Background/aims:
Ozone (O3
) is a highly reactive
molecule with antimicrobial properties against
bacteria, fungi and viruses, which have been
proven in different studies. We examined the effect
of ozonated water on Candidaalbicans on acrylic
denture plate and compared its efficacy with
commercially available denture cleansing tablets
Methods:
The heat-cured acrylic resins were cultured
with C. albicans. After treatment of ozonated water
and denture cleansing tablet dissolved solution the
number of attached C. albicans was counted by
digital colony counter in terms of Colony Forming
Units CFU/ ml.
Results:
After treatment with ozonated water (4
mg/l) for 60 min, the colony forming units were
markedly depleted as compared with the control
group but aseptic. The antimicrobial activity against
C. albicans between plates immersed in ozonated
water were slightly greater than those treated with
commercially available denture cleaners.
Conclusion:
Our results suggest that application
of ozonated water may be useful in reducing the
number of C. albicans on denture plates.
Ozone is known to be a potent oxidizer and has
the ability to oxidize any known biological entity.
A recent study demonstrated that denture plaque
control is essential for the prevention of denture
stomatitis associated with Candidaalbicans1
. In
addition, the fitted surfaces of dentures have been
shown to be reservoirs of Candida albicans, which
is associated with stomatitis and disseminated
fungal infectious diseases, and it is well known that
elderly hospitalised patients have a high risk of
aspiration pneumonia induced by microorganisms
on dentures2,3. Denture-cleansing methods are
generally divided into mechanical and chemical
cleaning methods. However, it has been reported
that mechanical cleaning methods are insufficient
for a complete reduction of microorganisms
on denture plates4
. Thus, it is considered that
chemical cleaning methods are more effective,
in which hypochlorites, peroxides, enzymes, and
acids are generally employed as immersion-type
chemical solutions for denture cleaning. However,
some chemical agents used for denture cleaning
are known to damage acrylic resin and metal alloy materials, and are also relatively expensive
compared to ozonated water used in the present
study5
.
Ozonated water has been shown to be a powerful
antimicrobial agent against bacteria, fungi,
protozoa, and viruses6
. It is recognized that dental
plaque is a kind of bacterial biofilm7,8 in which
bacterial growth is the primary factor governing
the relative abundance of different microorganisms
including C. albicans. In a recent study by
Nagayoshi et al he stated that ozonated water was
useful in reducing the number of infections caused
by oral microorganisms. This suggested that oral
microorganisms might be inactivated by ozonated
water at different doses9
. It has been reported that
ozonated water can be mutagenic if used for a long
period and in high concentrations10. In the present
study, we evaluated the antimicrobial efficacy
of ozonated water against Candida albicans
on resin denture plates in vitro by counting the
colony forming units and compared it with the
effectiveness of commercially available denture
cleansing tablets with sodium mono phosphate
perborate as its main content.
Growth conditions for Candida albicans and
preparation of test plates
The experimental denture plates were prepared
using heat-cured acrylic resin (25 mm x 2 mm x
2mm) (Asian Acrylates Acrylin H Heat Cure, Dbm
Pink, Mumbai) according to the manufacturer’s
instructions. The resin plates were ground with#180
emery paper, sonicated in water for 60 min, and
immersed in water for 1 day to remove the residual
monomer, then dried in air.
Sterile 1 X PBS (Phosphate Buffered Saline); pH 7.4
24g Nutrient broth was suspended in 1000ml of
distilled water and was autoclaved at 121°C; 15lbps
for 15 minutes.
The medium was prepared by dissolving 39g of
the commercially available Potato Dextrose Agar
Medium (HiMedia-GM096) in 1000ml of distilled
water. The dissolved medium was autoclaved
at 15 lbs pressure, 121°C. After autoclaving, the
media (20ml) was allowed to cool to 60°C and was
poured to pre sterilized Petriplates. The plates were
allowed to solidify in a laminar air flow chamber.
Candida albicans (ATCC 10231) was used for the
study.
Candida albicans was prepared (growth adjusted
to 0.5% McFarland’s standard). PMMA Blocks
were incubated in respective test tubes containing
Candida albicans for 5 days at room temperature
for the formation of biofilm.
Contaminated samples (n =10) were grouped
using simple random allocations as follows:
GROUP A is incubated samples (n =10) that left
untreated were considered as the control group
GROUP B is incubated samples (n =10) which
were immersed in ozonated water.
GROUP C is incubated samples (n =10) which
were treated with commercially available denture
cleansing tablet dissolved solution.
Untreated remained as control group and was
considered as Group A.
Ozonated water was generated by portable ozone
generator DENT OZONE, ADC Inc. Mumbai) (Fig
1). The concentration of ozonated water in the
aqueous phase is adjusted to concentrations of
4 mg/l for 60mint and this group was considered
as Group B
Commercially available denture cleansing tablet
with sodium mono phosphate perborate as its main
content dissolved in 150ml of distilled water for
24hrs (as per manufacturer’s `instructions) and
this group was considered as Group C.
After treatment with Ozonated water and Denture
cleansing tablets, the treated blocks were rinsed
three times with sterile 1 X PBS (pH 7.4) to remove
unattached Candida sp and debris. Then the
blocks were added with 3 ml sterile 1 X PBS (pH
7.4) and vortexed for 10 minutes, the supernatant
containing detached bio film was transferred to
sterile eppendrof tubes, respectively. 10µL was
plated uniformly on potato dextrose agar plates
and incubated at 370
C for 24 hrs.
The plates were checked for colony forming units
(CFUs) after 24 hours of incubation. The CFUs were
then enumerated using a Digital Colony Counter.
The CFUs in Control and test was compared for
determining the inhibition of cell adherence which
was expressed in terms of CFUs/mL.
Statistical analysis was performed using software
STATISTICA (Version 7.0; StatSoft Inc.). Significant
statistical differences between treatments were
examined using Student–Newman–Keuls test.
Differences were considered statistically significant
at P < 0.05.
We examined the effects of different cleaning
methods on the adherence of Candida albicans
to resin plates (Table 1). This showed that heat
cured PMMA blocks treated with ozonated water
of 4mg/l concentration for 60 minutes shows less
number of colony forming units per millilitre of
Candida albicans. They are most effective in
reducing the growth of Candida albicans, but
was not completely aseptic.
The control group had the highest colony count
of Candida albicans.
We compared the microbicidal efficacy of ozonated
water with commercially available
Denture cleanser tablet [sodium monophoshate
per borate]. Our results showed that denture
cleanser tablets are effective in removing Candida albicans from PMMA, but its potency is less than
that of ozonated water. They showed more CFU/
ml than ozonated water and less than that of
control group.
Figure 1 shows scanning electron microscopic
picture of control group (Mag 100x50µm), which
was the untreated group showing high number
of colony forming units per millilitre. (Group A)
Figure 2 shows scanning electron microscopic
picture (Mag 100x50µm), of Group B, the group
which was treated with ozonated water showing
least number of CFUs/ ml of candida albicans.
Figure 3 shows scanning electron microscopic
picture (Mag 100x50µm), of Group C, the group
which was treated with commercially available
denture cleansing tablet, showing less number of
CFUs / ml of candida albicans than control group
and less effective in removing candida than group
treated with ozonated water.
In this study, two different denture hygiene methods
are presented, which differed in their ability to
remove Candida albicans from denture samples,
thus supporting a rejection of the null hypothesis for C albicans removal.
With the onset of the 21st century, infectious disease
specialists are being asked to manage a greater
number of patients with serious infections who
are over 65 years old, and are gaining a new
perspective on the emerging problem of geriatric
infectious diseases.
Recently, because of growing interest, there have
been a great number of clinical studies of a variety
of infectious diseases in geriatric populations,
and it has come to be generally accepted that
the etiology, clinical manifestations, therapy, and
prognosis of pneumonia in elderly patients are
quite different than in younger adults11.
In the dental field, it is necessary to find innovative
methods and techniques that can prevent, reduce,
or eliminate oral microorganism colonization
in elderly patients who have denture plates12.
Further, it has been reported that there is a strong
association between poor denture hygiene and oral
candida colonization13. Results of another recent
study indicated that compromised elderly patients
above 70 years of age had a higher number of
C.albicans than patients in their 60s14. These
findings suggest that plaque accumulation on
the dentures of handicapped elderly patients could
create an appropriate environment for the growth of
C. albicans. Many researchers have reported that
surface roughness is a key factor in the entrapment
of microorganisms on denture surfaces. Verran and Maryan15 found that an increase in surface
roughness facilitated C. albicans retention on
acrylic resin surfaces. However, few microbiological
studies have been conducted to develop a cheap
and reliable apparatus that is easy to use. In the
present study, we prepared the resin plates (25
mm x 2 mm x 2 mm) with a rough surface to create
an appropriate environment for the growth of C.
albicans, and examined the effect of ozonated
water on the viability of C. albicans in order to
understand its effect on dentures and compare its
effectiveness with commercially available denture
cleansing tablet with sodium mono phosphate per
borate as its main content.
And the data we obtained from the study is that
the cleansing efficiency is higher in ozonated
water treated group than that of group treated with
commercially available denture cleansing tablet.
Arita et al. observed that accumulation of
microorganisms, especially C. Albicans, on acrylic
resin denture surfaces was significantly reduced
by rinsing with ozonated water accompanied
by ultrasound. This effect depends on the
concentration of ozone and the highest effect is
with ozone concentration of 4mg/ml.16
A denture cleanser releasing ozone bubbles with a
concentration of 10 ppm has been developed and
is effective in reducing the number of C. albicans
following a 30–60 min exposure.17
Suzuki et al. showed that ozone is successfully
used in dental casting alloys, such as a removable
partial denture which unlike other detergent
materials in the markets, it does not have any
negative effect on the surface and weight properties
of these alloys.18
Oizumi et al. showed that ozone gas is more
effective in removing denture microorganisms
than ozonated water.19 A study by Ekren et al.
showed the effect of ozone on bond strength of soft
liners to acrylic resin materials. Ozone reduces
the bond strength of soft liners such as mollosil
and molloplast B to acrylic resin dentures and the higher the exposure to ozone is, less bond
strength will be obtained.20 It has been reported
that ozone has astrong oxidizing power with a
reliable microbicidal effect21, 22, 10, and it is generally
accepted that oxidation mediated by ozone
destroys cell wall and cytoplasmic membranes
of bacteria and fungi23. After a membrane is
damaged by oxidation, its permeability increases,
causing the microorganism to die.
There are several applications for ozone in the
field of prosthodontics like patients suffering from
periimplantitis were investigated by Karapetian
et al.24 They compared the effectiveness of
conventional, surgical and ozone therapy methods
to cure periimplantitis. They reported that the main
challenge seems to be the decontamination of
the implant surface, its surrounding tissue and
the prevention of recolonization with periodontal
pathogenic bacteria. And the most effective
bacterial reduction was recorded in the ozone-treated patient group.
Although several studies have examined ozone in
various fields of dentistry, this is still a novelty and
further studies are needed. It seems that ozone can
effectively remove bacterial plaques and create
a suitable environment for the performance of
different dental restorations and dentures.
In this study we propose that it is possible to apply
ozonated water for denture cleaning, and this
method effectively saves expense at home for
the aged.
Ozone therapy has a wide range of applications in
almost every field of dentistry. Its unique properties
include immunostimulant, analgesic, antihypnotic,
detoxicating, antimicrobial, bioenergetic and
biosynthetic actions. It is atraumatic, painless, non-invasive nature and relative absence of discomfort
increase patient’s acceptability and compliance.
However it is necessary to pay attention to the side
effects of ozone, that include epiphoria, rhinitis, cough, headache, nausea and vomiting25.
This study was conducted in Biogenix Research
Center, Trivandrum. Candida albicans (ATCC®
10231TM) was used for the study.
Since this culture was prepared in vitro without
any patient involvement, there is no scope for
ethical issues.