Impact of oral lozenges salivary mutans streptococci, plaque

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Clin Oral Invest
DOI 10.1007/s00784-014-1221-6
ORIGINAL ARTICLE
Impact of orally administered lozenges with Lactobacillus
rhamnosus GG and Bifidobacterium animalis subsp. lactis BB-12
on the number of salivary mutans streptococci, amount of plaque,
gingival inflammation and the oral microbiome in healthy adults
Aino Toiviainen & Heli Jalasvuori & Emilia Lahti & Ulvi Gursoy & Seppo Salminen &
Margherita Fontana & Susan Flannagan & George Eckert & Alexis Kokaras &
Bruce Paster & Eva Söderling
Received: 19 November 2013 / Accepted: 26 February 2014
# The Author(s) 2014. This article is published with open access at Springerlink.com
Abstract
Objectives The aim was to evaluate the effects of orally
administered Lactobacillus rhamnosus GG (LGG) and
Bifidobacterium animalis subsp. lactis BB-12 (BB-12) on
the number of salivary mutans streptococci (MS), amount of
plaque, gingival inflammation and the oral microbiota in
healthy young adults.
Materials and methods The study was a randomised, controlled, double-blind trial. Healthy volunteers used lozenges
containing a combination of LGG and BB-12 (test group, n=
29) or lozenges without added probiotics (control group, n=
31) for 4 weeks. At baseline and at the end of the test period,
Aino Toiviainen and Heli Jalasvuori have equal contribution to this study.
A. Toiviainen : H. Jalasvuori : E. Lahti : U. Gursoy :
E. Söderling (*)
Institute of Dentistry, University of Turku, Lemminkäisenkatu 2,
20520 Turku, Finland
e-mail: [email protected]
S. Salminen
Functional Foods Forum, University of Turku, Itäinen Pitkäkatu 4 A,
20520 Turku, Finland
M. Fontana : S. Flannagan : G. Eckert
Department of Cariology, Restorative Sciences & Endodontics,
School of Dentistry, University of Michigan, 1011 N University Ave,
Ann Arbor, MI 48109, USA
A. Kokaras : B. Paster
Department of Microbiology, The Forsyth Institute, 245 1st St,
Cambridge, MA 02142, USA
B. Paster
Department of Oral Medicine, Infection & Immunity, Harvard
School of Dental Medicine, Boston, MA 02115, USA
the plaque index (PI) and gingival index (GI) were determined, and stimulated saliva was collected. The microbial
composition of saliva was assessed using human oral microbe
identification microarray (n=30). MS and lactobacilli (LB)
were plate cultured.
Results The probiotic lozenge decreased both PI and GI
(p<0.05) while no changes were observed in the control
group. However, no probiotic-induced changes were
found in the microbial compositions of saliva in either
group.
Conclusions The probiotic lozenge improved the periodontal
status without affecting the oral microbiota.
Clinical relevance Short-term consumption of LGG and
BB-12 decreased the amount of plaque which was associated with a clinical impact: a decrease in gingival
inflammation.
Keywords Lactobacillus rhamnosus GG . Bifidobacterium
lactis BB-12 . Probiotic . HOMIM . Mutans streptococci .
Lactobacilli . Plaque index . Gingival index
Introduction
Recent studies have demonstrated the health impact of specific probiotic bacteria for humans and several recommendations
for probiotic use have appeared. The most widely studied
species belong to the genera Lactobacillus and
Bifidobacterium. Lactobacillus rhamnosus GG (LGG) has
been reported to be effective in preventing and treating rotavirus diarrhea, atopic eczema and upper respiratory infections
[1–3]. The most thoroughly investigated Bifidobacterium
strain Bifidobacterium lactis BB-12 (BB-12) has been used
Clin Oral Invest
in prevention/treatment of diarrhea and respiratory infections
[4, 5]. In addition, the combination of these two probiotics
appears to possess additional efficacy in prevention and treatment of allergic disorders, acute respiratory infections and
acute otitis media [6–8]. Recently, the stability of probiotics
has been reported to be improved by enhancing the tolerance
to processing conditions, and both L. rhamnosus GG and
B. lactis could be improved without altering their basic probiotic properties [9, 10].
Most probiotics colonise the gut only temporarily [11].
Thus, to obtain health benefits from consuming probiotics,
the consumption should take place on a daily basis. From
a dental point of view, however, acidogenic and aciduric
lactobacilli and bifidobacteria are generally considered cariogenic [12] and could be regarded as a risk for dental
health. Even though some probiotics like LGG and Lactobacillus reuteri can form biofilm [13, 14], most studies
suggest that intake of probiotics leads only to a transient
oral colonisation [15–18]. Furthermore, the existing clinical studies suggest that probiotics are safe for teeth and
they may even have beneficial effects on dental health
[19–23].
There are several mechanisms by which probiotics may
influence oral health, e.g. immune modulation, impact on
the oral microbiome, production of antimicrobial substances by the probiotic or the modified microbiome,
competitive exclusion and hindering adhesion of oral pathogens [24]. All available data demonstrate that effects of
probiotics are both species and strain specific. Most studies on probiotics and oral health have focused on measuring changes in mutans streptococci (MS) counts [25–27].
Even though high counts of MS do not necessarily mean
an increased caries risk, decreasing MS without affecting
the microbiota should make the plaque less virulent. Several studies have suggested that L. reuteri decreases counts
of MS [27] even though also contradictory results have
been published [28]. LGG consumption lasting from
2 weeks to 7 months has been associated with decreases
or no changes in MS counts [19, 28, 29]. Short-term
B. lactis consumption showed decreases in MS counts
[27]. However, apart from MS, very little is known about
effects of probiotics on the microbiota. Effects of
probiotics on periodontal pathogens has received little
interest so far, even though, administration of some
probiotics has been associated with improved gingival
and periodontal conditions [24].
Based on the health benefits reported for the combination
of LGG and BB-12, we decided to assess the impact of this
probiotic combination on plaque accumulation, gingival
health and the oral microbiota in healthy subjects. The null
hypothesis was that the consumption of the LGG-BB-12
combination has no effect on the number of salivary MS,
plaque index, gingival index or oral microbiota.
Materials and methods
Subjects
Altogether 77 volunteer students of the University of Turku,
Finland, were screened for the presence of salivary MS
(Dentocult® SM Strip Mutans, Orion Diagnostica, Espoo,
Finland). The inclusion criteria were good general health,
willingness to participate and salivary MS counts ≥103 colony-forming units (CFU)/ml. The 62 subjects showing salivary
MS counts of ≥103 CFU/ml were invited to participate and
they volunteered for the study. The study was approved by the
ethics committee of the Hospital District of Southwest Finland: ETMK 22/180/2012. A written informed consent was
obtained from all subjects. The ethics committee defined the
stopping rules. The ClinicalTrials.gov identifier of the study is
NCT01577485.
Subjects were randomly divided into two groups: group 1
with a mean age (SD) of 24.6 (2.7)years (3 males, 28 females), and group 2 with a mean age (SD) of 24.0 (3.0)years
(4 males, 27 females). Fifty-nine subjects had normal flow
rates of paraffin-stimulated saliva (>1 ml/min); three subjects
had flow rates <1 ml/min (1/group 1 and 2/group 2). All
subjects had good oral hygiene with no immediate need for
major restorative dentistry.
None of the subjects smoked. All subjects in group 2
(control group) completed the study. In group 1 (probiotic
group), one female subject dropped out during the wash-out
period, and another female subject dropped out during the test
period. Both subjects reported gastrointestinal problems as the
reason for dropping out.
Study design
The study was a randomised, controlled, double-blind trial. A
4-week run-in period was chosen based on earlier studies
[15–17]. At the beginning of the run-in period, the subjects
were given written instructions as follows: commercial products containing L. rhamnosus GG or B. lactis BB-12 were not
allowed but otherwise the subjects were to maintain their
normal tooth brushing and dietary habits. None of the subjects
were habitual consumers of such products before the run-in
period. All subjects used xylitol products (mostly chewing
gum) on daily basis before the study. The subjects were
instructed not to use their regular xylitol products during the
study. Most subjects brushed their teeth twice a day. Throughout the study, the subjects used the same brand of fluoridated
toothpaste provided to them free of charge. The drinking water
in the city of Turku contains 0.3 mg/l fluoride. Antimicrobial
mouthrinses were not allowed. Two of the subjects had been
on a brief course of antimicrobial medication 3 weeks before
the study started, and one subject used low-concentration
tetracycline medication throughout the study. Compliance to
Clin Oral Invest
given instructions was checked at both sampling visits (baseline and 4 weeks after). At these visits, the subjects were also
interviewed for dietary habits and oral/general health. These
data were used to check for compliance, possible adverse
effects and other confounding factors.
The subjects were randomly allocated into two groups
(groups 1 and 2): both groups used the same run-in chewing
gum for 4 weeks before the test period started. The recommendation for the use of the run-in gum was 4 pieces per day.
After the run-in, group 1 used green-coded test lozenges and
group 2 used yellow-coded control lozenges for 4 weeks. Also
for the lozenges, the recommendation was 4 pieces per day.
The recommended number of gums/lozenges resulted in a
daily xylitol dose of approximately 2 g which, according to
several studies, is too low to exert any “xylitol-effects” on the
oral microbiota [30]. Since most of the subject regularly
consumed xylitol prior to the study, to ensure compliance both
the wash-out chewing gum and test lozenge contained xylitol
as one of the bulk agents. The daily amount of probiotics was
approximately 2×109 cells for LGG and BB-12 each. The first
of four lozenges was instructed to be taken in the morning, and
the last one in the evening following brushing of the teeth. All
but one of the subjects, who used only two lozenges per day,
reported using four lozenges per day. The subjects were asked
to chew the lozenge thoroughly to disrupt its structure before
swallowing.
Test products
The run-in chewing gum was a mild tasting, noncommercial
chewing gum manufactured free of charge for the study by
Karl Fazer AB (Vantaa, Finland). The gum pieces weighed
1.2 g and contained 42 % xylitol, 18 % sorbitol and 5 %
maltitol. The 1-g lozenges used during the test period were
also manufactured by Fazer. They were compressed from
50 % xylitol and 46 % sorbitol. The L. rhamnosus GG (ATCC
53103) used in the probiotic lozenges was from Probiotical
S.p.A., Novara, Italy. The B. lactis BB-12®(DSM15954) was
from Chr. Hansen A/S, Hoersholm, Denmark. Each probiotic
lozenge contained LGG 4.4×108 and BB-12 4.8×108. The
stability of the probiotics was improved before tableting as
described earlier [9], thus the viability of the probiotics did not
change during manufacturing or storage at room temperature.
The control lozenge had the same polyol composition but
contained no probiotics. The appearance and solubility properties of the probiotic lozenge were similar to that of the
control lozenge with no probiotics. The chewing gums and
lozenges were supplied in plastic bottles containing adsorbent
pads to control humidity. The control/test lozenge bottles were
marked with a yellow/green sticker. A 4.5-week supply was
given to all subjects, along with instructions to store the bottles
at room temperature.
Outcome measures, sample size and blinding
The primary outcome measure was the MS level in saliva and
the secondary outcome measure the plaque index. As demonstrated in an earlier study, a significant probiotic-induced
decrease in MS levels could be detected with as few as 10
MS-positive subjects; thus, sample size calculation was not
used here [31]. Furthermore, since we could compare the
baseline MS levels with the values obtained after the consumption of the control/probiotic lozenge, paired sample analysis, sensitive for even small changes in the variables, could
be used.
The second and third authors (HJ, EL), who recruited the
subjects and collected all samples, as well as the technical
assistants analysing the samples, were blinded to the study
assignment. The group codes were revealed to the researchers
only after the statistical analyses had been performed.
Determination of plaque and gingival index
Plaque was allowed to accumulate for 24 h with no oral
hygiene at baseline and at the end of each tablet period. On
the morning of the plaque collection day, the subjects were
instructed not to use the run-in chewing gum/test tablet. The
Silness-Löe plaque index (PI; [32]) and the Löe-Silness gingival index (GI; [33]) were determined in the afternoon using
a periodontal probe. Care was taken to avoid bleeding.
Saliva collection for human oral microbe identification
microarray analyses and plate culturing
Following the determination of PI and GI, 4 ml paraffinstimulated saliva was collected and the collection time was
recorded. For the human oral microbe identification microarray (HOMIM) analyses, the saliva samples of 15 randomly
chosen subjects per group were selected. One milliliter samples of the saliva were pipetted onto 10 μl TE buffer (SigmaAldrich, St. Louis, MO, USA) and stored at −70 °C before
shipment on dry ice. Genomic DNA was purified using
MasterPure Gram Positive DNA Purification kit (Epidentre
Biotechnologies, Madison, WI, USA), with modifications
(http://mim.forsyth.org). For plate culturing, samples
(100 μl) of the saliva were added into 900 μl Tryptic Soy
Broth with 10 % glycerol (TSB; Scharlau Chemie S.A., Barcelona, Spain) and stored at −70 °C before microbial analyses.
HOMIM
At the Forsyth Institute, microbial profiles were generated
from image files of scanned HOMIM microarrays (http://
bioinformatics.forsyth.org/homim/). In brief, concentration
levels of approximately 300 oral taxa were determined by
microarray hybridisation using a fluorescent readout reverse-
Clin Oral Invest
capture method [34]. Fluorescently labelled sample microbial
DNA was captured by 16S rRNA-based probes attached to
glass slides. The fluorescent intensity for each probe was
scanned, normalised and scaled as previously reported [34].
Signals of 2× background were considered to be negative and
assigned a HOMIM level score of 0. Positive hybridisation
signals were categorised into five levels, with 1 indicating a
signal that was just detectable, and 5 indicating maximum
signal intensity.
Plate culturing of MS and lactobacilli
The TSB tubes were thawed and vortexed for 1 min. The
content was pumped back-and-forth using a disposable pipette
and the resulting suspension was mildly sonicated. After serial
tenfold dilutions, the bacteria were plated on DifcoTM Mitis
salivarius (Becton Dickinson and Company, Sparks, MD,
USA) agar containing bacitracin (MSB). The plates were
incubated at +37 °C in a 7 % CO2 atmosphere for 2 days.
MS were identified as described earlier [35]. The number of
MS was identified based on colony morphology and counted
by means of a stereomicroscope. The identification of Streptococcus mutans was based on consistent findings of “rough”
colony morphology; positive fermentation with sorbitol, mannitol, raffinose and melibiose; and negative dextran agglutination. Identification of Streptococcus sobrinus was based on
“smooth” colonies, positive fermentation with mannitol but
negative with raffinose and melibiose, and positive dextran
agglutination. The lactobacilli and were grown for 2 days
anaerobically (80 % N2, 10 % CO2, 10 % H2) at +37 °C on
DifcoTM Rogosa SL agar (Becton Dickinson and Company).
The results were expressed as colony-forming units per
millilitre. The detection limit of the plate culturing was
100 CFU/ml.
Plaque collection and determination of the polysaccharide
content of the plaque samples
After the saliva collection, all available supragingival plaque
was collected with dental curettes from the left half of the
mouth and suspended in tubes containing 0.5 ml 1 N NaOH.
The plaque samples were stored at −70 °C before analyses.
The sugars of hydrolysed plaque reflect the polysaccharide
content of plaque and the proteins reflect the levels of
Table 1 Impact of probiotic
treatment on plaque and gingival
indices at baseline and 4 weeks
after the use of the LGG +
BB12/control lozenge. Means ±
SD given
microbes in the plaque [36, 37]. For determining the sugar/
protein ratio, the test tubes with the plaque samples in NaOH
were heated for 1 hour in a boiling water bath. Thereafter,
sugar [38] and protein [39] concentrations were determined
and their ratio was calculated.
Statistical analysis
t test for paired samples and Wilcoxon signed ranks test were
used to study differences in PIs, GIs and salivary MS and
lactobacilli counts between baseline and the end of the test
period. Independent samples t test was used to compare values
between the two groups at baseline and after the test period.
Pearson’s test was used to calculate the correlations. A multinomial logistic regression analysis was performed to study the
associations of the GI and PI in the study groups. The program
package SPSS 19.0 for Windows was used. The level of
statistical significance was set at p<0.05.
The HOMIM results were analysed at the University of
Michigan. Change from pretreatment to post-treatment was
tested within each group using Wilcoxon signed rank tests.
Comparisons between groups for differences pretreatment and
for differences in the change from pretreatment to posttreatment were made using Wilcoxon rank sum tests. To
control the overall level of significance for the large number
of tests in the HOMIM analyses, p values were evaluated
using an false discovery rate (FDR) adjustment. SAS version
9.3 was used for these analyses.
Results
The mean PI and GI values decreased significantly in the
probiotic group while no change was observed in the control
group (p=0.016 for PI and p=0.012 for GI; Table 1). The
number of cases with decreased PI and GI values in the
probiotic group showed a positive correlation (r=0.389; p=
0.037) and they were also significantly associated (OR 5.8,
95 % CI 1.0–30). In the control group, the number of subjects
with decreased PI and GI values did not correlate and no
association was found.
The HOMIM analysis included probes for about 300
predominant oral bacterial taxa. According to the results, microbiota at baseline did not differ between the
Plaque index
Gingival index
Group
Baseline
4 weeks
p
Baseline
4 weeks
p
Control (n=31)
LGG + BB-12 (n=29)
0.94±0.40
1.10±0.36
0.82±0.35
0.94±0.35
n.s.
0.016
0.70±0.44
0.71±0.28
0.61±0.37
0.57±0.23
n.s.
0.012
Clin Oral Invest
detected in the sugar/protein ratios of the plaques (data not
shown).
Only two subjects dropped out of the study: one during the
wash-out period and one during the test period. Both reported
that gastrointestinal problems were the reason, which did not
appear to be connected with the consumption of the lozenge.
Compliance in the study was good with only one of the
subjects reporting that she used less than the four recommended products per day.
Discussion
Fig. 1 Individual HOMIM profiles for 27 selected predominant microbes representing the oral microbiota of the 15 subjects of the probiotic
group at baseline and after 4-week use of the probiotic lozenge. The color
intensity reflects relative proportions of the species present in the sample
groups, and none of the subjects harboured Porphyromonas
gingivalis, Prevotella intermedia or Aggregatibacter
actinomycetemcomitans. Only three subjects had lactobacilli
present in their saliva. After the FDR adjustment, the analyses
did not reveal any statistically significant changes in the
microbiota when baseline values were compared with values
obtained after the test period. Figure 1 shows individual
HOMIM profiles for 27 selected predominant microbes
representing the oral microbiota of the subjects of the probiotic group at baseline and after 4-week use of the probiotic
lozenge.
At baseline, all subjects harboured S. mutans in their saliva.
Of these, 14 subjects in the probiotic group and 19 subjects in
the control group had high salivary MS counts (>105 CFU/
ml). Only one subject showed a combination of S. mutans and
S. sobrinus; thus, results for mutans streptococci (MS) are
shown. No study-induced changes in the MS counts were
detected either in the probiotic or the control group (Table 2).
At baseline, 15 subjects in the probiotic group and 14 subjects
in the control group had detectable levels of lactobacilli in
their saliva. The use of the LGG-containing lozenges was not
reflected in the salivary lactobacilli counts of the subjects. No
changes were detected in the counts of lactobacilli when
baseline values were compared with the values obtained after
the test period.
The polysaccharide content of plaque may reflect plaque
adhesion and virulence. No study-induced differences were
Table 2 Counts of mutans streptococci and lactobacilli (log CFU/
ml saliva) at baseline and 4 weeks
after the use of the LGG +
BB12/control lozenge. Means ±
SD given
Our results demonstrated a LGG-BB-12-induced decrease in
the plaque index, which promoted a decrease in the gingival
index. To our knowledge, this is the first study to demonstrate
such an effect on gingival health of this probiotic combination.
Importantly, the result was obtained in healthy subjects.
Most studies present the suggestion that probiotics could
control plaque by decreasing periodontopathogens or MS [24,
27]. Recently, L. reuteri was shown to decrease counts of
selected periodontopathogens both with and without a clinical
impact [40, 41]. No studies have connected LGG and/or BB12 consumption with decreases in counts of
periodontopathogens. Our subjects possessed a microbiota
typical for healthy subjects [42]. The subjects did not harbor
any major periodontopathogens at baseline; thus, a decrease in
PI and GI could not be associated to these microorganisms.
In addition to the HOMIM analyses of the microbiota, we
also cultured the saliva samples since a multitude of reference
data exist for plate-cultured MS and lactobacilli (LB). No
study-induced effects on salivary MS counts were found in
the probiotic group. Earlier studies detected decreases or no
changes in MS in association with LGG consumption [19, 28,
29]. BB-12 consumption has been associated with decreases
in MS counts [27], but this effect could not be detected in our
study. The method we used to culture the LB could detect
salivary LB as well as the LGG of the probiotic product, but
not BB-12. The consumption of the combination of LGG and
BB-12 had no effect on the salivary LB counts. As for LGG,
the result is in agreement with our earlier study [28]. Clinical
studies have shown for both LGG [15, 17] and BB-12 [17, 18]
poor retention to the oral cavity. Thus, even though we did not
determine BB-12, it should not have been present in the saliva
Mutans streptococci
Lactobacilli
Group
Baseline
4 weeks
p
Baseline
4 weeks
p
Control (n=31)
LGG + BB-12 (n=29)
4.22±1.77
4.63±0.69
4.42±1.61
4.55±0.91
n.s.
n.s.
1.74±2.04
2.14±2.18
2.47±2.09
2.50±2.24
n.s.
n.s.
Clin Oral Invest
samples. The negative result of the HOMIM supports this
consideration.
The sugars of hydrolysed plaque reflect the polysaccharide
content of plaque and the proteins the amount of microbes in
the plaque. Polysaccharides of plaque have been used as
indicators of the virulence of plaque, a.o. adhesivity [36,
37]. In our study, the polysaccharide content of the plaque
did not change. This suggests that the decreases found for PI
and GI in the probiotic group were not associated with changes in the polysaccharide contents of the plaques.
Immune modulation of the host could be a possible explanation for the improved periodontal status in the probiotic group,
since no change in the oral microbiota or the adhesion properties
of plaque could be demonstrated. Earlier, consumption of
L. reuteri reduced pro-inflammatory cytokines in the crevicular
fluid of adults with gingival inflammation [43]. This finding may
reflect a local effect oral immune responses, since L. reuteri is
effective in treating diarrhea but does not improve the immune
response of the host [2]. However, LGG and BB-12 have even in
short-term use shown beneficial effects on the immune responses
of children and adults [44–46], which should be reflected thus
also in the composition of the crevicular fluid. LGG and B. lactis
were both evaluated in a consensus opinion to have the effectiveness of grade A in improving the immune response of the
host [2]. The combination of LGG and BB-12 appears to be even
more effective in this respect compared to LGG or BB-12 alone
[6–8].
Taken together, the combination of LGG and BB-12 improved periodontal health in healthy subjects, without affecting the composition of the oral microbiota or adhesion properties of plaque.
Acknowledgments The excellent technical assistance of biomedical
research technician Oona Hällfors is gratefully acknowledged. The
STABPRO project (funded by TEKES, Finland), Finnish Dental Society
Apollonia and Turku University Foundation are acknowledged for providing funding for the study.
Conflict of interest The authors declare that they have no conflicts of
interest.
Open Access This article is distributed under the terms of the Creative
Commons Attribution License which permits any use, distribution, and
reproduction in any medium, provided the original author(s) and the
source are credited.
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