1. health and fitness
2. disease

Int J Entric Pathog. 2013 November; 1(2): 36-42.
Research Article
Published Online 2013 Navember 1.
Probiotic Therapy, What is the most Effective Method for Host Protection
Against Enteric Pathogen
1
2
Sayyed Mohammad Hossein Ghaderian , Mahboobeh Mehrabani Natanzi , Mahdi
3
2, *
Goudarzvand , Zohreh Khodaii
1 Department of Medical Genetics, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, IR Iran
2 Dietary Supplements and Probiotic Research Center, Alborz University of Medical Sciences, Karaj, IR Iran
3 Physiology and Pharmacology Department, Faculty of Medicine, Alborz University of Medical Sciences, Karaj, IR Iran
*Corresponding author: Zohreh Khodaii, Department of Nutrition-Biochemistry, Faculty of Medicine, Alborz University of Medical Sciences, Karaj, IR Iran. Tel: +98-2634336007, Fax:
+98-2634319188, E-mail: [email protected]
Received: August 07, 2013; Revised: August 11, 2013; Accepted: August 31, 2013
Background: Prevention of adverse microbial colonization is supposed to be the most important beneficial effect of the gut microflora.
Objectives: The aims of the present study were to compare the effect of co-incubation, pre-incubation and supernatant of sixteen probiotic
strains on prevention of enteroinvasive E. coli adhesion.
Materials and Methods: Probiotic strains were added to Caco-2 cells followed by E. coli in pre-incubation assay. Tested strains and
enteroinvasive E. coli were added to cell lines at the same time in co-incubation assay. Finally, enteroinvasive E. coli was treated with bacteria
free supernatant of test strains then added to cell line in treatment with bacteria free supernatant assay.
Results: This study showed that the most effective assays in prevention of pathogen adherence were treatment with bacteria free
supernatant and pre-incubation respectively.
Conclusions: The effect of probiotic bacteria by-products on pathogen exclusion may be of more importance in protecting the host.
Therefore, gut colonization or at least persistent presence of probiotics may be helpful in infection prevention.
Keywords: Probiotics; Pathogen Exclusion; Co-Incubation; Pre-Incubation; Caco-2 Cells
1. Background
Prevention of adverse microbial colonisation is supposed to be the most important beneficial effect of the
gut microflora. Probiotic bacteria can change the intestinal normal flora from a potentially harmful microflora towards a beneficial composition (1, 2). Adhesion,
competitive exclusion capacity, immunomodulation,
and prevention of gastrointestinal epithelium from infection are important criteria for selection of probiotic
bacteria (3-5). The effectiveness of probiotics on pathogen inhibition has been reported in vitro and in vivo (4).
Co-incubation of probiotics and pathogens decreased
the number of ureolytic pathogen and completely inhibited its urease activity (6). Pre-incubation of human
intestinal cell lines, HT29 and Caco-2, with Lactobacillus
acidophilus reduced adhesion and invasion of enteroinvasive E. coli to the cell lines, whereas co-incubation of
probiotic and pathogen had less significant preventative effects (7). Pre-incubation of Bifidobacterium strains
were effective on pathogens exclusion from human
intestinal mucus (8). The bactericidal activity and adhesion prevention of bacteria free supernatant (bfs) of
lactic acid bacteria (LAB) against Helicobacter pylori
was reported by Lin and co-workers (9). The potential
mechanisms by which probiotics can exert their beneficial effects were reported limiting the access of harmful
bacteria to host mucosal surfaces, by steric hindrance
and altering the response of the host to microbial attack
(10). The alteration of the microenvironment or interference of probiotic bacteria with the signaling cascades
triggered by the pathogen, are important as well (11).
For investigation of the mechanisms by which probiotic
can inhibit pathogen adhesion, different methods and
cell lines has been used. However, there are no comparisons between different methods on pathogen adhesion
Implication for health policy/practice/research/medical education:
Probiotics are a group of microorganisms that beneficially affect the host. Adhesion, competitive exclusion capacity, immunomodulation, and prevention of gastrointestinal epithelium from infection are important criteria for selection of probiotic bacteria. For investigation of the mechanisms by
which probiotic can inhibit pathogen adhesion, different methods and cell lines has been used. However, there are no comparisons between different
methods on pathogen adhesion prevention. In the present study, the effect of probiotic bacteria on Enter invasive E. coli (EIEC) adhesion was evaluated to
find the most preventive underlying mechanism of these strains against EIEC.
Copyright © 2014, Alborz University of Medical Sciences.; Published by Safnek. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Ghaderian SMH et al.
prevention.
2. Objectives
In the present study, the effect of sixteen putative probiotic bacteria on enteroinvasive E. coli (EIEC) adhesion
was evaluated to find the most preventive underlying
mechanism of these strains against EIEC.
3. Materials and Methods
3.1. Bacterial Strains, Cultivation Conditions and
Preparation
Eleven adhesive strains were isolated either from
pharmaceutical or dairy products and identified using
biochemical and molecular methods. Methods of probiotic isolation, identification and adherence were used
according to Haeri et al. (12). Lactobacilli strains were
L. acidophilus isolated from advanced acidophilus plus
Solgar Ltd. (named 1C2), Quest Digestive Aids Quest Vitamines Ltd. (2C1), Multibionta Seven Seas Ltd. (4C1) and
Health Aid acidophilus Pharmadas Ltd. (5C1), L. Plantarum isolated from children chewy Acidophilus/ Chewy
Bears and friends American Health Ltd. (6C3), L. Brevis
Betta buy low fat fruit flavour yoghurt Morrison’s (1D2),
L. Sanfrancisco Low fat natural yoghurt Morrison’s (2D3),
L. Casei (Shirota) Yakult fermented drink (6D2) and
three bifidobacterium spps isolated from Active Danone
France (7D1), Vitality yogurt Müller (8D1) and Probiotic
low fat yogurt Tesco (9D1). Five type strains purchased
from NCIMB were Lactobacillus acidophilus (L. acidophilusT) NCIMB 701748, Lactobacillus casei rhamnosus (L.
rhamnosusT) NCIMB 8010, Lactobacillus casei subspecies
casei (L. casei T) NCIMB 11970, Bifidobacterium bifidum (B.
bifidumT) NCIMB 702715 and Bifidobacterium longum (B.
longumT) NCIMB 702259. Lactobacilli strains were cultivated on MRS broth at 37 °C on air for 24 hours. Bifidobacteria were cultivated on TPY broth at 37 °C anaerobically
using Genbox for 24 hours.
E. coli G24 enteroinvasive (EIEC) with localised pattern of adherence kindly donated by Dr. J. Fletcher from
teaching collection of University of Bradford, UK. Pathogen strains were cultured on LB broth at 37 °C on air for
24 hours. All test strains were centrifuged, washed twice
with PBS and resuspend on culture medium at density
of 1×107/ ml before each assay.
3.2. Cell Line Culture
Caco-2 cells (CB No: 02D052) were bought at passage
number 7 from ECCAC, grown in Minimum Essential Medium (Sigma M 2279), Foetal calf serum (Lablech 4-101500) 10%, Non-essential amino acid solution (Sigma M
7145) 1%, L-Glutamine (GIBCO 25030-024) 1% and penicilInt J Entric Pathog. 2014;1(2)
lin 10,000 unit/ml & streptomycin 10,000 µg/ml (GIBCO
15140-122) 1% (all concentrations are v/v).
After second passage from thawed cells, Caco-2 cells
were sub-cultured at a density of 5 × 103 cells / ml into
12-well plates (Corning / Costar 3513) plus 2 ml cell culture medium, with a coverslip (16 mm diameter, BDH,
406/89/22) at the bottom of each well and incubated at
37 °C / 5% CO2. The cell culture medium was replaced every other day. Cells were ready for use after reaching the
confluecy, between days 13 - 21 after cultivation.
A total of at least 100 tissue culture cells in the fields at
the corners (not near the edge of the coverslip) and center of a coverslip were used to count the number of adherent pathogen and putative probiotic bacteria. These
values were used for further analysis. Wells with either
only pathogen, only test strain, only Caco-2 cells or no
bacteria-no cell line, acted as controls and the number
of adherent bacteria per 100 tissue culture cells was recorded. All experiments were carried out in duplicate
on three separate occasions.
3.3. Prevention of Pathogen Adherence by Probiotics
3.3.1. Co-incubation Assay
In this assay potential probiotics and the pathogen,
EIEC E. coli G24, were added to the tissue culture monolayer simultaneously to provide a competitive adherence assay. One hundred micro liters of each of the two
prepared bacterial strains, contained 1×106 colony forming unit (cfu), were resuspended in 1800 micro liters
tissue culture medium without antibiotics, then added
simultaneously to the cell monolayers and incubated at
37 °C in 5% CO2 for 3 hours. Wells with either only pathogen acted as controls. All experiments were carried out
in duplicate on three separate occasions.
The media and unattached bacteria were aspirated
and the coverslips were washed, fixed, and stained using a standard Gram stain method (13).
The stained coverslips were removed from the wells,
dehydrated and then allowed to dry. The side of the coverslip with the attached cells was mounted on a light
microscope slide using Histomount (VWR 362622L) (14).
3.3.2. Pre-incubation Assay
To study the effect of probiotic bacteria already adherent to the tissue culture cells on the prevention of
pathogen adherence, lactobacilli or bifidobacteria were
incubated with the tissue culture cells, before pathogen
was added. Cells lines were prepared as described previously. The medium was removed and cells were washed
twice with PBS. Then 150 µl of overnight culture of potential probiotics, prepared as described previously,
were added to each well and incubated at 37 °C in 5 %
CO2. After 3 hours the well content was aspirated and
37
Ghaderian SMH et al.
cells were washed twice with PBS then 2 ml standard cell
culture media was added. One hundred and fifty microliters of overnight culture of E. coli G24 contained 1×106
cfu was prepared as before, added to each well and incubated for a further 3 hrs at 37 °C in 5 % CO2 after which
coverslips were prepared for analysis.
3.4. Treatment of Pathogen with bfs
To evaluate the effect of bacteria free supernatant from
lactobacilli and bifidobacteria on adherence of E. coli G24,
15 ml of an overnight culture of lactobacilli or bifidobacteria in MRS contained 1×109 cfu, was centrifuged at
8000g for 30 min. The supernatant was filter sterilized
using a 0.2-µm pore-size filters (SARSTED 83.1826.001).
Then 150 µl of E. coli G24 (EIEC) was added to each sterile
supernatant and incubated at 37 °C in a shaking incubator (Gallenkamp Orbital Incubator, England) for 2 hours.
The bacteria were then harvested by centrifugation
(8000 g / 15 min), washed 3 times with PBS, and aliquots
used for either a viability check or added to T84 cells or
Caco-2 cell monolayers. Cell lines were then incubated at
37 °C in 5 % CO2 for 3 hours and coverslips were prepared
and analyzed. EIEC with no treatment acted as control.
The viability of E. coli was checked after incubation of
E. coli with bfs of all test strains by preparation of decimal dilutions and plating out of 0.1 ml of each dilution.
The colonies formed in each plate were counted visually
after overnight incubation.
3.5. Statistical Analysis
To test if there is any difference in the level of adherence of pathogen when it is co-incubated with probiotic
or if the probiotic is present beforehand, results were
analyzed using the unpaired Student’s t test. In this statistical analysis, the difference between the mean number of probiotic and pathogen adherent to the both assays were compared. Thus the effect of co-incubation or
pre-incubation on the number of probiotic or pathogen
cells attached to the cell line was evaluated. All results
were reported as mean ± SEM and a value of P < 0.05 was
considered as significant. All experiments were carried
out in duplicate on three separate occasions. All experiments were carried out in duplicate on three separate
occasions.
cubation or pre-incubation assays. The results showed
that after 3 hours of co-incubation the number of E. coli
attached was significantly reduced compared to the
control with all strains except 5 out of 16 strains (Figure
1 A). These strains were 2C1 (L. acidophilus), 7D1 (Bifidobacterium sp.), 8D1 (Bifidobacterium sp.) and 9D1 (Bifidobacterium sp.) and L. CaseiT (Table 1). In the pre-incubation assay format, the number of E. coli attached to Caco-2 cells
was significantly reduced with all strains compared to
the control except 3 out of 16 strains (Figure 1 B). These
strains were 5C1 (L. Acidophilus), L. acidophilus T , B. Longum T (Table 1). The number of adhering pathogen significantly decreased after treatment of E. coli G24 with
bacteria free supernatant of all test strains. Incubation
of E. coli G24 with bacteria free supernatant of test strain
for 2 hours at 37 °C resulted in a significant decrease in
adherence of pathogen. Isolates 5C1 (L. acidophilus) and
6C3 (L. plantarum) gave less inhibitory effects compared
to other isolate (Table 1).
All results are mean of six experiments. Values are expressed as mean ± SEM,
4.2. Comparison of Co-Incubation and Pre-Incubation Methods
The isolates 2C1 (L. acidophilus), 7D1 (Bifidobacterium
sp.), 8D1 (Bifidobacterium sp.), 9D1 (Bifidobacterium sp.)
and L. casei decreased the number of pathogen cells adhering in the pre-incubation but not co-incubation assay (Table 2). 7 out of 16 strains, the number of adherent
E. coli to the cell lines after pre incubation was significantly less than after co-incubation (Table 2). Co-incubation and pre-incubation had much reduced response
compared to treatment of E. coli with culture supernatant on pathogen adherence prevention.
Figure 1. Adhesion of Enteroinvasive E. Coli Attached to Caco-2 Cells, in
the Co-incubation
4. Results
4.1. Prevention of Pathogen Adherence
When reading slides it was noted that pathogen attached either on different sites on one cell, in different
cells or at the same place as probiotic bacteria. Precise
observation around the slide did not show any particular pattern of co-adherence. Lactobacilli or bifidobacteria
were not able to exclude pathogen completely in co-in38
(A) and pre-incubation (B) assays of 5C1 (L. acidophilus), observed using
light microscopy (x100 magnification), after Gram staining, (E.coli: gram
negative, light red rod and L. acidophilus: Gram positive, dark purple
rod).
Int J Entric Pathog. 2014;1(2)
Ghaderian SMH et al.
Table 1. The Number of Adhered E. coli after Co-incubation, Pre-incubation and Treatment with bfs of Probiotic Strains
Code (strain)
1C2 (L. acidophilus)
2C1 (L. acidophilus)
04C1 (L. acidophilus)
5C1 (L. acidophilus)
6C3 (L.plantarum)
1D2 (L.brevis)
2D3 (L.sanfrancisco)
6D2 (L.caseiShirota)
7D1 (Bifidobacteriumsp.)
8D1 (Bifidobacteriumsp.)
9D1 (Bifidobacteriumsp.)
L. acidophilusT
L.rhamnosus
T
L.caseiT
B.bifidum
T
B.longumT
a P < 0.05
b P < 0.001
Adhered E. coli without
Probiotic (control)
Adhered E. coli After
Co-incubation
Adhered E. coli After
Pre-incubation
Adhered E. coli After
Treatment with bfs
220 ± 42
162 ± 23a
220 ± 42
210 ± 30
160 ± 25a
190 ± 26a
210 ± 31
5 ± 1b
110 ± 21b
6 ± 1b
220 ± 42
220 ± 42
220 ± 42
220 ± 42
220 ± 42
156 ± 17a
117 ± 22b
90 ± 20b
150 ± 27a
180 ± 31a
3 ± 2b
105 ± 12b
3 ± 2b
112 ± 18b
155 ± 15a
4 ± 1b
5 ± 2b
220 ± 42
100 ± 19b
220 ± 42
220 ± 37
90 ± 17b
220 ± 42
210 ± 31
90 ± 21b
12 ± 2b
220 ± 42
225 ± 25
56 ± 9b
15 ± 3b
220 ± 42
115 ± 30b
211 ± 25
6 ± 2b
220 ± 42
22 ± 10b
220 ± 42
205 ± 31
70 ± 24b
3 ± 0b
118 ± 27b
204 ± 21
220 ± 42
220 ± 42
96 ± 33b
80 ± 20b
4 ± 1b
19 ± 6b
85 ± 16b
22 ± 2b
17 ± 3b
4 ± 0b
7 ± 1b
8 ± 2b
Table 2. Comparison of the Effect of Co-incubation and Pre-incubation on the Number of Adhered Probiotic Test Strains and E. coli
Code (strain)
1C2(L. acidophilus)
2C1(L.acidophilus)
4C1(L.acidophilus)
5C1 (L. acidophilus)
6C3(L.plantarum)
1D2(L.brevis)
2D3(L.sanfrancisco)
6D2(L.caseiShirota)
7D1(Bifidobacterium
sp)
8D1(Bifidobacteriu
msp)
9D1(Bifidobacteriu
msp)
L. acidophilusT
L.rhamnosusT
L.caseiT
B.bifidumT
B.longumT
a P < 0.05
b P < 0.001
Co vs Pre-Incubation for E. coli
Adhered E. coli after
co-incubation
162 ± 23
210 ± 30a
156 ± 17b
190 ± 26
Adhered E. coli after
pre-incubation
180 ± 31
160 ± 25
Co vs Pre-Incubation for Probiotic Test Strain
Adhered probiotic
after co-incubation
34 ± 12
45 ± 7b
105 ± 12
55 ± 8b
210 ± 31
32 ± 10
50 ± 6b
Adhered probiotic
after pre-incubation
45 ± 10
95 ± 15
96 ± 15
38 ± 16
117 ± 22b
112 ± 18
90 ± 20
110 ± 21
25 ± 5b
150 ± 27
155 ± 15
66 ± 13
70+18
100 ± 19
80 ± 20
40 ± 11
42 ± 15
115 ± 36
80 ± 14
220 ± 37b
90 ± 17
60 ± 21b
210 ± 31b
90 ± 21
35 ± 10b
157 ± 18
225 ± 25b
56 ± 9
55 ± 7b
209 ± 6
115 ± 30
211 ± 25
28 ± 7a
36 ± 4
22 ± 10
19 ± 6
70 ± 20
70 ± 24
9 ± 3b
70 ± 15
96 ± 33
85 ± 16
35 ± 5
39 ± 7
118 ±27
204 ± 21
24 ±6
25 ± 8
205 ± 31b
Int J Entric Pathog. 2014;1(2)
155 ± 15
70 ± 15
39
Ghaderian SMH et al.
4.3. Comparison of the Number of Adhered Probiotic after Co and Pre-incubation
Not surprisingly, the number of cells of the test strains
adhered to the Caco-2 cells after pre-incubation com-
pared to co-incubation was in most cases significantly
increased (P < 0.05). All test strains had better adherence with the pre-incubation method when they had
opportunity to adhere in the absence of a pathogen
compared to the co-incubation method (Table 3).
Table 3. Results of Comparison of the Number of Adhered Probiotic Test Strains after Co-Incubation and Pre-Incubation
Code (strain)
1C2 (L. acidophilus)
2C1 (L. acidophilus)
4C1 (L. acidophilus)
5C1 (L. acidophilus)
6C3 (L.plantarum)
1D2 (L.brevis)
2D3 (L.sanfrancisco)
6D2 (L.caseiShirota)
7D1 (Bifidobacteriumsp.).
8D1 (Bifidobacteriumsp.)
9D1 (Bifidobacteriumsp.)
L. acidophilusT
L.rhamnosus
T
L.casei
T
B.bifidum
T
B.longumT
a P < 0.05
b P < 0.001
Mean ± SEM of Adhered Probiotic after
Co-Incubation
Mean ± SEM of Adhered Probiotic after
Pre-Incubation
34 ± 12
45 ± 10a
45 ± 7
55 ± 8
32 ± 10
50 ± 6
25 ± 5
66 ± 13
40 ± 11
60 ± 21
35 ± 10
55 ± 7
96 ± 15a
38 ± 16a
115 ± 36b
80 ± 14b
70 ± 18a
42 ± 15a
155 ± 15b
157 ± 18b
209 ± 6b
28 ± 7
36 ± 4a
70 ± 20
70 ± 15
9±3
35 ± 5
24 ± 6
All results are mean of six experiments. Values are expressed as mean ± SEM
5. Discussion
An important aspect of the function of probiotic bacteria
is the protection of the host gastrointestinal micro-environment from invading pathogens (15). It is believed that
the gastrointestinal microflora in vivo provides protection
for the host against colonization by pathogenic bacteria
(15, 16). The methodology used in the present study helps
us to distinguish between the effects of substances in the
culture media or effects due to live bacteria. For evaluation
of test strains on pathogen adherence to the cell lines, we
used light microscopy of stained cell sheets and counted
the number of bacteria adherent to 100 cells of the cell
line. The microscopic method was used previously by
others (17, 18). Lee et al. (19) indicated that the direct microscopic counting and radioactive label measurement
gave comparable results. This method allows the differentiation of the different bacterial types on the cell culture
surface without the risk of radiolabel usage. Counting is
time consuming and may not be as accurate as radiolabeling methods. The present study showed that bacteria free
40
95 ± 15b
70 ± 15b
39 ± 7a
25 ± 8a
supernatant of all isolates either prevent completely or
considerably decreased the adherence of E. coli to Caco-2
cells. The methodology used in this work and the results
were similar to that of other authors (20). This antibacterial effect could be due to bacteriocin-like substances, but
exhibiting much broader activity and produced by a range
of species of lactobacilli. Also we cannot exclude the effect
of low pH, production of organic acids and hydrogen peroxide (21-23). So it is possible that use of probiotic strains
as starter cultures for fermented foods may subsequently
prevent pathogen adherence to the host cells.
Pre-incubation was more effective than co-incubation
in prevention of pathogen adherence, where co-incubation was not effective in prevention of pathogen adherence, pre-incubation assay was effective in that. The exceptions were type strains L. acidophilus and B. longum.
The adherence of these strains to the Caco-2 cells were
poor, so, in the final part of the assay there were few of
that type strains but high numbers of pathogen. The
effect of co-incubation and pre-incubation is in agreement with other authors' results (3).
Comparison of the number of adhered probiotic to the
cell line, in the presence or absent of pathogen was not
noted by other authors. In the present work we enumerInt J Entric Pathog. 2014;1(2)
Ghaderian SMH et al.
ated the adherent probiotic strains after co-incubation
assays and compared them with the number of adherent bacteria when added alone. Statistical analysis of results showed that the values for test strains were mainly
higher for pre-incubation than co-incubation assay.
In the other words, pathogen may exclude or compete
with probiotics for adhering. One possible reason is put
forward by Lee et al. (19). They co-incubated L. casei Shirota and L. rhamnosus GG with E. coli TG and observed
that these lactobacilli were excluded when incubated
together by E. coli adhering to cells. They explained this
effect by the theory that the turnover of some bacteria
adhering to cells is more rapid compared to others and
any detached strain were readily replaced by surrounding E. coli in the culture medium (14). This observation is
in agreement with in vitro, human and animal studies
where lactobacilli are gradually replaced by enterobacteria after the intake of lactobacilli was discontinued
(24, 25). So it can be hypothesized that administration of
probiotics with infected food may be less effective than
if the gut has previously been colonized with probiotics.
The method described here was investigated as an in
vitro model whilst the gut is a very complicated ecosystem and the effectiveness of an isolate will be affected by
bacterial interactions, host immunity and diet and antibiotics. Luyer et al. investigated two probiotic strains of
L. rhamnosus and L. fermentum in vitro and in vivo. Both
strains were able to inhibit the adherence of E. coli to
the Caco-2 cell line and in rat, they reported that there
is a correlation between in vivo and in vitro study (26).
In conclusion, using probiotic bacteria to colonize gut
with adhering probiotics or at least persistence presence of probiotics before probable infection may be
helpful in infection prevention. The effectiveness of probiotics may depend upon the actual pathogen ingested.
To investigate the true effectiveness of an isolate, in vivo
assays are necessary, but this work enables selection of
strains with well-defined properties for specific use.
Acknowledgements
There is no acknowledgment.
Authors’ Contribution
All authors have participated equally in this study.
Financial Disclosure
There is no conflict of interest.
Funding/Support
The study is self-funded.
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