Published Online 2013 November 1. Research Article
Probiotic Therapy, What is the most Effective Method for Host Protection
Against Enteric Pathogen
Sayyed Mohammad Hossein Ghaderian
1, Mahboobeh Mehrabani Natanzi
2, Mahdi
Goudarzvand
3, Zohreh Khodaii
2, *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
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 preven-tion of gastrointestinal epithelium from infecpreven-tion are important criteria for selecpreven-tion of probiotic bacteria. For investigapreven-tion 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 © 2013, Alborz University of Medical Sciences. 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.
1. Background
Prevention of adverse microbial colonisation is sup-posed to be the most important beneficial effect of the gut microflora. Probiotic bacteria can change the intes-tinal normal flora from a potentially harmful micro-flora towards a beneficial composition (1, 2). Adhesion, competitive exclusion capacity, immunomodulation, and prevention of gastrointestinal epithelium from in-fection are important criteria for selection of probiotic bacteria (3-5). The effectiveness of probiotics on patho-gen inhibition has been reported in vitro and in vivo (4). Co-incubation of probiotics and pathogens decreased the number of ureolytic pathogen and completely in-hibited its urease activity (6). Pre-incubation of human intestinal cell lines, HT29 and Caco-2, with Lactobacillus
acidophilus reduced adhesion and invasion of enteroin-vasive E. coli to the cell lines, whereas co-incubation of
probiotic and pathogen had less significant preventa-tive effects (7). Pre-incubation of Bifidobacterium strains were effective on pathogens exclusion from human intestinal mucus (8). The bactericidal activity and ad-hesion 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 benefi-cial 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 interfer-ence 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 compari-sons between different methods on pathogen adhesion
prevention.
2. Objectives
In the present study, the effect of sixteen putative pro-biotic 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 probi-otic 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 Vi-tamines Ltd. (2C1), Multibionta Seven Seas Ltd. (4C1) and Health Aid acidophilus Pharmadas Ltd. (5C1), L. Planta-rum 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. acidophi-lusT) 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
culti-vated on MRS broth at 37 °C on air for 24 hours. Bifidobac-teria were cultivated on TPY broth at 37 °C anaerobically using Genbox for 24 hours.
E. coli G24 enteroinvasive (EIEC) with localised pat-tern of adherence kindly donated by Dr. J. Fletcher from teaching collection of University of Bradford, UK. Patho-gen 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 Me-dium (Sigma M 2279), Foetal calf serum (Lablech 4-101-500) 10%, Non-essential amino acid solution (Sigma M 7145) 1%, L-Glutamine (GIBCO 25030-024) 1% and
penicil-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 cul-ture 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 ev-ery 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 cen-ter of a coverslip were used to count the number of ad-herent 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 re-corded. 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 mono-layer simultaneously to provide a competitive adher-ence assay. One hundred micro liters of each of the two prepared bacterial strains, contained 1×106 colony form-ing 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 patho-gen 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 us-ing a standard Gram stain method (13).
The stained coverslips were removed from the wells, dehydrated and then allowed to dry. The side of the cov-erslip 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 ad-herent 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 previ-ously. The medium was removed and cells were washed twice with PBS. Then 150 µl of overnight culture of po-tential 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
cells were washed twice with PBS then 2 ml standard cell culture media was added. One hundred and fifty micro-liters of overnight culture of E. coli G24 contained 1×106
cfu was prepared as before, added to each well and in-cubated 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 bifido-bacteria 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 incuba-tor (Gallenkamp Orbital Incubaincuba-tor, 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 deci-mal 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 adher-ence 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 sta-tistical analysis, the difference between the mean num-ber of probiotic and pathogen adherent to the both as-says 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 experi-ments were carried out in duplicate on three separate occasions.
4. Results
4.1. Prevention of Pathogen Adherence
When reading slides it was noted that pathogen at-tached 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 particu-lar pattern of co-adherence. Lactobacilli or bifidobacteria
were not able to exclude pathogen completely in
co-in-cubation or pre-inco-in-cubation 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 ( Bifidobac-terium sp.), 8D1 (Bifidobacterium sp.) and 9D1 ( Bifidobacte-rium sp.) and L. CaseiT (Table 1). In the pre-incubation as-say 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.
Long-um T (Table 1). The number of adhering pathogen sig-nificantly 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 ex-pressed as mean ± SEM,
4.2. Comparison of Co-Incubation and
Pre-Incu-bation Methods
The isolates 2C1 (L. acidophilus), 7D1 (Bifidobacterium
sp.), 8D1 (Bifidobacterium sp.), 9D1 (Bifidobacterium sp.) and L. casei decreased the number of pathogen cells ad-hering in the pre-incubation but not co-incubation as-say (Table 2). 7 out of 16 strains, the number of adherent
E. coli to the cell lines after pre incubation was signifi-cantly less than after co-incubation (Table 2). Co-incu-bation and pre-incuCo-incu-bation had much reduced response compared to treatment of E. coli with culture superna-tant on pathogen adherence prevention.
Figure 1. Adhesion of Enteroinvasive E. Coli Attached to Caco-2 Cells, in the Co-incubation
(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).
Table 1. The Number of Adhered E. coli after Co-incubation, Pre-incubation and Treatment with bfs of Probiotic Strains
Code (strain) 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
1C2 (L. acidophilus) 220 ± 42 162 ± 23a 180 ± 31a 3 ± 2b
2C1 (L. acidophilus) 220 ± 42 210 ± 30 160 ± 25a 4 ± 1b
04C1 (L. acidophilus) 220 ± 42 156 ± 17a 105 ± 12b 3 ± 2b
5C1 (L. acidophilus) 220 ± 42 190 ± 26a 210 ± 31 5 ± 1b
6C3 (L.plantarum) 220 ± 42 117 ± 22b 112 ± 18b 4 ± 1b
1D2 (L.brevis) 220 ± 42 90 ± 20b 110 ± 21b 6 ± 1b
2D3 (L.sanfrancisco) 220 ± 42 150 ± 27a 155 ± 15a 5 ± 2b
6D2 (L.caseiShirota) 220 ± 42 100 ± 19b 80 ± 20b 22 ± 2b
7D1
(Bifidobacteri-umsp.) 220 ± 42 220 ± 37 90 ± 17
b 17 ± 3b
8D1
(Bifidobacteri-umsp.) 220 ± 42 210 ± 31 90 ± 21
b 12 ± 2b
9D1
(Bifidobacteri-umsp.) 220 ± 42 225 ± 25 56 ± 9
b 15 ± 3b
L. acidophilusT 220 ± 42 115 ± 30b 211 ± 25 6 ± 2b
L.rhamnosusT 220 ± 42 22 ± 10b 19 ± 6b 4 ± 0b
L.caseiT 220 ± 42 205 ± 31 70 ± 24b 3 ± 0b
B.bifidumT 220 ± 42 96 ± 33b 85 ± 16b 7 ± 1b
B.longumT 220 ± 42 118 ± 27b 204 ± 21 8 ± 2b
a P < 0.05 b P < 0.001
Table 2. Comparison of the Effect of Co-incubation and Pre-incubation on the Number of Adhered Probiotic Test Strains and E. coli
Co vs Pre-Incubation for E. coli Co vs Pre-Incubation for Probiotic Test Strain Code (strain) Adhered E. coli after
co-incubation Adhered E. coli after pre-incubation Adhered probiotic after co-incubation Adhered probiotic after pre-incubation
1C2(L. acidophilus) 162 ± 23 180 ± 31 34 ± 12 45 ± 10
2C1(L.acidophilus) 210 ± 30a 160 ± 25 45 ± 7b 95 ± 15
4C1(L.acidophilus) 156 ± 17b 105 ± 12 55 ± 8b 96 ± 15
5C1 (L. acidophilus) 190 ± 26 210 ± 31 32 ± 10 38 ± 16
6C3(L.plantarum) 117 ± 22b 112 ± 18 50 ± 6b 115 ± 36
1D2(L.brevis) 90 ± 20 110 ± 21 25 ± 5b 80 ± 14
2D3(L.sanfrancisco) 150 ± 27 155 ± 15 66 ± 13 70+18
6D2(L.caseiShirota) 100 ± 19 80 ± 20 40 ± 11 42 ± 15
7D1(Bifidobacterium
sp) 220 ± 37
b 90 ± 17 60 ± 21b 155 ± 15
8D1(Bifidobacteriu
msp) 210 ± 31
b 90 ± 21 35 ± 10b 157 ± 18
9D1(Bifidobacteriu
msp) 225 ± 25
b 56 ± 9 55 ± 7b 209 ± 6
L. acidophilusT 115 ± 30 211 ± 25 28 ± 7a 36 ± 4
L.rhamnosusT 22 ± 10 19 ± 6 70 ± 20 70 ± 15
L.caseiT 205 ± 31b 70 ± 24 9 ± 3b 70 ± 15
B.bifidumT 96 ± 33 85 ± 16 35 ± 5 39 ± 7
B.longumT 118 ±27 204 ± 21 24 ±6 25 ± 8
a P < 0.05 b P < 0.001
4.3. Comparison of the Number of Adhered
Probi-otic 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 adher-ence 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) Mean ± SEM of Adhered Probiotic after
Co-Incubation Mean ± SEM of Adhered Probiotic after Pre-Incubation
1C2 (L. acidophilus) 34 ± 12 45 ± 10a
2C1 (L. acidophilus) 45 ± 7 95 ± 15b
4C1 (L. acidophilus) 55 ± 8 96 ± 15a
5C1 (L. acidophilus) 32 ± 10 38 ± 16a
6C3 (L.plantarum) 50 ± 6 115 ± 36b
1D2 (L.brevis) 25 ± 5 80 ± 14b
2D3 (L.sanfrancisco) 66 ± 13 70 ± 18a
6D2 (L.caseiShirota) 40 ± 11 42 ± 15a
7D1 (Bifidobacteriumsp.). 60 ± 21 155 ± 15b
8D1 (Bifidobacteriumsp.) 35 ± 10 157 ± 18b
9D1 (Bifidobacteriumsp.) 55 ± 7 209 ± 6b
L. acidophilusT 28 ± 7 36 ± 4a
L.rhamnosusT 70 ± 20 70 ± 15
L.caseiT 9 ± 3 70 ± 15b
B.bifidumT 35 ± 5 39 ± 7a
B.longumT 24 ± 6 25 ± 8a
a P < 0.05 b P < 0.001
All results are mean of six experiments. Values are ex-pressed as mean ± SEM
5. Discussion
An important aspect of the function of probiotic bacteria is the protection of the host gastrointestinal micro-envi-ronment 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 mi-croscopic counting and radioactive label measurement gave comparable results. This method allows the differen-tiation 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 radiolabel-ing methods. The present study showed that bacteria free
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 antibacte-rial 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 per-oxide (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-incuba-tion was not effective in prevenco-incuba-tion of pathogen adher-ence, pre-incubation assay was effective in that. The ex-ceptions 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 agree-ment 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
enumer-ated the adherent probiotic strains after co-incubation assays and compared them with the number of adher-ent bacteria when added alone. Statistical analysis of re-sults 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 Shi-rota 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 surround-ing 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 enterobac-teria 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 ecosys-tem and the effectiveness of an isolate will be affected by bacterial interactions, host immunity and diet and anti-biotics. 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 pres-ence of probiotics before probable infection may be helpful in infection prevention. The effectiveness of pro-biotics 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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