Previous Article | Next Article 
Applied and Environmental Microbiology, September 2000, p. 3692-3697, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Quantitative Approach in the Study of Adhesion of Lactic Acid
Bacteria to Intestinal Cells and Their Competition with
Enterobacteria
Y. K.
Lee,1,*
C. Y.
Lim,1
W. L.
Teng,1
A. C.
Ouwehand,2
E. M.
Tuomola,3 and
S.
Salminen2
Department of Microbiology, Faculty of
Medicine, National University of Singapore, Singapore 117597, Republic of Singapore,1 and Department
of Biochemistry and Food Chemistry, University of Turku, FIN-20014
Turku,2 and Novatreat Inc., FIN-20700
Turku,3 Finland
Received 10 May 2000/Accepted 20 June 2000
 |
ABSTRACT |
To describe the phenomena of bacterial adhesion to intestinal cells
and the competition for adhesion between bacteria, mathematical equations based on a simple dissociation process involving a finite number of bacterial receptors on intestinal cell surface were developed. The equations allow the estimation of the maximum number of
Lactobacillus sp. and Escherichia coli cells
that can adhere to Caco-2 cells and intestinal mucus; they also
characterize the affinity of the bacteria to Caco-2 cells and
intestinal and fecal mucus and the theoretical adhesion ratio of two
bacteria present in a mixed suspension. The competition for adhesion
between Lactobacillus rhamnosus GG and E. coli
TG1 appeared to follow the proposed kinetics, whereas the competition
between Lactobacillus casei Shirota and E. coli
TG1 may involve multiple adhesion sites or a soluble factor in the
culture medium of the former. The displacement of the adhered Lactobacillus by E. coli TG1 seemed to be a
rapid process, whereas the displacement of E. coli TG1 by
the Lactobacillus took more than an hour.
| |
INTRODUCTION |
Probiotics are viable bacterial cell
preparations or components of bacterial cells that have beneficial
effects on the health and well being of the host (9, 17).
Many of the probiotic bacteria are lactic acid bacteria and are useful
in the treatment of dysfunctions with disturb intestinal microflora and
abnormal gut permeability (10). Successful probiotic
bacteria are usually able to colonize the intestine, at least
temporarily, by adhering to the intestinal mucosa (1, 11, 19,
20). Studies have also suggested that adhesive probiotic bacteria
could prevent the attachment of pathogens, such as coliform bacteria
and clostridia, and stimulate their removal from the infected
intestinal tract (1, 11, 19, 20).
Laboratory models using human intestinal cell lines such as Caco-2
(2, 5, 8, 15, 22) and intestinal mucus (13) have
been developed to study the adhesion of probiotic lactic acid bacteria
and their competitive exclusion of pathogenic bacteria. In this study,
a quantitative approach is proposed for the design of experiments and
interpretation of data in laboratory studies using cell line and mucus
models. This approach provides a better insight to the mechanism of
competition between probiotic bacteria and pathogens, and thus allows
development of more efficient probiotic products.
| |
MATERIALS AND METHODS |
Bacterial strains.
Two commercial probiotic strains were
used: Lactobacillus casei Shirota obtained from Yakult
Singapore Pty., Ltd., and Lactobacillus rhamnosus GG
(ATCC 53103) obtained from the National Collection of Industrial and
Marine Bacteria Ltd. (Aberdeen, Scotland). Both bacterial strains have
clinically demonstrated probiotic properties (9). The
bacteria were cultured in MRS broth (BBL Cockeysville, Md.) at 37°C
with 5% CO2 for 18 h before the study.
Escherichia coli TG1 (Gibson, 1984) was obtained from
C. K. Lim (of the Microbiology Department) and it was grown in
Luria-Bertani broth (BBL) at 37°C for 18 h before the study.
E. coli TG1 was chosen for this bacterium has a maximum
adhesion to Caco-2 cells which falls between that of L. casei Shirota and that of L. rhamnosus GG. For the
mucus assay (see below) (methyl,1',2'-3H) thymidin was added to the media at a concentration of 10 µl ml
1 (117 Ci
mmol1) to radiolabel the bacteria.
Intestinal cell culture.
The Caco-2 cell culture
(7) was used in the adhesion assay. This human colon
adenocarcinoma cell line was obtained from the American Type Culture
Centre (Manassas, Va.). The cells were cultured in Dulbecco's modified
Eagle's minimal essential medium (DMEM) (GIBCO-BRL), containing 25 mM
glucose, 20% (vol/vol) heated inactivated fetal calf serum
(GIBCO-BRL), and 1% nonessential amino acids (GIBCO-BRL). The cells
were grown at 37°C in 5% CO2. For the adhesion assay,
monolayers of Caco-2 cells were prepared in two-chamber slides (Lab-Tek
chamber slide; Nunc Inc.) by inoculating 2.8 × 105
viable cells into 2 ml of culture medium. The medium was replaced every
two days.
Intestinal mucus.
Intestinal mucus was isolated from feces
of healthy adult volunteers as described earlier (13). In
short, fecal extracts were prepared by homogenizing feces in
phosphate-buffered saline (PBS) (pH 7.2) containing protease inhibitors
and sodium azide and centrifuging the suspension at 15,000 × g. The mucus was isolated from the clear fecal extract by dual
ethanol precipitation. The crude mucus was further purified by size
exclusion chromatography.
Human ileostomy glycoproteins were a generous gift from J. G. H. Ruseler-van Embden of the Erasmus University, Rotterdam, The Netherlands.
Adhesion assay. (i) On Caco-2 cells.
Fifteen-day-postconfluent Caco-2 monolayers were washed twice with 1 ml
of sterile PBS before the adhesion assay. One ml of the test bacteria
at concentrations between 1 × 105 and 4 × 108 CFU ml1 were added to 1 ml of complete
Caco-2 medium. This suspension (2 ml) was added to each chamber of the
two-chamber slide and incubated at 37°C, in a 5%
CO2-95% air atmosphere, with gentle rocking. After
incubation for 60 min, the monolayers were washed twice with sterile
PBS (pH 7.2), fixed with methanol, Gram stained, and examined
microscopically. Visual counting of adhered cells was adopted in this
study, for it allows the differentiation of the gram-positive
Lactobacillus and gram-negative E. coli. Each adherence assay was conducted in triplicate by two students (Y.Y.L. and
W.L.T.), and the number of adherent bacteria was counted on about 1,000 Caco-2 cells, in 60 randomly selected microscopic fields. To stimulate
the physiological pH condition of the gastrointestinal tract, all
experiments were done at pH 7.
In the study of the competition for adhesion on Caco-2 cells,
Lactobacillus and
E. coli were added
simultaneously or sequentially
to the Caco-2 cultures before counting.
In the latter case, free
cells of the first bacterium were removed by
washing with PBS
(pH 7.2) before the second bacterium was added. The
lactic acid
bacteria have the tendency to form chains and aggregates.
It was
necessary to disperse the chains and aggregates of the bacterial
cells before the adhesion study, to ensure that cells observed
under
the microscope in the adhesion assay were cells adhered
to Caco-2 and
ileosotomy glycoprotein surfaces (
13).
(ii) On immobilized mucus and ileostomy glycoproteins.
The
study of adhesion of the microorganisms on mucus and ileostomy
glycoproteins was performed as described earlier (13). In
short, human intestinal mucus or human ileostomy glycoprotein was
passively immobilized in microtiter plate wells. Bacteria were allowed
to bind to the mucus or ileostomy glycoproteins at concentrations
between 4.4 × 106 and 4.1 × 108
CFU · ml1. The radioactivity was assessed by
liquid scintillation. The relation between the measured radioactivity
and the number of bacteria was determined by plate counting.
Theory.
In many studies on the adhesion of bacterial cells
to intestinal epithelial cells, when the number of bacterial cells
adhered to intestinal cells is plotted against the concentration of
bacterial culture, a section of a rectangular hyperbola is obtained, as shown in Fig. 1. Such a relationship implies a process
of simple dissociation. That is,
where
k + 1 and
k 
1 represent
the dissociation constants for the reaction. The process is similar to
the reaction between
a substrate and an enzyme that forms a
substrate-enzyme complex,
without the formation of a product.
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 1.
Adhesion of L. rhamnosus GG, L. casei Shirota, and E. coli TG1 to human intestinal cell
line Caco-2, presented as the number of bacteria bound per 100 Caco-2
cells versus the concentration of bacteria added (CFU per
milliliter).
|
|
The relationship is based on the assumption that the interaction
between the bacterial cells and the intestinal cells or mucus
remains
in equilibrium. This condition should be achieved if the
bacterial
cells do not penetrate the intestinal
cells.
It is also assumed that the concentration of the bacterial culture
remained essentially unchanged throughout the study, so
that the
concentration of the bacterial culture can be considered
equal to the
initial bacterial concentration. This condition is
usually achieved
when the total number of bacterial cells is much
greater than the
number of bacterial cells adhering to the intestinal
cells. This is
usually the case in most of the adhesion studies,
where the
concentration of the bacterial cells added is in the
range of
10
5 to 10
8 per ml, whereas that of the
intestinal cell culture (e.g., Caco-2
cells) is about 10
2
per ml and the number of bacterial cells adhered to the Caco-2
cells is
fewer than 10 per
cell.
In the equation described above, if
x is the concentration
of the bacterial culture added,
e is the intestinal
epithelial
cell or mucus concentration, and
ex
is the concentration of the
bacterium-intestinal cell-mucus complex,
then the concentration
of free bacterial cells will be (
x ex).
Because the process is in equilibrium, the dissociation constant for
the process (
kx) can be defined as
kx = (
k 1)/(
k +
1) = (
x ex)
x/
ex. This equation can be
rearranged to give an
expression for the concentration of the
bacterium-intestinal cell-mucus
complex,
ex =
e ·
x/(
kx +
x). When
x is very
much larger than
kx,
ex approaches
e. The maximum value of
ex obtained
when the
intestinal cells or mucus is saturated with bacteria as
em, which
may be written as
|
(1)
|
When
x is equal to
kx,
ex is equal to
em/2; thus, the
value of
kx could be experimentally obtained
from the value of
x, which
gives half the maximum
ex (i.e.,
em/2).
Equation
1 can be rearranged to give a linear relationship.
|
(2)
|
Hence, a plot of 1/
ex against
1/
x will give a straight line, in which the intercept on the
ordinate gives a value of 1/
em,
and that on the
abscissa gives a value of
1/
kx.
In the case where two types of bacteria are present in the system and
they compete for the same receptors or adhesion sites
(through steric
hindrance of cells in close vicinity), the competition
for adhesion of
each of the bacterial types is determined by the
affinity of the
bacteria to the intestinal cells or the intestinal
mucus
(
kx) and the concentration of the bacterial
culture (
x).
Thus, the ratio of
ex
for bacterium 1 and bacterium 2 can be described
as
|
(3)
|
Statistics.
Differences between treatments were examined for
the level of significance by Student's t test after
analysis of variance.
| |
RESULTS |
Adhesion of bacteria to Caco-2 cells.
When the concentration
of adhered bacterial cells (cells per 100 Caco-2 cells) was plotted
against the concentration of bacterial cells added, a hyperbolic
relation was observed for all three of the bacteria tested (Fig. 1).
The plots of the reciprocal of adhered cell concentration versus the
reciprocal of the concentration of cells added, for
L. casei
Shirota and
L. rhamnosus GG are given in Fig.
2, and that
for
E. coli is given in Fig.
3. In all the cases a linear relationship
was observed.
It follows from equation 2 that the intercept on
the ordinate gives the
value of the reciprocal of the maximum
number of bacterial cells
adhered to 100 Caco-2 cells (
em). The
intercept
on the abscissa is
1/
kx, where
kx is the dissociation
constant for the adhesion
process. Thus, the values of
em and
kx for the three bacteria were calculated, and
they are summarized
in Table
1.
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 2.
Double-reciprocal representation of the adhesion of
L. rhamnosus GG and L. casei Shirota to human
intestinal cell line Caco-2. The lines indicate the linear fit
according to the least-squares method.
|
|
View larger version (13K):
[in this window]
[in a new window]
|
FIG. 3.
Double-reciprocal representation of the adhesion of
E. coli TG1 to human intestinal cell line Caco-2. The lines
indicate the linear fit according to the least-squares method.
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Maximum number of adhered bacterial cells on 100 Caco-2
cells and dissociation constant of the adhesion process for
various strainsa
|
|
As shown in Table
1, among the three bacteria studied, the maximum
number of
L. rhamnosus GG cells that can adhere to Caco-2
cells is about 10 times that of the
L. casei Shirota,
whereas
the maximum adhesion number of
E. coli TG1 is
between those of
the two lactobacilli.
E. coli TG1 was
chosen for this study as
it would allow us to understand the
competition of an enterobacterium
with a lactic acid bacterium which
has a higher adhesion capacity
(i.e.,
L. rhamnosus GG) and
with one whose adhesion capacity is
lower (i.e.,
L. casei Shirota).
L. casei Shirota's having the lowest
kx implies that it has a higher affinity for
adhesion to Caco-2 cells than do
L. rhamnosus GG and
E. coli TG1; i.e., adhered
L. casei Shirota
dissociates
less easily than the other two
bacteria.
Competition between Lactobacillus and E. coli for adhesion.
In this study, various concentrations of
a Lactobacillus strain (2 × 108 to 6 × 108 cells ml1) were mixed with an equal
volume of E. coli (2 × 108 cells
ml1) and then added onto the Caco-2 cells. The final
concentration of the respective bacterial strains is thus half of the
original concentration. The Gram-stained Lactobacillus and
E. coli adhered on Caco-2 cells could be easily
differentiated and counted microscopically. The observed concentrations
of the adhered Lactobacillus and E. coli and the
predicted ratio of the two bacteria based on equation 3 are given in
Tables 2 and 3.
In the case of
L. rhamnosus GG, the predicted ratio of
E. coli and lactobacilli counted on Caco-2 cells
(
eE/
eL) is comparable
to the
observed values (Table
2). In the case of
L. casei Shirota,
the predicted values of
eE/
eL are
1.169 to 1.806 for the three
concentrations of
Lactobacillus
added. A value of >1 indicates
that the
Lactobacillus has
been excluded by
E. coli for adhesion
to Caco-2 cells. The
observed values of
eE/
eL are 8 to 23 times
lower than the predicted values, ranging from 0.062 to 0.135.
Exclusion of E. coli by adhered lactobacilli.
In
the study, the lactobacilli (108 cells ml1)
were allowed to adhere to Caco-2 cells. Nonadhered
Lactobacillus cells were removed by PBS, and then the
E. coli TG1 (108 cells ml1) was
added and incubated with Caco-2 cells for 1 h. The respective adhesion number of the two bacteria was counted and is shown in Table
4.
View this table:
[in this window]
[in a new window]
|
TABLE 4.
Results of the exclusion study in which
Lactobacillus cells were allowed to adhere to Caco-2 cells
before E. coli was addeda
|
|
In the case of
L. rhamnosus GG, the concentration of the
E. coli TG1 counted (160.56 ± 14.14 cells/100 Caco-2
cells) was not
statistically different (
P > 0.05)
compared with
E. coli when
it was added alone and incubated
with Caco-2 cells (169.16 ± 15.63
cells/100 Caco-2 cells),
whereas in the case of
L. casei Shirota,
the
concentration of the
E. coli TG1 counted (122.26 ± 15.66 cells/100
Caco-2 cells) was significantly lower
(
P < 0.05) than that of
E. coli
(169.16 ± 15.63 cells/100 Caco-2 cells). However, in both
of the
studies involving
L. rhamnosus GG (62.36 ± 11.10 cells/100
Caco-2 cells) and
L. casei Shirota (53.95 ± 2.82 cells/100 Caco-2
cells), the counts of the lactobacilli were much
lower than those
when
L. rhamnosus (397.7 ± 53.2 cells/100 Caco-2 cells) and
L. casei (78.94 ± 9.60)
alone were incubated with Caco-2 cells. The
values of
eE/
eL were greater than 1; i.e.,
there were more
E. coli cells than
lactobacilli.
Displacement of adhered E. coli TG1 by
lactobacilli.
In the study, the E. coli TG1
(108 cells ml1) was allowed to adhere on
Caco-2 cells. Nonadhered E. coli cells were removed by PBS,
and then the lactobacilli (108 cells ml1) was
added and incubated with Caco-2 cells for 1 h. The adhesion numbers of the two bacteria were counted and are shown in Table 5.
In both cases, the concentrations of the
E. coli TG1 counted
are statistically lower (
P < 0.05) than those when the
E. coli alone was incubated with Caco-2 cells (169.16 ± 15.63 cells/100
Caco-2 cells). However, the values of
eE/
eL were greater than
1; i.e.,
there were more
E. coli cells than
lactobacilli.
Adhesion of bacteria to immobilized intestinal mucus and ileostomy
glycoproteins.
When the number of bacterial cells adhered to
intestinal mucus or human ileostomy glycoproteins per well
(0.1-cm2 wells) was plotted against the number of
bacteria added per well, a near-linear to hyperbolic relationship
was observed for all three of the bacteria tested (Fig.
4 and 5).
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 4.
Adhesion of L. rhamnosus GG, L. casei Shirota, and E. coli TG1 to immobilized human
intestinal mucus presented as the number of bacteria bound per
microtiter plate well (surface area, 0.1 cm2) versus the
concentration of bacteria added.
|
|
View larger version (31K):
[in this window]
[in a new window]
|
FIG. 5.
Adhesion of L. rhamnosus GG (A) and L. casei Shirota and E. coli TG1 (B) to immobilized human
ileostomy glycoproteins presented as the number of bacteria bound per
microtiter plate well (surface area, 0.1 cm2) versus the
concentration of bacteria added.
|
|
Double-reciprocal plots of adhered
L. rhamnosus GG and
E. coli TG1 against the concentration of bacteria added are
presented
in Fig.
6 and
7. A linear
relation was observed for both plots.
A double-reciprocal plot of
adhered
L. casei Shirota is shown
in Fig.
8
and
9. This plot appears to be composed of two linear
parts with a break at around 1.7 × 10
7 CFU
ml
1 for the intestinal mucus and 9.3 × 10
7 CFU ml
1 for the human ileostomy
glycoprotein. The calculated
em and
kx values for the bacteria are shown in Table
5. For
L. casei Shirota,
the maximum number
of bacterial cells that can adhere to intestinal
mucus at low cell
concentrations (5 × 10
4 cells well
1)
was four times lower than that measured at high cell concentrations
(2 × 10
5 cells well
1), while the
maximum number of bacteria that can adhere to ileostomy
glycoprotein at
low cell concentrations (1.96 × 10
5 cells
well
1) was 5.1 times lower than that at high cell
concentrations (10
6 cells well
1). However,
its adhesion affinities on intestinal mucus and ileostomy
glycoproteins were higher at low cell concentrations.
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 6.
Double-reciprocal representation of the adhesion of
L. rhamnosus GG and E. coli TG1 to immobilized
human intestinal mucus. The lines indicate the linear fit according to
the least-squares method.
|
|
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 7.
Double-reciprocal representation of the adhesion of
L. rhamnosus GG and E. coli TG1 to immobilized
human ileostomy glycoproteins. The lines indicate the linear fit
according to the least-squares method.
|
|
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 8.
Double-reciprocal representation of the adhesion of
L. casei Shirota to immobilized human intestinal mucus. The
lines indicate the linear fit according to the least-squares method.
|
|
View larger version (14K):
[in this window]
[in a new window]
|
FIG. 9.
Double-reciprocal representation of the adhesion of
L. casei Shirota to immobilized human ileostomy
glycoproteins. The lines indicate the linear fit according to the
least-squares method.
|
|
| |
DISCUSSION |
Our studies indicate that the direct microscopic counting and
radioactive label counting as measures of bacteria adhesion gave
comparable results. The microscopic method was adopted in the studies
for competition for adhesion, for it allows the differentiation of the
different bacterial types on the Caco-2 cell surface. The present study
demonstrated that the adhesion process of bacteria on Caco-2 cells
follows the mathematical relationship (equation 2) developed for a
simple dissociation process involving a finite number of adhesion sites
or receptors on Caco-2 cells (Fig. 2 and 3). Thus, this allows the
maximum number of adhesion sites (em) to be
estimated for each bacterial strain and the adhesion affinity of
respective bacteria for Caco-2 cells to be estimated.
Among the bacteria studied, L. rhamnosus GG has the highest
adhesion at saturation cell concentration, which is about 10 times higher than that for L. casei Shirota and three times higher
than that for E. coli TG1 (Table 1). Thus, the calculations
suggest that if L. casei Shirota and E. coli TG1
are competing for the same adhesion sites or surface receptors on
Caco-2 cells, L. casei Shirota would not be able to prevent
the adhesion of E. coli.
L. casei Shirota, on the other hand, has a higher affinity
(lower dissociation constant [kx]) than
L. rhamnosus and E. coli. That is, L. casei Shirota adheres more readily to and dissociates less easily
from Caco-2 cells.
A similar approach, applying the principles of Michaelis-Menten enzyme
kinetics to the study of E. coli adhesion to intestinal cell
monolayers, was proposed (6). It was concluded that the adhesion of E. coli involved an initial reversible binding
step that was followed by an irreversible step. In the present study and others (4, 11, 19, 20), we had observed competition between adhered cells and free cells for adhesion sites on intestinal cells. The discrepancy between the former and the latter studies may be
due to the use of undifferentiated cells in the former study.
In the studies, each of the lactobacilli was mixed with an equal number
of the E. coli TG1 cells, and the mixed culture suspension was incubated with Caco-2 cells. If the lactobacilli and E. coli were competing for the same adhesion sites or surface
receptors, the ratio of adhered E. coli
(eE) to lactobacilli (eL)
would be expected to be determined by their em,
cell concentration added (x), and kx
as described by equation 3. This was the case when the L. rhamnosus GG and the E. coli TG1 were competing for
adhesion on Caco-2 cells (Table 2). The predicted
eE/eL values were close to the
observed values obtained experimentally. The results of this study
suggest that to achieve the lowest
eE/eL, the concentration of L. rhamnosus should approach the saturation concentration, i.e.,
>100 × kx or >2.08 × 1010 cells/ml. A concentration of 105 viable
cells per ml of a probiotic product has been suggested as the
therapeutic minimum for humans (16). Consumption of
106 to 1010 viable cells per day is necessary
for a beneficial effect to develop (9, 16, 18). Equation 3
developed in this study suggests that the concentration of the
probiotic bacterium is a critical parameter in determining
eE/eL. It is, nevertheless, expected
that a sufficient volume of a probiotic cell suspension must be
consumed so that the probiotic cells can saturate the entire
gastrointestinal (GI) tract, especially the lower part of the GI tract.
In the study of the competition between L. casei Shirota and
E. coli TG1, calculations from equation 3 predicted that the E. coli would compete well with L. casei for
adhesion (eE/eL > 1), because
of the lower em of L. casei (137.9 cells/100 Caco-2 cells) compared with E. coli (500 cells/100
Caco-2 cells). Even if L. casei occupied all of the 137.9 adhesion sites, it could not prevent the E. coli from
adhering to the balance of the 362.1 sites. The observed
eE/eL ranged between 0.135 and 0.062 (Table 3); i.e., the Lactobacillus prevented the adhesion of
E. coli to Caco-2 cells. It is likely that the interaction
of L. casei and E. coli involved more than
competition for the adhesion sites on Caco-2 cells.
In the exclusion study, it was observed that L. rhamnosus GG
did not prevent the adhesion of E. coli to Caco-2 cells.
This may be due to the relatively high dissociation constant of
L. rhamnosus (2.08 × 108 cells
ml1), and any dissociated Lactobacillus cells
were readily replaced by surrounding E. coli cells in the
culture medium. The concentration of E. coli determined
(122.26 ± 15.66 cells/100 Caco-2 cells) in the study involving
L. casei was slightly, though statistically significantly
(P < 0.05) lower than the concentration determined when E. coli was present alone (169.16 ± 15.63 cells/100 Caco-2 cells). The lower dissociation constant (8.62 × 107 cells ml1) and possibly a soluble protein
factor produced by L. casei Shirota may have hindered their
displacement by E. coli cells. This observation is in
agreement with studies of humans and animals, where
Lactobacillus cells were found to be replaced gradually by
enterobacteria after the intake of lactobacilli had stopped (11,
19). Commercially available probiotic organisms have not been
reported to establish themselves permanently in the human GI tract. Our
results suggest that the concentration of free (nonadhering)
Lactobacillus in the GI contents needs to be maintained at a
high level to prevent the adhered lactobacilli from being replaced by
other bacteria; alternatively, the lactobacilli need to divide rapidly
to maintain a high local cell concentration. The observation that
adhered Lactobacillus cells in the GI tract were gradually
replaced by enterobacteria suggests that the lactobacilli used were not
able to grow sufficiently rapidly to establish permanent residence in
the GI tract.
There is ample laboratory and clinical evidence to demonstrate that
oral administration of lactobacilli could be used to treat GI bacterial
infection (9). The efficacy of lactobacilli in displacing
adhered E. coli on Caco-2 cells was investigated in the
displacement study (Table 6). The process of displacing
adhered E. coli with Lactobacillus appears to be
slow. After 1 h of incubation with lactobacilli alone, less than
half of the adhered E. coli cells were displaced. In the
studies using fermented milk containing lactobacilli to treat diarrheal
disorders in human patients (4, 12, 15), it was necessary
that patients consume the fermented milk for 2 to 3 days, before
significant improvement in clinical symptoms of the illness was
observed. It is important to recognize the slow process of displacing
E. coli with lactobacilli in the in vitro study, which could
not simply be explained by dissociation phenomena.
View this table:
[in this window]
[in a new window]
|
TABLE 6.
Results of the displacement study in which E. coli TG1 cells were allowed to adhere to Caco-2 cells before
lactobacilli were addeda
|
|
Although L. rhamnosus GG had the highest saturation cell
concentration, its adhesion affinity to intestinal mucus and ileostomy glycoproteins was considerably lower than that of L. casei
Shirota or E. coli TG1. The double-reciprocal plots of
L. casei Shirota added versus bound, allowed two linear
curve fits (Fig. 8 and 9). This may mean that two binding mechanisms
are involved for this bacterium, one for a high bacterial concentration
(lower affinity) and one for a lower concentration (higher affinity). A
possible explanation is that multiple adhesion sites on the mucous
layer are involved in the adhesion of an L. casei Shirota cell. At low bacterial concentrations, a bacterium adheres to a maximum
number of sites on mucus, whereas at high bacterial concentrations, a
minimum number of adhesion sites are involved. The data in Table 5
suggest that up to four adhesion sites (2 × 105/5 × 104 = 4) on intestinal mucus
and five adhesion sites on ileostomy glycoproteins could be involved in
the adhesion of L. casei Shirota on intestinal mucus. This
explains the observation that at low bacterial concentrations the
affinity for mucus was high, whereas at high bacterial concentrations
the adhesion affinity was lower (Table 5). In either case, the adhesion
affinity is much higher than those for the other two tested bacteria.
The quantitative approach developed in this study has proven useful in
the understanding of the mechanism and kinetics of the adhesion process
of bacteria on intestinal cells and mucus and the competition between
different bacteria for adhesion. Our results may provide help in
estimating the numbers of bacteria needed for future competitive
exclusion studies.
| |
ACKNOWLEDGMENTS |
The skillful technical assistance of Satu Tölkkö is
gratefully acknowledged.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, National University of Singapore, 5 Science Drive 2, Singapore 117597, Republic of Singapore. Phone: 65-874 3284. Fax:
65-776 6872. E-mail: MICLEEYK{at}NUS.EDU.SG.
| |
REFERENCES |
| 1.
|
Benno, Y., and T. Mitsuoka.
1992.
Impact of Bifidobacterium longum on human fecal microflora.
Microbiol. Immun.
36:683-694.
|
| 2.
|
Bernet, M.-F.,
D. Brassart,
J.-R. Neeser, and A. L. Servin.
1993.
Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions.
Appl. Environ. Microbiol.
59:4121-4128[Abstract/Free Full Text].
|
| 3.
|
Bernet, M.-F.,
D. Brassart,
J.-R. Neeser, and A. L. Servin.
1994.
Lactobacillus acidophilus LA1 binds to cultured human intestinal cell lines and inhibits cell attachment and cell invasion by enterovirulent bacteria.
Gut
35:483-489[Abstract/Free Full Text].
|
| 4.
|
Boudraa, G.,
M. Touhami,
P. Pochart,
R. Soltana,
J. Y. Mary, and J. F. Desjeux.
1990.
Effect of feeding yogurt versus milk in children with persistent diarrhea.
J. Pediatr. Gastroenterol. Nutr.
11:509-512[Medline].
|
| 5.
|
Chauviere, G.,
M.-H. Coconnier,
S. Kerneis,
J. Fourniat, and A. L. Servin.
1992.
Adhesion of human Lactobacillus acidophilus strain LB to human enterocyte-like Caco-2 cells.
J. Gen. Microbiol.
138:1689-1696.
|
| 6.
|
Cohen, P. S.,
A. D. Elbein,
R. Solf,
H. Mett,
J. Regos,
E. B. Menge, and K. Vosbeck.
1981.
Michaelis-Menten kinetic analysis of Escherichia coli SS142 adhesion to intestine 407 monolayers.
FEMS Microbiol. Lett.
12:99-103.
|
| 7.
|
Fogh, J.,
J. M. Fogh, and T. Orfeo.
1977.
One hundred and twenty-seven cultured human tumour cell lines producing tumours in nude mice.
J. Natl. Cancer Inst.
59:221-226.
|
| 8.
|
Greene, J. D., and T. R. Klaenhammer.
1994.
Factors involved in adherence of lactobacilli to human Caco-2 cells.
Appl. Environm. Microbiol.
60:4487-4494[Abstract/Free Full Text].
|
| 9.
|
Lee, Y. K.,
K. Nomoto,
S. Salminen, and S. L. Gorbach.
1999.
Handbook of Probiotics.
John Wiley & Sons, New York, N.Y.
|
| 10.
|
Lee, Y. K., and S. Salminen.
1995.
The coming of age of probiotics.
Trends Food Sci. Technol.
6:241-244[CrossRef].
|
| 11.
|
Lidbeck, A.,
J.-A. Gustafasson, and C. Nord.
1987.
Impact of Lactobacillus acidophilus supplements on the human oropharyngeal and intestinal microflora.
Scand. J. Infect. Dis.
19:531-537[Medline].
|
| 12.
|
Majamaa, H.,
E. Isolauri,
M. Saxelin, and T. Vesikari.
1995.
Lactic acid bacteria in the treatment of acute rotavirus gastroenteritis.
J. Pediatr. Gastroenterol. Nutr.
20:333-338[Medline].
|
| 13.
|
Ouwehand, A. C.,
P. Niemi, and S. J. Salminen.
1999.
The normal fecal microflora does not affect the adhesion of probiotic bacteria in vitro.
FEMS Microbiol. Lett.
177:35-38[CrossRef][Medline].
|
| 14.
|
Pinto, M.,
S. Robine-Leon,
M.-D. Appay,
M. Kedinger,
N. Triadou,
E. Dussaulx,
B. Lacroix,
P. Simon-Assmann,
K. Haffen,
J. Fogh, and A. Zweibaum.
1983.
Enterocyte-like differentiation and polarisation of the human colon carcinoma cell line Caco-2 in culture.
Biol. Cell
47:323-330.
|
| 15.
|
Raza, S.,
S. M. Graham,
S. J. Allen,
S. Sultana,
L. Cuevas, and C. A. Hart.
1995.
Lactobacillus GG promotes recovery from acute nonbloody diarrhea in Pakistan.
Pediatr. Infect. Dis. J.
14:107-111[Medline].
|
| 16.
|
Robinson, R. K. (ed.).
1991.
Therapeutic properties of fermented milks, p. 23-43.
Elsevier Applied Science, London, United Kingdom.
|
| 17.
|
Salminen, S.,
A. Ouwehand,
Y. Benno, and Y. K. Lee.
1999.
Probiotics: how should they be defined?
Food Sci. Technol.
10:107-110[CrossRef].
|
| 18.
|
Salminen, S.,
M. Deighton, and S. Gorbach.
1993.
Lactic acid bacteria in health and disease, p. 199-225.
In
S. Salminen, and A. von Wright (ed.), Lactic acid bacteria. Marcel Dekker, New York, N.Y.
|
| 19.
|
Saxelin, M.,
T. Pessi, and S. Salminen.
1995.
Fecal recovery following oral administration of Lactobacillus strain GG (ATCC 53103) in gelatine capsules to healthy volunteerts.
Int. J. Food Microbiol.
25:199-203[CrossRef][Medline].
|
| 20.
|
Tamura, N.,
M. Norimoto,
K. Yoshida,
C. Hirayama, and R. Nakai.
1983.
Alteration of fecal bacterial flora following oral administration of bifidobacterial preparation.
Gastroenterol. Jpn.
18:17-24.
|
| 21.
|
Tuomola, E. M., and S. J. Salminen.
1998.
Adhesion of some probiotic and dairy Lactobacillus strains to Caco-2 cell cultures.
Int. J. Food Microbiol.
41:45-51[CrossRef][Medline].
|
Applied and Environmental Microbiology, September 2000, p. 3692-3697, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Ikeda, R., Saito, F., Matsuo, M., Kurokawa, K., Sekimizu, K., Yamaguchi, M., Kawamoto, S.
(2007). Contribution of the Mannan Backbone of Cryptococcal Glucuronoxylomannan and a Glycolytic Enzyme of Staphylococcus aureus to Contact-Mediated Killing of Cryptococcus neoformans. J. Bacteriol.
189: 4815-4826
[Abstract]
[Full Text]
-
Vesterlund, S., Karp, M., Salminen, S., Ouwehand, A. C.
(2006). Staphylococcus aureus adheres to human intestinal mucus but can be displaced by certain lactic acid bacteria. Microbiology
152: 1819-1826
[Abstract]
[Full Text]
-
Tao, Y., Drabik, K. A., Waypa, T. S., Musch, M. W., Alverdy, J. C., Schneewind, O., Chang, E. B., Petrof, E. O.
(2006). Soluble factors from Lactobacillus GG activate MAPKs and induce cytoprotective heat shock proteins in intestinal epithelial cells. Am. J. Physiol. Cell Physiol.
290: C1018-C1030
[Abstract]
[Full Text]
-
Salminen, S. J., Gueimonde, M., Isolauri, E.
(2005). Probiotics That Modify Disease Risk. J. Nutr.
135: 1294-1298
[Abstract]
[Full Text]
-
Kumura, H., Tanoue, Y., Tsukahara, M., Tanaka, T., Shimazaki, K.
(2004). Screening of Dairy Yeast Strains for Probiotic Applications. J DAIRY SCI
87: 4050-4056
[Abstract]
[Full Text]
-
Lee, Y. K., Ho, P. S., Low, C. S., Arvilommi, H., Salminen, S.
(2004). Permanent Colonization by Lactobacillus casei Is Hindered by the Low Rate of Cell Division in Mouse Gut. Appl. Environ. Microbiol.
70: 670-674
[Abstract]
[Full Text]
-
Lee, Y.-K., Puong, K.-Y., Ouwehand, A. C., Salminen, S.
(2003). Displacement of bacterial pathogens from mucus and Caco-2 cell surface by lactobacilli. J Med Microbiol
52: 925-930
[Abstract]
[Full Text]
-
Isolauri, E, Kirjavainen, P V, Salminen, S
(2002). Probiotics: a role in the treatment of intestinal infection and inflammation?. Gut
50: iii54-59
[Abstract]
[Full Text]
-
Ouwehand, A. C., Salminen, S., Tolkko, S., Roberts, P., Ovaska, J., Salminen, E.
(2002). Resected Human Colonic Tissue: New Model for Characterizing Adhesion of Lactic Acid Bacteria. CVI
9: 184-186
[Abstract]
[Full Text]
-
Asahara, T., Nomoto, K., Watanuki, M., Yokokura, T.
(2001). Antimicrobial Activity of Intraurethrally Administered Probiotic Lactobacillus casei in a Murine Model of Escherichia coli Urinary Tract Infection. Antimicrob. Agents Chemother.
45: 1751-1760
[Abstract]
[Full Text]
Top
Abstract
Introduction
