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Applied and Environmental Microbiology, November 1998, p. 4573-4580, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Antagonistic Activity against
Helicobacter Infection In Vitro and In Vivo by the Human
Lactobacillus acidophilus Strain LB
Marie-Helene
Coconnier,1
Vanessa
Lievin,1
Elisabeth
Hemery,2 and
Alain L.
Servin1,*
Institut National de la Santé et de la
Recherche Médicale, CJF 94.07, UFR de Pharmacie, Université
Paris XI, F-92296 Châtenay-Malabry,1
and
Anatomie Pathologie A, Centre Hospitalier Universitaire
de Nantes, F-44000 Nantes,2 France
Received 17 March 1998/Accepted 12 August 1998
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ABSTRACT |
The purpose of the present study was to examine the activity of the
human Lactobacillus acidophilus strain LB, which secretes an antibacterial substance(s) against Helicobacter pylori
in vitro and in vivo. The spent culture supernatant (SCS) of the strain LB (LB-SCS) dramatically decreased the viability of H. pylori in vitro independent of pH and lactic acid levels.
Adhesion of H. pylori to the cultured human mucosecreting
HT29-MTX cells decreased in parallel with the viability of H. pylori. In conventional mice, oral treatment with the LB-SCS
protected against infection with Helicobacter felis.
Indeed, at both 8 and 49 days post-LB-SCS treatment (29 and 70 days
postinfection), inhibition of stomach colonization by H. felis was observed, and no evidence of gastric histopathological lesions was found. LB-SCS treatment inhibits the
H. pylori urease activity in vitro and in H. pylori that remained associated with the cultured human
mucosecreting HT29-MTX cells. Moreover, a decrease in urease
activity was detected in the stomach of the mice infected with H. felis and treated with LB-SCS.
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INTRODUCTION |
Enterovirulent pathogens develop
pathogenicity by cell attachment, which is a prerequisite for
intestinal colonization and cell entry, and by production of cytotoxic
or cytotonic toxins (for reviews, see references 20
and 37). Helicobacter pylori is the
causal agent of type B gastritis, peptic ulcers, and gastric cancer
(for reviews, see references 9, 14, 17, 27, and 30). It is a spiral-shaped, gram-negative rod that
has developed sophisticated strategies to colonize epithelial cells
lining the antrum of the stomach and to survive in the acidic
environment. In antral and duodenal biopsy specimens, H. pylori has been shown to attach to epithelial cells and
occasionally penetrate the cells. Although their role in H. pylori colonization remains to be clarified, five adhesins that
recognize different receptors have been described. Binding of
H. pylori has been investigated in vitro with gastric cell cultures, all undifferentiated, which in particular lack polarization, in contrast to gastric epithelial cells. Attachment of
H. pylori to human gastric cells is followed by
attaching-effacing lesion formation in microvilli or microvillus-like
projections accompanied by cytoskeletal rearrangements and tyrosine
phosphorylation of host cell proteins. Moreover, the H. pylori cytotoxin CagA produces cellular vacuolation in a number of
different epithelial cell types in vitro.
Recent reports have documented the role of exogenous lactobacilli in
the prevention and treatment of gastrointestinal disorders. To explain
this effect, several authors have reported the production of
antimicrobial substances inhibiting the growth of pathogens. In
particular, it was recently demonstrated that probiotic lactobacilli can develop antagonistic activity against human pathogens (1, 2,
38). Certain lactobacilli synthesize antimicrobial compounds that
are related to the bacteriocin family (for reviews, see references 23 and 25). Others are well-known
metabolic end products of lactic acid fermentation, lactic and acetic
acids, and hydrogen peroxide (for a review, see reference
39) or remain unidentified (2, 12, 38).
It was recently reported that lactobacilli can inhibit the growth of
H. pylori in vitro and exhibit antagonistic activity
against H. pylori in vivo (4, 24, 33). And
in human volunteers, the spent culture supernatant of the human
Lactobacillus acidophilus strain LA1 is active against
H. pylori (32).
The aim of this report was to examine the antagonistic activity
developed by the human L. acidophilus strain LB against
H. pylori in vitro and in vivo. This strain was found
to adhere to cultured human polarized intestinal cells (8).
It displays antagonistic activity in vitro against noninvasive and
invasive enterovirulent pathogens (7, 11) and in vivo
against Salmonella typhimurium (12) and
Campylobacter jejuni (35) infections. The LB
strain secretes an antimicrobial substance(s) other than lactic acid
that is heat stable and only moderately sensitive to enzymatic
treatments (12). Moreover, several characteristics of the
component(s) supporting the antimicrobial activity suggest that it
could contain(s) an unusual acidic amino acid present in a novel
peptidic agent. Results presented here show that the spent culture
supernatant (SCS) of strain LB (LB-SCS) dramatically decreased the
viability of H. pylori in vitro independent of pH and
lactic acid levels, protects mice against Helicobacter felis infection, and inhibits the H. pylori and H. felis urease activity in vitro and in vivo, respectively.
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MATERIALS AND METHODS |
Bacteria.
Strains of H. pylori and
H. felis were provided by I. Corthesy-Theulaz
(Institute of Microbiology, Lausanne University, Lausanne, Switzerland). H. pylori 1101 was isolated from a
patient suffering from functional dyspepsia and erosive gastritis
(32). H. felis CS1 (ATCC 49179) was
originally isolated from the stomach of a cat (13, 31).
H. pylori was grown on brain heart infusion (BHI) agar
plates containing 0.25% yeast extract (Difco Laboratories, Detroit,
Mich.), 10% horse serum, and 0.4% Campylobacter selective complement (Skirrow supplement, SR 69; Oxoid Ltd., Basingstoke, England). H. felis was grown on BHI agar plates
containing 0.25% yeast extract supplemented with 10% horse serum.
Helicobacter culture was incubated upside down in a gas jar
with a microaerophilic atmosphere (gas-generating kit, CampyGen; Oxoid
Ltd.) at 37°C.
L. acidophilus LB was originally isolated from human stool
(Lacteol Laboratory, Houdan, France). Lactobacillus casei GG
was originally isolated from the fecal flora of a healthy human
volunteer and was obtained from S. L. Gorbach (Tufts University,
Boston, Mass.). LB and GG strains were grown in De Man Rogosa Sharpe
(MRS) broth (Biokar, Beauvais, France) for 18 h at 37°C. SCS was
obtained by centrifugation of 18-h-old LB or GG culture adjusted to
5 × 108 CFU of bacteria per ml at 10,000 × g for 30 min at 4°C. Centrifuged SCS was passed through a
sterile 0.22-µm-pore-size filter (Millex GS; Millipore, Molsheim,
France). The sterility of filtered SCS was verified by plating on BHI
agar. Since a pH ranging from 4 to 4.5 for different SCSs was observed,
the pH of cultures was adjusted to 4.5 with HCl for all experiments.
Cell culture.
The mucus-secreting HT29-MTX cell
subpopulation (28) selected from the parental, mainly
undifferentiated HT-29 cell line (21) by growth adaptation
to methotrexate (10
6 M) was used. This subpopulation
remains stable when it is subcultured under standard conditions, i.e.,
in standard glucose-containing medium. Cells were routinely grown in
Dulbecco modified Eagle's medium (DMEM) (25 mM glucose) (Life
Technologies, Cergy-Pontoise, France), supplemented with 10%
inactivated (30 min, 56°C) fetal bovine serum (Life Technologies).
Monolayers of cells were grown in six-well tissue culture plates (ATGC
Biotechnologies, Paris, France). Cells were seeded at a density of
2 × 104 cells per cm2. For maintenance
purposes, cells were passaged weekly by using 0.02% trypsin in
Ca2+Mg2+-free phosphate-buffered saline (PBS)
containing 3 mM EDTA. Experiments and maintenance of the cells were
carried out at 37°C in a 10% CO2-90% air atmosphere.
The culture medium was changed daily. Cultures were used at late
postconfluence, i.e., after 20 days in culture.
Antimicrobial testing.
The method used to determine the
viability of H. pylori subjected to LB- or GG-SCS has
been previously described (2, 12). Briefly, an 18-h-old
H. pylori culture in BHI agar was resuspended in PBS
and centrifuged at 5,500 × g for 5 min at 4°C. The
supernatant was discarded, and the bacteria were washed once with
sterile PBS and resuspended in BHI. Colony count assays were performed by incubating approximately 250 µl of H. pylori in
BHI (4 × 108 CFU of bacteria per ml) with 250 µl of
LB- or GG-SCS and 500 µl of DMEM at 37°C. Initially and then at
predetermined intervals, aliquots were removed, serially diluted, and
plated on BHI agar to determine bacterial colony counts.
Inhibition assays of H. pylori cell
association.
The inhibition of the H. pylori
association with HT29-MTX cells by LB-SCS or GG-SCS was determined by
preincubating the pathogen (250 µl of 108-CFU/ml BHI)
with 250 µl of LB- or GG-SCS for 1 h at 37°C. After centrifugation (5,500 × g, 10 min, 4°C), the
bacteria were washed with sterile PBS and resuspended in the HT29-MTX
cell culture medium. A total of 1 ml of the LB-SCS or GG-SCS treated
H. pylori was added to each well of the tissue culture
plate. The plates were incubated for 120 min at 37°C in 10%
CO2-90% air and then washed three times with sterile PBS.
The cell-associated H. pylori (extracellular plus
intracellular bacteria) was released by adding H2O to lyse
the HT29-MTX cells. Appropriate dilutions of the lysate were plated on
BHI agar to determine the number of viable cell-associated bacteria by
bacterial colony counts.
Infection of conventional mice by H. felis.
Adult
female conventional BALB/c mice previously used to examine
Helicobacter infection in vivo (13, 19, 31, 34,
36) were chosen. Conventional mice (Iffa Credo, L'Arbresle 69, France) were 6 weeks of age. They were housed and fed in accordance
with the relevant national legislation. Mice were fed a diet of
commercial RO3 ad libitum (UAR, Villemoisson/Orge, France) and
demineralized water. Mice (three groups of 12 to 13 mice) were infected
intragastrically with a fixed concentration of H. felis
(0.2 ml; 108 CFU/mouse). Three weeks postinfection, two
groups of H. felis-infected mice (12 mice per group)
were treated over a period of 7 days. The first received daily 1 ml of
LB-SCS per mouse. The second received a combined antibiotic and antacid
treatment (0.2 ml of water containing 62.5 mg of amoxicillin/ml plus
0.4 mg of omeprazole/ml per mouse (3).
Histology and assessment of H. felis
bacteria.
At 1 and 42 days posttreatment (29 and 70 days
postinfection, respectively), 6 or 7 mice in the H. felis-infected, H. felis-infected and
LB-SCS-treated, and H. felis-infected and amoxicillin
plus omeprazole-treated groups were killed. The stomach of each mouse was removed and dissected longitudinally. Urease activity was determined in one half, and the other half was fixed in 10%
formalin-buffered saline. The tissue was then trimmed to include all
areas of the stomach, processed by standard methods (13,
31), and embedded in paraffin. Five-micrometer sections were cut
and stained with hematoxylin-eosin and Giemsa. The sections were coded
by V. Liévin and examined in a blind manner by E. Hemery. The
following areas of the stomach were examined: cardia-like tissue,
nonglandular area, body, antrum-body border, and antrum. Gastritis and
inflammation were examined in hematoxylin-eosin-stained sections.
Gastritis and/or inflammation was defined by the presence of
lymphocytic or neutrophilic infiltration or by the presence of
epithelial cell dedifferentiation (31). Examining the
different anatomical regions of the H. felis-infected
and the LB-SCS-treated H. felis-infected mice in
Giemsa-stained sections, H. felis cells colonizing the glands were graded on a scale of 0 to 4 as follows: 0, absence of
cells; 1, cells isolated and randomly distributed; 2, reduced number of
cells; 3, large number of cells; 4, high number of cells.
Determination of urease activity.
Urease activity was
determined by a method based on the commercial rapid urease test
(Jatrox test; Röhm-Pharma GmbH, Weiterstadt, Germany) with a
sensitivity of 102 bacteria (31). Briefly, 10 µl of H. pylori culture was added to 1 ml of the
reaction solution (1 g of urea/ml [wt/vol] containing 850 µg of
phenol red/ml [wt/vol] as a pH indicator). For the determination of
urease activity in the stomachs of the H. felis-infected and LB-SCS-treated H. felis-infected mice, each half stomach was placed in 1 ml of the
reaction solution. The development of urease activity was measured as a
function of time by a spectrophotometric analysis at 550 nm. Urease
activity was standardized per weight unit of stomach material. The
standard deviation of the means of the weights of stomachs examined was
less than 5%.
Transmission and scanning electron microscopy.
A
108-CFU/ml inoculum of H. pylori was
treated with LB-SCS for 2 h at 37°C. After centrifugation
(5,500 × g, 10 min, 4°C), the bacteria were washed
with PBS. After negative staining with phosphotungstic acid (2%
[wt/vol] in H2O), the specimens were examined with a
Philips E-320 transmission electron microscope at 60 kV.
After the H. pylori adhesion assay, cultured HT29-MTX
cells were fixed with 2.5% (vol/vol) glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for 1 h at room temperature, washed with
phosphate buffer, postfixed for 30 min with 2% (wt/vol)
OsO4 in the same buffer, washed three times with the same
buffer, and dehydrated in a graded series of 30 to 100% ethanol
treatments. Cells were dried in a critical-point dryer (Balzers CPD030)
and coated with gold. The specimens were then examined with a JEOL scanning electron microscope.
Determination of lactic acid.
To determine the lactic acid
concentration in the SCS, a commercial kit for D- and
L-lactic acid was used (Test Combination D-lactic acid and L-lactic acid UV method;
Boehringer Mannheim GmbH, Mannheim, Germany).
Analysis.
Results are expressed as means ± standard
error of the mean. For statistical comparisons, the Student
t test was performed.
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RESULTS |
Activity of LB-SCS against H. pylori infecting the
cultured human mucosecreting polarized HT29-MTX cells.
The
antibacterial activity of the LB-SCS against H. pylori
was examined by using H. pylori 1011 as an indicator.
To study the impact of the LB-SCS treatment on the H. pylori interaction with the mucus secreting cells, cultured human
colon adenocarcinoma mucosecreting HT29-MTX cells were used.
H. pylori was treated with LB-SCS as a function of time
prior to the adhesion assay. Viability and binding of
H. pylori to
the human mucosecreting HT29-MTX cells was quantified.
As a control,
the MRS broth adjusted to pH 4.5 showed slight activity
compared
with the LB-SCS activity; after 1 h of contact there was
a 1-log
decrease in viable
H. pylori and binding was
observed (Fig.
1).
Because an identical
1-log decrease in viability was observed
with PBS adjusted to pH
4.5 (data not shown), the decrease in
the activity of the control
could be attributed to the autolysis
of
H. pylori
during incubation at 37°C. Fig.
1 shows the rapid
decrease in
viability and binding of
H. pylori as a function of
exposure to LB-SCS. After 0.5 h of contact, a 2.5-log decrease
in
both
H. pylori viability and binding was observed.
After 1
h of contact, a dramatic (6-log) decrease of
viability and binding
was obtained.
H. pylori binding
to HT29-MTX cells decreased dose
dependently in parallel with the
dose-dependent decrease in
H. pylori viability (data
not shown).
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FIG. 1.
Time-dependent activity of L. acidophilus
LB-SCS and two other treatments against H. pylori.
Closed symbols, viability of H. pylori; open symbols,
adhesion of H. pylori to the cultured human polarized
mucosecreting HT29-MTX cells. Each value shown is the mean ± standard error of the mean from three experiments. BHI, control
H. pylori in BHI broth; MRS, MRS broth.
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As observed by transmission electron microscopy (Fig.
2), the untreated
H. pylori presented the characteristic curved form
with sheathed
polar flagella (Fig.
2A). Upon LB-SCS treatment,
H. pylori showed a sequence of ultrastructural changes. The first
ultrastructural sign of the morphological transformation was the
formation of bleb-like structures (not shown) which increased
in size,
increasing the curvature of the bacterium (Fig.
2C).
In the end, the
bacteria took a U-shaped form that decreased in
size, ultimately
assuming a precoccoid form (Fig.
2D). As observed
by scanning electron
microscopy, the
H. pylori interacted with
the brush
border of the cell and the secreted mucin (Fig.
3A and
B). The altered form and the
coccoid form of LB-SCS-treated
H. pylori cells adhered
to the HT29-MTX cells (Fig.
2C and D).
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FIG. 2.
Transmission electron micrographs showing H. pylori bacteria that were controls (A), lactic acid treated (B),
and LB-SCS treated (C and D). The micrographs are representative of
three separate experiments.
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FIG. 3.
Scanning electron micrographs of adhesion of
H. pylori to the mucus-secreting HT29-MTX cells. Panels
show control (A and B) and LB-SCS-treated (C and D) H. pylori. Arrows in panel D show adhering coccoid forms of the
H. pylori. The micrographs are representative of three
separate experiments.
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In order to determine if the lactic acid produced by the LB strain
promoted the decrease in
H. pylori viability, an
additional
experiment was conducted. Enzymatic determination
revealed that
the level of lactic acid in the LB-SCS was 84 mM.
When
H. pylori 1101 was subjected to 100 mM
L- or
DL-lactic acid at pH 4.5, its
viability
remained unchanged (Table
1). Based
on transmission
electron microscopy (Fig.
2), it was apparent that the
morphology
of
H. pylori subjected to lactic acid
treatment was unchanged.
Altogether, these results suggest that the
substance produced
by the LB strain, which develops
H. pylori antagonistic activity,
was distinguishable from the lactic
acid produced by the strain.
L. casei GG was previously described as secreting an
antibacterial compound(s) (
38) that is active in vitro
and in vivo
against
S. typhimurium (
22). The
GG-SCS is active against
H. pylori but is less
potent in its inhibitory action than LB-SCS.
A 2.5-log decrease of
H. pylori viability occurred after 1 h of
contact
with GG-SCS, whereas a 5.5-log decrease occurred after
LB-SCS
treatment. In contrast to the LB-SCS, which lost only a
part of its
activity after heating, GG-SCS entirely lost its activity
against
H. pylori after heating (Table
1).
Activity of LB-SCS against H. felis-infected
mice.
The development of anti-Helicobacter activity of
the LB-SCS in vivo was examined. For this purpose, the conventional
BALB/c mouse model infected orally with H. felis CS1
was used. A group of 13 mice was infected with a single dose of
H. felis. At days 29 and 70 postinfection, seven and
six animals, respectively, were sacrificed and evaluated for infection
(Table 2 and Fig. 4). At 29 days postinfection, all of the
infected mice were positive for spiral bacteria. Moreover, five of
seven were found positive by histology that showed evidence of gastric
inflammation characterized by an inflammatory infiltrate of
lymphocytes, plasma cells, and polymorphonuclear cells in the antrum
and corpus mucosa. At 70 days postinfection, the overall prevalence of
infection remained high, with four of six mice being positive by
histology and all the mice being positive for H. felis.
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TABLE 2.
Histopathologic analysis and H. felis
evaluation in gastric mucosa of conventional BALB/c mice infected with
H. felis and treated with LB-SCS or amoxicillin
plus omeprazole
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FIG. 4.
Histopathologic analysis of the gastric mucosa of
conventional BALB/c mice infected with H. felis and
treated with LB-SCS or amoxicillin-omeprazole. (A) Gastric mucosa of a
21-day H. felis-infected mouse (hematoxylin and eosin
staining; original magnification, ×150). (B) H. felis
in gastric mucosa of a 21-day H. felis-infected mouse
(Giemsa staining; original magnification, ×300). (C) Gastric mucosa of
a 21-day H. felis-infected mouse treated for 7 days
with LB-SCS (hematoxylin and eosin staining; original magnification,
×150). (D) Gastric mucosa of a 21-day H. felis-infected mouse treated for 7 days with
amoxicillin-omeprazole (hematoxylin and eosin staining; original
magnification, ×150).
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Two groups of 12 or 13
H. felis-infected mice at 21 days postinfection were treated daily for 7 days with LB-SCS or an
amoxicillin-omeprazole
mixture. At days 1 and 42 posttreatment
(days 29 and 70 postinfection,
respectively), half of the animals were
sacrificed and evaluated
for infection as described above (Table
2 and
Fig.
4). In the
LB-SCS-treated group, all mice at both days 1 and
42 posttreatment
showed the presence of
H. felis in
their stomachs. However, a
highly significant decrease in the score of
H. felis infection
at day 1 posttreatment compared with
the control group was observed.
In contrast, none of the mice at day 1 posttreatment showed inflammatory
changes compared with the untreated
H. felis-infected mice. At
day 42 post-LB-SCS
treatment, the score in
H. felis remained unchanged,
and all the stomachs presented a noninflammatory pattern.
H. felis-infected
mice treated with amoxicillin plus omeprazole were
totally cured
of
H. pylori at day 1 posttreatment as no
bacteria were observed
in histologic sections and no inflammatory
lesions were found
(Table
2 and Fig.
4). At day 42 posttreatment,
H. felis reappeared
in the stomachs of mice without
histological signs of
inflammation.
LB-SCS inhibits H. pylori urease activity in vitro
and in vivo.
The activity of LB-SCS on the in vitro urease
activity of H. pylori was examined. As shown in Fig.
5, the urease activity in
H. pylori culture was inhibited in the presence
of LB-SCS. In comparison, GG-SCS expressed weak antagonistic
activity. The inhibitory effect of LB-SCS on H. pylori urease activity was heat stable, whereas the GG-SCS
inhibitory activity was totally abolished by heating (results not
shown). Examining whether lactic acid participates in inhibition of the
H. pylori urease activity, it was found that a
concentration of 200 mM DL-lactic acid totally inhibited
the H. pylori urease activity, whereas a range of
concentrations from 60 to 100 mM, similar to that determined in the
LB-SCS (84 mM), failed to inhibit urease activity of H. pylori. These results demonstrate that lactic acid does not
participate in the action of LB-SCS against the H. pylori urease.
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FIG. 5.
In vitro activity of LB-SCS and GG-SCS against the
urease activity of H. pylori. Each value shown is the
mean of three experiments (standard deviations are not shown but were
less than 5%). Statistical analysis performed with a Student
t test at 90 min between treatment with MRS broth and LB-SCS
or LB-SCS and GG-SCS showed a highly significant difference
(P < 0.01). O.D., optical density. MRS, MRS broth.
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Urease activity was detected in the human mucosecreting HT29-MTX cells
infected with
H. pylori (Fig.
6), consistent with the
observed presence
of 6 logs of viable adhering
H. pylori (Fig.
1). It was
observed that 2 logs of viable adherent
H. pylori
remained
present after LB-SCS treatment (Fig.
1). No urease activity
was
detected for these adherent
H. pylori bacteria. In
contrast, viable,
adherent, GG-SCS-treated
H. pylori
displayed a high level of urease
activity, comparable to that of
untreated viable and adherent
H. pylori.
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FIG. 6.
Urease activity of adherent LB-SCS- and GG-SCS-treated
H. pylori to cultured human mucosecreting HT-29MTX
cells. Each value shown is the mean from three experiments (standard
deviations are not shown but were less than 5%). Statistical analysis
performed with a Student t test at 175 min between MRS broth
and LB-SCS treatments shows a highly significant difference
(P < 0.01). No significant difference was found
between MRS broth and GG-SCS treatments. O.D., optical density.
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The urease activity in the
H. felis-infected mouse
stomachs was determined (Fig.
7). At 29 days postinfection, urease activity
developed rapidly in the
H. felis-infected mouse stomachs. In
the
H. felis-infected group that was treated with LB-SCS at day
1 posttreatment (29 days postinfection), the urease activity developed
slowly, and 50% inhibition was observed relative to the maximum
observed with the untreated
H. felis-infected mice. In
the
H. felis-infected mice treated with amoxicillin
plus omeprazole,
urease activity was not detected in any of the
stomachs at day
1 posttreatment (29 days postinfection). At day 42 posttreatment
(70 days postinfection),
H. felis urease
activity was present
in the stomachs of both the
H. felis-infected LB-SCS-treated and
the
amoxicillin-plus-omeprazole-treated mice (not shown).
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FIG. 7.
Urease activity in the stomachs of conventional BALB/c
mice infected with H. felis and treated with LB-SCS or
amoxicillin plus omeprazole. Urease activity was measured in the
stomachs of mice 29 days postinfection. Each value shown is the mean
from 6 or 7 mice (the standard deviations are not shown but were less
than 5%). Statistical analysis performed with a Student t
test between treatment with the control and LB-SCS or amoxicillin plus
omeprazole showed a highly significant difference (P < 0.01). O.D.; optical density.
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DISCUSSION |
The selected human L. acidophilus strains LB and LA1
isolated from human stools inhibit in vitro association with and entry into cultured human enterocytic cells by enterovirulent bacteria (1, 7, 11). These strains produce an antimicrobial
substance(s) active in vitro and in vivo against S. typhimurium infection (2, 12). The results of this
study show that L. acidophilus LB secretes a
heat-stable antimicrobial substance(s) active against
Helicobacter infection in vitro and in vivo. It is known
that lactobacilli secrete metabolic products (39) such as
lactic acid exerting activity against H. pylori
(4, 24, 33). Our results provide evidence that the
anti-Helicobacter substance(s) found in LB-SCS was different
from lactic acid. Species in human gut microflora are known to exert
antagonistic activities referred to as the barrier effect against
pathogens by the production of metabolites (15). Moreover,
it is well established that an ecological disequilibrium in the human
colon results from the alteration of the endogenous microflora by
antibiotics, which allows the emergence and the development of the
virulence mechanisms of Clostridium difficile, the
etiological agent of pseudomembraneous colitis (29).
Lactobacilli are components of the normal intestinal flora of healthy
humans. In particular, it has been reported that the primary
microorganisms associated with the stomach belong to the genus
Lactobacillus (16). This could result from the
particular capacity of these bacteria to survive and develop in an
acidic environment. Recently, Kabir et al. (24) reported
that a strain of Lactobacillus salivarius develops an in
vitro and in vivo antagonistic effect against H. pylori. The inhibitory effect of L. acidophilus on
the growth of H. pylori has been observed in vitro
resulting from a secreted product(s) (4). A recent clinical
study demonstrates that the SCS of L. acidophilus LA1,
secreting an antibacterial compound(s) (2), used as an
adjuvant of antibiotic-antacid treatment prevents the reemergence of
infecting H. pylori in humans (32).
LB-SCS exhibits antagonistic activity in vivo against H. felis infecting the mouse. H. felis is a bacterium
closely related to H. pylori, capable of producing
urease, colonizing the stomach, and inducing gastric lesions (13,
19, 31, 34, 36). When examining the cell damage, the results
demonstrated the therapeutic efficacy of LB-SCS in mice infected with
H. felis, although H. felis was
observed in the stomachs of LB-SCS-treated mice. The reappearance
of H. felis in the stomachs of the
amoxicillin-plus-omeprazole-treated mice was observed 42 days
after the treatment was stopped. Michetti et al. (32) also
reported the reappearance of H. pylori in humans subjected to combined antibiotic-antacid treatment. This
phenomenon could be explained by the fact that H. pylori could survive in pockets of the apical cell membrane
(13). Transformation of H. pylori into
coccoid forms could explain this survival, although this point remains
controversial. Indeed, H. pylori has a helical bacillary appearance in favorable conditions which undergoes
transformation into coccoid forms under unfavorable conditions
(5, 6). The role of H. pylori coccoid forms
in the transmission of H. pylori and in the
relapse of infection after antimicrobial therapy (3, 18) is
still a matter of debate. Several authors have proposed that coccoid
forms are a mechanism by which H. pylori can survive
harsh environmental conditions or is able to convert to resistant forms
under conditions of therapeutical use. Others believe the coccoid form
of H. pylori is the morphological manifestation of
bacterial cell death (26). The H. pylori
ultrastructural changes observed upon LB-SCS treatment resemble the
sequence of those related to H. pylori cell death
observed by Kusters et al. (26).
The acidic environment plays an important role in colonization of the
stomach by Helicobacter (10). The urease of
Helicobacter is a surface protein component of
H. pylori producing ammonia which allows survival by
neutralizing the acidic environment (17, 27). When examining
the mechanism by which the LB strain exerts activity against
Helicobacter, urease activity of H. pylori
was inhibited in vitro. Moreover, mice infected with H. felis were protected when LB-SCS was administered orally.
McGowan et al. (30) reported that a dramatic decrease
in H. pylori viability occurs in vitro at pH 2, and
survival of H. pylori at this pH is markedly
enhanced in the presence of urea resulting from the urease activity of
H. pylori. Considering this, it is tempting to
speculate that the in vivo inhibitory activity of the LB strain against
H. felis infection could result both from the
inhibition of the H. felis urease activity and from the
secreted antibacterial compound(s).
In conclusion, the data presented here clearly show that the
human L. acidophilus strain LB exerts
antagonistic activity against Helicobacter infection in
vitro and in vivo.
| |
ACKNOWLEDGMENTS |
This work was supported by a research contract between the
Institut National de la Santé et de la Recherche Médicale
(INSERM), the Université Paris XI, and Lactéol Laboratory
(Houdan, France).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: UPS
Faculté de Pharmacie Paris XI, CJF 94.07, INSERM, F-92296
Châtenay-Malabry, France. Phone and fax: 33.1.46.83.56.61. E-mail: alain.servin{at}cep.u-psud.fr.
| |
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Abstract
Introduction
