LWT - Food Science and Technology 98 (2018) 329–334

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LWT - Food Science and Technology

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Synbiotic combination of Lactobacillus rhamnosus NCDC 298 and short chain T

fructooligosaccharides prevents enterotoxigenic Escherichia coli infection
Santosh Ananda, Surajit Mandala,∗, Kumar Siddharth Singhb, Prasad Patila, Sudhir Kumar Tomara
a
Dairy Microbiology Division, ICAR- National Dairy Research Institute, Karnal, Haryana, India
b
Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, Haryana, India

A R T I C LE I N FO A B S T R A C T

Keywords: This study aimed to examine the role of fructooligosaccharides (FOS), inulin and maltodextrin as prebiotics on
Probiotics aggregation characteristics of putative probiotic Lactobacillus rhamnosus NCDC 298 strain. Same prebiotics were
Enterotoxigenic Escherichia coli assessed alone for their anti-adhesive effect against enterotoxigenic Escherichia coli (ETEC) followed by its sy-
Prebiotics nergistic synbiotic effect on ETEC virulence to HT-29 intestinal cell lines. The effect of prebiotics on auto-
Synbiotic
aggregation and coaggregation property of probiotic strain was studied by comparing absorbance over an in-
Diarrhea
terval of time. FOS was found to enhance autoaggregation and coaggregation characteristics of L. rhamnosus
NCDC 298 with ETEC. Anti-adherence effect of prebiotics towards ETEC was determined by cultural method.
Adherence inhibition percentage was occurred maximum in presence of inulin followed by FOS, having non-
significant relationship in-between. Synergistic effect of both probiotics and prebiotics against ETEC virulence
were seen by comparing cyclic AMP (cAMP) and cyclic GMP (cGMP) levels with controls. Significant decrease in
both the cyclic nucleotides were recorded in presence of FOS with L. rhamnosus NCDC 298.
Fructooligosaccharide influenced adhesion characteristics of L. rhamnosus NCDC 298 with anti-virulence activity
against ETEC, both alone and as synbiotic combination. Hence this combination can be further used to develop
prophylactic anti-diarrheal formulation.

1. Introduction The prophylactic treatment for secretory diarrhea mainly involves

Adhesion is an early key step for colonization of human gastro-
intestinal tract by bacteria. Adhesion of enteropathogens leads to dis-
ruption of the intestinal barrier to cause intestinal infection (Collado,
Meriluoto, & Salminen, 2008). Interference with the adhesion of pa-
thogens on mucosa helps to prevent diseases and dysbiosis. In secretory
diarrheal cases, ETEC was found to be the most common culprit among
all diarrheal pathogens. ETEC is estimated to cause 280–400 million
diarrheal episodes annually in children less than 5 years of age, re-
sulting upto 5 lakhs deaths (Gupta et al., 2008). It colonize the surface
of the small bowel mucosa using their colonization factors and then
release their powerful enterotoxins, the heat labile toxin (LT) and heat
stable toxin (ST). Both LT and ST causes increase in cAMP and cGMP
levels in enterocytes respectively. This phenomenon mediates a net
efflux of electrolytes and water into the lumen of the proximal small
intestine, through activation of cystic fibrosis transmembrane con-
ductance regulator which is clinically known as watery diarrhea
(Croxen & Finlay, 2010).
standard use of antibiotics and other antimicrobial drugs as fast luminal
acting agent, which on routine may develop antibiotic resistance among
gut microflora (Anand, Mandal, Patil, & Tomar, 2016). On the other
hand, probiotics have been implicated in a number of health benefits
and were considered as good prophylactic agents, which when deliv-
ered orally act by production of inhibitory compounds, boosting im-
mune system and providing nutritional benefits. Other than these,
through bacterial interference at intestinal wall, it interfere with the
establishment of a pathogen by competing for host-extracellular matrix-
binding sites, thereby blocking adhesion and spread of the pathogens
(Lebeer, Vanderleyden, & De Keersmaecker, 2008). Probiotic strains
like L. rhamnosus GG, L. reuteri and Saccharomyces boulardii showed
beneficial results in various meta-analyses of randomized controlled
studies for diarrhea in different age groups (McFarland, 2007; Sanders
et al., 2013). Functionality of probiotics can be further boosted by the
presence of non-digestible oligosaccharides known as prebiotics like
fructooligosaccharides (FOS). They are not only found to enhance anti-
pathogenic ability but a few of them have an immense capability to

∗
Corresponding author. Present Address: Department of Dairy Microbiology, Faculty of Dairy Technology, West Bengal University of Animal and Fishery Sciences,
Kolkata, West Bengal, India.
E-mail address: mandalndri@rediffmail.com (S. Mandal).

https://doi.org/10.1016/j.lwt.2018.08.061
Received 1 June 2018; Received in revised form 28 August 2018; Accepted 30 August 2018
Available online 31 August 2018
0023-6438/ © 2018 Published by Elsevier Ltd.
S. Anand et al.
mimic adhesion sites of pathogens thereby providing protection from
infection to intestinal enterocytes (Licht, Ebersbach, & Frøkiær, 2012).
Presence of prebiotic like FOS has been shown to enhance both ad-
herence and antagonistic property of probiotic cultures (Likotrafiti,
Tuohy, Gibson, & Rastall, 2013). It has been also demonstrated that the
diarrheal dysbiosis could be decreased by consumption of synbiotics in
a more effective way relative to the ingestion of probiotics alone
(Preidis & Versalovic, 2009).
Therefore, due to their alone as well as synergistic effects, the use of
probiotics and prebiotics is emerging as a strategy for the prevention of
gut infections, both by disturbing pathogenic adhesion and mitigating
virulence factors. Earlier we reported that short chain FOS was found
preferentially utilizable prebiotic to L. rhamnosus NCDC 298 and able to
enhance its antagonistic action against ETEC (Anand, Mandal, & Tomar,
2017). In the present study, we evaluated the effect of prebiotics on L.
rhamnosus NCDC 298 strain in terms of its autoaggregation and coag-
gregation property, anti-adhesive effect of prebiotics alone and sy-
nergistic effect of both on virulence of ETEC to HT-29 cells.
2. Material and methods
2.1. Bacterial strains and chemicals used
The standard strain of ETEC MTCC 723 was procured from the
Microbial Type Culture Collection (MTCC), Institute of Microbial
Technology, Chandigarh (India). Probiotic culture of L. rhamnosus
NCDC 298 were procured from the National Collection of Dairy
Cultures (NCDC), National Dairy Research Institute, Karnal, India. The
cultures were grown in de Mann Ragosa Sharpe (MRS; HiMedia,
Mumbai, India) and Luria Bertani (LB; HiMedia, Mumbai, India)
medium for Lactobacillus strain and ETEC respectively for usage and
preservation as glycerol stocks at −80 °C for further use. Short-chain
fructooligosaccharides [scFOS, (greater than 95% oligosaccharides, less
than 5% glucose, fructose and sucrose) Zytex Biotech, Mumbai, India],
Inulin and Maltodextrins (HiMedia, Mumbai, India) were used as pre-
biotics. Glucose was used as a non-selective sugar. All the prebiotics
were received in powder form, which was further diluted, filter ster-
ilized and stored at 4 °C.
2.2. Autoaggregation and coaggregation assays
The specific cell–cell interactions were determined using modified
autoaggregation assay (Del Re, Sgorbati, Miglioli, & Palenzona, 2000)
and coaggregation assay (Handley et al., 1987), as described below.
2.2.1. Autoaggregation
Lactobacillus rhamnosus NCDC 298 was grown for 18 h at 37 °C in
basal All-Purpose Tween (APT) growth medium (composition includes
(g/L) casein enzymic hydrolysate, 12.5; yeast extract, 7.5; sodium ci-
trate, 5; sodium chloride, 5; dipotassium phosphate 5; magnesium
sulphate, 0.8; manganese chloride, 0.14; ferrous sulphate, 0.04; poly-
sorbate 80, 0.2; thiamine hydrochloride, 0.001; pH adjusted to
6.7 ± 0.2 at room temperature) in presence of either glucose or dif-
ferent prebiotics (as single carbon source at 1%). The cells were har-
vested by centrifugation (5000 rpm for 15 min at 4 °C). Cell pellet was
washed twice, suspended in phosphate buffer saline (PBS) and the op-
tical density (OD600) of cell suspension was adjusted to 0.7 (which
corresponds to 108 CFU/ml cells). Cell suspension (10 ml) was taken in
clean glass test tube, mixed by vortexing for 10 s and incubated at 37 °C.
Upper most portion of the suspension (1 ml) was collected after 5 h and
optical density (OD 600) was determined. Auto-aggregation (%) was
expressed as follows:
Auto-aggregation (%) = {1- (At/Ao)} x 100
Where, At = OD at 5 h; Ao = OD at 0 h.
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LWT - Food Science and Technology 98 (2018) 329–334
2.2.2. Coaggregation
Lactobacillus rhamnosus NCDC 298 and ETEC were grown in basal
APT growth medium in presence of either glucose or different pre-
biotics (as single carbon source at 1%) for 18 h at 37 °C. Cells were
harvested by centrifugation (5000 × g for 15 min at 4 °C), pellet was
suspended and washed twice in phosphate buffer saline (PBS). Optical
density (OD600) of cell suspension was adjusted to 0.7 (which corre-
sponds to 108 CFU/ml). Cell suspension of L. rhamnosus NCDC 298
(2.5 ml) and ETEC (2.5 ml) were mixed in a clean glass test tube by
vortexing for 10 s and incubated at 37 °C. Cell suspension of L. rham-
nosus NCDC 298 (5 ml) and ETEC (5 ml) were transferred in clean glass
test tubes separately, mixed by vortexing and incubated at 37 °C. The
OD600 of the upper most portion of cell suspension (1 ml) was measured
after 5 h of co-incubation. Coaggregation (%) was calculated as follows:
Coaggregation (%) = [{(AX + AY)/2} – A X+Y]/(AX + AY)/2
Where,
AX, AY = OD600 of L. rhamnosus NCDC 298 and ETEC of cell sus-
pensions, respectively
AX+Y = OD600 of the mixed cell suspension
2.3. Establishment of HT-29 intestinal cell line monolayer
Human intestinal epithelial cell lines (HT-29) was procured from
National Centre of Cell Sciences (NCCS), Pune, India. Cells were rou-
tinely grown in Dulbecco‘s modified eagle‘s minimal essential medium
(DMEM; Sigma, USA), containing glucose (4.5 g/l), supplemented with
10% fetal bovine serum (FBS; Sigma, USA), 100 mg streptomycin per ml
(Sigma, USA) and 100 U penicillin per ml (Sigma, USA) at 37 °C in a
humidified 5% CO2 atmosphere. Approximately, 104−5 cells per ml
were seeded in each well of 6-well tissue culture plates (Corning, USA)
and were allowed to differentiate. The growth medium was changed
every alternate day and 90–100% confluence were observed 15 days
after seeding. These confluent HT-29 cell monolayer were used for ex-
amining the effect of prebiotic alone and as synbiotic preparations on
adhesion of ETEC to epithelial cells. Cells were maintained overnight in
antibiotic-free DMEM media supplemented with 5% FBS and was wa-
shed twice with unsupplemented DMEM (without antibiotics and FBS)
before each experiment.
2.3.1. Adhesion of ETEC to cultured intestinal cell monolayer in presence of
prebiotics
ETEC was grown in LB broth (37 °C for 18 h) and cells were har-
vested by centrifugation at 8000 × g. Cell pellet was washed twice with
PBS and resuspended in minimal DMEM (without antibiotics, glucose
and FBS) to achieve multiplicity of infection (MOI) of 100 with epi-
thelial cells. Each of the sugar (prebiotic or glucose) were mixed with
ETEC cells before addition to intestinal cell monolayer. As a control,
same volume of water was used in place of prebiotic solution. The sugar
solutions represented 10% of the final volume of the suspension, having
concentration of 16 mg/ml of prebiotic or glucose in the assay mixtures
(Quintero et al., 2011). After 3 h, of incubation in tissue culture con-
dition, wells were then washed 3 times with PBS to remove loosely
adhered bacteria cells and HT-29 cells were detached by trypsinization.
ETEC cells were collected and enumerated on VRBA medium. Total
ETEC cells found adhered without any sugar was assumed as hundred
percent adhesion, relative to which anti-adhesive score of prebiotics
was calculated as follows:
% Adherence inhibition = [(number of adhered bacteria (CFU/ml) in
control - number of adhered bacteria (CFU/ml) in treated)/(number of
adhered bacteria (CFU/ml) in control] * 100
S. Anand et al.
2.3.2. Cyclic AMP and cyclic GMP levels during ETEC infection of HT-
29 cells
Lactobacillus rhamnosus NCDC 298 and ETEC cultures were grown in
MRS broth and LB broth respectively. Cells were harvested by cen-
trifugation (8000 × g) for 10 min at 4 °C, pellet was washed thrice with
PBS and diluted to appropriate concentrations in minimal DMEM
media. Confluent HT-29 monolayers were incubated under serum de-
prived condition, with ETEC at a MOI of 100 for 2 h in minimal DMEM
media. Following infection, these monolayers were treated with freshly
prepared gentamicin solution (50 μg/ml) for 30 min to remove adherent
ETEC and L. rhamnosus NCDC 298 cells from the HT-29 cell surface.
After gentamicin treatment, HT-29 cells were washed twice with
minimal DMEM media, lysed by sonication and processed to detect the
levels of cAMP and cGMP using cyclic cAMP and cGMP EIA kit
(Cayman, USA) as per manufacturer's instructions. A Victor ×3 plate
reader (Perkin Elmer, Singapore) was used to record the absorbance at
410 nm wavelength and the sample readings were compared with those
of standards.
2.4. Statistical analysis
All statistical analyses were performed using MS-Excel 2013 and
GraphPad Prism 5.0 statistical tool packages. Results were presented in
mean ± standard deviation (SD), and statistical significance was set at
p < 0.05.
3. Results and discussion
3.1. Autoaggregation
The tested probiotic strain (L. rhamnosus NCDC 298) showed higher
autoaggregation percentages than the pathogenic strain (ETEC) as
shown in Table 1. L. rhamnosus NCDC 298 exhibited significantly higher
autoaggregation percentage in presence of FOS (32.60%) followed by
glucose (29.79%), inulin (21.87%) and maltodextrin (14.25%). The
growth of L. rhamnosus NCDC 298 was slow in minimal medium and the
cells showed lower autoaggregation percentages. On the other hand,
auto-aggregative ability of ETEC pathogen was highest (9%) in pre-
sence of inulin, followed by FOS (7.11%) and maltodextrin (6.94%).
Inulin and maltodextrins were not found to influence aggregation in
comparison to FOS, although it was slightly higher than what was ob-
tained with minimal growth.
Autoaggregation of probiotic strains is postulated to be necessary
for adhesion to intestinal epithelial cells that prevents colonization by
pathogenic microorganisms. Some earlier studies suggest that the pre-
sence of prebiotics can increase, decrease or have no effect on adhesion
of beneficial bacteria such as bifidobacteria or lactobacilli, to intestinal
cells (Kadlec & Jakubec, 2014), whereas some documented that auto-
aggregation property of probiotics can be influenced by the presence of
carbohydrates (Kimoto-Nira et al., 2015; Schär-Zammaretti, Dillmann,
D’Amico, Affolter, & Ubbink, 2005). Likotrafiti et al. (2013) reported,
probiotic culture L. fermentum 907 exhibiting high levels of adhesion to
cell lines when grown in both glucose and Actilight 950P (scFOS) which
could act as potential antimicrobial synbiotics with antimicrobial ac-
tivity against both E. coli O157:H7 and E. coli O86 strains in vitro.
Glucose was taken as a non-selecting additive, but displayed significant
Table 1
Autoaggregation of Lactobacillus rhamnosus NCDC 298 and E. coli MTCC 723 in presence of prebiotics.
Cultures Autoaggregation (%)
Minimal Dextrose
a b
Lactobacillus rhamnosus NCDC 298 10.98 ± 1.35 29.79 ± 2.22
Escherichia coli MTCC 723 4.36 ± 0.62a 7.25 ± 0.64a
a-c
Means ( ± standard deviation; n = 3) with different superscripts in same row differ significantly (P < 0.05).
331
LWT - Food Science and Technology 98 (2018) 329–334
scores during auto/co-aggregation. This observation was found in
agreement with that obtained by Tomás, Wiese, & Nader‐Macías, 2005
who reported increase in autoaggregation with the concentration of
glucose in the growth medium.
Non-specific mechanisms like passive forces, electrostatic interac-
tions, hydrophobic, and steric forces are influenced by the hydro-
phobicity or hydrophilicity of bacteria as well as the surface charge of
the substratum (Xu, Jeong, Lee, & Ahn, 2009). Kadlec and Jakubec
(2014) reported increase in surface negative charge and adherence of L.
rhamnosus CCDM 150 to epithelial cells on addition of prebiotic like
Orafiti P95, suggesting an action mechanism for the particular prebiotic
involves a change in the charge of surface. Subsequently, the hydro-
phobic and hydrophilic attributes due to proteins and polysaccharides
on the bacterial cell surface also helps in the adherence to intestinal cell
wall (Wang, Meng, Zhang, Wang, & Shang, 2010). Kushal (2001) re-
ported a higher cell surface hydrophobicity of L. acidophilus NCDC 13 in
presence of inulin. Recently, Pan, Kumaree, and Shah (2017) observed
greater probiotic adhesion and autoaggregation with relatively greater
membrane hydrophobicity, when grown in prebiotic containing
medium. The presence of prebiotics influenced changes in the mem-
brane surface functional groups with higher amide area/sugar surface
area ratio, indicating greater amount of protein than sugar on surface,
playing crucial role in adhesion.
Our results concur with the previous reports and support our ob-
servations that adhesion property like autoaggregation of probiotics can
be influenced by the presence of prebiotics, which eventually pre-
venting physical contact between the ETEC cells and the cultured in-
testinal cell monolayer.
3.2. Coaggregation
Results were expressed as percentage reduction in the absorbance of
a mixed suspension compared with the individual suspension in
Table 2. L. rhamnosus NCDC 298 showed similar percent sedimentation
values of 24.26 and 22.22 with ETEC in presence of FOS and glucose
respectively. Presence of Inulin (10.25) and Maltodextrins (12.10) in
the medium was not found to influence the co-aggregation ability of the
culture combinations.
Co-aggregation is the phenomenon of clustering of two or more
different types of bacteria, and some workers have expressed it in terms
of binding of pathogenic bacteria by probiotic bacteria (Tareb,
Bernardeau, Gueguen, & Vernoux, 2013). In the study, coaggregation
results revealed quite greater coaggregation within 5 h, when L. rham-
nosus NCDC 298 was grown with FOS. Our results regarding co-ag-
gregating property of probiotic strains were in agreement with an
earlier study performed by Saran Bisht, Singh, & Teotia (2012), where
greater co-aggregation rate in presence of honey and inulin as prebiotic
sugars have been reported. In our case, co-aggregation of the L. rham-
nosus NCDC 298 (in presence of the prebiotic FOS) with ETEC would
ultimately prevent its adhesion to the epithelial cells. This is due to the
actual decrease in number of free ETEC cells which would have
otherwise adhered to their receptors sites present on the enterocytes.
3.3. Effect of prebiotics on adhesion of ETEC on intestinal cell line
The effect of prebiotics on adhesion of ETEC to intestinal cell line
FOS Inulin Maltodextrin
b c
32.60 ± 2.64 21.87 ± 1.47 14.25 ± 1.44a
7.11 ± 0.48ab 9.65 ± 0.87ab 6.94 ± 0.83a
S. Anand et al.
Table 2
Co-aggregation of Lactobacillus rhamnosus NCDC 298 with E. coli MTCC 723 in presence of prebiotics.
Lactobacillus culture Co-aggregation (%)
Minimal Dextrose
c a
Lactobacillus rhamnosus NCDC 298 7.94 ± 0.78 22.22 ± 1.13
a-c
Means ( ± standard deviation; n = 3) with different superscripts in same row differ significantly (P < 0.05).
Fig. 1. Anti-adhesive property of different prebiotics on HT-29 cell lines
against enterotoxigenic Escherichia coli MTCC 723.
a-c
Means ( ± standard deviation; n = 3) with different superscripts differ sig-
nificantly (P < 0.05); FOS-Fructooligosaccharides; MD- Maltodextrin.
(HT-29) has been summarized in Fig. 1. Percentage inhibition of ETEC
adhesion on intestinal cells was greatest when inulin (45.3%) was
present followed by FOS (41.75%), Maltodextrins (30.86%) and Glu-
cose (13.08%). Percentage inhibition scores between inulin and FOS
was found non-significant.
Adherence to host cell surfaces is the primary step before coloni-
zation and infection. Pathogens recognize the carbohydrate binding
sites present on enterocytes which are structurally similar to exogenous
prebiotic oligosaccharides (Buddington, Kelly-Quagliana, Buddington,
& Kimura, 2002). These prebiotic oligosaccharides might compete with
the cognate sugars on enterocytes or may be utilized as decoy receptors
for binding to the pathogen adhesins and inhibit pathogen adhesion
(Searle et al., 2009; Shoaf-Sweeney & Hutkins, 2009). Microbial enu-
meration from trypsinized monolayer cells revealed that the tested
oligosaccharides reduced the adhesion of ETEC to HT-29 cells. Inhibi-
tion of adhesion was maximum with inulin followed by FOS and mal-
todextrins. However, percentage inhibition by inulin and FOS were
found to be non-significant. The observed differences in inhibition of
adhesion due to the tested oligosaccharides were likely due to their
structural differences. Subsequently, preferable use of such prebiotics
having good anti-adherence property with probiotics can prove more
effective against a broad range of gastrointestinal pathogens. Earlier
studies performed on HEp-2 and Caco-2 cell lines by Shoaf, Mulvey,
Armstrong, and Hutkins (2006) evaluated anti-adhesion property in the
presence of FOS, inulin, galactooligosaccharides (GOS), lactulose, and
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LWT - Food Science and Technology 98 (2018) 329–334
FOS Inulin Maltodextrin
a bc
24.26 ± 0.70 10.25 ± 0.41 12.10 ± 0.66b
raffinose (at the level of 10% each) and found maximum anti-adhesion
percentage in case of GOS followed by inulin and FOS. In another ex-
periment by Quintero et al. (2011), anti-adhesive effect on C. sakazakii,
as pathogen in presence of GOS and polydextrose on Caco-2 cell lines
was successfully depicted. However, certain oligosaccharides, including
globotriose (Galα1-4Galβ1-4Glu), globotetraose (GalNAcβ1-3Galα1-
4Galβ1-4Glu), fucosylated sugar and sialic acid have been reported to
inhibit the adherence of toxins produced by virulent E. coli strains and
thus are potential anti-infective agents against these toxins (Paton,
Morona, & Paton, 2006; Yu, Chen, & Newburg, 2013). Recently, pre-
sence of lactulose showed inhibition in adherence of anaerobic patho-
gens like Peptostreptococcus anaerobius, Clostridium perfringens, and
Bacteroides fragilis to Caco-2 and HT-29 intestinal cell lines (Sharma &
Kanwar, 2018). However, glucose did not fared well in terms of pre-
venting pathogen (ETEC) adhesion to epithelial cells. This might be due
to its simple structure and preferential utilization by ETEC. It was also
reported that glucose presence at an optimal concentration for LT ex-
pression can enhances ETEC adherence through the promotion of LT
production which may affect the severity of ETEC infection (Wijemanne
& Moxley, 2014).
3.4. cAMP and cGMP levels in enterocytes after infection
The levels of cAMP and cGMP in HT-29 cells upon infection by ETEC
alone and in presence of probiotics and prebiotics has been shown in
Fig. 2 and Fig. 3. In general, the presence of probiotics and prebiotics
during infection with ETEC was found to stabilize the level of these
cyclic nucleotides in cells. cAMP levels were found to be lowest when
cells were infected by ETEC in presence of L. rhamnosus NCDC 298 with
glucose (50.33 pmol/ml), followed by FOS (51.28 pmol/ml), inulin
(54.55 pmol/ml), L. rhamnosus NCDC 298 strain alone without prebiotic
(66.63 pmol/ml) and with maltodextrin (68.12 pmol/ml) relative to the
uninfected (37.34 pmol/ml) and ETEC infected (84.09 pmol/ml;
without probiotic and prebiotic) controls. Similarly, cGMP levels were
found to be lowest in presence of glucose (5.70 pmol/ml), followed by
FOS (9.98 pmol/ml), inulin (14.37 pmol/ml), L. rhamnosus NCDC 298
alone with ETEC (17.48 pmol/ml) relative to negative (4.73 pmol/ml;
uninfected) and positive (19.79 pmol/ml; ETEC infection without pro-
biotic and prebiotic) controls. Cells infected by ETEC in presence of L.
rhamnosus NCDC 298 with maltodextrin displayed highest levels of
cGMP (24.65 pmol/ml) production.
The immediate effect of ETEC invasion is due to its released en-
terotoxins (LT and ST) and can be visualized as disturbed cAMP and
cGMP levels inside the intestinal cells. There may be a significant fall in
Fig. 2. cAMP nucleotides concentrations obtained
after different synbiotic treatments to en-
terotoxigenic E. coli MTCC 723 infected HT-29
epithelial cell line.
a-d
Means ( ± standard deviation; n = 3) with dif-
ferent superscripts differ significantly (P < 0.05);
ETEC – E. coli MTCC 723; 298- L. rhamnosus NCDC
298; GLU- glucose, FOS – Fructooligosaccharides,
INU – Inulin, MD- Maltodextrin.
S. Anand et al.
these cyclic nucleotides levels, if there is decrease in toxin production
or adhesion of toxin vesicles produced by ETEC (Croxen & Finlay, 2010)
and could be considered as a measure of extent of pathogen invasion.
Here in the study, presence of probiotics during infection with ETEC
was found to control the levels of cyclic nucleotides in cells when
compared to positive control, and the efficacy was much pronounced in
presence of glucose and FOS. Greater amount of cyclic nucleotides in
MD containing group may be due to its chemical structure which
consists of D-glucose only, which may enhance adhesion and toxin se-
cretion in enterotoxigenic E. coli under lower acidic conditions (Hegde,
Bhat, & Mallya, 2009; Wijemanne & Moxley, 2014). Decrease in cAMP
and cGMP levels may be due to phenomena such as catabolite repres-
sion in presence of glucose (Perlman, De Crombrugghe, & Pastan,
1969). Our results indicates that autoaggregation and co-aggregation
property of probiotics could be enough to prevent the ETEC from in-
fection, which produces toxin vesicles and further fuses with intestinal
cells membrane by endocytosis (Kesty, Mason, Reedy, Miller, & Kuehn,
2004). Presence of prebiotics and their interaction with vesicular re-
ceptors in path of produced toxin may hinder their movement towards
enterocytes.
Probiotics also have been found to suppress toxin gene expression in
some enterovirulent strains including ETEC and enterohemorrhagic E.
coli (EHEC) (Dubreuil, 2017). Other than organic acids, specialized
peptides and quorum sensing (QS) signal inhibitor molecules produced
by probiotic strains, were also found to modify toxin transcription
process (Peng, Reichmann, & Biswas, 2015). Medellin-Pena, Wang,
Johnson, Anand, and Griffiths (2007) reported down regulation in
virulence related genes in response to Lactobacillus acidophilus La-5 cell-
free spent medium (CFSM) containing QS signal inhibitor molecules
which directly interacts with bacterial transcriptional regulators. Li,
Wang, Xu, Magarvey, and McCormick (2011) reported Lactobacillus
reuteri producing cyclic dipeptides which quench agr-mediated expres-
sion of toxic shock syndrome toxin-1 in Staphylococcus aureus.
4. Conclusion
We tried to address the role of prebiotics alone and in synbiotic
combination with probiotics in preventing pathogen adhesion to host
gut. Fructooligosaccharides as prebiotic, was found to enhance auto-
aggregation and coaggregation property of L. rhamnosus NCDC 298 and
alone was found to prevent the adhesion of ETEC on cultured intestinal
cell lines. In synbiotic combination, both L. rhamnosus NCDC 298 and
FOS have been found to further decrease the virulent effect of ETEC
strain in in vitro conditions. We propose that this synbiotic combination
can be further used to develop prophylactic anti-diarrheal formulation
validated through clinical trials.
Acknowledgments
We thank Dr. J. K. Kaushik, Animal Biotechnology Centre, ICAR-
NDRI, Karnal for providing cell culture facility.
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LWT - Food Science and Technology 98 (2018) 329–334
Fig. 3. cGMP nucleotides concentrations obtained
after different synbiotic treatments to en-
terotoxigenic E. coli MTCC 723 infected HT-29
epithelial cell line.
a-e
Means ( ± standard deviation; n = 3) with dif-
ferent superscripts differ significantly (P < 0.05);
ETEC – E. coli MTCC 723; 298- L. rhamnosus NCDC
298; GLU- glucose, FOS – Fructooligosaccharides,
INU – Inulin, MD- Maltodextrin.
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