bi¢dobacteria
isolated from allergic infants were found to adhere signi¢cantly less to the human mucus compared to the strains
from healthy infants (P 6 0.0001). Also the composition of
the Bi¢dobacterium £ora was di¡erent for the two study
groups. These results indicate a correlation between allergic diseases and the human intestinal Bi¢dobacterium £ora.
Based on these results, it may be suggested that the decrease of the intestinal bi¢dobacteria in the allergic disorders previously reported could be explained by the loss of
the mucus adhesive endogenous bi¢dobacteria, due to the
presence of low adhesive B. adolescentis strains. Adhesive
strains have also been observed to be more likely to modulate the immune system [31]. The low adhesion of the
predominant bi¢dobacteria in the feces of allergic infants
may thereby be related to the aberrant immune response
in allergic infants. However, more detailed investigations
should be performed to characterize the mucosal adhesion
of endogenous bi¢dobacteria and their role in the etiology
of allergic disorders.
The results from the current study suggest that speci¢c
species of more adhesive bi¢dobacteria, typically for
healthy infants, may need to be administered to allergic
infants in order to `normalize' the intestinal Bi¢dobacterium £ora. Merely increasing the level of colonization by
bi¢dobacteria may not be enough, the levels of speci¢c
Bi¢dobacterium species may need to be altered.
Acknowledgements
Ms. Karita Peltonen, M.Sc., Research Scientist at the
Department of Biochemistry and Food Chemistry, University of Turku, is thanked for skilful technical assistance
in the identi¢cation of the bi¢dobacteria. Financial support was obtained from the Academy of Finland.
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