In Vitro Functional and Immunomodulatory Properties of the
Lactobacillus helveticus MIMLh5-Streptococcus salivarius ST3
Association That Are Relevant to the Development of a Pharyngeal
Valentina Taverniti,aMario Minuzzo,bStefania Arioli,aIlkka Junttila,c,eSanna Hämäläinen,c,dHannu Turpeinen,dDiego Mora,a
Matti Karp,gMarko Pesu,d,e,fand Simone Guglielmettia
Department of Food Science and Microbiology, Universita ` degli Studi di Milano, Milan, Italya; Department of Biomolecular Sciences and Biotechnology, Universita ` degli
Studi di Milano, Milan, Italyb; School of Medicine, University of Tampere, Tampere, Finlandc; Institute of Biomedical Technology, Biomeditech, University of Tampere,
Tampere, Finlandd; Fimlab Ltd., Tampere, Finland,ePirkanmaa Hospital District, Tampere, Finlandf; and Institute of Environmental Engineering and Biotechnology,
Tampere University of Technology, Tampere, Finlandg
tory tract infections. In this context, we characterized in vitro the functional and immunomodulatory properties of the strains
Lactobacillus helveticus MIMLh5 and Streptococcus salivarius ST3 that were selected during previous investigations as promis-
ing pharyngeal probiotics. In this study, we demonstrated in vitro that strains MIMLh5 and ST3, alone and in combination, can
efficiently adhere to pharyngeal epithelial cells, antagonize Streptococcus pyogenes, and modulate host innate immunity by in-
helveticus MIMLh5 and S. salivarius ST3 for the preparation of novel products that display probiotic properties for the pharyn-
“probiotic approach.” Traditionally, probiotic products are ex-
However, several probiotics have been designed for the vaginal
mucosa (35), the urinary tract (5), the skin (20), and the oral
cavity (38). The use of probiotics for the oropharyngeal tract
(OPT) is particularly promising. OPT dysfunctions are often re-
lated to the presence of microbial pathogens (for instance, Strep-
lis) or to microbial dysbiosis. Furthermore, compared with the
exogenous origin. For these reasons, the OPT is a potential target
for new, specifically designed probiotic products.
An excellent example demonstrating the potential of the
probiotic approach for the OPT is represented by the research
activity of J. R. Tagg and coworkers, who isolated the Strepto-
coccus salivarius strain K12 (45). Strain K12 has a marked abil-
of three different bacteriocins, salivaricins A2, B, and 9 (44).
Strain K12 was also demonstrated to be able to colonize the
upper respiratory tract (17) and to downregulate the innate
immune responses of human epithelial cells (8). The scientific
results from the Tagg lab studies supported the creation of a set
of probiotic pharmaceutical products (lozenges, powders, and
he administration of living microbial cells to humans or ani-
mals to promote or maintain health is commonly known as a
chewing gum), commercialized under the name of BLIS, that
were specifically designed for the prevention or treatment of
dysfunctions such as cavities, periodontitis, halitosis, and
pharyngitis. In July 2011, the oral probiotic products BLIS K12
were granted generally recognized as safe (GRAS) status by the
United States Food and Drug Administration (FDA), enabling
this probiotic to be included as an ingredient in food products
within the United States.
biotic and dairy bacteria and new oral isolates were screened in
keratinocytes, antagonize Streptococcus pyogenes, and modulate
host immune system responses (13, 14). As a result of these stud-
didates for the pharyngeal mucosa. In this report, we show the
Received 3 February 2012 Accepted 5 April 2012
Published ahead of print 13 April 2012
Address correspondence to Simone Guglielmetti, firstname.lastname@example.org.
Supplemental material for this article may be found at http://aem.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
June 2012 Volume 78 Number 12Applied and Environmental Microbiologyp. 4209–4216aem.asm.org
results of further investigations of these bacteria. We analyzed in
ployed in combination. Specifically, we examined their ability to
cus pyogenes, and to modulate the immune response of macro-
phages. Particularly, since the immunological study of the in-
teraction between probiotic bacteria and the oropharyngeal
mucosa has been based exclusively on epithelial cells, in this
study we assessed the responses induced by bacteria in host
cells properly belonging to the immune system. To explore the
potential of combining strains ST3 and MIMLh5 in a fer-
mented food product, we also studied the growth of S. sali-
varius ST3 in the presence of different sugars and in milk in
coculture with strain MIMLh5. Our results support the possi-
bility of producing a novel milk-based fermented food product
to be used as probiotic for the oropharyngeal mucosa.
MATERIALS AND METHODS
cillus helveticus MIMLh5 was grown at 42°C in MRS broth (Difco Labo-
ratories Inc., Detroit, MI). Streptococcus salivarius ST3 was cultivated at
37°C in M17 broth (Difco Laboratories) supplemented with 2% glucose.
plates using a BioTek Synergy HT multimode microplate reader (AHSI
with 2%, 1%, 5‰, 2.5‰, 1.25‰, or 0.625‰ of one of the following
sugars: fructose, galactose, glucose, inulin, lactose, lactulose, sucrose, or
fructooligosaccharides (FOS). All sugars were purchased from Sigma-Al-
tion of 384-well microtiter plates was conducted with an epMotion auto-
mated pipetting system (Eppendorf, Milan, Italy). Acidification curves
were determined by inoculating skim milk (Difco) (1% inoculum) with
an overnight culture of MIMLh5 and ST3 alone or in coculture and re-
cording the pH every hour for 24 h with a pH meter recorder (XS instru-
ments pH 2100; Opto-Lab, Concordia, Italy). Recombinant S. pyogenes
FaDu cell layer preparation. In vitro cultivation. FaDu cells (human
pharynx carcinoma cell line; ATCC HTB-43) were routinely grown in
Eagle’s minimum essential medium supplemented with 10% (vol/vol)
heat-inactivated (30 min at 56°C) fetal calf serum, 100 U ml?1penicillin,
L-glutamine (EMEM) and were incubated at 37°C in a water-jacketed
incubator in an atmosphere of 95% air and 5% carbon dioxide.
confluence. Next, the cell layers were carefully washed twice with phos-
phate-buffered saline (PBS), pH 7.3. The bacterial cell concentrations of
many). Bacterial cells were recovered by centrifugation and resuspended
in PBS to obtain a concentration of 108cells per ml. Two milliliters of the
37°C. Afterward, the monolayers were washed 3 times with PBS, fixed for
8 min with 2 ml of methanol, and stained with 2 ml of Giemsa’s stain
(Carlo Erba Reagenti S.p.A., Rodano, Italy). The cover glasses were then
washed 5 times with 1 ml of PBS, dried, and examined microscopically
randomly selected microscopic fields were counted, and average counts
Bacterial antagonism assay on the FaDu cell layer. Antagonism
against Streptococcus pyogenes was studied using the bacterial exclusion
FaDu cell layer for 1 h. The FaDu cells were then washed with PBS and
incubated with 2 ? 108cells ml?1of the indicator strain (S. pyogenes
bioluminescent recombinant strain C11LucFF) for 1 h. After the second
incubation, FaDu cell layers were quickly washed twice with 1 ml of PBS
tester strain was analyzed in triplicate in at least two independent experi-
lines were generated by transfecting cells with the plasmid pNiFty2-Luc
(Invivogen, Labogen, Rho, Italy) as described by Guglielmetti et al. (13).
?B)-binding sites and the luc firefly luciferase reporter gene. Stimuli that
activate NF-?B promote its binding to the vector promoter, resulting in
ml?1zeocin, the FaDu cells were detached by trypsinization and then
resuspended in EMEM at a concentration of 250,000 cells ml?1in the
presence of 100 mM HEPES (pH 7.4). Subsequently, 50 ?l of a tester
bacterial suspension containing 2.5 ? 109or 2.5 ? 108cells ml?1was
added to 450 ?l of the FaDu cell suspension, resulting in a multiplicity of
infections (MOI) of approximately 1,000 or 100, respectively. After incu-
bation at 37°C for 4 h, the samples were kept in ice and sonicated at
maximum power for 5 s using a Bandelin Sonoplus ultrasonic homoge-
nizer (Bandelin Electronic GmbH & Co., Berlin, Germany). Insoluble
particles were removed by centrifugation, and 100 ?l of the supernatants
were dispensed in duplicate into the wells of a 96-well white microtiter
system (Eppendorf). Next, 12.5 ?l of a 10 mM ATP solution (1 mM final
and bioluminescence was immediately measured with a Victor31420
curve was considered for comparison of results. All conditions were ana-
dent’s t test was used to detect statistically significant differences.
macrophage cell line. The cell line U937 (ATCC CRL-1593.2) was de-
rived from a human histiocytic lymphoma (37). These cells are main-
macrophages, including similar isoenzyme patterns (33) and other phe-
notypic markers (15). The normal growth medium for the U937 cells
10% (vol/vol) fetal bovine serum (FBS) (Gibco-BRL, Life Technologies,
Milan, Italy), 2 mM L-glutamine, 100 units ml?1penicillin, and 100 ?g
ml?1streptomycin (Sigma-Aldrich). U937 cells were seeded at a density
ified atmosphere of 95% air and 5% CO2. Differentiation was induced by
the addition of PMA (Sigma-Aldrich) into the cellular medium at a final
cells were washed once with sterile PBS buffer to remove all nonadherent
cells. One hour before the bacteria were added to the cells, the culture
media was replaced with RPMI 1640 medium supplemented with 1%
from Escherichia coli 0127:B8 (Sigma-Aldrich) was used as the positive
i.e., only RPMI 1640 medium with 1% (vol/vol) FBS, was used as the
Inhibition assay with Toll-like receptor neutralizing antibodies.
Human anti-Toll-like receptor 2 (anti-TLR-2) antibody (Invivogen) was
Taverniti et al.
aem.asm.org Applied and Environmental Microbiology
added to U937 cells 1 h before the stimulation with bacterial cells. A
human immunoglobulin A2 (IgA2) isotype control (Invivogen) was used
as the control to exclude nonspecific binding and blocking activity of the
antibody. Both the anti-TLR2 and IgA2 isotype control were used at 5 ?g
ml?1. This concentration was determined by examining the neutralizing
efficacy of anti-TLR2 with zymosan from Saccharomyces cerevisiae (Invi-
vogen) as the ligand.
Preparation of RNA and reverse transcription. After incubating
U937 cells at 37°C for 4 h, the supernatant was carefully removed from
each well and the total cellular RNA was isolated from the adhered U937
cells with an RNeasy Minikit (Qiagen, Inc., Valencia, CA). Afterward,
RNA concentration and purity were determined with a NanoDrop spec-
trophotometer (ND-1000, Thermo Fischer Scientific), and reverse tran-
scription to cDNA was performed with an iScript Select cDNA synthesis
kit (Bio-Rad Laboratories, Hercules, CA) using the following thermal
cycle: 5 min at 25°C, 30 min at 42°C, and 5 min at 85°C.
Real-time quantitative PCR. The mRNA expression levels of cyto-
kines were analyzed with SYBR green technology in real-time quanti-
tative PCR (qPCR) using SsoFast EvaGreen Supermix (Bio-Rad) on a
Bio-Rad CFX96 system according to the manufacturer’s instructions.
The primers used are as follow (5=¡3=): 18srRNA forward, ATCCCT
GAAAAGTTCCAGCA; 18srRNA reverse, CCCTCTTGGTGAGGTCA
ATG; interleukin-10 (IL-10) forward, AGCAGAGTGAAGACTTTCT
TTC; IL-10 reverse, CATCTCAGACAAGGCTTGG; tumor necrosis
CCCAGGCAGTCAGATCAT; cyclooxygenase 2 (COX-2) forward, CCC
TTGGGTGTCAAAGGTAA; COX-2 reverse, TGAAAAGGCGCAGTTT
ACG. All primers were selected using Primer3Plus software (http://www
.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi), and the specificity
agarose gels. Quantitative PCR was carried out according to the following
5 s. Gene expression was normalized to that of the 18S ribosomal DNA
fold of induction (FOI) respective to the control (namely, unstimulated
RESULTS AND DISCUSSION
In previous studies, we selected Streptococcus salivarius ST3 and
Lactobacillus helveticus MIMLh5 from several oral bacterial iso-
lates and probiotic/dairy bacteria as potential probiotics for the
pharyngeal mucosa (13, 14). In this study, we evaluated the pos-
sibility of using strains ST3 and MIMLh5 in combination by as-
sessing their in vitro probiotic and immunologic properties.
ST3/MIMLh5 cosuspension did not affect adhesion and an-
tagonistic properties of the individual strains on the FaDu cell
layer. In previous studies, strains ST3 and MIMLh5 showed sim-
different behaviors both in the activation of NF-?B and in the
profile of induced cytokines (13, 14). Therefore, we evaluated in
vitro the potential probiotic properties of strains ST3 and
MIMLh5 used in combination. We first studied the interaction
with FaDu human pharyngeal cell line in terms of adhesion and
ability to antagonize Streptococcus pyogenes (Fig. 1). The adhesion
of each strain was not affected when the other was also present,
indicating that there is no competition for adhesion sites between
MIMLh5 and ST3 in our test conditions (MOI of 1000 for each
adhered per 100 FaDu cells) for the two strains, in fact, was not
significantly different when a strain was alone or in coculture (P
value of 0.835 or 0.507, respectively); both conditions resulted in
an adhesion index of approximately 2,000 (data not shown).
In the following series of experiments, we measured the biolu-
minescence produced by S. pyogenes C11LucFFas an indication of
the exclusion antagonism exerted by strains ST3, MIMLh5 and
their mixed suspension. L. helveticus MIMLh5 showed better an-
tagonism against S. pyogenes (72% reduction of luminescence),
but S. salivarius strain ST3 could also effectively antagonize the
pathogen (53% luminescence reduction). When the tester bacte-
tic activity was maintained at the highest level (72% reduction),
indicating that the two strains can coexist without reducing their
in vitro exclusion ability (Fig. 1B).
The effect of strain MIMLh5 on NF-?B activation in FaDu
epithelial cells is predominant over that of strain ST3. An in-
creasing number of in vitro and in vivo experiments support the
by interacting with the immune system (7, 23). The ability to
bacteria that have been proposed as probiotics for the OPT, such
as Streptococcus salivarius K12 (8) and the two strains included in
this study (13, 14). Particularly, in vitro immunological tests with
FaDu cells revealed different responses to S. salivarius ST3 and L.
helveticus MIMLh5 (13, 14). While strain MIMLh5 reduced IL-8
and IL-6 secretion (14), ST3 had only a limited effect on these
cytokines but drastically inhibited IL-1? and stimulated macro-
phage inflammatory protein 1? (MIP-1?) (13). It has been pro-
exposure to strain MIMLh5 can be attributed to the inhibition of
activation of NF-?B (13). In this study, we explored the effect of
strains ST3 and MIMLh5 in combination on NF-?B activation in
FaDu cells. As observed in previous investigations (13, 14), L.
helveticus MIMLh5 decreased the NF-?B-dependent production
of bioluminescence, while ST3 showed a stimulatory effect (Fig.
1C). When the two bacterial strains were used in combination at
the same multiplicity of infection (MOI of 1,000 each), activation
of NF-?B was reduced to an extent similar to that determined by
MIMLh5 alone (Fig. 1C), indicating that the inhibitory effect of
this bacterium on NF-?B-mediated responses is predominant
over the outcome induced by strain ST3.
Effect of the strains MIMLh5 and ST3 on activation of U937
trated mainly on immune responses at the epithelial cell level (8,
13). An evaluation of the responses by cells involved in the im-
mune system is thus required. In this study, we quantified via
cytokine involved in inflammatory responses ) and IL-10 (a
of proinflammatory cytokines in several cell types [9, 11]) in
PMA-differentiated U937 human macrophages. We tested the
strains MIMLh5 and ST3 alone and in combination (coincuba-
tion) at MOIs of 100 and 1,000. In all tested conditions, strains
MIMLh5 and ST3 induced a higher IL-10/TNF-? ratio than lipo-
polysaccharide (LPS) from Escherichia coli, a potent proinflam-
matory stimulus. Unlike LPS, the induction of IL-10 by MIMLh5
ST3, in particular, triggered profound IL-10 expression. In coin-
cubation experiments, we used a total MOI (i.e., the sum of the
bacterial cell numbers of both strains) corresponding to the MOI
of the bacterial strains employed individually to see possible syn-
Potential Probiotic Properties of MIMLh5/ST3
June 2012 Volume 78 Number 12aem.asm.org 4211
ergistic effects. At the lower MOI tested, the mixed suspension of
MIMLh5 and ST3 resulted in an intermediate cytokine induction
compared to that of the single strains (Fig. 2A). In contrast, syn-
ergism was observed at the MOI of 1,000; the combination of
both TNF-? and IL-10 (Fig. 2A). These data show that the bacte-
rial cell concentration affects the immune response of U937 cells;
a dose-dependent approach should be therefore considered when
defining a specific immunological effect of a bacterial strain, as
demonstrated in previous works (10).
In this study, the strains MIMLh5 and ST3 significantly in-
duced the expression of the proinflammatory cytokine TNF-?.
Previous studies have shown that different Lactobacillus species
can trigger pronounced proinflammatory activity in DCs (6, 43)
and in macrophages (26). Macrophages are professional phago-
cytes that reside in the secondary lymphoid organs as well as in
detect microbial invaders (1). Upon the recognition of pathogen-
associated molecular patterns (PAMPs ), macrophages pro-
that alert the immune system to the infection of injury. A proin-
(Th1-type) immunity, like that observed for MIMLh5 and ST3,
could thus be crucial in combating intracellular pathogen attacks
studies have reported that most lactic acid bacterial strains that
have been tested could induce a Th1 response, even though this
(29, 31). Notably, in the same studies, some strains of S. thermo-
philus, a species phylogenetically close to S. salivarius, induced a
high level of IL-10 (29). In our experiments, S. salivarius ST3 in-
duced a higher level of IL-10 than TNF-?. Furthermore, the in-
creased production of the anti-inflammatory cytokine was more
pronounced in ST3 than in MIMLh5 at the highest MOI tested
(Fig. 2A). Therefore, we speculate that the presence of both bac-
teria, even at high doses (MOI of 1,000), might not result in det-
rimental effects. The regulatory effect of the significant IL-10 ex-
ditions such as rheumatic disease (32).
The strains MIMLh5 and ST3 induce cyclooxygenase 2
FIG 1 Probiotic properties of Lactobacillus helveticus MIMLh5 and Streptococcus salivarius ST3 on a FaDu human pharyngeal cell layer. (A) Bacterial adhesion
as observed with Giemsa staining under a light microscope; FaDu cell nuclei appear in red. (B) Antagonistic exclusion activity against bioluminescent Strepto-
cell). IL-1? was used as the positive control for NF-?B activation. Data in histograms are the means (? standard deviations) from at least three independent
significant differences compared to the control (P ? 0.001).
Taverniti et al.
aem.asm.orgApplied and Environmental Microbiology
ase (COX), or prostaglandin synthase H (PGH), is a homodimer
enzyme involved in the synthesis of prostaglandins (PGs) from
arachidonic acid (12). PGs are involved in several physiological
processes and contribute to the protection of the gastrointestinal
mucosa (46, 27). Two isoforms of the COX enzyme have been
identified and described (21). COX-1 is constitutively expressed
in a wide range of tissues, whereas COX-2 is constitutively ex-
pressed in very few tissues but is induced by several stimuli, in-
cluding bacterial components (16). We investigated the effect of
the strains MIMLh5 and ST3 on COX-2 gene activation in the
U937 cell model. L. helveticus MIMLh5 and S. salivarius ST3 in-
duced COX-2 gene expression to similar levels. The expression
levels for MIMLh5 and ST3 were 5.20 and 5.75, respectively, at an
(Fig. 2B). The association of the two bacteria resulted in an en-
we also observed a strong increase in COX-2 gene expression in
murine bone marrow-derived dendritic cells (BMDCs) (see Fig.
S1 in the supplemental material).
Although other studies have suggested that lactic acid bacteria
could affect COX-2 secretion, there is no clear consensus in the
to reduce the amount of prostaglandins and levels of COX-2 gene
expression at the intestinal level in murine models (3, 41) and in
30) and in vivo (19, 22). Lactobacillus acidophilus, a species phylo-
genetically related to L. helveticus, was reported to significantly
increase COX-2 expression and PGE2secretion in the human co-
lon cancer cell line Colo320 (30). Differences in tested strains and
model systems likely explain the lack of consensus found in the
The ability of the strains MIMLh5 and ST3 to induce COX-2
expression suggests that these bacteria could directly affect in-
flammatory processes not only by modulating cytokine secretion
but also by inducing prostaglandin production. COX-2 is in-
in guiding T cells toward an immunosuppressive phenotype (28)
and in resolving inflammation (42). The observed effect of
of immediate benefit for the host’s mucosa; furthermore, rapid
COX-2 upregulation in response to injury or inflammation has
been reported to restore mucosal integrity (40).
pathways involved in the immune responses induced by L. helve-
ticus MIMLh5 and S. salivarius ST3, we investigated which Toll-
like receptor (TLR) recognizes these bacteria. TLRs are a class of
transmembrane proteins involved in innate immunity (2); in
participate in detecting various conserved microbial molecules,
such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), pepti-
FIG 2 Transcription analysis of cytokine genes in U937 cells stimulated with Lactobacillus helveticus MIMLh5 and Streptococcus salivarius ST3 after 4 h of
of 1 ?g ml?1. The values are the means (? standard deviations) for a result representative of three independent experiments, expressed as the fold change in
induction relative to the result for the control (U937 unstimulated cells), which was set at a value of 1. Asterisks indicate statistically significant differences
of IL-10 and TNF-? in the presence of a neutralizing antibody against TLR-2 (anti-TLR2). Anti-TLR2 was added to U937 cells 1 h before stimulation with
bacteria. Immunoglobulin-A2 isotype (IgA2) was used as the control for nonspecific blocking activity.
Potential Probiotic Properties of MIMLh5/ST3
June 2012 Volume 78 Number 12 aem.asm.org 4213
doglycan (PGN), bacterial lipoproteins, lipoarabinomannan, and
zymosan (25). More specifically, TLR-2 forms heterodimers with
TLR-1 or TLR-6 and primarily interacts with Gram-positive bac-
teria by recognizing PGN, LTA, and lipoproteins (39).
In this study, as preliminary information needed to set up the
experiment, we observed that 5 ?g ml?1of zymosan induced in
U937 cells a 5-fold reduction in the expression level of TNF-?, a
cytokine known to be induced in the downstream pathway acti-
vated by TLR-2. We then performed experiments with a neutral-
ability to bind ligands. An IgA isotype control was also used to
exclude nonspecific binding to U937 cell receptors. After a 1-h
RT-qPCR. When TLR-2 was blocked with anti-TLR2, we ob-
served significantly less TNF-? mRNA, both when strains
in combination (Fig. 2C). These results indicate that TLR-2 is
involved in mediating the immunostimulatory activity of both
strains. These data are consistent with previous studies that
showed that the immunomodulatory activity elicited by lactoba-
49). However, preincubation with anti-TLR2 resulted in a reduc-
tion of IL-10 expression only with strain ST3, whereas MIMLh5-
mediated expression of IL-10 was not affected (Fig. 2C), indicat-
induction of this cytokine. The interference of the anti-TLR2-Ab
in a TLR2-dependent manner have been already described (34,
48). It is plausible that a similar response mechanism is triggered
by S. salivarius ST3 in U937 cells.
When the strains were combined, the reduction of the proin-
flammatory cytokine TNF-? was similar to that of ST3 alone
(1.43- and 1.34-fold reduction, respectively). Even though anti-
TLR2 generally reduced TNF-? induction, the relative increase of
TNF-? gene expression caused by the bacteria in combination
when TLR-2 was blocked with anti-TLR2 than that in control
the synergistic effect of using both strains was intensified by the
presence of the anti-TLR2 antibody. As a consequence, when
(approximately 18%) with bacteria in combination, whereas the
can therefore speculate that PRRs other than TLR-2 are involved
in the recognition of these bacteria. It might also be hypothesized
that blocking TLR-2 can impact the formation of heterodimers
of receptors (such as scavenger receptors), therefore causing dif-
ferent cytokine responses.
ment bovine milk. In light of the in vitro functional properties of
L. helveticus MIMLh5 and S. salivarius ST3 in combination, we
examined the possibility of cocultivating these bacteria in bovine
milk and potentially employing MIMLh5 and ST3 in dairy prod-
ucts. L. helveticus MIMLh5 is a natural whey starter culture that is
well adapted to grow in bovine milk. In contrast, S. salivarius is a
human oral commensal isolate that cannot proliferate when cul-
tivated in milk due to its limited ability to use lactose as the sole
carbon and energy source. We determined the growth curve of
ST3 with eight different carbohydrates at six concentrations in
M17 medium (see Fig. S2 in the supplemental material). Strain
ST3 displayed optimal growth in the presence of glucose, sucrose,
fructose and fructooligosaccharides (FOS); its growth was signif-
galactose and lactitol (Fig. 3A). Interestingly, strain ST3 metabo-
lized Actilight (Fig. 3B), a commercial product consisting of a
tose) that is frequently used as a prebiotic supplement. When we
added one of the four sugars that are efficiently metabolized by
ST3 to skim milk, ST3 growth became similar to that in M17
supplemented with the same sugar. Subsequently, we performed
acidification curves by monitoring the pH in skim milk supple-
mented with 2% glucose after a 1% inoculum of the overnight
cultures of strains ST3 and MIMLh5 alone and in coculture. The
association of the two strains resulted in a faster acidification of
FIG 3 Growth curves of Streptococcus salivarius ST3. (A) Growth in M17
medium supplemented with 2% of 8 different carbon sources. (B) Growth in
M17 medium supplemented with 6 different concentrations of Actilight fruc-
tooligosaccharides (FOS). The curves are representatives of two experiments
carried out in sextuplicate.
Taverniti et al.
aem.asm.org Applied and Environmental Microbiology
the medium (Fig. 4). In fact, after 6 h of incubation at 42°C, ST3
and MIMLh5 monocultures reached pH 5.2 and 5.5, respectively,
whereas their coculture reduced the pH to 4.9 in skim milk sup-
plemented with 2% glucose or FOS (Fig. 4). After 6 h of coincu-
bation in skim milk supplemented with glucose at 42°C, ST3 and
MIMLh5 reached a number of viable cells of 9.5 ? 108and 8.9 ?
107CFU ml?1, respectively (Fig. 4), indicating that both strains
contributed to the milk acidification. These data support the po-
tential of using a combination of strains ST3 and MIMLh5 to
ferment milk for the production of a novel food product.
Conclusion. There is an increasing interest in prophylactic
strategies effective for upper respiratory tract infections, which
represent a major cause of medical prescription for antibiotics,
especially in children. The identification of bacterial strains that
could be used as probiotics for the pharyngeal mucosa is one po-
This study is part of an ongoing research project designed to
identify new potential probiotic bacteria for the pharyngeal mu-
cosa and to develop novel food products for an effective adminis-
tration of these probiotics. We presented a further characteriza-
tion of the strains MIMLh5 and ST3 that were selected during
previous investigations as the most promising pharyngeal probi-
ST3, alone and in combination, can efficiently adhere to pharyn-
geal epithelial cells, antagonize S. pyogenes, and modulate host
innate immunity by inducing potentially protective effects. We
gave particular attention to bacterial immunomodulatory prop-
erties because modulation of the host’s immunity is one of the
otics and is supported by an increasing number of in vitro and in
vivo studies (7). We found that the combination of MIMLh5 and
ST3 resulted in a synergistic effect, according to cytokine induc-
tion, that might help the host immune system react to potential
pathogens while maintaining a balance between pro- and anti-
inflammatory cytokines, thus preventing possible exaggerated re-
sponses. Finally, we observed that these microorganisms grow ef-
ficiently when cocultured in milk, suggesting that the preparation
ST3 can be a practicable solution for the administration of these
In conclusion, we propose the combined use of Lactobacillus
helveticus MIMLh5 and Streptococcus salivarius ST3 for the prep-
aration of novel products that display probiotic properties for the
pharyngeal mucosa. A clinical study will soon be carried out to
confirm that the in vitro properties exerted by strains MIMLh5
of preventing oropharyngeal infections.
We thank Kati Pulkkinen and Zsuzsanna Ortutay for the important tech-
Part of this study was funded by Fondazione Cariplo (grant 2010-
0678). I.J. is financially supported by Competitive Research Funding of
dation. This study was also financially supported by the Academy of Fin-
land (M.P., projects 128623 and 135980), a Marie Curie International
Reintegration Grant within the 7th European Community Framework
Programme (M.P.), the Emil Aaltonen Foundation (M.P.), the Sigrid Ju-
pere University Hospital (M.P., grants 9M080 and 9N056).
1. Aderem A, Underhill DM. 1999. Mechanisms of phagocytosis in macro-
phages. Annu. Rev. Immunol. 17:593–623.
2. Akira S, Hemmi H. 2003. Recognition of pathogen-associated molecular
patterns by TLR family. Immunol. Lett. 85:85–95.
damage associated with collagen-induced arthritis (CIA) by reducing the
4. Belardelli F. 1995. Role of interferons and other cytokines in the regula-
tion of the immune response. APMIS 103:161–179.
5. Borchert D, et al. 2008. Prevention and treatment of urinary tract infec-
tion with probiotics: review and research perspective. Indian J. Urol. 24:
6. Christensen HR, Frokiaer H, Pestka JJ. 2002. Lactobacilli differentially
rine dendritic cells. J. Immunol. 168:171–178.
7. Corthesy B, Gaskins HR, Mercenier A. 2007. Cross-talk between probi-
otic bacteria and the host immune system. J. Nutr. 137:781S–790S.
8. Cosseau C, et al. 2008. The commensal Streptococcus salivarius K12
promotes host-microbe homeostasis. Infect. Immun. 76:4163–4175.
9. D’Andrea A, et al. 1993. Interleukin 10 (IL-10) inhibits human lympho-
ulatory factor/IL-12 synthesis in accessory cells. J. Exp. Med. 178:1041–
10. Evrard B, et al. 2011. Dose-dependent immunomodulation of human
11. Fiorentino DF, Zlotnik A, Mosmann TR, Howard M, O’Garra A. 1991.
IL-10 inhibits cytokine production by activated macrophages. J. Immu-
12. Funk CD. 2001. Prostaglandins and leukotrienes: advances in eicosanoid
biology. Science 294:1871–1875.
13. Guglielmetti S, et al. 2010. Oral bacteria as potential probiotics for the
pharyngeal mucosa. Appl. Environ. Microbiol. 76:3948–3958.
14. Guglielmetti S, et al. 2010. A dairy bacterium displays in vitro probiotic
properties for the pharyngeal mucosa by antagonizing group A strepto-
cocci and modulating the immune response. Infect. Immun. 78:4734–
15. Harris P, Ralph P. 1985. Human leukemic models of myelomonocytic
development: a review of the HL-60 and U937 cell lines. J. Leukoc. Biol.
16. Herschman HR. 1996. Prostaglandin synthase 2. Biochim. Biophys. Acta
17. Horz HP, Meinelt A, Houben B, Conrads G. 2007. Distribution and
FIG 4 Growth of Lactobacillus helveticus MIMLh5 and Streprococcus thermo-
philus ST3 (single culture and coculture) at 42°C in skim milk supplemented
with 2% glucose or FOS. (A) Bacterial plate counts of coculture; T0, immedi-
ately after 1% inoculum; T3 and T6, after 3 and 6 h, respectively. (B) Acidifi-
Potential Probiotic Properties of MIMLh5/ST3
June 2012 Volume 78 Number 12aem.asm.org 4215
persistence of probiotic Streptococcus salivarius K12 in the human oral
Oral Microbiol. Immunol. 22:126–130.
18. Janeway CA, Jr, Medzhitov R. 2002. Innate immune recognition. Annu.
Rev. Immunol. 20:197–216.
19. Khailova L, et al. 2010. Bifidobacterium bifidum reduces apoptosis in the
intestinal epithelium in necrotizing enterocolitis. Am. J. Physiol. Gastro-
intest. Liver Physiol. 299:G1118–G1127.
20. Krutmann J. 2009. Pre- and probiotics for human skin. J. Dermatol. Sci.
21. Kujubu DA, Fletcher BS, Varnum BC, Lim RW, Herschman HR. 1991.
TIS10, a phorbol ester tumor promoter-inducible mRNA from Swiss 3T3
J. Biol. Chem. 266:12866–12872.
22. Kwon HK, et al. 2010. Generation of regulatory dendritic cells and
CD4?Foxp3?T cells by probiotics administration suppresses immune
disorders. Proc. Natl. Acad. Sci. U. S. A. 107:2159–2164.
23. Lebeer S, Vanderleyden J, De Keersmaecker SC. 2010. Host interactions
of probiotic bacterial surface molecules: comparison with commensals
and pathogens. Nat. Rev. Microbiol. 8:171–184.
24. Lee JM, Hwang KT, Jun WJ, Park CS, Lee MY. 2008. Antiinflammatory
nuclear factor-kappaB in Raw264.7 macrophage cells. J. Microbiol. Bio-
25. Means TK, Golenbock DT, Fenton MJ. 2000. The biology of Toll-like
receptors. Cytokine Growth Factor Rev. 11:219–232.
26. Morita H, et al. 2002. Cytokine production by the murine macrophage
cell line J774.1 after exposure to lactobacilli. Biosci. Biotechnol. Biochem.
27. Morteau O, et al. 2000. Impaired mucosal defense to acute colonic injury
28. Newberry RD, Stenson WF, Lorenz RG. 1999. Cyclooxygenase-2-
dependent arachidonic acid metabolites are essential modulators of the
intestinal immune response to dietary antigen. Nat. Med. 5:900–906.
29. Ongol MP, et al. 2008. Potential of selected strains of lactic acid bacteria
to induce a Th1 immune profile. Biosci. Biotechnol. Biochem. 72:2847–
30. Otte JM, et al. 2009. Probiotics regulate the expression of COX-2 in
intestinal epithelial cells. Nutr. Cancer 61:103–113.
31. Perdigon G, Maldonado GC, Valdez JC, Medici M. 2002. Interaction of
lactic acid bacteria with the gut immune system. Eur. J. Clin. Nutr.
32. Puliti M, et al. 2002. Regulatory role of interleukin-10 in experimental
group B streptococcal arthritis. Infect. Immun. 70:2862–2868.
33. Radzun HJ, Parwaresch MR, Sundstrom C, Nilsson K, Eissner M. 1983.
Monocytic origin of the human hematopoietic cell line U-937 and its
convertibility to macrophages evidenced by isoenzyme mapping. Int. J.
34. Re F, Strominger JL. 2004. IL-10 released by concomitant TLR2 stimu-
induced by TLR4 or TLR3 in human dendritic cells. J. Immunol. 173:
35. Reid G, Dols J, Miller W. 2009. Targeting the vaginal microbiota with
probiotics as a means to counteract infections. Curr. Opin. Clin. Nutr.
Metab. Care 12:583–587.
36. Shida K, Kiyoshima-Shibata J, Kaji R, Nagaoka M, Nanno M. 2009.
Peptidoglycan from lactobacilli inhibits interleukin-12 production by
macrophages induced by Lactobacillus casei through Toll-like receptor
2-dependent and independent mechanisms. Immunology 128(Suppl. 1):
37. Sundstrom C, Nilsson K. 1976. Establishment and characterization of a
38. Tagg JR, Dierksen KP. 2003. Bacterial replacement therapy: adapting
‘germ warfare’ to infection prevention. Trends Biotechnol. 21:217–223.
39. Takeda K, Kaisho T, Akira S. 2003. Toll-like receptors. Annu. Rev.
40. Tan XD, Chen YH, Liu QP, Gonzalez-Crussi F, Liu XL. 2000. Prostan-
oids mediate the protective effect of trefoil factor 3 in oxidant-induced
intestinal epithelial cell injury: role of cyclooxygenase-2. J. Cell Sci. 113:
41. Urbanska AM, Paul A, Bhathena J, Prakash S. 2010. Suppression of
tion of microencapsulated probiotic yogurt formulation. Int. J. Inflam.
42. Wallace JL. 2006. COX-2: a pivotal enzyme in mucosal protection and
resolution of inflammation. ScientificWorldJournal 6:577–588.
43. Weiss G, et al. 2010. Lactobacillus acidophilus induces virus immune
defence genes in murine dendritic cells by a Toll-like receptor-2-
dependent mechanism. Immunology 131:268–281.
44. Wescombe PA, et al. 2011. Salivaricin 9, a new lantibiotic produced by
Streptococcus salivarius. Microbiology 157:1290–1299.
45. Wescombe PA, et al. 2006. Production of the lantibiotic salivaricin A and
its variants by oral streptococci and use of a specific induction assay to
46. Williams CS, Mann M, DuBois RN. 1999. The role of cyclooxygenases in
inflammation, cancer, and development. Oncogene 18:7908–7916.
47. Yamamoto Y, Okato S, Takahashi H, Takeda K, Magari S. 1988.
Distribution and morphology of macrophages in palatine tonsils. Acta
Otolaryngol. Suppl. 454:83–95.
48. Yamazaki S, et al. 2011. TLR2-dependent induction of IL-10 and Foxp3?
CD25?CD4?regulatory T cells prevents effective anti-tumor immunity
induced by Pam2 lipopeptides in vivo. PLoS One 6:e18833. doi:10.1371/
49. Zeuthen LH, Fink LN, Frokiaer H. 2008. Toll-like receptor 2 and nucle-
otide-binding oligomerization domain-2 play divergent roles in the rec-
ognition of gut-derived lactobacilli and bifidobacteria in dendritic cells.
Taverniti et al.
aem.asm.org Applied and Environmental Microbiology