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R E S E A R C H Open Access
Antiviral activity of Lactobacillus reuteri
Protectis against Coxsackievirus A and
Enterovirus 71 infection in human skeletal
muscle and colon cell lines
Lei Yin Emily Ang
1,2†
, Horng Khit Issac Too
1,2†
, Eng Lee Tan
3,4
, Tak-Kwong Vincent Chow
1
, Pei-Chi Lynette Shek
3
,
Elizabeth Tham
3
and Sylvie Alonso
1,2*
Abstract
Background: Recurrence of hand, foot and mouth disease (HFMD) pandemics continues to threaten public health.
Despite increasing awareness and efforts, effective vaccine and drug treatment have yet to be available. Probiotics
have gained recognition in the field of healthcare worldwide, and have been extensively prescribed to babies and
young children to relieve gastrointestinal (GI) disturbances and diseases, associated or not with microbial infections.
Since the faecal-oral axis represents the major route of HFMD transmission, transient persistence of probiotic bacteria in
the GI tract may confer some protection against HFMD and limit transmission among children.
Methods: In this work, the antiviral activity of two commercially available probiotics, namely Lactobacillus reuteri Protectis
(L. reuteri Protectis) and Lactobacillus casei Shirota (L. casei Shirota), was assayed against Coxsackieviruses and Enterovirus
71 (EV71), the main agents responsible for HFMD. In vitro infection set-ups using human skeletal muscle and colon cell
lines were designed to assess the antiviral effect of the probiotic bacteria during entry and post-entry steps of
the infection cycle.
Results: Our findings indicate that L. reuteri Protectis displays a significant dose-dependent antiviral activity
againstCoxsackievirustypeA(CA)strain6(CA6),CA16andEV71,butnotagainstCoxsackievirustypeBstrain2.
Our data support that the antiviral effect is likely achieved through direct physical interaction between bacteria
and virus particles, which impairs virus entry into its mammalian host cell. In contrast, no significant antiviral
effect was observed with L. casei Shirota.
Conclusions: Should the antiviral activity of L. reuteri Protectis observed in vitro be translated in vivo, such
probiotics-based therapeutic approach may have the potential to address the urgent need for a safe and effective
means to protect against HFMD and limit its transmission among children.
Keywords: Hand, Foot and mouth disease, Probiotics, Lactobacillus reuteri, Coxsackievirus, Enterovirus 71
* Correspondence: micas@nus.edu.sg
†
Equal contributors
1
Department of Microbiology and Immunology, Yong Loo Lin School of
Medicine, National University of Singapore, Centre for Life Sciences, 28
Medical Drive, #03-05, Singapore 117456, Singapore
2
Immunology programme, Life Sciences Institute, National University of
Singapore, Singapore, Singapore
Full list of author information is available at the end of the article
© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Ang et al. Virology Journal (2016) 13:111
DOI 10.1186/s12985-016-0567-6
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Background
Hand, foot and mouth disease (HFMD) is a common
viral infection that affects mostly infants and children
below 5 years of age. The main causative agents respon-
sible for HFMD belong to a group of enteroviruses from
Picornaviridae family, and consist predominantly of cox-
sackievirus type A (CA) strain 16 (CA16) and entero-
virus 71 (EV71) [1]. Other enteroviruses such as CA6,
CA7, CA10, CA14 and coxsackievirus type B strain 2
(CB2) may also associate with the disease. In most cases,
the disease is mild and self-limiting, with major clinical fea-
tures manifesting as HFMD and herpangina [2, 3]. How-
ever, more severe clinical manifestations with neurological
complications including aseptic meningitis, brainstem en-
cephalitis, acute flaccid paralysis and cardiopulmonary
dysfunction resulting from acuteEV71infection,havealso
been reported [3, 4]. Furthermore, co-infection with CA16
and EV71 has been detected in patients [5]. A growing
bodyofevidencesuggeststhatoverwhelmingproduc-
tion of inflammatory mediators associated with high
viral titer plays a critical role in the pathogenesis of
EV71 infection [3, 6, 7].
In the past decade, epidemiology studies of HFMD out-
breaks resulting in morbidity and mortality with neuro-
logical complications have been increasingly reported in
countries across the Asia-Pacific region and sometimes in
Europe [8–11]. However, there is still no effective vaccine
and specific antiviral treatment available currently. Infec-
tion risk control is mainly achieved through good hygiene
practices, closure of childcare centres and schools, and
adopting distancing measures. However, these measures
imply a substantial socio-economic burden [7]. Efforts in
developing suitable vaccines have been pursued to address
the urgent need to control HFMD epidemics [12, 13].
So far three inactivated EV71 whole-virus vaccine can-
didates have completed Phase III clinical trials. These
C4 genotype-based vaccines showed high immunogen-
icity and good protective efficacy by preventing herpan-
gina and EV71-associated hospitalization. In addition,
they were shown to cross-neutralize the circulating
EV71 predominant genotypes and subgenotypes B1, B5
and C4A which have been associated with epidemics in
recent years. However, no cross-protection against CA16
was observed [14, 15].
Probiotics, as defined by the Food and Agricultural
Organization of the United Nations and World Health
Organization, are “live microorganisms which, when ad-
ministered in adequate amounts, confer a health benefit
on the host”[16]. Lactic acid bacteria (LAB) and bifido-
bacteria are the most common types of probiotics. They
are widely consumed as part of fermented foods with
specially added active live cultures; such as in yogurt,
soy yogurt, or as dietary supplements. Probiotics were
initially thought to exert a beneficial effect on the host
by improving intestinal microbial balance, through in-
hibition of, or competition with pathogens and toxin-
producing bacteria. It was later shown that probiotics
seem to display more specific health effects that are
being increasingly investigated and documented [17]. An
extensive scientific literature is available on the effects
of probiotics in alleviating chronic intestinal inflamma-
tory diseases [18], preventing and treating pathogen- or
antibiotic-induced diarrhoea [19], urogenital infections
[20], and atopic diseases [21]. Immuno-modulatory ac-
tivities were reported for some LAB strains through the
regulation of cytokine production, by increasing the
number of IgA-producing plasma cells or the propor-
tion of T lymphocytes and Natural Killer cells, or by
improving phagocytosis [22, 23]. Clinical trials have fur-
ther demonstrated that probiotics may decrease the
incidence of respiratory tract infections [24] and dental
cariesinchildren[25].
Since the faecal-oral axis represents the major route of
HFMD transmission [7], transient persistence of pro-
biotic bacteria in the gastrointestinal (GI) tract may con-
fer some protection against HFMD and limit
transmission among children. Consistently, a previous
publication has reported the anti-EV71 activity of me-
tabolites secreted by Lactobacillus plantarum and Bifi-
dobacterium bifidum in Vero cells [26]. Here, we studied
the potential antiviral activity of two commercially avail-
able LAB probiotic, namely Lactobacillus reuteri Protectis
(L. reuteri Protectis) and Lactobacillus casei Shirota (L.
casei Shirota), against coxsackieviruses type A and B, and
EV71 in infection assays using human skeletal muscle and
colon cells. L. reuteri Protectis, commercialized by BioGaia,
was shown to improve gut health in infants and children
[27–30]. L. casei Shirota contained in Yakult products has
also been demonstrated to relieve gastrointestinal symp-
toms, prevent viral infections and reduce risk for vari-
ous cancers [31–35]. Most importantly, both probiotics
are safe to consume in clinically ill children.
Methods
Bacteria strains and growth conditions
L. reuteri Protectis (Deutsche Sammlung von Mikroor-
ganismen 17938) [36] was re-activated from freeze-dried
BioGaia ProTectis tablet. L. casei Shirota is a kind gift
from A/Prof Lee Yuan Kun (Department of Microbiol-
ogy and Immunology, National University of Singapore).
Both L. reuteri Protectis and L. casei Shirota were grown in
MRSbrothoronMRSagar(Oxoid,UnitedKingdom)at
37 °C. All LAB cultures were incubated under microaerobic
condition (closed cap without agitation) to stationary phase,
between 16 and 18 h, to achieve a bacterial concentration
of 10
12
colony-forming unit per mL (CFU/mL). Bacteria
cultures were passaged twice from frozen stock ( −80 °C).
Formaldehyde-inactivated L. reuteri Protectis was obtained
Ang et al. Virology Journal (2016) 13:111 Page 2 of 12
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by resuspending live bacteria pellet in phosphate buff-
ered saline (PBS) containing 4 % v/v formaldehyde
(Sigma-Aldrich, United States) overnight. Prior to in-
fection assays bacteria were washed extensively with
PBS to remove traces of MRS broth or formaldehyde,
and serially diluted in Dulbecco’s Modified Eagle
Medium (DMEM) supplemented with 2 % (v/v) heat-
inactivated fetal bovine serum (FBS).
Cultures of cell lines and virus strains
Human rhabdomyosarcoma (RD) cells (ATCC® CCL-
136™) were maintained in DMEM supplemented with
10 % v/v heat-inactivated FBS. Human Caco-2 cells
(ATCC® HTB-37™) were cultured in DMEM containing
20 % v/v heat-inactivated FBS, 1 % v/v non-essential amino
acids (100X), 1 % v/v GlutaMAX supplement (100X),
1.5 % v/v sodium bicarbonate (7.5 %) and 1 % v/v sodium
pyruvate (100 mM). Both cell lines were culture in 5 %
CO
2
atm at 37 °C, and were sub-cultured every 2–3days.
RD cells and Caco-2 cells were used between passages 13
and 40. All the virus strains, CA6 (NUH0026/SIN/08, Ac-
cession No. GU198758.1), CA16 (CA16-G-10, Accession
No. U05876) [37], CB2 (KOR 04-279, Accession No.
EF174469) and EV71 strain 41 (5865/SIN/00009, Accession
No. AF316321) [38], used for this study were propagated in
RD cells. All the reagents used to maintain cell cultures
were purchased from Thermo Fisher Scientific (Gibco).
Immunostaining assay
L. reuteri Protectis bacteria (10
11
CFU) were co-
incubated with CA16 (10
5
PFU) in 2 % DMEM at 37 °C
for one hour. The mixture was then added to RD cells
(10
5
cells) for another hour to allow viral entry. CA16-
infected RD cells served as control. After one hour incu-
bation, the cells were washed thrice with PBS to remove
unbound bacteria and viruses, and immediately fixed
with ice cold methanol. The cells were then stained with
mouse anti-CA16 antibody (MAB979, Merck, 1:1000
dilution) and rabbit anti-beta actin antibody (ab8227,
Abcam, 1:1,000 dilution) followed by incubation with
anti-mouse AF488-conjugated (A-11001, Invitrogen, 1:500
dilution) and anti-rabbit AF594-conjugated (R37117, Invi-
trogen, 1:500 dilution) secondary antibodies, respectively.
Cell nuclei were stained with 4',6’-diamidino-2-phenylin-
dole (DAPI) (Invitrogen) (1:100,000 dilution) at room
temperature for 30 min in the dark. Images were captured
using Olympus IX81 microscope. Cell fluorescence (viral
signals, green) was measured using ImageJ software and
the corrected total cell fluorescence (CTCF) was calculated
using the equation: CTCF = Raw integrated Density –
(Area of selected cell × Mean fluorescence of background
readings).
Cell viability assay
RD cells (2.5 × 10
4
cells/well) and Caco–2 cells (5 × 10
3
cells/well) were seeded onto 96 well plates (Nunc,
United States) and incubated overnight and for 6 days,
respectively. The culture medium was changed every 2-
3 days. Monolayers were incubated with various concen-
trations of live bacteria for an hour and washed thrice
with PBS before fresh 2 % DMEM supplemented with
50 μg/mL gentamicin (Sigma-Aldrich, United States)
was added. After 24 h incubation at 37 °C and CO
2
,
2 % v/v alamarBlue reagent (Invitrogen, United States)
diluted in 2 % DMEM was added to each well. Fluorescence
intensity was then measured at excitation wavelength of
570 nm and emission wavelength of 585 nm using a micro-
plate reader (Infinite 200, Tecan). The relative percentage
(%) of cell survival with respect to control wells containing
untreated cells was calculated. Values were corrected for
background fluorescence obtained with media only.
Virus quantification
RD cells (1.25x10
5
cells/well) were seeded onto 24 well
plates (Nunc) and infected with 200 μL of 10-fold seri-
ally diluted viral supernatant. After 1 h incubation at
37
0
C and CO
2
, 1 % w/v sodium carboxymethyl cellulose
(Sigma-Aldrich) in Minimum Essential Medium (MEM)
(Invitrogen, United States) supplemented with 2 % v/v
heat-inactivated FBS and 1.5 % v/v sodium bicarbonate
(7.5 %) was added to the wells. After 3 days incubation,
the cells were fixed with 4 % v/v formaldehyde and
stained with 1 % w/v crystal violet (Sigma-Aldrich). Pla-
ques were scored visually and the virus titers were
expressed as plaque-forming units per mL (PFU/mL).
Three technical replicates were performed for each dilu-
tion of a biological sample. The limit of detection for the
plaque assay was set at 10 PFU/mL.
Antiviral activity assays
Four experimental set-ups were designed namely, pre-
incubation, pre-treatment, co-treatment and post-treatment.
Virus infection was carried out at a multiplicity of infec-
tion (MOI) of 1 (10
5
PFU/mL) for all the set-ups. Cell
monolayers were washed with PBS thrice and were main-
tained in 2 % DMEM supplemented with 50 μg/mL of
gentamicin after contact time with bacteria. Culture
supernatant was harvested at 12 (CB2-infection) or 24
(CA6, CA16, or EV71-infection) hours post-infection.
Samples were stored in −80 °C and plaque assay was per-
formed subsequently to determine the viral titer. The virus
titers were compared with the titer obtained with cells
exposed to virus only.
Virus-bacteria binding assay
Live L. reuteri Protectis bacteria were co-incubated with
CA6, CA16, EV71 or CB2 virus in 2 % DMEM at 37 °C
Ang et al. Virology Journal (2016) 13:111 Page 3 of 12
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and CO
2
for an hour. The bacteria-virus mixtures were
then spun down at 4,000 xgfor 5 min at 4 °C. The
culture supernatant was filtered with 0.22 μm syringe
filter unit (Millipore, United States) before viral titer
determination in RD cells (section 2.4).
Statistical analysis
The results were expressed as mean ± standard deviation
(SD) of three technical replicates. All the experiments
were performed twice independently. Comparison be-
tween control and different treatment groups was statisti-
cally analyzed by one-way analysis of variance (ANOVA)
with Dunnett’s post-test or by Mann–Whitney Utest as
indicated, using GraphPad Prism version 5.00 for Win-
dows, GraphPad Software (San Diego California USA,
www.graphpad.com). Probability values (p) of < 0.05 were
considered statistically significant.
Results
Live L. reuteri Protectis bacteria significantly reduced CA6,
CA16, EV71 but not CB2 virus titers in infected RD and
Caco-2 cells
Prior to evaluating the antiviral activity of L. reuteri Pro-
tectis bacteria in human RD and intestinal Caco-2 cell
lines, a cell viability assay was performed to assess the
cytotoxicity of these probiotic bacteria. Results indicated
that 1 h incubation of up to 10
11
CFU of live L. reuteri
Protectis bacteria with RD and Caco-2 cell monolayers
did not lead to significant cell viability loss (≥80 % via-
bility) (Fig. 1). Next, various incubation conditions were
performed to test the antiviral activity of L. reuteri Pro-
tectis (Fig. 2). In the pre-incubation set-up, bacteria
and virus particles were incubated together for 1 h
prior to infection of mammalian cells with the mixture.
In the pre-treatment set-up, cell monolayers were incu-
bated with bacteria for 1 h, and washed with PBS prior
to virus infection. In the co-treatment set-up, cell
monolayers were incubated for 1 h with virus and bac-
teria concomitantly. Finally, in the post-treatment set-
up, cell monolayers were incubated with the virus for
1 h, and washed with PBS prior to incubation with bac-
teria for another hour. The culture supernatants were
sampled at 12 or 24 h post-infection to determine the
virus titers. CA6, CA16, CB2 and EV71 strains were
tested. Virus alone, treated under the same experimen-
tal conditions, was used as a positive control (POS).
The results indicated that L. reuteri Protectis bacteria
displayed a significant antiviral activity against CA6,
CA16 and EV71 but not against CB2 virus (Fig. 3a-d).
Furthermore, the pre-incubation set-up where bacteria
and virus are co-incubated prior to incubation with
mammalian cells, led to the strongest reduction in virus
titers compared to the positive control. This observa-
tion suggested a direct interaction between bacteria and
virus particles, which may impair virus entry. The
inhibitory effects observed were dose-dependent and
virus-dependent whereby the greatest antiviral activity
was observed against CA16 with more than 2–3logre-
duction in virus titers when pre-mixed with 10
11
bac-
teria (Fig. 3b). In the same conditions, reduction in
CA6 and EV71 virus titers was approximately 2 log and
1 log, respectively (Fig. 3a and c). A dose-dependent
antiviral activity was also observed in the co-treatment
set-up where virus and bacteria were added concomi-
tantly to the cell monolayers. However, reduction in
virus titers was less dramatic than those obtained in the
pre-incubation set-up. In contrast, no dose-dependent
antiviral activity was clearly observed in the pre-treatment
and post-treatment set-ups with CA6 and CA16 (Fig. 3a
and b), but was seen with EV71 (Fig. 3c). Furthermore,
comparable observations were made with both cell lines,
suggesting that the antiviral activity of L. reuteri Protectis
is not cell type-dependent and likely affects an important
step of the infection cycle.
Fig. 1 Cell viability in the presence of live L. reuteri Protectis bacteria. Different concentrations of L. reuteri Protectis bacteria were added to RD
cells (a) and Caco-2 cells (b) and incubated for 1 h, then washed with PBS thrice before 50 μg/ml gentamicin-supplemented maintenance media
was added to the cells. Alamar blue assay was performed at 24 h post-treatment according to the manufacturer’s instructions. Data are expressed
as the mean ± standard deviation of technical triplicates. Two biological repeats were conducted. One representative is shown
Ang et al. Virology Journal (2016) 13:111 Page 4 of 12
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Coxsackievirus type A and EV71 interact with live L.
reuteri Protectis bacteria
The data obtained indicated that the pre-incubation set-
up led to the greatest reduction in virus titers, thus
suggesting that L. reuteri Protectis bacteria interfere with
the virus entry step. To further test this hypothesis, L.
reuteri bacteria and CA16 virus were co-incubated for
1 h in a cell free environment prior to infection of RD
cells as described for the pre-incubation experimental
setup. After 1 h infection, the cell monolayer was washed
thoroughly, fixed and processed for immunostaining using
anti-CA16 and anti-actin antibodies, while nuclei were
stained with DAPI. The corrected total cell fluorescence
(CTCF) specific to the virus signal (green) was calculated
and indicated significantly lower signal intensity with the
(CA16 + L. reuteri)-infected cells compared to CA16-
infected cells only (Fig. 4). These data therefore further
supported that L. reuteri bacteria interfere with CA16
entry into mammalian cells.
We next asked whether L. reuteri bacteria physically
interact with the virus particles during the pre-
incubation phase, thereby compromising viral entry
into the mammalian cells subsequently. To test this hy-
pothesis, a dose range of live L. reuteri Protectis
bacteria were co-incubated with a fixed amount of virus
particles for one hour in a mammalian cell-free system.
The mixtures were then centrifuged, the supernatants
were collected and filtered to remove intact bacteria,
and the amount of virus particles was determined by
plaque assay. The results indicated a reduction in con-
centration of virus particles in the supernatant com-
pared to the virus alone control (Fig. 5). This reduction
was dependent on the amount of bacteria that were co-
incubated with the virus and was seen with CA6, CA16
and EV71, but not with CB2 virus, thus correlating with
the observation that L. reuteri Protectis bacteria do not
impact CB2 infectivity (Fig. 3). Together, these data
support that L. reuteri Protectis bacteria interact dir-
ectly and physically with CA6, CA16 and EV71 virus
particles and likely interfere with viral entry into the
mammalian cells.
Dead intact L. reuteri Protectis inhibits coxsackievirus type
A infection
We next asked whether live L. reuteri Protectis bacteria
were necessary to display a significant antiviral activity.
To test this hypothesis, formaldehyde-treated L. reuteri
Protectis bacteria were pre-incubated with CA6 or
Fig. 2 Schematic diagram of experimental in vitro set-ups. In the pre-incubation set-up, live bacteria and virus were pre-incubated for 1 h at 37 °C,
before being incubated with the mammalian cells for 1 h. In the pre-treatment set-up, the mammalian cells were incubated with live bacteria for 1 h,
washed with PBS and infected with virus for 1 h. In the co-treatment set-up, live bacteria were added to the cells at the same time of virus infection
for 1 h. In the post-treatment set-up, the mammalian cells were infected with virus for 1 h, then washed with PBS and incubated with live bacteria for
another hour. In all four set-ups, 50 μg/ml gentamicin-supplemented maintenance media was eventually added to the cell monolayers.
Sample supernatants were harvested 12 or 24 h post-infection and plaque assay was performed using RD cells to determine the viral titer
Ang et al. Virology Journal (2016) 13:111 Page 5 of 12
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Fig. 3 (See legend on next page.)
Ang et al. Virology Journal (2016) 13:111 Page 6 of 12
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CA16 virus according to the pre-incubation set-up
described above. The data showed that a bacteria dose-
dependent reduction in virus titers was observed that was
comparable to that observed with live L. reuteri Protectis
bacteria (Fig. 6). Therefore, the data indicated that the
antiviral activity of L. reuteri Protectis bacteria does not
depend on bacteria replication and/or bacterial product
secretion. They support that the antiviral mechanism re-
lies on a physical interaction between bacteria and virus
particles which likely interferes with the ability of the virus
to bind to its mammalian receptor(s).
L. casei Shirota bacteria do not display significant antiviral
activity against CA16 and EV71
The potential antiviral activity of another widely con-
sumed probiotic bacterium namely L. casei Shirota was
explored. First, the cytotoxicity of L. casei Shirota was de-
termined by incubating a dose range of bacteria with RD
and Caco-2 cells. The results indicated that 10
11
CFU of
live L. casei Shirota bacteria appears to be toxic (< 80 %
viability) to both RD cells and Caco-2 cells (Fig. 7). This
cytotoxicity is likely due to the high amounts of lactic acid
produced by L. casei Shirota bacteria which results in
(See figure on previous page.)
Fig. 3 Antiviral effect of L. reuteri Protectis. Pre-incubation, pre-treatment, co-treatment and post-treatment setups were performed as detailed in
Fig. 1. Virus titers in the supernatant of CA6- a, CA16- b, EV71- cand CB2- dinfected RD cells and Caco-2 cells were determined by standard
plaque assay in RD cells. A one-way ANOVA test with Dunnett’s posttest was performed (* p<0.05, ** p<0.005, *** p<0.0005). Data are
expressed as the mean ± standard deviation of technical triplicates. Two biological repeats were conducted. One representative is shown
a
CA16 + L. reuteri CA16 only
4
5
6
7
8
CTCF/cell
****
b
Fig. 4 Immunostaining of RD cells infected with (L. reuteri + CA16) mixture. aL. reuteri Protectis bacteria (10
11
CFU) were co-incubated with CA16
(10
5
PFU) for 1 h in 37 °C prior to adding the mixture onto RD cells (10
5
cells) for another 1 h. A control with RD cells infected with CA16 only was also
performed. The monolayers were then washed thrice before methanol fixation and immunostained with anti-CA16 and anti-beta actin antibodies. Nuclei
were also stained with DAPI. Images were taken using Olympus IX81 microscope. bCorrected total cell fluorescence (CTCF) (viral signal) was calculated
from each cell originated from three random microscopic views. Statistical analysis was performed using Mann–Whitney Utest (
****
,p< 0.0001)
Ang et al. Virology Journal (2016) 13:111 Page 7 of 12
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acidification of the culture medium as evidenced by
the colour shift of the pH indicator (data not shown).
Therefore, the antiviral assays were conducted with
concentrations of L. casei Shirota ranging from 10
9
to
5×10
10
CFU.
The antiviral activity against CA16 and EV71 of L.
casei Shirota was assayed in the pre-incubation, pre-
treatment, co-treatment and post-treatment set-ups.
The results indicated significantly lower CA16 virus ti-
ters with L. casei Shirota concentrations of 10
10
and/
or 5 × 10
10
CFU in all the experimental set-ups
(Fig. 8a). However, significant cytotoxicity was clearly
observed at these bacterial concentrations, with cells
lifting off from the bottom of the wells (data not
shown). Similar observations were made with EV71-
infected cells (Fig. 8b). Therefore, these data indicate
that L. casei Shirota does not appear to display a sig-
nificant antiviral activity against CA16 and EV71.
Discussion
Despite increasing interests from the scientific com-
munity, development of effective prophylactic and
therapeutic strategies against HFMD remains overdue.
Thanks to their high safety profile, probiotics have
been reported as an alternative preventive and thera-
peutic approach to treat a number of illnesses in in-
fants and young children, in particular those affecting
the GI tract [39].
Upon ingestion, coxsackieviruses and EV71 establish
infection at the gastric mucosa, the primary site of
infection, from where the viral particles transiently
disseminate systemically and accumulate in muscles
where the virus multiplies extensively [7, 40]. Subse-
quently, EV71 is believed to gain access to the CNS at
the neuromuscular junctions and migrate to the brain-
stem via retrograde axonal transport [40, 41]. Based on
this model of infection, the antiviral effect of probiotic
Fig. 5 Virus-bacteria binding assay. Different quantities of live L. reuteri Protectis bacteria were incubated with a fixed amount of EV71, CA6, CA16
or CB2 virus particles for 1 h. The mixtures were then centrifuged and filtered to obtain free virus in the supernatant. Virus titers were determined
by plaque assay using RD cells. A one-way ANOVA test with Dunnett’s posttest was performed (* p<0.05, ** p<0.005, *** p<0.0005). Data are
expressed as the mean ± standard deviation of technical triplicates. Two biological repeats were conducted. One representative is shown
Fig. 6 Antiviral effect of formaldehyde-inactivated L. reuteri Protectis. Formalin-fixed L. reuteri Protectis bacteria were bacteria were pre-incubated
with CA6 or CA16 virus according to the pre-incubation set-up with Caco-2 cells. Virus titers in the supernatant of CA6- (a) and CA16- (b) infected
cells were determined by standard plaque assay in RD cells. A one-way ANOVA test with Dunnett’s posttest was performed (* p<0.05, ** p<0.005, ***
p<0.0005). Data are expressed as the mean ±standard deviation of technical triplicates. Two biological repeats were conducted. One representative
is shown
Ang et al. Virology Journal (2016) 13:111 Page 8 of 12
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bacteria against coxsackieviruses and EV71 was tested
in vitro using relevant cell lines namely human skeletal
muscle RD and intestinal Caco-2 cells. Our data clearly
demonstrate a significant antiviral activity of L. reuteri
Protectis against CA16, CA6 and EV71 but not CB2
virus. In contrast, L. casei Shirota did not display any
significant antiviral effect against CA16 or EV71, indi-
cating that the antiviral effect observed with L. reuteri
Protectis is specific and limited to this probiotic
bacterium.
L. reuteri is a commensal bacterium that can be
found in the gut flora of some mammals and birds.
Administration of L. reuteri to human babies, children
and adults (including HIV patients) is safe and has
been used for more than 10 years as probiotics. L. reu-
teri wasshowninanumberofclinicaltrialstoen-
hance protection against a variety of diseases of
microbial (including rotavirus, Gardnerella vaginalis,
and Helicobacter pylori infection), chemical and envir-
onmental origin. In addition to maintaining the bal-
ance among the GI microbiota, which is the primary
role of probiotics, L. reuteri has been reported to dis-
play some immuno-modulatory properties through the
modulation of inflammatory cytokines and chemokines
production by enterocytes and immunocytes, thereby
influencing the host mucosal immune responses [22,
23]. Furthermore, studies have shown that reuterin se-
creted by L. reuteri has antimicrobial properties
against Gram positive and Gram negative bacteria, as
well as yeast, moulds and protozoa [42, 43]. The mode
of action of reuterin remains speculative although in-
hibition of DNA synthesis and induction of oxidative
stress in the target microorganisms have been pro-
posed. A few studies have reported the antiviral effect
of other probiotics through the production of anti-
microbial molecules such as bacteriocins [39] or cell
wall components [44]. However, in our study, two main
lines of experimental evidence support that the antiviral
effect of L. reuteri Protectis against CA and EV71 is un-
likely to be mediated by the production of a soluble
antimicrobial molecule such as reuterin. Firstly, filtered
culture supernatant from L. reuteri Protectis harvested
during the exponential or stationary phase, failed to
show antiviral effect in the various experimental set-
ups (data not shown). Secondly, dead bacteria (forma-
lin-fixed) retain their antiviral property. Furthermore,
pre-incubation of L. reuteri Protectis bacteria with CA
or EV71 showed a significant dose-dependent reduction
of virus titers which suggests a physical interaction be-
tween bacteria and viral particles that may interfere
with virus entry into the mammalian host cell. This hy-
pothesis is further supported by the observation of re-
duced virus titers in the supernatant of L. reuteri
Protectis-virus mixtures after centrifugation and filtra-
tion. In addition to a direct binding of bacteria to the
viral particles that likely interferes with the entry step
(pre-incubation set-up), competition for attachment
sites on cell surface between bacteria and virus (anti-
viral activity observed in co-treatment and post-
treatment set-ups) could also contribute to the antiviral
effect observed. Further work is necessary to elucidate
the mechanisms by which L. reuteri Protectis interacts
physically with CA16, CA6 and EV71.
The next logical step would be to test the antiviral
activity of L. reuteri Protectis in animal models of CA
and EV71 infection. So far mouse models of HFMD
have employed the intraperitoneal, intramuscular or
intracranial routes to establish infection [45, 46]. These
routes of infection are not suitable to test the antiviral
efficacy of L. reuteri Protectis which likely relies on a
direct and local interaction between bacteria and the
virus particles in the GI-tract. The oral route of
Fig. 7 Cell viability in the presence of live L. casei Shirota. Different concentrations of bacteria were added to RD (a) and Caco-2 (b) cells as indicated
and incubated for 1 h, then washed with 1xPBS thrice before 50 μg/ml gentamicin-supplemented maintenance media was added to the cells. Alamar
blue assay was performed at 24 h post-treatment according to the manufacturer’s instructions. Data are expressed as the mean ±standard deviation of
technical triplicates. Two biological repeats were conducted. One representative is shown
Ang et al. Virology Journal (2016) 13:111 Page 9 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
infection in these animal models has proven less suc-
cessful due to the existence of specific oral bottlenecks
represented by physical barriers (colonic epithelium)
that limit virus trafficking from the gut to other body
sites [47]. Alternatively, EV71 oral infection of non-
human primates has been reported [46, 47] and could
be employed to test the antiviral effect of L. reuteri
Protectis. However, economic and ethical aspects must
be carefully considered.
Conclusion
In conclusion, our work indicates a significant antiviral
activity of L. reuteri Protectis against the main agents
responsible for HFMD. Should these in vitro findings
be translated in vivo, they would strongly suggest that
L. reuteri Protectis has the potential to significantly
impact positively on HFMD epidemics. However, due
to the lack of a suitable in vivo model, and owing to
the excellent safety profile of this probiotic in babies
and young children, direct translation of this pre-
clinical work to human application may be possible
and could be quickly implemented in relevant commu-
nities. Such probiotics-based therapeutic approach
may address the urgent need for a safe and effective
means to protect against HFMD and limit its transmis-
sion among children.
Fig. 8 Antiviral effect of L. casei Shirota. Pre-incubation, pre-treatment, co-treatment and post-treatment setups were performed as detailed in
Fig. 1. Virus titers in the supernatant of CA16- (a) and EV71- (b) infected RD cells and Caco-2 cells were determined by standard plaque assay in
RD cells. A one-way ANOVA test with Dunnett’s posttest was performed (* p<0.05, ** p<0.005, *** p<0.0005). Data are expressed as the mean
± standard deviation of technical triplicates. Two biological repeats were conducted. One representative is shown
Ang et al. Virology Journal (2016) 13:111 Page 10 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Abbreviations
ANOVA, one-way analysis of variance; CA, coxsackievirus type A; CB2, coxsackievirus
type B strain 2; CFU/mL, colony-forming unit per mL; DMEM, Dulbecco’smodified
eagle medium; EV71, enterovirus 71; FBS, fetal bovine serum; GI, gastrointestinal;
HFMD, hand, foot and mouth diseases; L. casei Shirota, Lactobacillus casei
Shirota; L. reuteri Protectis, Lactobacillus reuteri Protectis; LAB, lactic acid bacteria;
MOI, multiplicity of infection; p, probability values; PBS, phosphate buffered
saline; PFU/mL, plaque-forming unit per mL; POS, positive control; RD,
rhabdomyosarcoma; SD, standard deviation
Acknowledgements
L. reuteri Protectis was used with permission from BioGaia AB. L. casei Shirota
was a kind gift from A/P Lee Yuan Kun (Department of Microbiology and
Immunology, National University of Singapore). Human Caco-2 cells were
obtained from A/P Kevin Tan (Department of Microbiology and Immunology,
National University of Singapore).
Funding
This work was supported by a CS-NIG grant from National Medical Research
Council awarded to ET.
Authors’contributions
ELT, ET and SA supervised the work. LYEA and HKIT designed and performed
the experiments. LYEA, HKIT and SA performed literature review and data
analysis. ELT and TKVC contributed materials. LYEA, HKIT and SA wrote the
paper. TKVC, PCLS provided inputs to the manuscript. All authors have read
and approved the final version of the manuscript.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Microbiology and Immunology, Yong Loo Lin School of
Medicine, National University of Singapore, Centre for Life Sciences, 28
Medical Drive, #03-05, Singapore 117456, Singapore.
2
Immunology
programme, Life Sciences Institute, National University of Singapore,
Singapore, Singapore.
3
Department of Paediatrics, National University
Hospital, Singapore, Singapore.
4
Centre for Biomedical & Life Sciences,
Singapore Polytechnic, Singapore, Singapore.
Received: 4 April 2016 Accepted: 20 June 2016
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