Original Article

Effervescent tablets and ultrasonic devices against Candida and

mutans streptococci in denture biofilm
Ingrid Machado de Andrade1, Patricia Costa Cruz1, Claudia Helena Lovato da Silva1, Raphael
Freitas de Souza1, Helena de Freitas Oliveira Paranhos1, Regina Celia Candido2, Jose Moacyr
Marin3 and Maria Cristina Monteiro de Souza-Gugelmin2
1

Department of Dental Materials and Prosthodontics, Ribeirao Preto School of Dentistry, University of Sao Paulo, Ribeirao Preto,
Brazil;2Department of Clinical, Toxicological and Bromatological Analyses, Ribeirao Preto Faculty of Pharmaceutical Sciences, University of Sao
Paulo, Ribeirao Preto, Brazil;3Department of Morphology, Stomatology and Physiology, Ribeirao Preto School of Dentistry, University of Sao
Paulo, Ribeirao Preto, Brazil

doi: 10.1111/j.1741-2358.2010.00378.x
Effervescent tablets and ultrasonic devices against Candida and mutans streptococci in denture
biofilm
Objective: To evaluate the antimicrobial action of effervescent tablets and ultrasound on Candida spp. and
mutans streptococci from denture biofilm.
Background: It is not uncommon for edentulous patients to be elderly and find it difficult to brush their
dentures. Hence, auxiliary methods are required for cleansing dentures as well as treating oral infections.
Materials and methods: Seventy-seven complete denture wearers were randomly assigned into four
groups: (A) Brushing with water (control); (B) Effervescent tablets; (C) Ultrasonic device (Ultrasonic
Cleaner, model 2840 D); (D) Effervescent tablets and ultrasonic device. All groups brushed their dentures
with a specific brush and water, three times a day, before applying their treatments. Denture biofilm was
collected at baseline and after 21 days. The samples were collected by brushing the dentures with saline
and the detached microbial cells were quantified by plating. Counts [log (CFU+1) ml)1] of total aerobes,
Candida spp. and mutans streptococci were compared by one-way ANOVA or KruskalWallis test (a = 0.05).
Results: No significant difference was found among the methods from C. albicans (p = 0.76), C. tropicalis
(p = 0.94) and C. glabrata (p = 0.80). Lower counts were found for methods B and D when compared with
the other methods against mutans streptococci (p < 0.001). Method B showed lower total aerobic counts
than A, whereas C and D showed intermediate results (p = 0.011).
Conclusion: The effervescent tablets significantly reduced mutans streptococci and total aerobes from
denture biofilm. However, they was not as effective against C. albicans. Ultrasonic cleansing presented a
discrete antimicrobial effect and was less effective than the tablets for complete denture disinfection.
Keywords: complete denture, Streptococcus mutans, Candida albicans, denture cleansers, ultrasonics.
Accepted 16 January 2010

Introduction
Several reports have emphasised the precarious
conditions of use and maintenance of complete
dentures1. Poor denture hygiene is a risk factor for
oral infections and systemic dissemination of these,
given that denture base resins become colonised by
several microbial species in the oral cavity25.
Denture hygiene methods can be divided into
mechanical or chemical procedures1,6. Chemical
264

methods are classified according to their composition and mechanism, i.e. hypochlorites, peroxides,
enzymes, acids, crude drugs and mouth washes for
dentures7. Immersion of complete dentures in
alkaline peroxide is a simple hygiene method; they
are a combination of active ingredients, primarily
designed to attack the organic constituents of
deposits on dentures. When these peroxides are
dissolved in water, they become alkaline hydrogen
peroxide, which decomposes when it comes into

 2010 The Gerodontology Society and John Wiley & Sons A/S, Gerodontology 2011; 28: 264270

Chemical cleanser and ultrasound vs. denture biofilm

contact with certain substances and releases small

oxygen bubbles with the mechanical action of
detaching the biofilm from the denture surface. The
oxidant agents help to remove stains and have
some antibacterial action8. This type of solution can
be used alone or in combination with a mechanical
method1,7,9,10.
Mechanical methods comprise brushing, sonic
vibrators and ultrasonic7. Although brushing is the
most widely used method11,12, patients with
limited motor co-ordination find it difficult to
perform11. In addition, it is possible for wear of the
acrylic resin and superficial damage to relining
materials to occur13,14 and therefore, auxiliary
methods can be considered advantageous1518.
With regard to mechanical denture hygiene
methods, ultrasonic devices are mechanical aids,
generally used by professionals16,1921. The
mechanical cleansing activity of the device is
complemented with the concomitant use of a
chemical solution12. Ultrasound has two action
mechanisms, the first being the movement of liquid
resulting from sound waves transferred to the
liquid (vibration) and the second, the collapse of
bubbles formed by vibration of the unit22. There is
some controversy about the effectiveness of ultrasonic devices8,16, as the result may be attributed to
the mechanical action of the device19,2325 or to the
action of the chemical solutions used2628. The
combination of this method with brushing or with
a method of immersion in a chemical solution has
been suggested as an effective alternative for
cleansing complete dentures, particularly in
hospitals and older care institutions or for patients
with limited motor co-ordination. However the
effectiveness of the ultrasonic device has not been
clinically tested.
Although the comparison of chemical methods
and brushing is relatively common1,6,2932, the
comparison of soaking solutions, ultrasound and
their association has not been previously
described33.
Due to the complexity of the composition of
denture biofilms, the microbial count could be an
appropriate variable for assessing denture hygiene
methods. Candida spp. yeasts are the most common
species associated with denture biofilm12,13,34,35.
Although C. albicans is the most commonly detected
yeast, other species can regularly be identified, i.e. C.
tropicalis, C. krusei, C. glabrata and C. parapsilosis36,37.
Mutans streptococci and other bacterial species
are also important denture biofilm components.
They facilitate the adherence of Candida cells to the
denture base and mucosa34,38,39, mainly by means
of extracellular polymer production40. Bacteria also

265

increase acidity, which generates environmental

conditions for yeast growth41.
Therefore, the objective of this trial was to
compare the effectiveness of sodium perborate
effervescent tablets, an ultrasound device and a
combination of the two procedures as oral hygiene
methods for use by complete denture wearers.

Materials and methods

After approval by the Institutional Ethics Committee and obtaining a signed term of informed
consent from the participants in this study, 113
edentulous patients (26 men and 87 women)
4884 years of age (mean age 64 years) were
selected. They had requested denture replacements
and were under treatment at the Department of
Dental Materials and Prosthodontics of the Ribeirao
Preto Dental School. The participants presented
good overall health and were wearing conventional
maxillary and mandibular complete dentures. The
complete dentures were made of heat-activated
acrylic resin, had been worn for a period of time of
510 years and 1 scores, in agreement with the
Additive Index of Ambjrnsen et al.42
The exclusion criteria were: (i) subjects taking
antifungal agents or using antiseptic mouthwashes;
(ii) subjects taking medication known to predispose
them to oral candidiasis, such as antibiotics or steroid therapy; (iii) subjects with a medical history of
any disease or medical condition that predisposed
them to oral candidiasis or led to the subjects being
carriers of oral Candida spp.; (iv) participants who
showed no evidence of at least one of the investigated micro-organisms (Candida spp. or mutans
streptococci).
Hygiene methods and experimental design
The experimental period was 21 days. Initially, the
internal surfaces of the maxillary complete dentures were disclosed (by 1% neutral red application) and cleaned by the researcher with a specific
brush (Medic Denture Condor S.A., Sao Bento do
Sul, Santa Catarina, Brazil) and liquid soap (ErvaDoce JOB, Ribeirao Preto, Sao Paulo, Brazil), with
the purpose of completely eliminating the biofilm.
Subsequently, the patients were divided into four
groups:
(1) Control (n = 25): (a) Denture brushing three
times a day, after each meal (breakfast, lunch
and dinner) for 2 min, using a brush designed
specifically for cleansing complete dentures
(Bitufo, Itapeva, Sao Paulo, Brazil) and tap

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266

I. M. de Andrade et al.

water; (b) Rinsing the oral cavity with running

water after brushing; (c) Keeping the dentures
immersed in water while sleeping.
(2) Experiment 1 (n = 29): (a) and (b) As in (1); (c)
As in (1) but by soaking the dentures in a
container with warm water (37C) and one
alkaline peroxide-effervescent tablet (Corega
tabs Block Drug Company, Inc., Jersey City,
New Jersey-NJ, USA) for 5 min, after dinner;
(d) Rinsing the dentures before inserting them
into the oral cavity; (e) Keeping the dentures
immersed in water while sleeping.
(3) Experiment 2 (n = 28): (a), (b) and (c) As in
(1); (d) At the end of the experimental period
(21 days), immersion of the dentures in a
sterilised glass vial with 250 ml of sterilised
water and ultrasonic vibration (Ultrasonic
Cleaner, model 2840 D Odontobras Ind. and
Com. Equip. Med. Odont. Ltda, Ribeirao Preto,
Sao Paulo-SP, Brazil) for 15 min, performed by
the technician.
(4) Experiment 3 (n = 31): combination of experiment methods 2 and 3.
Of 113 denture wearers that were initially selected
for the present study, 25 were allocated to the control group, 29 to the experiment 1, 28 to experiment
2 and 31 participated in experiment 3 group. Of these
113 individuals, 36 were excluded for the following
reasons: (i) samples that did not present Candida spp.
in the first collection (baseline); (ii) samples that did
not present any mutans streptococcus in the first
collection (baseline). Therefore, samples of only 77
denture wearers were considered for statistical
analysis. These 77 samples were divided into groups
according to the treatment performed and the
micro-organism presented, as shown in Table 1.
Collecting procedures
The biofilm was collected twice for each participant, before and after the 21-day period. Each
maxillary complete denture was removed and
placed on a sterile Petri dish (15 100 mm) by the
patient. The dentures were immediately rinsed

with 10 ml of saline solution and the internal surfaces were brushed (Denture; Condor S.A.) for
5 min. These procedures were carried out using
two dental lamps placed in front of the operator to
prevent sample contamination. The resultant
suspension was vortexed for 2 min and diluted in
decimal series from 100 to 10)5. Aliquots of 50.0 ll
from the decimal dilutions were cultured in
12 60 mm Petri dishes. Plating of the biofilm
suspension was done in duplicate. The dishes contained either Bacitracin Sucrose Agar/SB-20 or
Chromagar Candida agar (CHROMagar Microbiology, Paris, France) and Brain Heart Infusion Agar
(Difco, Detroit, MI, USA) for mutans streptococci,
Candida spp. and total aerobes, respectively. The
culture mediums were incubated at 37C according
to each respective condition: (a) candle jar for 48
72 h, or (b) aerobiosis for 48 h, (c) aerobiosis for
24 h.
Microbial counting
Colonies characteristic of the mutans group of
streptococci and Candida spp. were counted after
the incubation period, by means of a stereoscopic
microscope (LEICA S-6E, Heerbrug, Switzerland). Identification of the mutans group of
streptococci was carried out by observing the
morphological features of the colonies, according to
Davey and Rogers43. Yeast identification was
performed by the following tests: 45C growth
temperature; micromorphology in cornmeal agar
with Tween-80; hydrolysis of urea; fermentation of
carbohydrates; assimilation of different sources of
carbon and hydrogen; and reduction of triphenyltetrazolium chloride.
The number of colony forming units (CFU) per
ml for each participant was obtained for mutans
streptococci and Candida spp. It should be noted
that lower values mean greater antimicrobial effect.
It is important to note that the first collection
(baseline) was not performed in order to compare it
with the number of micro-organisms quantified in
the second collection. In other words, the outcome

Table 1 Number of subjects according to the evaluated treatments and microbial species.
Treatments

Species

Control
(21 patients)

Corega tabs
(21 patients)

Ultrasound
(17 patients)

Ultrasound
+ Corega tabs
(18 patients)

Candida spp.
Mutans group Streptococci

21
21

21
15

17
13

18
18

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Chemical cleanser and ultrasound vs. denture biofilm

variable was not the difference between the second

and the first collection. The first collection only
served as a parameter for the exclusion of individuals who did not present the studied microorganisms. The antimicrobial action was evidenced
by non-detection or the detection of a small
number of micro-organisms found in the second
collection, after the denture hygiene procedures
had been implemented for 21 days.
Data analysis
The data obtained for microbial counts were
expressed as log (CFU + 1) per ml. The statistical
significance of differences among the four tested
treatments was compared within species by means
of one-way ANOVA followed by Tukeys HSD test or
KruskalWallis test, according to the data distribution. All analyses were performed at a 0.05 level
of significance. The data were analysed with SPSS
software for Windows version 12.0.0 for Windows
(SPSS Inc., Chicago, IL, USA).

Results
Table 1 shows the sample distribution according to
each group and species, and Fig. 1 shows the
transformed values for.C. albicans, mutans group of
streptococci and total aerobes. By means of the
ANOVA test (Table 2), it was confirmed that none of
the three tested treatments modified C. albicans
counts when compared with the control group. The
mutans group of streptococci was significantly
reduced after using the effervescent tablets and also
after their use associated with an ultrasonic method. However, the use of the latter did not significantly reduce the mutans group of streptococci
10
a

9
8
Log (CFU+1)/ml

ab

ab

7
6

5
4

3
2
1
0
C. albicans

Control

Corega tabs

Mutans group
streptococci
Ultrasound

Total aerobes

Corega tabs + ultrasound

Figure 1 Mean results and standard deviation for

C. albicans and mutans group Streptococci and total aerobic
counts. Similar letters indicate no difference between
treatments, within each species (Tukeys HSD test;
a = 0.05).

267

counts. There was a significant reduction of total

aerobes with the use of effervescent tablets and the
other denture hygiene methods presented intermediate levels of antimicrobial action when
compared to the control group.
The other species presented more heterogeneous
results and did not conform to normality, even
after logarithmic transformation. C. krusei was
found only once and the results for C. tropicalis can
be found in Fig. 2. Among the other Candida
species, C. glabrata was more frequently isolated
and its respective results are shown in Fig. 2. The
yeast counts were not significantly influenced by
the tested treatments (C. tropicalis: KW = 0.38,
p = 0.944; C. glabrata: KW = 0.99, p = 0.803).

Discussion
The findings of the present study showed that the
effervescent tablets demonstrated significant action
against the mutans group of streptococci, but this
was not found for C. albicans. These results are in
agreement with previous studies, such as those of
Dills et al. 31 and Drake et al.44, which reported
important antimicrobial activity of alkaline peroxides against Streptococcus spp. and ineffectiveness
against C. albicans. These results also are in agreement with those of Ferreira et al.45, who assessed
the effect of denture cleansers (Polident; Efferdent
and sodium hypochlorite) on Candida adherence to
denture liners. Their study showed that Polident
and Efferdent were not effective in preventing
initial Candida adherence to denture liners when
compared with immersion in water; only sodium
hypochlorite was effective.
Paranhos et al.46 evaluated the effect of alkaline
peroxide (Bonyplus tablets) and other denture
hygiene methods against Candida spp. and mutans
Streptococcus biofilms formed on acrylic resin specimens. As was shown in the present findings, the
alkaline peroxide was not very effective for Candida
spp. Yeast counts were high after the use of alkaline
peroxide, but further reduction was attained by
brushing with a dentifrice. However, unlike the
present study, the alkaline peroxide was also
unable to reduce mutans Streptococcus (field strain)
to low levels. This discrepancy is probably due to
the fact that their study was performed in vitro and
tested isolated mutans Streptococcus.
The present results were also in agreement with
those of Silva et al.47, who evaluated the effectiveness of sodium perborate-based denture
cleanser tablets and 3.8% sodium perborate on the
disinfection of acrylic resin specimens contaminated in vitro by C. albicans, mutans Streptococcus and

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268

I. M. de Andrade et al.

Table 2 One-way

ANOVA

for C. albicans, mutans group Streptococci and total aerobes.

Species

Source

SS

df

C. albicans

Treatment
Error
Total
Treatment
Error
Total
Treatment
Error
Total

6.32
394.92
401.23
260.94
459.46
720.40
26.94
114.91
141.84

3
73
76
3
61
64
3
52
55

Mutans group Streptococci

Total aerobes

MS

2.11
5.41

0.39

0.761 (ns)

86.98
7.53

11.55

<0.001*

8.98
2.21

4.06

0.011*

ns: no significant difference (p > 0.05); *significant difference (p < 0.05).

Figure 2 Candida tropicalis and C. glabrata counts.

other micro-organisms. It was observed that the

commercial tablets tested did not present satisfactory antimicrobial activity against C.albicans,
although they showed antimicrobial activity
against mutans Streptococcus.
Studies with different results also found strong
antifungal activity of peroxides against C. albicans48,49. These discrepancies may be explained by
methodological characteristics, since the latter
reports were obtained in in vitro studies that tested
isolated Candida spp. In vivo studies provide different
conditions, mainly because yeasts are associated
with oral bacteria. Moreover Nakamoto et al.48
added 5 ml of C. albicans cells in suspension directly

to 5 ml of alkaline peroxide solution contained in a

tube. In the study of Nikawa et al.49 samples colonised by fungi were immersed in 200 ml each of the
tested alkaline peroxide solutions and were incubated together in solutions for a long period of 2 h.
Oxidising agents found in alkaline peroxide
solutions are active antimicrobial components27,50.
Hydrogen peroxide is one of these agents and can
affect the vital structures of cells by means of in situ
generation of the highly reactive hydroxyl groups51.
Probably, as Drake et al.44 suggested, the production
of a glucan barrier by mutans Streptococcus may have
limited the exposure of other species, such as Candida spp., to the active components. It is possible that
C. albicans form the first adherent layer of biofilm,
covered by mutans Streptococcus layers. Thus, the
substantial reduction of mutans Streptococcus is possibly a consequence of its location in the more
superficial layers than those in which the yeast cells
are found. In other words, Candida spp. remains
protected by the biofilm structure. As yeasts are not
significantly affected by alkaline peroxides, the latter should be indicated for used in association with
other denture hygiene methods.
The ultrasonic results were contrary to those of
other studies that reported better antimicrobial
effects when compared with those of the effervescent tablets23,27. Both Gwinnet and Caputo23 and
Palenik and Miller27 compared these methods
in vitro for cleansing contaminated complete
dentures. Furthermore, the first reported study
tested the treatment action on the denture biofilm
accumulated in 72 h and the second study27 used a
mouthrinse as a means to apply the ultrasound.
The in vivo design and other features could also be
stated as a reason for these discrepancies. In addition to the fact that the biofilm is a complex
microbial community, the ultrasonic method was
instituted only after the 21-day period and a relatively inert conducting fluid (distilled water) was

 2010 The Gerodontology Society and John Wiley & Sons A/S, Gerodontology 2011; 28: 264270

Chemical cleanser and ultrasound vs. denture biofilm

used. Other studies also found satisfactory results

for ultrasonic methods when compared with
chemical methods19,25. However, one of the studies
used a short test period (3 days) and chemicals
were used in the ultrasonic device19. The other
study used another outcome variable (SEM imaging parameters) and the participants did not have
their dentures cleaned by the researchers at the
beginning of the trial25.
The reduced effectiveness of the ultrasonic
methods was probably due to the absence of
chemical substances within the ultrasonic cleanser.
Budtz-Jorgensen8 stated that complete denture
hygiene by this method could be associated with
the chemical activity of the immersion liquid rather
than with the mechanical action. This may be the
best explanation for the results in the present
study, given that the ultrasonic method was used
with distilled water and was not able to affect
C. albicans or the mutans group of streptococci. The
impact of cavitation bubbles in the water was not
sufficient to eliminate the tested microbial species.
Mutans Streptococcus exhibits a thick cell wall
composed of peptidoglycan39, which may be the
main reason for its resistance. Moreover, C. albicans
also exhibits a thick multi-layered cell wall and
may be highly resistant to ultrasound.
The combination of alkaline peroxide and ultrasonic methods provided results that were similar to
using only peroxides, irrespective of the species.
This confirms that the antimicrobial effects were a
consequence of the use of the chemical method
tested. Although one method was not enhanced by
the other, an important limitation of this study was
that the peroxides were not used as conducting fluid
in the ultrasonic device. Thus, future comparisons
could assess the use of different chemicals within
the ultrasonic device, i.e. surfactants or antimicrobial agents, by means of a clinical trial design.

Conclusions
In summary, the use of effervescent tablets as a
denture hygiene method significantly reduced
mutans Streptococci and other aerobic species in the
denture biofilm. However, it was not effective
against C. albicans. Ultrasonic cleansing presented a
discrete antimicrobial effect and was less effective
than cleansing tablets for complete denture
disinfection.

Acknowledgements
This research was supported by FAPESP (grant no.
2005/55705-2).

269

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Correspondence to:
Ingrid Machado de Andrade, Department of Dental
Materials and Prosthodontics, Ribeirao Preto Dental
School, University of Sao Paulo, Av. do Cafe s/n,
14040-904 Ribeirao Preto, SP, Brazil.
E-mail: ingridma76@yahoo.com.br

 2010 The Gerodontology Society and John Wiley & Sons A/S, Gerodontology 2011; 28: 264270