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Beneficial Microbes, 2018

2016;online
##(##): 1-10 ARTICLE IN PRESS P u b l i s h e r s

The effects of a probiotic milk drink on bacterial composition in the supra- and
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subgingival biofilm: a pilot study

A. Becirovic, J.F. Abdi-Dezfuli, M.F. Hansen, S.A. Lie, E.N. Vasstrand and A.I. Bolstad*

Department of Clinical Dentistry, Faculty of Medicine, University of Bergen, Årstadveien 19, 5009 Bergen, Norway;
anne.bolstad@uib.no

Received: 24 January 2018 / Accepted: 26 May 2018

© 2018 Wageningen Academic Publishers

RESEARCH ARTICLE
Abstract

Probiotics can convert a dysbiotic bacterial environment into a healthy one. The aim of the present study was to
assess the effect of daily intake of a probiotic milk drink on the composition of bacterial species in dental supra- and
subgingival biofilms. Sixteen dental students were enrolled into this study with a crossover, within subject, design. The
participants were asked to allow plaque accumulation by refraining from cleaning their molars during two separate
periods, each lasting three weeks. Each period consisted of an initial professional dental cleaning procedure done
at the university clinic, then a 3 week plaque accumulation period, followed by a return to the clinic for supra- and
subgingival plaque sampling. The first period served as a control, and during the second plaque accumulation period
the participants drank 200 ml probiotic milk beverage each day. The accumulated plaque removed at the end of the
accumulation period was later tested against a panel of 20 oral bacterial species using the checkerboard method.
Three weeks consumption of a probiotic beverage led to a significant reduction in 15 of 20 bacterial species present
in supragingival plaque and a reduction in 4 of 20 bacterial species in subgingival plaque (all P<0.05). This study
showed a favorable effect of probiotics on periodontopathic bacteria in dental biofilms. The potential influence of
this kind of probiotic in prevention or treatment of periodontal inflammation deserves further study.

Keywords: probiotics, biofilm, periodontitis, Lactobacillus, Bifidobacterium

1. Introduction (Utter et al., 2016). Dental and periodontal diseases are

Biofilms on teeth (dental plaque) harbour a very diverse
collection of oral microorganisms. Immediately after
eruption or cleaning, the tooth surfaces become coated
with biologically active proteins and glycoproteins, derived
mainly from saliva or from gingival crevicular fluid (GCF).
Only a few bacterial species are able to adhere to this
aquired pellicle, mainly the early colonising streptococci
(Marsh, 2010b). At the time that the metabolism makes the
environment more anaerobic, secondary colonisers adhere
and the biofilm becomes more complex by interspecies
coaggregation (Mark Welch et al., 2016).
Periodontal inflammation is initiated by microorganisms
in dental biofilm (Loe et al., 1965; Socransky and Haffajee,
2005; Theilade et al., 1966). Oral microbiota in dental
biofilm in healthy individuals are quite stable over time
considered to be initiated by a detrimental shift in the
balance of the normally stable resident oral microbiome
(Rosier et al., 2018).
The traditional treatment of periodontitis is thorough
supra- and subgingival mechanical debridement of the
teeth, combined with periodontal surgery when indicated.
In some cases antibiotics are implemented as a supplement
in treatment of serious cases, or in patients with refractory
disease. In such situations, a combination of the broad-
spectrum antibiotics amoxicillin and metronidazole are
typically prescribed (Cionca et al., 2009; McGowan et al.,
2018; Van Winkelhoff et al., 1989). However, as antibiotic
resistance is increasing worldwide and has become a
major global health problem, a very restrictive approach in
prescribing antibiotics has been recommended (Bronzwaer
et al., 2004; Cionca, 2017; World Health Organisation,

ISSN 1876-2883 print, ISSN 1876-2891 online, DOI 10.3920/BM2018.0009 1

 A. Becirovic et al.
2012). In consequence, there is a need for new drugs or
other effective treatment alternatives.
The Nobel laureate Élie Metchnikoff argued that as bacteria
are dependent on food, it should be possible to modify
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the microflora in our bodies, and to exchange harmful
bacteria for the more helpful bacteria found in yoghurt
(Metchnikoff, 1910). Such beneficial microorganisms
were termed probiotics (Lilly and Stillwell, 1965). World
Health Organisation (WHO) has the following definition for
probiotics: ‘live microorganisms, which when administered
in adequate amounts, confer a health benefit on the host
(Araya et al., 2002). Probiotic bacteria may alter the
microbiological composition of the mouth by outcompeting
pathogenic organisms, interacting with their virulence
factors, mitigating host immune response and possibly
decrease periodontal inflammation (Rosier et al., 2018).
The effects of probiotics on cariogenic and periodontitis
associated bacteria have been studied both in vitro (Baca-
Castanon et al., 2015; Ince et al., 2015; Nissen et al., 2014;
Teanpaisan et al., 2011; Wu et al., 2015; Zhao et al., 2012),
and in vivo with probiotics administered as tablets (Laleman
et al., 2015; Mayanagi et al., 2009; Nishihara et al., 2014;
Shimauchi et al., 2008; Tekce et al., 2015; Teughels et al.,
2013; Toiviainen et al., 2015), sachets (Morales et al., 2016),
or milk drink (Staab et al., 2009).
The most common probiotics consist of lactic acid
producing bacteria, such as Lactobacillus, which can
produce different antimicrobial components, including
organic acids, hydrogen peroxide, bacteriocins, and
adhesion inhibitors (Pangsomboon et al., 2006; Silva et al.,
1987), and Bifidobacterium species (Matsubara et al., 2016).
The hypothesis of this study was that a milk drink containing
Lactobacillus spp. and Bifidium species would affect dental
biofilm in vivo. The aim of this pilot study was therefore to
determine if intake of a probiotic milk drink had an effect
on the composition of microbiota in supra- and subgingival
plaque in a group of healthy volunteers.
Plaque sampling
Polishing Polishing
Plaque-accumulation Healing; wash-out
3 weeks 5 weeks
T01 T1
Figure 1. Time line for the experimental part of the study.
2
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2. Materials and methods
Study population
First-year dental students at the University of Bergen,
Bergen, Norway, were recruited for this pilot study.
Seventeen students volunteered to participate after having
received thorough oral and written information about the
project and signed informed consent. The project was
approved by the Regional Committee for Medical and
Health Research Ethics in Western Norway (REC-West
2014/62), and was conducted according to the guidelines
of Helsinki. There was one drop-out after the first visit (for
personal reasons) and 16 students completed the study.
Inclusion criteria were healthy dental students without
systemic diseases, no drug consumption, minor or no caries
experience, and no periodontal disease. Exclusion criteria
were systemic diseases, untreated caries or periodontitis,
pregnancy, or having used antibiotics within the last 6
months. All students who volunteered for the study fulfilled
these criteria.
Study design
The project had a cross-over, within-subject design, where
the volunteers acted as both test and control individuals.
Mean age of this convenience sample was 21.3±2.2 years,
and 87.5% were women. All volunteers had excellent oral
hygiene and no gingivitis. All sampled sites had probing
pocket depth ≤3 mm.
The time line for this research project is shown in Figure 1.
At the first visit (T01) the dental students were examined
to ensure that they fulfilled the inclusion criteria. Then
thorough professional tooth cleaning and polishing were
performed with curettes and pumice in rubber cups to
remove plaque on all teeth in each individual. Over the
next 3 weeks the participants restrained from all dental
and interdental cleaning of molars in the upper and lower
jaw. Being dental students, they were all very aware of
which teeth not to brush during the periods, and followed
up very well. After 3 weeks (T1) the students returned to
the dental clinic for bacterial sampling of the supra- and
subgingival palatal and lingual surfaces of the 1st and 2nd
molars, followed by professional tooth cleaning. A wash-
out period of 5 weeks followed, where the participants
Plaque sampling
Polishing Polishing
Probiotics
Plaque-accumulation
3 weeks Time
T02 T2
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performed optimal dental and interdental cleaning with
toothbrush and dental floss on all teeth twice a day. After
this intermediate reset period, the participants again came
to the clinic for cleaning and polishing (T02). Thereafter
they again refrained from cleaning the molars for 3 weeks,
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during which time they drank 200 ml of probiotic milk
drink as their last meal each evening. This time point was
chosen to obtain as similar conditions as possible between
the participants, and to avoid interference from rinsing or
eating directly after intake of the milk drink. After these 3
weeks (time point T2), plaque was again sampled supra-
and subgingivally from the molars, after which the teeth
were again properly cleaned.
The supragingival plaque was sampled by passing a curette
along the supragingival lingual/palatal tooth surfaces of the
1st and 2nd molars. The subgingival plaque was sampled
similarly, except for placing the curette subgingivally to
the bottom of the periodontal pocket. In total, four tubes
were sent for checkerboard analyses per individual; one
supragingival and one subgingival sample at time T1 and
at time T2.
The supra- and subgingival plaque samples collected at
time T1 and T2, in total 64 samples, were transferred
immediately after sampling into a pre-reduced sterile
transport medium (PRAS Dental Transport Medium,
Morgan Hill, CA, USA), and sent to Microbiological
Diagnostic Services, Department of Oral Biology, Faculty
of Dentistry, University of Oslo, Norway, for DNA-DNA
checkerboard analysis against a panel of 20 different oral
bacterial species (specified in the tables) (Socransky et
al., 1994, 2004). The personnel performing the bacterial
identification were blinded to the origin of the samples.
The probiotic drink (BIOLA, Tine® Meieriet Bergen,
Bergen, Norway) consisted of skimmed milk, 5% sugar,
8% blueberries juice, 3% blackcurrants juice, 1% raspberries
juice, yoghurt culture, 200×109 bacteria of each of the three
species Lactobacillus acidophilus La-5, Bifidobacterium
Bb-12 (Bifidobacterium animalis subsp. lactis) and
Lactobacillus rhamnosus GG per l milk drink (40×109 of
each probiotic bacteria per 200 ml daily dose), in addition
to aroma, pectin, potassium sorbate and vitamin D.
The following comparisons were done: comparison between
bacterial concentration in supragingival plaque with and
without consumption of probiotics, between bacterial
concentration in subgingival plaque with and without
consumption of probiotics, between bacterial concentration
in supra- and subgingival plaque without consumption of
probiotics, and between bacterial concentration in supra-
and subgingival plaque with consumption of probiotics.
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Probiotic milk drink changes the dental biofilm
Statistics
The results are presented in tables as median (with lower
and upper quartile shown) for the two test-time points,
with P-value for difference in distribution. The figures
give a visual presentation of the bacterial concentrations.
The concentrations of the different bacteria were coded as
0, <105, 105, between 105 and 106, 106, or >106 cells. Due
to skewed concentrations, the non-parametric Wilcoxon
matched pair sign rank test was used. The statistic software
package STATA (version IC 13.1; StataCorp LLC, College
Station, TX, USA) was used for analyses, and P-values less
than 0.05 was regarded as statistically significant.
3. Results
From the statistical analyses it is clear that the concentration
of most of the tested bacteria was reduced in supragingival
plaque by use of the probiotic milk drink, and for 15 of the
20 species the difference reached statistical significance
(Table 1 columns A/B, Figure 2A and 2B). Bacteria
distinctive to supragingival flora as well as bacteria
considered as periodontal pathogens were found in lower
concentrations after 3 weeks plaque accumulation with
consumption of probiotics than in plaque accumulated
without use of probiotics (Table 1). Examples are
Aggregatibacter actinomycetemcomitans, Campylobacter
rectus, Fusobacterium nucleatum ssp. nucleatum, Prevotella
intermedia, Porphyromonas gingivalis, Parvimonas
micra, Treponema denticola and Tannerella forsythia.
The concentrations of the supragingival bacterial species
without and with intake of probiotics are better illustrated
in Figure 2A and 2B, respectively. A strong shift to the left
towards fewer microorganisms can be observed for almost
all bacteria.
The influence of probiotics on bacteria in subgingival
plaque was less than in supragingival plaque (Table 1). In
subgingival plaque only 4 of 20 bacteria showed statistically
significant reduction (Table 1 columns C/D, Figure 3A and
3B). However, among those reduced were F. nucleatum ssp.
nucleatum, P. intermedia and Treponema socranskii ssp.
socranskii which are considered as periodontal pathogens.
When analysing species in subgingival plaque before and
after use of probiotics, the tendency to shift to the left was
much less than what was found in supragingival plaque
(Figure 3A and 3B compared with Figure 2A and 2B).
When comparing the presence of bacterial species in
supragingival plaque with their presence in subgingival
plaque, several significant differences were detected.
Most of these differences appeared when comparing
supra- and subgingival plaque without use of probiotics
(Table 1 columns A/C). All bacteria were present in higher
concentrations in supragingival than in subgingival plaque
without use of probiotics, with 17 of 20 showing significant
Please cite this article as 'in press'3
 A. Becirovic et al.

Table 1. Bacterial concentration in supra- and subgingival plaque before and after consumption of probiotics, and P-values for
the respective comparisons.1

Bacterial species Supragingival plaque Subgingival plaque P-value

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– probiotics + probiotics – probiotics + probiotics A/B C/D A/C B/D

A B C D

Aggregatibacter 1.5 (0.5, 2.0) 1.0 (0.0, 1.0) 0.0 (0.0, 0.0) 0.0 (0.0, 0.0) 0.0180 n.s 0.0009 0.0027
actinomycetemcomitans
Actinomyces gerencseriae 3.0 (1.5, 4.5) 2.0 (2.0, 3.0) 1.0 (1.0, 1.0) 1.0 (1.0, 2.0) n.s n.s 0.0032 0.0039
Actinomyces israelii 1.0 (0.0, 3.0) 0.0 (0.0, 0.0) 0.0 (0.0, 0.0) 0.0 (0.0, 0.0) 0.0053 n.s 0.0053 n.s
Actinomyces viscosus 2.5 (1.5, 4.0) 1.5 (0.5, 2.0) 1.0 (1.0, 1.0) 0.0 (0.0, 0.5) 0.0023 0.0039 0.0009 0.0013
Campylobacter rectus 2.0 (1.0, 3.0) 1.0 (0.0, 1.0) 0.0 (0.0, 0.0) 0.0 (0.0, 0.0) 0.0030 n.s 0.0007 n.s
Eikenella corrodens 2.0 (1.5, 3.0) 1.0 (1.0, 2.0) 1.0 (1.0, 1.0) 1.0 (1.0, 1.0) 0.0132 n.s 0.0089 n.s
Eubacterium saburreum 2.0 (1.0, 3.0) 0.0 (0.0, 0.5) 0.0 (0.0, 0.5) 0.0 (0.0, 0.5) 0.0032 n.s 0.0009 n.s
Fusobacterium nucleatum ssp. 2.0 (1.0, 3.0) 0.5 (0.0, 1.0) 1.0 (1.0, 1.0) 0.0 (0.0, 0.0) 0.0013 0.0045 0.0112 0.0446
nucleatum
F. nucleatum ssp. 1.5 (1.0, 3.0) 1.0 (1.0, 2.0) 1.0 (0.0, 1.0) 1.0 (0.0, 1.0) n.s n.s 0.0394 0.0432
polymorphum
F. nucleatum ssp. vincentii 1.0 (0.0, 2.0) 1.0 (0.0, 2.0) 0.0 (0.0, 1.0) 1.0 (0.0, 1.0) n.s n.s n.s n.s
Porphyromonas endodontalis 2.0 (0.0, 2.5) 0.0 (0.0, 0.0) 0.0 (0.0, 0.0) 0.0 (0.0, 0.0) 0.0020 n.s 0.0042 n.s
Porphyromonas gingivalis 3.0 (1.0, 3.0) 0.0 (0.0, 1.0) 0.0 (0.0, 2.0) 0.0 (0.0, 0.0) 0.0218 n.s 0.0082 n.s
Prevotella intermedia 2.5 (1.0, 3.5) 0.0 (0.0, 0.0) 2.0 (1.0, 2.0) 0.0 (0.0, 1.0) 0.0023 0.0011 n.s n.s
Prevotella melaninogenica 0.5 (0.0, 2.0) 0.0 (0.0, 0.5) 0.0 (0.0, 0.0) 0.0 (0.0, 0.0) n.s. n.s 0.0133 n.s
Parvimonas micra 1.0 (0.0, 2.0) 0.0 (0.0, 0.0) 0.0 (0.0, 0.0) 0.0 (0.0, 0.0) 0.0033 n.s 0.0080 n.s
Prevotella nigrescens 2.0 (0.0, 2.0) 0.0 (0.0, 0.0) 0.0 (0.0, 0.0) 0.5 (0.0, 1.0) 0.0291 n.s 0.0312 n.s
Streptococcus intermedius 3.0 (1.0, 3.0) 1.0 (0.0, 2.5) 1.0 (0.0, 1.0) 1.0 (0.5, 2.0) 0.0346 n.s 0.0007 n.s
Treponema. denticola 2.0 (1.0, 3.0) 1.0 (1.0, 1.0) 1.0 (0.0, 1.0) 1.0 (1.0, 1.0) 0.0058 n.s 0.0009 n.s
Tannerella forsythia 1.0 (1.0, 2.0) 1.0 (0.0, 1.0) 1.0 (0.0, 1.0) 0.0 (0.0, 1.0) 0.0064 n.s 0.0471 n.s
Treponema socranskii ssp. 1.5 (0.5, 2.5) 1.0 (0.0, 2.5) 0.0 (0.0, 1.0) 1.0 (0.5, 1.5) n.s 0.0247 0.0099 n.s
socranskii

1 The results are presented as median (with lower and upper quartile) for the two test-time points, with P-values for difference in distribution. P<0.05 was

regarded as statistical significant. n.s. = not significant. Score 1 means <105 cells, score 2 means ~105 cells, score 3 means between 105 and 106 cells,
score 4 means ~106 cells, and score 5 means >106 cells.

differences, and most of the values from the comparison
were highly significant.
After use of probiotics, the differences between the species
in supragingival plaque compared to subgingival plaque
(Table 1 columns B/D) were only significant for 5 of 20
species, including A. actinomycetemcomitans, F. nucleatum
ssp. nucleatum and F. nucleatum ssp. polymorphum.
4. Discussion and conclusions
In this study we have shown that daily intake of a readily
available probiotic milk drink containing L. acidophilus
La-5, Bifidobacterium Bb-12 and L. rhamnosus GG
for three weeks reduced the concentration of bacteria
in supragingival plaque, and also to a lesser degree in
subgingival plaque. Although reduced in number, most
species were not totally eradicated by use of the probiotic
containing milk drink. However, this may be an advantage,
because to be a successful antimicrobial agent, the aim is
not to eradicate all bacteria in the biofilm, but to maintain
an oral biofilm at levels compatible with oral health without
loosing the natural and beneficial properties of the resident
oral microflora (Marsh, 2010a).
Of the more than 700 different bacterial species in the
oral cavity, about 10-15 of the species identified in dental
biofilm have been highly associated with periodontal disease
and considered as periodontal pathogens (Socransky and
Haffajee, 2005; Ximenez-Fyvie et al., 2000a,b). These were
among the bacteria analysed with checkerboard in the
present study.

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 Probiotic milk drink changes the dental biofilm

A
A. actinomycetemcomitans A. gerencseriae A. israelii A. viscosus C. rectus
100
80
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60
40
20
0
E. corrodens E. saburreum F. nucleatum F. polymorphum F. vincentii
100
80
60
40
20
Percentage (%)

0
P. endodontalis P. gingivalis P. intermedia P. melaninogenica P. micra
100
80
60
40
20
0
P. nigrescens S. intermedius T. denticola T. forsythia T. socranskii
100
80
60
40
20
0

0 <105 ~105 105<X<106 ~106 >106

B
A. actinomycetemcomitans A. gerencseriae A. israelii A. viscosus C. rectus
100
80
60
40
20
0
E. corrodens E. saburreum F. nucleatum F. polymorphum F. vincentii
100
80
60
40
20
Percentage (%)

0
P. endodontalis P. gingivalis P. intermedia P. melaninogenica P. micra
100
80
60
40
20
0
P. nigrescens S. intermedius T. denticola T. forsythia T. socranskii
100
80
60
40
20
0

Figure 2. Distribution of bacterial cells at different concentrations in supragingival plaque of the test persons illustrated by
colour code along the x-axis. (A) Without consumption of probiotics. (B) After consumption of probiotics. The y-axis indicates
the percentage of individuals.

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 A. Becirovic et al.

A
A. actinomycetemcomitans A. gerencseriae A. israelii A. viscosus C. rectus
100
80
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60
40
20
0
E. corrodens E. saburreum F. nucleatum F. polymorphum F. vincentii
100
80
60
40
20
Percentage (%)

0
P. endodontalis P. gingivalis P. intermedia P. melaninogenica P. micra
100
80
60
40
20
0
P. nigrescens S. intermedius T. denticola T. forsythia T. socranskii
100
80
60
40
20
0

0 <105 ~105 105<X<106 ~106 >106

B
A. actinomycetemcomitans A. gerencseriae A. israelii A. viscosus C. rectus
100
80
60
40
20
0
E. corrodens E. saburreum F. nucleatum F. polymorphum F. vincentii
100
80
60
40
20
Percentage (%)

0
P. endodontalis P. gingivalis P. intermedia P. melaninogenica P. micra
100
80
60
40
20
0
P. nigrescens S. intermedius T. denticola T. forsythia T. socranskii
100
80
60
40
20
0

Figure 3. Distribution of bacterial cells at different concentrations in subgingival plaque in the test persons illustrated by colour
code along the x-axis. (A) Without consumption of probiotics. (B) After consumption of probiotics. The y-axis indicates the
percentage of individuals.

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The most commonly used probiotics in the dental and
periodontal field belong to the genera Lactobacillus and
Bifidobacterium (Haukioja, 2010; Matsubara et al., 2016),
both regarded as natural habitants of the human microbiota
(Hojo et al., 2007). Because bifidobacteria are strict
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anaerobes and rather slow growers, the feasibility of their
use in probiotics previously has been questioned (Meurman,
2005). However, later on it was shown in a RCT, that the
use of probiotic yoghurt supplemented with B. animalis
subsp. lactis resulted in lower plaque and gingivitis scores,
less bleeding on probing (BoP), decreased GCF volume and
interleukin 1β in GCF (Kuru et al., 2017).
Oral lozenges containing L. rhamnosus GG and B. animalis
subsp. lactis BB12 were reported to decrease both plaque
index and gingival inflammation in a group of healthy
volunteers (Toiviainen et al., 2015). The milk drink used
in our study contained these two bacteria in addition to L.
acidophilus La-5. L. rhamnosus GG, initially isolated from
the human intestine, is the most widely studied probiotic
bacterium. It produces a substance with potential inhibitory
effect on different species, a.o. Streptococcus spp. (Silva et
al., 1987), which are the important first colonisers of the
dental biofilm (plaque). Eight of 13 Lactobacillus species
showed competent biofilm degradation of ≥90% reduction
in biofilm values of A. actinomycetemcomitans species in
vitro (Jaffar et al., 2016), among them were Lactobacillus
casei subsp. rhamnosus and L. acidophilus. Furthermore,
L. salivarius and L. gasseri were shown to down regulate
A. actinomycetemcomitans exotoxin expression (Nissen
et al., 2014). Based on an in vitro study, it was suggested
that biofilm degradation of A. actinomycetemcomitans
by Lactobacillus spp. was probably not due to the lactic
acid and low pH condition, but rather due to the enzymes;
proteases, lipase and amylase (Jaffar et al., 2016).
In a 1 year follow-up study of chronic periodontitis patients
treated non-surgically, oral administration of L. rhamnosus
SP-1 containing sachets once a day for three months
combined with scaling and root planning (SRP) resulted
in significant reductions in the number of patients with
probing depth ≥6 mm in the test group compared to SRP
alone, thereby decreasing the need for a surgical phase after
initial therapy (Morales et al., 2016). Even larger reduction
in pocket depths were reported in RCTs when Lactobacillus
reuteri were used (Teughels et al., 2013; Vivekananda et
al., 2010). L. reuteri had also an inhibitory effect against T.
forsythia in vitro (Baca-Castanon et al., 2015). In clinical
studies, L. reuteri has been found to induce significant
reduction in pro-inflammatory cytokine response and
improvement of clinical parameters in chronic periodontitis
patients (Szkaradkiewicz et al., 2014), in addition to reduce
plaque amount and gingival inflammation (Krasse et al.,
2006). Oral administration of L. salivarius WB21 in tablets
for 8 weeks reduced the number of periodontopathic
bacteria in subgingival plaque from healthy individuals,
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Probiotic milk drink changes the dental biofilm
especially T. forsythia (Mayanagi et al., 2009), whereas oral
tablets of Lactobacillus brevis, Lactobacillus plantarum and
Pediococcus acidilactici did not lead to significant changes
in treatment of gingivitis (Montero et al., 2017).
An important issue is how long the probiotic bacteria
can survive in the mouth. This may influence the
usefulness of probiotics to improve oral conditions. An
in vitro study tested the ability of 17 Lactobacillus strains
and 7 Bifidobacterium strains to bind to saliva coated
hydroxyapatite, and a great variation in binding patterns
among the different strains was reported, which may
indicate a variation in persistence also in vivo (Haukioja
et al., 2006).
The bacterial growth conditions are very different
supragingivally compared to subgingivally, with access
to oxygen and saliva supragingivally, compared to the
anaerobic conditions and GCF in the periodontal pocket.
The difference in bacterial concentration between supra-
and subgingival plaque bacteria in the present study was
highly significant for almost all examined bacteria before
use of probiotics, which is in line with previous reports
(Socransky and Haffajee, 2005). However, this was not the
case after use of probiotics. Then only a few bacteria were
found in statistically significant different concentrations
supra- compared to subgingivally, mostly due to the
change in bacterial concentration supragingivally. This
may reflect that probiotics distributed as a liquid drink has
better accessibility to the supragingival plaque than to the
subgingival plaque in the periodontal pocket. It is possible
that a longer experimental period would have influenced
on the subgingival biofilm composition.
Some experimental animal studies are available. Probiotic
therapy with B. animalis subsp. lactis on ligature-induced
experimental periodontitis in rats showed benefits in
reduction or protection of alveolar bone loss (Oliveira
et al., 2017; Ricoldi et al., 2017). In addition, oral
administration of L. gasseri was found to be effective in
preventing P. gingivalis-accelerated periodontal disease
in mice (Kobayashi et al., 2017). Elegantly, a recombinant
form of L. acidophilus expressing the immunogenic FomA
protein was constructed, and oral administration of the
recombinant L. acidophilus reduced the risk of periodontal
infection with P. gingivalis and F. nucleatum in a mice model
(Ma et al., 2013).
The milk drink used in the present study contained 8%
blueberries juice, 3% blackcurrants juice and 1% raspberries
juice. An additive effect of the berry extracts in the juice
cannot be excluded. RCTs on the effect of berry extracts
on periodontal health are to our knowledge lacking. A few
in vitro studies have been reported on the effect of berry
fractions on oral bacteria or biofilms, some of which studied
cranberries (Duarte et al., 2006; Lagha et al., 2018; Riihinen
Please cite this article as 'in press'7
 A. Becirovic et al.
et al., 2011; Yamanaka et al., 2007). It is possible that the
polyphenols in the berry extracts may be involved in non-
specific antiadhesion effects against dental and periodontal
bacteria such as Streptococcus mutans, F. nucleatum, A.
actinomycetemcomitans and P. gingivalis (Duarte et al.,
${protocol}://www.wageningenacademic.com/doi/pdf/10.3920/BM2018.0009 - Thursday, July 26, 2018 5:58:56 AM - Gothenburg University Library IP Address:130.241.16.16
2006; Yamanaka et al., 2007). In the future, it is a need for
in vivo studies comparing the separate effect of probiotics
and berry extracts. However, in our study, intake of milk
and juice during the day was allowed, and to measure any
additional effect of intake of the probiotic containing milk
drink product on bacterial composition in plaque was the
aim of this study.
From systematic reviews and meta-analyses it has been
deduced that in general, oral administration of probiotics
in humans improved the clinical signs of chronic and
aggressive periodontitis, such as probing pocket depth,
BoP and attachment loss, together with a reduction in
levels of major periodontal pathogens (Gruner et al., 2016;
Martin-Cabezas et al., 2016; Matsubara et al., 2016; Polak et
al., 2015). However, whether probiotics may interfere with
the relatively stable dental biofilm of sufficient degree to be
a valuable tool for caries or periodontal disease prevention
or treatment, is not confirmed.
To conclude, although the microbiota constituting the
biofilm on teeth is regarded as rather stable, three weeks of
daily intake of a probiotic milkdrink significantly reduced
the concentration of bacteria in supragingival plaque, and
to a lesser degree also in subgingival plaque. In view of the
increasing problem with antibiotic resistance and ease of
providing probiotics, their potential to influence dental
plaque composition in a positive way requires further
studies.
Conflict of interest
The authors have no conflict of interest to declare.
Acknowledgements
Funding from the University of Bergen is appreciated. We
thank Morten Enersen for organising the checkerboard
analysis at Microbiological Diagnostic Services, Department
of Oral Biology, Faculty of Dentistry, University of Oslo,
Norway. The language editing by Michele Cottler-Fox, MD,
is highly appreciated.
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