Biofilm Formation

IBIMA Publishing
Journal of Research and Practice in Dentistry

http://www.ibimapublishing.com/journals/DENT/dent.html
Vol. 2014 (2014), Article ID 112424, 9 pages
DOI: 10.5171/2014.112424
Research Article
Biofilm Formation in Denture Base Acrylic Resins
and Disinfection Method Using Microwave
Denture Base Acrylic Resins and Microwave Disinfection
Ana Patricia Fernandes Macedo Dantas1, Rafael Leonardo Xediek Consani1,

Janaina de Cassia Orlandi Sardi2, Marcelo Ferraz Mesquita1, Manoela Capla de
Vasconcellos dos Santos da Silva1 and Mario Alexandre Coelho Sinhoreti3
1
Department of Prosthodontics and Periodontology, Piracicaba Dental School, State University of
Campinas, Piracicaba, SP, Brazil
2
Department of Clinical Analysis, Laboratory of Clinical Mycology, Sao Paulo State University,
Araraquara, SP, Brazil
3
Department of Restorative Dentistry, Piracicaba Dental School, State University of Campinas,
Piracicaba, SP, Brazil
Correspondence should be addressed to: Rafael Leonardo Xediek Consani; rconsani@fop.unicamp.br
Received 31 October 2013; Accepted 19 December 2013; Published 21 January 2014
Academic Editor: Arife Doǧan
Copyright © 2014 Ana Patricia Fernandes Macedo Dantas, Rafael Leonardo Xediek Consani, Janaina
de Cassia Orlandi Sardi, Marcelo Ferraz Mesquita, Manoela Capla de Vasconcellos dos Santos da
Silva and Mario Alexandre Coelho Sinhoreti. Distributed under Creative Commons CC-BY 3.0
Abstract
The purpose of this study was to verify the effect of the microwave disinfection in denture base
acrylic resins with Candida sp biofilm formation. Samples (n=90) of each resin were submitted
to ethylene oxide sterilization, and then infected by inoculum of yeast (C. albicans, C. tropicalis
and C. dubliniensis) with 200 μL of cultures adjusted to 108 CFU/mL, placed in specific media
and aerobically incubated at 37°C for 24 hours for biofilm formation. For each resin and
microorganism, samples were irradiated by microwave energy at 650 W for 3 minutes. Non-
irradiated samples and without microorganisms samples were considered as control. The
samples were washed with saline solution and plated in Sabouraud dextrose agar (SDA) for
analysis of the number of colony forming units (CFU/mL). Data were submitted to Kruskal-
Wallis followed by Student-Newman-Keuls test (α=0.05). The results demonstrated a
significant decrease in the number of viable colonies for all biofilms formed by Candida species.
It was demonstrated an effective disinfection of the samples contaminated by Candida biofilm
when microwave irradiated. This study demonstrated that microwave disinfection was able to
significantly decrease the amount of microorganisms from denture base acrylic resins
contaminated and therefore could be used as a preventive method of candidiasis caused by
dentures.
Keywords: Acrylic resin, Candida sp, biofilm, disinfection, microwave.
_____________
Cite this Article as: Ana Patricia Fernandes Macedo Dantas, Rafael Leonardo Xediek Consani, Janaina de
Cassia Orlandi Sardi, Marcelo Ferraz Mesquita, Manoela Capla de Vasconcellos dos Santos da Silva and
Mario Alexandre Coelho Sinhoreti (2014), “Biofilm Formation in Denture Base Acrylic Resins and
Disinfection Method Using Microwave, Denture Base Acrylic Resins and Microwave Disinfection,” Journal of
Research and Practice in Dentistry, Vol. 2014 (2014), Article ID 112424, DOI: 10.5171/2014.112424
Journal of Research and Practice in Dentistry 2
Introduction Various drugs have been used for the

treatment of denture stomatitis, and
Complete or partial removable dental systemic antifungal therapies are also
prostheses unadapted and poorly cleaned commonly indicated. However, previous
may induce some pathological changes on study has reported the appearance of
the mucosa of the oral cavity (Mandali et resistant microorganisms during long-term
al., 2011). Prosthetic stomatitis by treatments or prophylactic. Although these
infestation of Candida species is the most drugs are effective in relieving the signs
frequent and it is characterized by and symptoms of denture stomatitis,
moderate or severe mucosal inflammation Candida biofilm-associated infections are
underneath the denture base. Candida difficult to treat and to fully eradicate with
albicans is not only able to adhere to the standard antifungal therapy (Ramage &
mucous surfaces, but also to colonize the Lopez-Ribot, 2005). Topical antimycotic
acrylic resins of the dental prosthesis. Both treatment is used to prevent spread of the
the plaque accumulated on the denture and infection that occurs on the fitting denture
the poor oral hygiene contribute to the surface by Candida species; however,
virulence of Candida promoting a clinical recurrences are frequent if the local or the
picture of Candida-associated denture systemic predisposing conditions are not
stomatitis (Salerno et al., 2011). Classic corrected (Budtz-Jorgensen, 1990).
study has shown that this oral inflamation Candida associated denture stomatitis has
may occur on the maxilla and mandible; also been associated with a lack of denture
however, it is more often associated with hygiene (Webb et al., 2005).
the maxilla, sometimes found under maxilla
partial dentures, but only rarely beneath An important predisposing factor for
mandibular dentures (Wilson, 1998). After stomatitis associated with Candida is the
colonization and adhesion of Candida permanence of high levels of
species to the epithelial surface, the microorganisms in saliva (Pires et al.,
subsequent mucosal lesion is due to tissue 2002). By this reason, a proper oral
destruction by potent proteolytic enzymes hygiene and efficient denture cleaning
or toxins and an inflammatory response to must be continuous for the bacterial
Candida antigens. biofilm control present on the denture base
and oral soft tissue (Salerno et al., 2011).
There are other factors that favour the However, preventive post-insertion to
develop of oral candidiasis, such as denture adjust defective dentures and providing
base fit, metabolic disorder, patient’s age, information on cleaning methods and
mucosa conditions, epithelial changes, poor instructions to maintain oral tissues
diet, appropriate denture hygiene, healthy is also important in relation to oral
xerostomia and salivary flow (Mandali et care (Coelho et al., 2004).
al., 2011; Wilson, 1998; Budtz-Jorgensen,
1990; Pires et al., 2002; Torres et al., 2002 Biofilm present on dentures can be
and Gendrau & Loewy, 2011). Study had controlled by mechanical methods,
shown that oral infestation by Candida chemical and mechanical-chemical
species occurs commonly in the presence sanitization. Several methods are available
of denture that creates an environment for disinfection of dental prostheses.
that favours the localization and Chemical solutions, such as glutaraldehyde,
development of potentially virulent sodium hypochlorite, chlorine dioxide,
organisms (Pusateri et al., 2009). In iodine, alcohol and chlorhexidine are
addition, persistent denture stomatitis is frequently used for disinfection purposes;
due to immature antimicrobial host however, there are several drawbacks in
defenses, acquired suppression of immune the use of such chemical agents for
defense mechanisms, or changes of the disinfection of prosthesis.
environmental conditions of the oral cavity
(Budtz-Jorgensen, 1990).
_______________
Ana Patricia Fernandes Macedo Dantas, Rafael Leonardo Xediek Consani, Janaina de Cassia Orlandi Sardi,
Marcelo Ferraz Mesquita, Manoela Capla de Vasconcellos dos Santos da Silva and Mario Alexandre Coelho
Sinhoreti (2014), Journal of Research and Practice in Dentistry, DOI: 10.5171/2014.112424
3 Journal of Research and Practice in Dentistry
Despite possessing bactericidal action, included in plastic (Classico Dental

these different chemical disinfectants have Products, Sao Paulo, SP, Brazil) or metallic
different effects on the surface (Safrany Metallurgy, Sao Paulo, SP, Brazil)
characteristics and color of denture resins flasks with type III dental stone
(Ma et al., 1997). A classic study has shown (Herondent; Vigodent, Petropolis, RJ,
that the association between brushing with Brazil). In the dental stone molds left by the
toothpaste and commercial cleanser wax patterns were conventionally made 30
soaking is a popular method of cleaning samples for each type of acrylic resin (n=
dentures (Jagger & Harrison, 1995). 90). Classico (Classico Dental Products),
However, the use of cleaning tablets for Onda-Cryl (Classico Dental Products) and
acrylic denture overnight storage reduces QC-20 (Dentsply, Petropolis, RJ, Brazil)
denture biofilm mass and pathogenicity heat-curing acrylic resins were
when compared to dry and water proportioned, manipulated and
preservation, and may contribute to the polymerized according to the
overall systemic health (Duyck et al., 2013). manufacturers’ instructions.
The procedures used in clinical therapy for The acrylic resin samples were sterilized
denture stomatitis comprises mechanical with ethylene oxide (ACECIL Sterilization
removal of biofilm from the surface of the Center, Campinas, SP, Brazil). Afterwards,
denture base and underlying mucosa the samples were manipulated up to 15
associated to antiseptics and disinfectants. days after sterilization because beyond this
Furthermore, it is also recommended the period the sterilization would be
use of systemic antifungal drugs and compromised due to expiration date.
denture disinfection by microwave energy
(Salerno et al., 2011). Although the Microorganisms and Growth Conditions:
microwave irradiation is still restricted, Three species of Candida were tested for a
microwave irradiation has been suggested more understandable study in relation to
in the literature by classic studies (Rohrer the disinfection of microorganisms
& Bulard, 1985; Baysan et al., 1998; Webb responsible for oral candidiasis: C. albicans
et al., 1998 and Dixon et al., 1999) and ATCC 90028, C. dubliniensis ATCC 7987 and
more recent works (Brondani et al., 2012; C. tropicalis ATCC 4563. Candida species
Silva et al., 2012 and Silva et al., 2013) as a are the most common pathogens form of
promising method for sterilizing acrylic oral infection for the development of
resin denture bases contaminated with denture stomatitis (Dorko et al., 2001; Dar-
Candida albicans. Odeh & Shehabi, 2003; Web et al., 2005;
Ganguly & Mitchell, 2011 and Salerno et al.,
Based on these considerations, the purpose 2011). The microorganisms were originally
of this study was to verify the effect of the obtained from the Microbiology and
microwave disinfection on different types Immunology Laboratory, Department of
of denture base acrylic resins Oral Diagnosis, Piracicaba Dental School,
(conventional, microwaved and boiled) in UNICAMP. Candida species were cultivated
presence of Candida biofilms (C. albicans, C. on Sabouraud Dextrose Agar (Difco
dubliniensis and C. tropicalis). The work Laboratories, Detroit, MI, USA) at 37ºC for
hypothesis was that microwave energy 24 hours in aerobic condition. Microbial
should cause similar effects in the biofilms suspensions were obtained from single
formed on different acrylic resins for colonies isolated on agar plates, inoculated
denture bases. in the appropriate broth for overnight
cultures at 37ºC. Candida spp. was grown
Materials and Methods in Sabouraud Dextrose Broth (SDB). Cells
of the resultant cultures were harvested,
Preparation and Sterilization of Samples: washed twice with phosphate-buffered
saline (PBS, pH 7.2), centrifuged at 5000x g
Circular wax patterns (40 mm in diameter for 5 minutes and re-suspended in
and 4 mm in high) were conventionally phosphate-buffered saline (PBS, pH 7.2).
_______________
Microbial suspensions were Microwave Irradiation:

spectrophotometrically (BioPhotometer
plus, Eppendorf, Hauppauge, NY, USA) For each type of acrylic resin, three groups
standardized to a concentration of 1x108 (n=30) were obtained: 1) samples
cells/mL. inoculated with C. albicans; 2) samples
inoculated with C. dubliniensis and 3)
Assays Biofilm Formation in Samples: samples inoculated with C. tropicalis. After
biofilm growth, the samples of each group
Pre-sterilized polystyrene flat-bottomed were submitted to microwave irradiation,
microtiter plates were used for Candida and this procedure was carried out for the
species biofilm production. Two mL of following subgroups (n=10): I – resin
standard cell suspensions (108 cells/mL) samples irradiated in triplicate, II – Non-
were added to each well containing the irradiated resin samples (positive control)
sterile resin specimen. The plate was and III – resin samples without
incubated at 37ºC for 90 minutes to contamination (negative control).
promote microorganism adherence on the
resin specimen. Afterwards, the specimens Subsequent to the incubation period, the
were transferred to other wells and the samples were removed from the wells of
non-adherent cells were removed by plastic plates containing culture media,
washing with 2 mL PBS. Two mL of washed in PBS and immersed individually
sabouraud dextrose fresh broth was added in sterile beaker containing 10 mL of sterile
to each well to promote biofilm growth. water. Each beaker was microwave
After incubation at 37ºC for 48 hours under irradiated (Continental; Amazonas, MA,
aerobic conditions, the plates were Brazil) at 650 W for 3 minutes (Sanitá et al.,
removed from the incubator and the wells 2008). After irradiation, each sample was
washed with PBS. Afterwards, the aseptically removed from the beaker,
specimens were transferred to a tube washed, transferred to a tube containing
containing PBS and sonicated for 20 PBS, and sonicated for 20 minutes to
minutes to disrupt the cells aggregate of disrupt the biofilm cell aggregates. The
the biofilm. The suspension containing the solution with detached cells of the biofilm
detached biofilm cells was vortexed, was vortexed and diluted, and plated on
diluted and plated on sterile Petri dishes sterile Petri dishes containing medium for
containing selective media for Candida spp. Candida spp. at 37ºC for 48 hours under
at 37ºC for 48 hours under aerobic aerobic. The media used for plating for
conditions. Candida spp. were Sabouraud dextrose
agar with 5 µg/mL of chloranphenicol.
Samples of resin were transferred to a tube After incubation, colony counts of each
containing 1 ml PBS and then sonicated to Petri dish were quantified using a digital
disrupt the cell pellet biofilm. The colony counter (CP 600 Plus).
suspension containing cells isolated from
the biofilm were vortexed and 100 µl of Statistical Analysis:
this suspension was plated and dilutions
(10x, 100x, 1000x) were also performed Kruskal-Wallis test was used for evaluated
and seeded in sterile Petri plates the mid-points of the microorganism
containing Sabouraud Dextrose Agar accounts in each one of the experimental
medium (SDA) (Silva et al., 2010 and Marra and control groups. The data were
et al., 2012). Count colonies on each Petri submitted to nonparametric multiple
dish were quantified using a CP 600 more comparison of paired means for Student-
digital colony counter (Phoenix, Newman-Keuls test. A significance level of
Araraquara, SP, Brazil). The results were 5% was used to analyze the difference
given as colony forming unit per milliliter among average positions.
(CFU/mL).
_______________
Results same resin was not significant, as well as

between resins in the same Candida specie.
The non-irradiated samples (control) When non-irradiated (control) and
revealed microbial growth in all resins disinfection treatments were compared,
after incubation for 48 hours. In each microwaved specimens showed decrease
treatment, the comparison between in the number of viable colonies formed by
Candida species in same resin was not C. tropicalis on all resin types, C.
significant, as well as between resins in the dubliniensis on the Classico and QC-20
same Candida specie. The disinfected resins, and C. albicans on the Onda-Cryl and
samples showed no viable colonies in all QC-20 resins (Table 1).
Candida species assessed after incubation,
for all acrylic resins. In each treatment, the
comparison between Candida species in
Table 1 - Means (SD) of CFU/mL in Acrylic Resins Contaminated with Candida Species for
Control and Microwaved Treatments
Treatment Acrylic resin C. albicans C. dubliniensis C. tropicalis

Classico 6.45 x 107 (6.80 x 107) 762.50 x 107 (816.70 x 162.00 x 107 (24.04 x
(conventional) a,A 107) a,A* 107) a,A*
Control Onda-Cryl 62.05 x 107 (69.22 x 7.70 x 107 (8.90 x 107) 71.50 x 107 (41.71 x
(microwaved) 107) a,A* a,A 107) a,A*
QC-20 19.75 x 107 (7.28 x 107) 9.30 x 107 (5.37 x 107) 47.95 x 107 (46.73 x
(boiled) a,A* a,A* 107) a,A*
Classico 0 (0) aA 0 (0) a,A 0 (0) a,A
(conventional)
Disinfection Onda-Cryl 0.16 x 107 (0.23 x 107) 0 (0) a,A 0.19 x 107 (0.02 x 107)
(microwaved) a,A a,A
QC-20 0 (0) a,A 0 (0) a,A 0.06 x 107 (0.04 x 107)
(boiled) a,A
Means followed by distinct letters are minutes showed different decreases in the
statistically different (5%). Lowercase number of viable colonies for biofilms
letters in each row compare Candida formed by C. albicans, C. dubliniensis and C.
species in each treatment for same resin tropicalis (Table 1). Based on these results,
(Kruskal-Wallis). Uppercase letters in each the work hypothesis that microwave
column compare different resins in the energy should cause similar effects in all
same Candida species and treatment biofilms formed on different acrylic resins
(Kruskal-Wallis). (*) Asterisks compare was partially confirmed.
treatments in the same Candida species
and resins. (Kruskal-Wallis/Student- The findings of the present investigation
Newman-Keuls). are in agreement with classic studies in
which was verified that microwave
Discussion irradiation is effective for cleaning and
disinfecting dental instruments (Rohrer &
The current study evaluated the effect of Bulard, 1985), resilient relines and denture
microwave disinfection in biofilms of bases (Baysan et al., 1998; Dixon et al.,
Candida species (C. albicans, C. dubliniensis 1999), and oral prosthesis (Webb et al.,
and C. tropicalis) formed in acrylic resin 1998). More recent studies also verified
(Classico, Onda-Cryl and QC-20). It was that microwave energy was efficient for
demonstrated that the non-irradiated denture disinfection (Brondani et al.,
samples presented microbial growth after 2012), denture disinfection associated to
incubation at 37°C for 48 hours. antifungal therapy (Silva et al., 2012) and
Microwaved samples at 650 W for 3 as an alternative method for disinfection of
_______________
different types of denture base acrylic time the greater alterations and the
resins (Silva et al., 2013). Based on these microorganism fracture occurs after 9
aforementioned studies, the microwave minutes (Rosaspina et al., 1994). The
disinfection of dentures infected with these candidal adhesion to denture base acrylic
microorganisms appears to contribute resins differs depending on the quality of
significantly for prevention of oral the material used as well as the Candida
stomatitis and cross-contamination. species in question. Thus, it was also
observed that, in general, fewer cells
There was no previous sterilization of the adhere to the heat-polymerized resin when
samples in this current investigation compared to auto- polymerized resin (He
because the aim was to verify the effect of et al., 2006).
the microwave disinfection on acrylic
resins contaminated by Candida biofilms In the present study, statistically significant
and not affected by planktonic cells of these differences were showed between
microorganisms. It has been noted in treatments for the number of viable
previous study that the symptoms of colonies formed by C. tropicalis on all
denture stomatitis often return shortly resins, C. dubliniensis on the Classico and
after treatment, suggesting that C. albicans QC-20 resins, and C. albicans on the Onda-
found in infestations is more resistant to Cryl and QC-20 resins (Table 1). This result
antifungal drugs action (Chandra et al., is significant considering that re-infestation
2001). This allegation reinforces the of the denture surface and infection of the
assumption that microwave energy should adjacent soft tissue were delayed
be used aiming the disinfection of acrylic dramatically in patients whose dentures
resin denture base contaminated by were microwaved compared with those
Candida species. whose dentures were chemically
disinfected (Banding & Hill, 2001).
An in vitro study showed microwave
sterilization for all dentures contaminated All acrylic resins studied are thermo-
by different species of Candida (C. albicans, polymerized; however, they have different
C. glabrata, C. tropicalis, C. krusei and C. temperature and time of polymerization.
dublinensis) isolated from standard Previous study has shown that this
cultures or from HIV positive pattern condition can cause different effects on the
(Sanitá et al., 2008). In general, these cell adhesion levels and the candidal
findings are in accordance with the results adherence to denture base differs
of the present study showing microwave depending on the quality of the acrylic
disinfection of acrylic resins contaminated resin used, as well as the Candida species in
by biofilms of C. albicans, C. dublinensis and question (He et al., 2006).
C. tropicalis. In addition, results from an in
vitro investigation showed that microwave Dental prostheses disinfection methods are
irradiation was also effective in disinfection intended to eliminate pathogenic
of dental prosthesis contaminated with S. microorganisms no causing adverse effects
aureus and P. aeruginosa species (Dovigo et on physical and mechanical properties of
al., 2009). acrylic resins. Exposure to microwaves for
5 minutes of samples immersed in water
Study reported in the literature had did not result in a significant change in
recommend that the microwave hardness for denture resin-based materials
disinfection should be with the samples (Dixon et al., 1999), and simulated
immersed in water (Dixon et al., 1999), microwave disinfection at 650 W for 3
indicating that the action on the pathogens minutes did not cause misfit of acrylic resin
can be purely thermal. Scanning electron denture bases immersed in water (Consani
microscopy analysis showed that et al., 2007). Based on these considerations,
microwave irradiation induced a it is possible to assume that the microwave
morphological modification in the Candida disinfection method used in this study
cells. Therefore, the longer the exposure should not cause deleterious effects on the
_______________
mechanical properties of resins for denture from the present study, which investigated
base. However, further studies should be the disinfection of various Candida species
conducted to analyze the effect of repeated biofilms. Thus, different energy,
microwave disinfection in Candida biofilms frequencies and periods of irradiation must
infesting different types of acrylic resins, be assessed in the future to identify ideal
and the possible changes in their relation time-energy for microwave
mechanical properties. disinfection of biofilms formed by Candida
species.
Reinforcing this assumption, it was showed
accumulative effects due to two protocols Clinical assessments of the effects of
of disinfection by microwave energy on microwave disinfection on the retention
surface roughness and adaptation of and stability of denture base are also
complete denture bases made with required. Investigations should be made
microwaved and boiled resins. The results about the long term effects of disinfection
showed that microwave irradiation at 690 techniques on the hardness and roughness
W for 6 minutes promoted changes on of acrylic resins used in complete and
surface roughness and adaptation of the partial removable dentures. Although there
denture made with both resin types, but is need for further confirmatory
the clinical use would not be affected by the investigations, the results of this study
magnitude of the changes (Campos et al., suggest that denture microwave
2009). disinfection seems to be a promising
method on the prevention of Candida
Inoculed by different Candida species (C. infestation.
albicans, C. dubliniensis and C. tropicalis),
previous study has shown that the Conclusion
microwave irradiation at 650 W for 3
minutes was effective for disinfection of Microwaved samples at 650 W for 3
different acrylic resin types from a same minutes showed different decreases in the
manufacturer (Silva et al., 2013). In the number of viable colonies for biofilms
same methodological conditions, the formed by C. albicans, C. dubliniensis and C.
current study showed that the microwave tropicalis. In general, microwave
effectivity was also evident for biofilms irradiation at 650 W for 3 minutes can be
from Candida species (C. albicans, C. an alternative method to disinfect different
dubliniensis and C. tropicalis) inoculated denture acrylic resins contaminated by
on acrylic resins from different different Candida species.
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Biofilm Formation

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Journal of Research and Practice in Dentistry

Ana Patricia Fernandes Macedo Dantas1, Rafael Leonardo Xediek Consani1,

Correspondence should be addressed to: Rafael Leonardo Xediek Consani; rconsani@fop.unicamp.br

Received 31 October 2013; Accepted 19 December 2013; Published 21 January 2014

Academic Editor: Arife Doǧan

Keywords: Acrylic resin, Candida sp, biofilm, disinfection, microwave.

Introduction Various drugs have been used for the

Despite possessing bactericidal action, included in plastic (Classico Dental

Microbial suspensions were Microwave Irradiation:

Results same resin was not significant, as well as

Treatment Acrylic resin C. albicans C. dubliniensis C. tropicalis

Budtz-Jögersen, E. (1990). “Etiology, with Selected Bacteria,” Journal of

Consani, R. L. X., Mesquita, M. F., Nobilo, M. Jagger, D. C. & Harrison, A. (1995).

Silva, M., Consani, R., Sardi, J., Mesquita, M.,

Silva, M. M., Mima, E. G., Colombo, A. L.,

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