367
Journal of Oral Science, Vol. 57, No. 4, 367-372, 2015
Original
Effects of different antibacterial agents on enamel
in a biofilm caries model
Selcuk Savas1), Ebru Kucukyılmaz1), Esra U. Celik2), and Mustafa Ates3)
1)Department
of Pediatric Dentistry, Faculty of Dentistry, Izmir Katip Celebi University, Izmir, Turkey
of Restorative Dentistry, Faculty of Dentistry, Izmir Katip Celebi University, Izmir, Turkey
3)Department of Biology, Fundamental and Industrial Microbiology Division, Faculty of Science,
Ege University, Izmir, Turkey
2)Department
(Received June 26, 2015; Accepted October 10, 2015)
Abstract: Using a mature biofilm model, the aim of
this study was to evaluate the effectiveness of different
antibacterial agents in comparison with silver diamine
fluoride (SDF). Forty-eight saliva-coated enamel slabs
were inoculated with Streptococcus mutans monospecies biofilm. The biofilms were then exposed to 10%
sucrose in tryptone yeast-extract culture medium,
8 times per day for 7 days. After the biofilm growth
period, the enamel slabs were treated with one of the
following substances: 1) distilled water; 2) SDF; 3)
acidulated phosphate fluoride (APF); 4) ammonium
hexafluorosilicate (AHF); 5) ammonium hexafluorosilicate + cetylpyridinium chloride (AHF+CPC); or
6) 0.2% chlorhexidine (CHX). After these treatment
procedures, the samples were incubated at 37ºC for
2 days, and the numbers of viable microorganisms
in the biofilms were counted. The number of viable
bacteria was significantly reduced by all of the antibacterial agents (P < 0.05). However, SDF showed
the highest antibacterial activity (P < 0.05), and the
effectiveness of the other agents was lower (P < 0.05).
SDF has a highly effective antibacterial action against
cariogenic Streptococcus mutans biofilm; none of the
other fluoride agents used in this study, or 0.2 CHX
agent, showed an antibacterial effect comparable to
that of SDF. (J Oral Sci 57, 367-372, 2015)
Correspondence to Dr. Selcuk Savas, Department of Pediatric
Dentistry, Faculty of Dentistry, Izmir Katip Celebi University,
Cigli, Izmir, Turkey
Fax: +90-2323252535 E-mail: selcuksavas1983@hotmail.com
doi.org/10.2334/josnusd.57.367
DN/JST.JSTAGE/josnusd/57.367
Keywords: dental caries; fluoride; oral biofilm;
Streptococcus mutans.
Introduction
The incidence of dental caries has declined in the
industrialized world and developing countries; however,
it is still the most prevalent chronic infectious disease,
especially in disadvantaged and poor populations (1-3).
Caries is a biofilm-related disease that can be identified by localized destruction of dental hard tissues by the
acidic products of oral bacteria (4,5). Because biofilm
bacteria are the driving force of demineralization and
caries development, control of dental plaque has become
an important strategy for caries prevention (6-8).
One of the most effective methods of preventing caries
progression is application of fluoride (6,9,10). The anticaries activity of fluoride-containing products can be
attributed to inhibition of the demineralization processes,
enhancement of enamel remineralization, and effects
on the biological activities of cariogenic microorganisms (6,9,10). Several different fluoride agents, such as
acidulated phosphate fluoride (APF), sodium fluoride,
stannous fluoride, and amine fluoride have been used
clinically (9-13). However, the antibacterial activity of
these materials is limited, and attempts to develop more
effective antibacterial materials are ongoing (11,14,15).
One of these materials, silver diamine fluoride (SDF), is
a safe, cost-effective, efficient, and noninvasive cariespreventive agent that has been used for treatment of
both primary and permanent teeth (16,17). It has been
hypothesized that SDF exerts an antibacterial action and
is able to reduce enamel surface mineral loss and increase
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the enamel surface microhardness (16,17). Although a
number of studies have indicated that SDF is effective,
its clinical use is restricted due to tooth staining caused
by silver deposition (18). For this esthetic reason, the
use of ammonium hexafluorosilicate (AHF) solution,
which replaces the silver component with silica, has
been suggested for prevention of caries progression (19).
Several studies have indicated that AHF treatment does
not produce tooth staining; upon application, a large
amount of precipitate forms on the tooth surface, and the
acidity of the solution causes etching of the hard tissues
of the tooth and re-precipitation of calcium and phosphate ions as silica-calcium-phosphate crystals (18-21).
It has been emphasized that the antibacterial activity of
AHF solution may be increased when it is used along
with several antibacterial agents, such as cetylpyridinium
chloride. However, only a limited number of studies have
investigated the antibacterial activity of AHF, and its
effects on dental biofilms are unclear (18).
Biofilm models are important tools for evaluating the
biochemical and microbiological composition of biofilms
formed under different conditions (6,14). Biofilm models
make it possible to create standardize procedures for
comparing the effectiveness of different materials. To our
knowledge, many of the previous studies have investigated the antibacterial activity of SDF solution, but none
have evaluated the effectiveness of different antibacterial
agents on mature dental biofilm in comparison with SDF.
Therefore, the aim of this in vitro study was to evaluate
the effects of various antibacterial agents in a validated
and tested S. mutans biofilm caries model simulating
highly cariogenic conditions on enamel. The null hypothesis was that there would be no statistically significant
differences among the agents tested.
Materials and Methods
Sample preparation
Forty-eight enamel blocks were prepared from sound,
caries-free bovine incisors. The roots of the teeth were
removed by sectioning approximately 2 mm below the
cement-enamel junction and perpendicular to the long
axis, using a water-cooled diamond disk. The buccal
surface of the crown was polished and flattened using
600, 800, and 1,200 sanding paper under water. Fortyeight standardized enamel specimens (7 × 4 × 1 mm)
were prepared from the buccal surfaces of the teeth.
After preparation, the specimens were divided into six
groups of eight enamel slabs each, according to the test
agents employed. The tooth specimens were suspended
in the wells of two 24-well culture plates and sterilized
with ethylene oxide. Then, the slabs were immersed in
Fig. 1 Culture medium pH during the experimental biofilm
growth period.
filtered, pooled human saliva and agitated (60 rpm for 30
min at 37ºC) to simulate pellicle formation.
The present study was approved by the Medical Ethics
Committee of Izmir Katip Celebi University, under report
no. 2013/204.
Biofilm growth
Ultrafiltered tryptone–yeast extract broth (UTYEB) was
used as the culture medium. S. mutans UA159 (American
Type Culture Collection 700610, Rockville, MD, USA)
colonies were transferred to the UTYEB, containing
1% glucose, and incubated at 37ºC under 10% CO2 to
reactivate the microorganisms. For biofilm formation,
S. mutans UA159 was inoculated into 1% glucose
containing UTYEB and incubated (37ºC and 5% CO2)
until an optical density of 1.5 at 600 nm/mL was obtained.
The enamel samples with human salivary pellicles were
positioned individually in wells containing 2.0 mL of the
inoculum and incubated at 37ºC under 10% CO2 to allow
bacterial adhesion. The next day, the enamel slabs with
biofilms were transferred to fresh UTYEB containing 0.1
mM glucose and exposed for 1 min, 8 times per day (at
8:00, 9:30, 11:00, 12:30, 14:00, 15:30, 17:00, and 18:30),
to 10% sucrose for 7 days. This 7-day period allowed the
bacteria to grow and mature in the biofilm. After each
sucrose exposure, the enamel slabs were washed 3 times
in 0.01% NaCl. To assess the acidogenicity of the biofilm,
the pH of the culture medium was recorded before each
change of medium using a microelectrode coupled to a
pH meter (inoLab pH/ION 7320; WTW GmbH, Weilheim, Germany) directly inside the wells (Fig. 1). The
culture medium was changed daily, after the first and last
sucrose exposures. On the 7th day of the experimental
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Table 1 Chemical compositions and application procedures of the tested materials
Distilled water
38% Silver diamine fluoride
(Saforide; Toyo Seiyaku Kasei Co. Ltd., Osaka, Japan)
1.23% Acidulated phosphate fluoride
(Sultan Topex APF gel, Sultan Dental Products, Englewood,
NJ, USA)
Ammonium hexafluorosilicate (0.476 mol/L)
(Sigma Aldrich, St. Louis, MO, USA)
Ammonium hexafluorosilicate (0.9 wt%) + Cetylpiridinium
chloride (0.1 wt%)
(Sigma Aldrich, St. Louis, MO, USA)
0.2% Chlorhexidine
(Klorhex, Drogsan, Ankara, Turkey)
Chemical composition
Chlorine, ammonium, sulfate, magnesium,
calcium, sodium (<2 ppm)
AgFH6N2
Application methods
Apply with a brush to all surface for 4 min
Apply with a brush to all surface for 3 min
Sodium fluoride, orthophosphoric acid
Apply with a brush to all surface for 4 min
H8F6N2Si
Apply with a brush to all surface for 3 min
H8F6N2Si + C21H38CIN-H2O
Apply with a brush to all surface for 3 min
Water, alcohol, glycerin, sodium saccharin,
chlorhexidine gluconate
Apply with a brush to all surface for 4 min
Table 2 The total viable microorganism in the biofilm and the medium pH values after treatment procedures
Total viable microorganism in the biofilm
Mean (SD)
3.94 × 108 (3.29 × 107)a
2.62 × 108 (1.72 × 107)b
0c
8
2.65 × 10 (2.04 × 107)b
2.48 × 108 (2.42 × 107)b
3.21 × 108 (3.01 × 107)d
Distilled water (control)
Acidulated phosphate fluoride
Silver diamine fluoride
Ammonium hexafluorosilicate
Ammonium hexafluorosilicate + Cetylpiridinium chloride
0.2% Chlorhexidine
*SD: Standard deviation
*Lowercase letters indicate statistically significance differences within columns (P < 0.05).
growth period, it was decided that a sufficient cariogenic
and mature biofilm layer had been obtained (22,23).
Treatment
The 48 enamel blocks were removed for treatment
procedures, and the biofilms were treated with one
of the following: 1) distilled water (control); 2) SDF
(Saforide; Toyo Seiyaku Kasei Co. Ltd., Osaka, Japan);
3) acidulated phosphate fluoride (APF) (Sultan Topex
APF gel, Sultan Dental Products, Englewood, NJ, USA);
4) ammonium hexafluorosilicate (AHF) (Sigma Aldrich,
St. Louis, MO, USA); 5) ammonium hexafluorosilicate
[0.9 wt%] + cetylpyridinium chloride [0.1 wt%] (AHF +
CPC) (Sigma Aldrich); or 6) 0.2% chlorhexidine (CHX)
(Klorhex, Drogsan, Ankara, Turkey). The chemical
compositions, manufacturers, and application procedures
for the treatment agents are presented in Table 1.
Biofilm assessment
After the treatment procedures, all of the enamel blocks
were returned to the 24-well plates and incubated for
48 h, after which they were washed three times in 0.9%
NaCl and transferred individually to sterile glass tubes
containing 9 mL of 0.9 NaCl. The tubes were sonicated
to detach the biofilms that had formed on the slabs.
To determine the total bacterial colony forming units
Medium pH
Mean (SD)
5.14 (0.29)a
5.70 (0.16)b
7.46 (0.19)c
5.56 (0.10)d
5.40 (0.15)e
5.27 (0.12)e
(CFUs), an aliquot (1 mL) of the homogenized suspension was serially diluted in 0.9 NaCl (100–108) and a
1-mL suspension from each dilution was inoculated onto
the plates, which contained brain-heart infusion agar.
To determine the number of viable microorganisms, the
plates were incubated in 10% CO2 at 37ºC for 48 h, and
then the number of CFU was counted.
Statistical analysis
All of the data were assessed for a normal distribution
using the Shapiro-Wilk test for normality. A normal
distribution of all data was verified, and values obtained
for the different treatment groups were compared by
analysis of variance followed by Tukey’s test, using
SPSS 22.0 statistical software (SPSS, Chicago, IL, USA)
(α = 0.05).
Results
The total bacteria counts and the medium pH values for
all groups are shown in Table 2. All of the antibacterial
agents significantly reduced the number of viable bacteria
(P < 0.05). Among the tested agents, SDF showed the
highest antibacterial activity (P < 0.05); after the evaluation period, the bacterial count in the S. mutans biofilm
decreased to zero. The smallest reduction in the number
of live bacteria occurred in the 0.2% CHX group (P <
370
0.05), and there were no significant differences among
the AHF, APF, and AHF+CPC groups (P > 0.05).
Discussion
The process of biofilm development involves initial
bacterial adhesion and accumulation, causing enamel
demineralization, which indicates that the incidence of
dental caries can be reduced by control of biofilm formation and accumulation (4,6,7). Streptococcus mutans is
the most cariogenic microorganism in dental biofilm due
to its acidogenic and aciduric properties, as well as its
capacity to utilize dietary carbohydrates and synthesize
extracellular polysaccharides (24,25). Although the S.
mutans biofilm model used in the present study does not
exactly simulate the complex microbial community in
the oral cavity, previous studies have indicated that the
improved model of S. mutans biofilm growth is sufficiently sensitive to show biofilm changes in the presence
of antimicrobial substances (6,14). Therefore, in this
study, the antibacterial effectiveness of different agents
was tested on the validated cariogenic S. mutans biofilm
caries model. Based on the results obtained, the null
hypothesis was rejected, since differences were observed
in the antibacterial activities of the various agents tested.
The present results revealed significant differences
between the negative control and the five treatment
groups (SDF, APF, AHF, APF+CPC, and CHX), and the
number of viable microorganisms was reduced in the
samples treated with the antibacterial agents. The differences in the observed antibacterial activities between
the control and tested material groups suggested that the
number of viable microorganisms was affected by the use
of different antibacterial agents. The highest antibacterial
activity was observed in the SDF group, and no viable
microorganisms were detected in the biofilms treated
with this agent. This result is consistent with those of
many previous studies (16,23,25). Chu et al. reported
the bacterial count decreased to zero after treatment of
mature S. mutans biofilm with SDF (22). In contrast,
confluent growth of live S. mutans and high CFU counts
were observed in the negative control group. At the end
of the experiment, pH values were found to be higher
in the SDF treatment group than in the control group.
Similarly, Mei et al. mentioned that 38% SDF inhibited
multispecies cariogenic biofilm formation on dentin
carious lesions and reduced the process of demineralization (23). In the same study, the pH value in the negative
control group was 3.5–4.0 and that in the SDF group was
6.0–6.5. The antibacterial activity of SDF is attributable
to the silver and fluoride ions in the structure of the material (16,17,23,25). SDF contains high concentrations of
silver (253,870 ppm) and fluoride (44,800 ppm) ions
(16,17). Silver ions are bactericidal metal cations that
inhibit biofilm formation by inactivating and interfering
with the bacterial synthesis of cellular polysaccharides
through inactivation of the glucosyltransferase enzymes
responsible for the synthesis of soluble and insoluble
glucan (16,17). Fluoride ions can also bind to bacterial
cell constituents and influence enzymes, such as enolase
and proton-extruding adenosine triphosphatase. High
concentrations of fluoride can inhibit biofilm formation
(16,17).
When we evaluated the antibacterial efficacy of the
other tested fluoride-containing agents, all of them were
found to have higher activity than that in the control
group. However, in comparison with SDF, their antibacterial effect against the S. mutans biofilm was lower.
Although no reported study has compared the antibacterial effect of these agents with that of SDF on a mature
biofilm caries model, the findings are consistent to those
of previous differently designed studies (14,18,25). Shah
et al. reported that SDF application reduced the saliva S.
mutans count to a greater degree than APF gel or 6% NaF
fluoride varnish (15). They found that APF and fluoride
varnish had a similar effect on microorganisms. Another
study that tested the antibacterial activities of SDF,
AHF, and AHF+CPC solutions on bacterial adhesion
found that SDF was the most effective agent, while AHF
had limited efficacy (18). However, it was emphasized
that addition of CPC to the AHF solution significantly
increased the antibacterial activity of the agent, and
that the amount of bacterial adhesion was significantly
decreased to the same level as that with SDF. CPC is
a quaternary ammonium salt of the pyridinium group
and bears a positive charge. CPC exerts its antibacterial
action by combining with negatively charged proteins
on bacterial cells and destroying their cell membrane by
disturbing the electrical balance (18). In that study, the
antibacterial agents were applied just before seeding of
bacteria on hydroxyapatite pellets, allowing only 12 h
for plaque accumulation. It is thought that, unlike this
previous study, the lack of difference between the AHF
and AHF+CPC groups in the present study was because
the agents were unable to sufficiently penetrate the
mature biofilm within the seven-day period.
In addition to fluoride-containing materials, CHX solution, with proven antibacterial activity on oral bacteria,
was also used in the present study. Of all available
antimicrobials used in dentistry, CHX is still the agent
used most frequently to reduce plaque for the purpose
of caries control, due to its broad antimicrobial spectrum
(14,26-28). In the present study, application of CHX
371
solution to the S. mutans biofilm significantly reduced
the count of viable microorganisms in the dental biofilm.
However, its bactericidal activity was found to be significantly lower than that of the other agents tested. This low
antibacterial effect of a single application of CHX on S.
mutans biofilm is consistent with the findings of many
other published studies (26-28). Pratten et al. reported
that a five-minute application of 0.2% CHX had an insufficient ability to reduce the viability of microorganisms
in a mature biofilm (26). However, they emphasized that
in the group treated for 60 minute, a marked reduction of
bacterial viability was evident. Zaura-Arite et al. reported
that 0.2% CHX was able to affect 6-h biofilm and was
effective for inhibiting S. mutans; however, its effect
was limited to the outer layer of 48-h dental plaque (27).
Similarly, Vitkov et al. reported that application of 0.1%
CHX for 1 or 5 min had an insufficient effect on 24-h oral
biofilm (28). These consistent findings demonstrate that
at low concentrations and with short single-application
times, CHX has only a limited effect on mature dental
biofilm.
Acid production is an important virulence factor of
S. mutans biofilms that warrants attention in studies of
dental caries prevention (7). The present results showed
that all of the tested materials reduced acid production by
S. mutans biofilms during the experimental period; the
highest pH was detected in the SDF group. This reduction of acid production was closely related to reduction of
the CFU count. In addition, the differences in plaque pH
reduction among the other test agents may be explained
by their various anti-physiological effects on the biofilm
cells, as there were no differences in CFU counts among
the groups.
The present study is the first to have investigated
the antibacterial efficacy of four different fluoridecontaining topical agents and 0.2% CHX on a mature S.
mutans biofilm caries model in comparison with SDF.
SDF is known to be a highly effective topical fluoride
agent against bacterial dental biofilm, and the present
findings clearly demonstrated that none of the other
fluoride agents or 0.2% CHX showed an antibacterial
effect comparable to that of SDF. Although the present
study could not completely simulate the complex oral
environment, the results provide useful information on
the antibacterial effects of different agents.
Within the limitations of this study:
1. The present study showed that SDF is an effective
antibacterial material that can penetrate mature
biofilm, and it is suggested that it would be useful as
an anti-caries agent.
2. The lower antibacterial activity of the other fluoride-
containing materials in comparison with SDF
indicates that fluoride alone is not as effective as the
synergistic effect of fluoride and silver ions.
3. The plaque pH in the treatment groups was found to
correlate with the number of viable microorganisms
in the biofilm. This finding, along with the known
relationship between dental plaque pH and caries
formation, suggests that remineralization agents
would also have antibacterial effects.
Acknowledgments
This study received support from the Scientific and Technological
Research Council of Turkey (TUBITAK) (Project No. 213S108).
Conflict of interest
The authors have no conflicts of interest to declare.
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