Lasers Med Sci (2011) 26:341–348
DOI 10.1007/s10103-010-0852-3
ORIGINAL ARTICLE
Susceptibility of Candida albicans, Staphylococcus aureus,
and Streptococcus mutans biofilms to photodynamic
inactivation: an in vitro study
Cristiane Aparecida Pereira & Rogério Lima Romeiro &
Anna Carolina Borges Pereira Costa & Ana Karina Silva Machado &
Juliana Campos Junqueira & Antonio Olavo Cardoso Jorge
Received: 26 March 2010 / Accepted: 11 October 2010 / Published online: 11 November 2010
# Springer-Verlag London Ltd 2010
Abstract The purpose of this study was to evaluate specific
effects of photodynamic inactivation (PDI) using methylene
blue as photosensitizer and low-power laser irradiation on the
viability of single-, dual-, and three-species biofilms formed
by C. albicans, S. aureus, and S. mutans. Biofilms were
grown in acrylic discs immersed in sterile brain heart
infusion broth (BHI) containing 5% sucrose, inoculated with
microbial suspension (106 cells/ml) and incubated for 5 days.
On the fifth day, the effects of the methylene blue (MB)
photosensitizer at a concentration of 0.1 mg/ml for 5 min and
InGaAlP laser (660 nm) for 98 s, alone and conjugated were
evaluated. Next, the discs were placed in tubes with sterile
physiological solution [0.9% sodium chloride (NaCl)] and
sonicated for to disperse the biofilms. Ten-fold serial
C. A. Pereira : R. L. Romeiro : A. C. B. P. Costa :
A. K. S. Machado : J. C. Junqueira : A. O. C. Jorge (*)
Department of Biosciences and Oral Diagnosis,
School of Dentistry of São José dos Campos,
UNESP – Univ Estadual Paulista,
Francisco José Longo 777, São José dos Campos,
São Dimas CEP: 12245-000 SP, Brazil
e-mail: olavojorge@fosjc.unesp.br
C. A. Pereira
e-mail: cricabio@gmail.com
R. L. Romeiro
e-mail: rogerio.romeiro@terra.com.br
A. C. B. P. Costa
e-mail: carol_biolog@yahoo.com.br
A. K. S. Machado
e-mail: anakarinasm@ig.com.br
J. C. Junqueira
e-mail: juliana@fosjc.unesp.br
dilutions were carried and aliquots seeded in selective agar,
which were then incubated for 48 h. Then the numbers CFU/
ml (log10) were counted and analyzed statistically (ANOVA,
Tukey test, p<0.05). Scanning electron microscopy (SEM)
on discs treated with PDI and control biofilms groups was
performed. Significant decreases in the viability of all
microorganisms were observed for biofilms exposed to PDI
mediated by MB dye. Reductions (log10) of single-species
biofilms were greater (2.32–3.29) than the association of
biofilms (1.00–2.44). Scanning electron microscopy micro-
graphs suggested that lethal photosensitization occurred
predominantly in the outermost layers of the biofilms. The
results showed that PDI mediated by MB dye, might be a
useful approach for the control of oral biofilms.
Keywords Biofilm . Candida albicans . Staphylococcus
aureus . Streptococcus mutans . Photodynamic inactivation
Introduction
The oral cavity is heavily colonized by a complex, relatively
specific and highly interrelated range of microorganisms,
which are organized in biofilms. Microbial biofilms are
composed of microorganisms adhered both to each other
and/or to surfaces or interfaces and embedded in an
extracellular polymeric matrix, which includes water and
nutrient channels [1]. The presence of pathogenic micro-
organisms within biofilms can lead to pathological processes
such as caries [2] and periodontal disease [3, 4].
The mutans group of streptococci, mainly Streptococcus
mutans species, is the main etiological agent of smooth
surface caries [5]. The accumulation of streptococci in
342
dental surface is considered a critical factor for the
development of cariogenic biofilms, due to the production
of acids and a consequent oral pH reduction, leading to a
higher probability of dental tissues demineralization [2, 6].
Candida albicans can be frequently isolated from the
oral cavity of healthy patients. It is the main cause of
different forms of oral, superficial, or systemic candidiasis
[7]. Furthermore, some reports concerning the isolation of
C. albicans from dental biofilms [8] have demonstrated that
these yeast species are not only associated with the
development of oral candidiasis but also with the patho-
genesis of caries and periodontal diseases [4].
Staphylococcus aureus is another opportunistic patho-
genic microorganism that can be isolated from the oral
cavity and has developed antibiotic resistance to penicillin
by beta-lactamase plasmid [9]. Biofilms formed by S.
aureus are communities embedded in a matrix of extracel-
lular polymers, mainly composed of intercellular adhesion
polysaccharides [10], which reduce the penetration of
antimicrobial agents and represent a significant therapeutic
barrier for many antibiotics. In addition, it has the ability to
produce extracellular enzymes, which have shown very
aggressive behavior [11].
Mechanical removal of biofilms has been the most
frequently used method for periodontal disease treatment.
Antimicrobial agents have been also used, but biofilm
species exhibit several antibiotic-resistance mechanisms
[12, 13]. Additionally, disruption of oral microbiota and
the difficulty to maintain therapeutic concentrations of
antimicrobials in the oral cavity are concerns associated
with the use of these agents [14].
A possible alternative to reduce biofilms is the photo-
dynamic inactivation (PDI). This therapy is based on the
use of photosensitizers and an appropriate wavelength of
visible light. Once exposed to light, the photosensitizers are
activated to a short-lived excited state that then converts to
a long-lived triplet state. This state may generate free
radicals or superoxide ions resulting from hydrogen or
electron transfer (type I) and can produce oxygen singlets
(type II). These products are capable of reacting with
cellular components, such as proteins, nucleic acids, and
lipids, causing irreversible damage and cell death [15–17].
Light and drug are non-toxic by themselves; hence, only
cells containing photosensitizer and receiving light are
affected by the treatment. This is the opportunity to achieve
selectivity and target specific areas of the mouth/biofilm
with this treatment. Moreover, PDI is a simple non-
mechanical therapy to apply. It can be also used for the
treatment of medically compromised individuals, children,
and handicapped people [18].
The aim of this study was to evaluate the effects of PDI
on the viability of in vitro biofilms formed by C. albicans,
S. aureus, S. mutans, and their associations on acrylic resin.
Lasers Med Sci (2011) 26:341–348
Materials and methods
Production of biofilms
Three reference strains [American Type Culture Collection
(ATCC)], C. albicans (ATCC 18804), S. aureus (ATCC
6538), and S. mutans (ATCC 35688), were used in the
study.
Standard suspensions of each strain, with optical density
relative to 106 cells/ml were prepared. For this purpose, C.
albicans were seeded onto Sabouraud dextrose agar (Difco,
Detroit, MI, USA), S. aureus and S. mutans were seeded
onto Brain Heart Infusion agar (BHI, Difco, Detroit, MI,
USA), and incubated at 37°C for 24 h. All experiments
with strains of S. mutans were incubated at 37°C and a
partial pressure of 5% CO2.
After incubation, the growth was suspended in sterile
physiological solution [0.9% sodium chloride (NaCl)] and
the number of cells in suspension was counted in a
spectrophotometer (B582, Micronal, São Paulo, Brazil).
The parameters of optical density and wavelength used
were, respectively: 0.284 and 530 nm for C. albicans, 0.374
and 490 nm for S. aureus, and 0.620 and 398 nm for S.
mutans.
To grow the biofilms, we used 280 acrylic resin (AC) discs
11 mm in diameter (Clássico, São Paulo, Brazil) sterilized in a
20-kGy gamma radiation chamber (cobalt 60) for 6
h (Embrarad, São Paulo, Brazil). After sterilization, the AC
discs were randomly assigned to seven different groups (40
for each group): GI- C. albicans, GII- S. aureus, GIII- S.
mutans, GIV- C. albicans and S. aureus, GV- C. albicans
and S. mutans, GVI- S. aureus and S. mutans, and GVII- C.
albicans, S. aureus and S. mutans. Afterwards, they were
placed in the first row of plates, 24 wells (Costar Corning,
NY, USA) with 2 ml of sterile brain heart infusion broth
(BHI) added with 5% sucrose. For the formation of single-
species biofilms (GI, GII e GIII), the wells were inoculated
with 0.1 ml of C. albicans, S. aureus, or S. mutans
suspension. For dual and multi-species biofilms (GIV, GV,
GVI e GVII), the wells were inoculated with 0.1 ml of a
suspension of each microorganism used in the association.
The AC discs were then incubated at 37°C for 5 days. The
media was not changed during the period of incubation.
Photosensitizer and light source
Methylene blue (MB) (Sigma, Poole, UK), at a concentration
of 0.1 mg/ml was used for the sensitization of biofilms.
Photosensitizer solution was prepared by dissolution of the
methylene blue in sterile physiological solution (0.9% NaCl)
and filtration through a sterile 0.22-μm Millipore membrane
(São Paulo, Brazil). After filtration, the photosensitizer
solution was stored in the dark.
Lasers Med Sci (2011) 26:341–348 343

Fig. 1 Mean values (n=10) and

standard deviation of CFU/ml
(log10) of single-species bio-
films for different experimental
conditions: sensitized with MB
(S+L-), exposed to laser (S-L+),
control (S-L-), and association
of MB and laser (S+L+). Tukey
test for each group tested. Values
followed by different capital
letters indicate a significant dif-
ference between the four groups
S+L-, S-L+, S-L-, and S+L+

The light source used was an indium-gallium-alumini-
um-phosphide (InGaAlP) laser (Photon lase III, DMC
Equipamentos, São Carlos, Brazil), with a wavelength of
660 nm, output power of 100 mW, and energy density of
350 J/cm2. The laser was at a distance of 0.05 cm and AC
discs with biofilms illuminated the whole area of the discs
(0.94 cm2) for a period of 98 s.
Four experimental conditions were tested for each group:
(a) S-L+, biofilms exposed to laser light, but not sensitized
with MB (n=10); (b) S+L-, biofilms sensitized with MB, but
not exposed to laser (n=10); (c) S-L-, control biofilms not
sensitized with MB and not exposed to laser light (n=10);
and, (d) S+L+, biofilms sensitized with MB and exposed to
laser (n=10).
Photodynamic inactivation of biofilms
On the fifth day, the AC discs containing the biofilms were
aseptically transferred to the second and third rows of the
plate (24 wells) and washed twice with sterile physiological
solution (0.9% NaCl), in order to remove loosely bound
material. Following this, AC discs containing the biofilms
were placed in the fourth row of the plate (24 wells).
According to the experimental conditions described,
0.1 ml of the photosensitizer was added for groups S+L-
and S+L+, whereas 0.1 ml physiological solution (0.9%
NaCl) was added for groups S-L+ and S-L-, and subse-
quently left in the dark for 5 min (pre-irradiation time). The
biofilms of groups S-L+ and S+L+ was then irradiated
according to the protocol described. Irradiation was
performed under aseptic conditions under a laminar flow
hood in the dark.
At the end of the PDI experiments, the microbial biofilms
were detached from the discs in sterile physiological solution
(0.9% NaCl) with the aid of sonicator (Sonoplus HD 2200,
50 W, Bandelin Electronic) for 30 s. Ten-fold serial dilutions
were carried and aliquots of 0.1 ml were seeded in duplicate
onto agar. Suspensions of single-species C. albicans biofilms
were seeded onto Sabouraud dextrose agar, while the S.
aureus and S. mutans ones were plated onto BHI agar. To
identify and quantify the remaining species in mixed
suspensions of biofilms were used selective culture agar for
each microorganism: (a) Sabouraud dextrose agar with
50 mg/l chloramphenicol (União Química, São Paulo,
Brazil) to C. albicans; (b) Mannitol salt agar (Difco, Detroit,
MI, USA) to S. aureus; and, (c) Mitis Salivarius agar (Difco,

Fig. 2 Mean values (n=10) and

standard deviation of CFU/ml
(log10) of dual-species biofilms
formed by C. albicans and S.
aureus for different experimental
conditions: sensitized with MB
(S+L-), exposed to laser (S-L+),
control (S-L-), and association of
MB and laser (S+L+). Tukey test
for each group tested. Values
followed by different capital let-
ters indicate a significant differ-
ence between the four groups
S+L-, S-L+, S-L-, and S+L+
344 Lasers Med Sci (2011) 26:341–348

Fig. 3 Mean values (n = 10)

and standard deviation of
CFU/ml (log10) of dual-species
biofilms formed by C. albicans
and S. mutans for different
experimental conditions: sensi-
tized with MB (S+L-), exposed
to laser (S-L+), control (S-L-),
and association of MB and laser
(S+L+). Tukey test for each
group tested. Values followed
by different capital letters indi-
cate a significant difference
between the four groups S+L-,
S-L+, S-L-, and S+L+

Detroit, MI, USA) supplemented with 0.2 IU/ml bacitracin Results

(União Química, São Paulo, Brazil) and 15% sucrose to S.
mutans. After 48 h of incubation, the number of colony-
forming units per milliliter (CFU/ml) was determined. The
results were log-transformed (log10) and analyzed by
analysis of variance (ANOVA) and the Tukey test. A p
value <0.05 was considered to indicate a statistically
significant difference.
Scanning electron microscopy
Scanning electron microscopy (SEM) was used to illustrate
the effects, before (S-L-) and after photodynamic inactivation
was carried out (S+L+), on biofilms formed on the AC discs.
The discs were fixed for 1 h in 2.5% glutaraldehyde and
dehydrated in several ethanol washes (10, 25, 50, 75, and 90%
for 20 min and 100% for 1 h). The samples were dried
overnight in a bacteriological incubator at 37°C, and
afterwards they were mounted on aluminum stubs, with
copper tape, coated with gold in a low-pressure atmosphere
with an ion sputter coater (Denton Vacuum Desk II, Denton
Vacuum, USA). The surface topographies of the biofilms
were visualized and photographed using a scanning electron
microscope (JSM-5310, JEOL, Japan), operating at 15 kV in
increments of 1,000 and 5,000 times.
Mean and standard deviation values of the CFU/ml (log10)
obtained in the four experimental conditions tested for each
biofilms group are shown in Figs. 1, 2, 3, 4, and 5.
The means of reduction (CFU log10) and p values
obtained for the biofilms submitted to PDI mediated by
MB dye (S+L+) in relation to the untreated controls
biofilms (S-L-) are shown in Table 1.
Scanning electron microscopy (SEM) was used to evaluate
the biofilms as well as the effects of the PDI on these
microbial communities, and micrographs are shown in Fig. 6.
For biofilms of the control groups formed by C. albicans
(GI: A and B) alone, aggregated blastospores were observed.
They were covering the surface of the substratum containing
light extracellular matrix. The biofilms of the control groups
formed by S. aureus (GII: A and B) or S. mutans (GIII: A
and B) were composed of aggregated cocci and extracellular
matrix. However, the extracellular matrix in the S. mutans
biofilm was more abundant. The multi-species control
biofilms of C. albicans and S. aureus (GIV: A and B) and
C. albicans and S. mutans (GV: A and B) were formed by
yeasts cells and aggregated bacteria on the substratum with
abundant extracellular matrix and the biofilm formed by S.
aureus and S. mutans (GVI: A and B) was constituted by

Fig. 4 Mean values (n=10) and

standard deviation of CFU/ml
(log10) of dual-species biofilms
formed by S. aureus and S.
mutans associated-biofilms for
different experimental condi-
tions: sensitized with MB (S+L-),
exposed with laser (S-L+), con-
trol (S-L-), and association of
MB and laser (S+L+). Tukey test
for each group tested. Values
followed by different capital let-
ters indicate a significant differ-
ence between the four groups
S+L-, S-L+, S-L-, and S+L+
Lasers Med Sci (2011) 26:341–348 345

Fig. 5 Mean values (n=10) and

standard deviation of CFU/ml
(log10) of three-species biofilms
formed by C. albicans, S. aure-
us, and S. mutans for different
experimental conditions: sensi-
tized with MB (S+L-), exposed
to laser (S-L+), control (S-L-),
and association of MB and laser
(S+L+). Tukey test for each
group tested. Values followed by
different capital letters indicate
a significant difference between
the four groups S+L-, S-L+,
S-L-, and S+L+

abundant extracellular matrix covering the substratum and
the cells. For the multi-species biofilm control containing
three microorganisms (GVII: A and B), there was a
formation of a complex biofilm formed by yeasts and
bacteria embedded in an abundant extracellular matrix. The
biofilms formed by single species or multi-species were
damaged by PDI (C and D), exhibiting a few cells on the
substratum and a decrease of the extracellular matrix. These
effects were more prominent in biofilms formed by single
species.
Discussion
A series of advantages found for a microbial colony can be
attributed to biofilms. There is better communication
between the cells due to their continuity, which certainly
favors biochemical activities and greater proliferation and
easier access to niches and resources. These could not be
used by individual cells due to the group defense against
microorganisms by saliva and antimicrobial agents [19].
Therefore, it has been essential to investigate the action of
PDI in the control of biofilms.
The antimicrobial effect of MB (0.1 mg/ml) associated
with a InGaAlP laser light (660 nm) on the viability of
single-, dual-, and three-species biofilms of C. albicans, S.
aureus, and S. mutans has not been previously studied.
Therefore, the results of this investigation have been of
extreme relevance due to the significant reductions
achieved.
In the present study, biofilms formed by only one type of
species were more sensitive to PDI mediated by MB dye,
compared to the ones formed by two and three species of
microorganisms. These data demonstrate that the more
complex the composition of the biofilms, the more resistant
it seems to be to the PDI process. The interactions between
the different matrix polymers, produced by different
microorganism, might result in a more viscous matrix.
Such a finding has been reported by Skillman et al. [20]
during a study of mixed species bacterial biofilms of
Enterobacter agglomerans and Klebsiella pneumoniae;
increased matrix viscosity was advanced as a possible
explanation for the enhanced resistance to disinfection of
these mixed-species biofilms. Rheological interactions
between polysaccharides from Pseudomonas cepacia and
P. aeruginosa have also been shown to decrease the
diffusion and antimicrobial activity of antibiotics [21].
With regard to the biofilms containing only one type of
species, the ones formed by strains of C. albicans (GI) were
the most resistant to the PDI mediated by MB dye

Table 1 Means of reduction

(CFU log10) and p values Group Reductions microbial biofilms CFU log10
obtained for the biofilms sub-
mitted to PDI mediated by MB C. albicans p value* S. aureus p value* S. mutans p value*
dye (S+L+) in relation to the
untreated controls biofilms (S-L-) GI 2.32±0.13 0.001 – – – –
GII – – 3.29±0.23 0.001 – –
GIII – – – – 2.81±0.12 0.001
GIV 1.90±0.11 0.001 2.43±0.14 0.001 – –
GV 1.72±0.15 0.001 – – 2.21±0.15 0.001
GVI – – 2.22±0.17 0.001 2.10±0.16 0.001
*p<0.05, significant statistical GVII 1.00±0.14 0.001 1.47±0.23 0.001 1.25±0.09 0.001
difference (ANOVA, Tukey test)
346 Lasers Med Sci (2011) 26:341–348

Fig. 6 Scanning electron micro-

scope micrographs of different
biofilms. GI: C. albicans ; GII: S.
aureus; GIII – S. mutans; GIV:
C. albicans and S. aureus; GV:
C. albicans and S. mutans; GVI:
S. aureus and S. mutans; and,
GVII: C. albicans, S. aureus and
S. mutans. Images a and b refer
to control biofilms (S-L-) - not
sensitized with MB and not
exposed to laser light. Images c
and d show sensitized biofilms
with MB for 5 min and exposed
to laser for 98 s (S+L+). Magni-
fication: a and c = 5000×; b and
d = 10000×
Lasers Med Sci (2011) 26:341–348
(compared to the other groups GII and GIII). It has been
observed that C. albicans and other yeasts have shown a
greater death resistance, when treated with PDI, compared
to Gram-positive bacteria. This factor might happen due to
the presence of a nuclear membrane in the structure of
yeasts, and also the greater cell size and reduced number of
target of singlet oxygen [16, 22].
The average reduction of biofilms formed by C. albicans in
this study was 2.32 log10 compared to the control groups.
Donnelly et al. [23], reported that 5 mg/ml of toluidine blue
O (TBO), 30 min of pre-irradiation time, and energy density
of 200 J/cm2 with a wavelength of 660 nm showed high
death levels (1.5 log10 to 6.5 log10) of C. albicans in biofilms.
On the other hand, the authors stated that 2 mg/ml of TBO
and incubation time of 30 min were sufficient for total death
of C. albicans in planktonic cultures, after light irradiation.
Biofilms formed by S. aureus (GII) were more sensitive
to the PDI mediated by MB dye and showed an average
reduction of 3.29 log10. Sharma et al. [24] found reduction
of 4.5 log10 CFU of methicillin-resistant S. aureus biofilms,
using TBO, followed by irradiation with 640-nm laser
diode.
PDI mediated by MB dye promoted an average
reduction of 2.81 log10 of S. mutans (GIII) biofilms. Wood
et al. [25] found similar results, 1.5–2.6 log10 CFU of S.
mutans by using MB photosensitizer (0.008 mg/ml) and a
400-W source of light. Also, Zanin et al. [26] obtained
reduced values of 2.10–3.11 log10 CFU of S. mutans
organized in biofilms treated with TBO (0.1 mg/ml) and
He-Ne, followed by laser irradiation for 5, 15, and 30 min.
The average reduction values varied between 1.00 to
2.44 log10 CFU for the biofilms formed by two and three
species. Qin et al. [27] also found minimum reduction of
about 21% of microorganism death of biofilms collected
from patients with periodontitis, by means of TBO
application (1 mg/ml) and diode laser irradiation for 57 s.
Multi-species microbial biofilms developed from sam-
ples of dental plaque were exposed to MB (0.025 mg/ml)
and irradiated with a 665-nm diode laser for 5 min,
resulting in 32% bacterial reduction. PDT promoted a
reduction of 63% when observing isolated bacteria from the
same dental plaque and grown in planktonic cultures [28].
On the contrary, Müller et al. [29] reported a bacterial
reduction of less than 1 log10 of oral biofilm developed in
discs of bovine enamel after sensitization with MB,
followed by 665-nm red light irradiation for 60 s.
In the relation to isolated effects of the InGaAlP laser
irradiation (S-L+ group), reduction in the numbers of CFU/
ml from the biofilms were not observed, suggesting
microbial resistance of these species to a luminous source.
MB showed no cytotoxic effect for the microorganisms
tested. However, these data results are not in agreement
with the ones reported in previous investigations, where
347
MB by itself showed both antifungal and antibacterial
activity [30, 31].
Additionally, the divergent results found in our study might
have occurred due to the lack of a pre-defined protocol for
PDI use. The great variety of biofilm models, concentration,
period of incubation, type of photosensitizer, as well as the
physiological state of microorganisms, exposure time, and the
laser energy density, might also influence PDI results [32].
In SEM images, large cellular aggregates surrounded by
extracellular matrix could be observed in untreated
control biofilms (S-L-). However, the biofilms submitted
to PDI (S+L+) exhibited less celular aggregated. These data
suggest that lethal photosensitization occurred mainly in the
outermost layers of biofilms, which probably happened due
to the inability of the photosensitizer to diffuse through
these structures. Confocal laser scanning micrographs of
biofilms obtained from microorganisms found in saliva
showed that the photodestruction occurred predominantly
in the outermost layers of the biofilms, after being exposed
to TBO and light [33].
Based on the current results, it could be concluded that
biofilms formed in vitro by C. albicans, S. aureus, and S.
mutans, both isolated and associated, were sensitive to PDI
mediated by MB dye.
Acknowledgements This work was supported by the Fundação de
Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil (Grant
09/52048-1). The authors Cristiane A. Pereira and Anna Carolina B. P.
Costa are grateful to FAPESP by the scholarships provided (Processes
2010/00879-4 and 2009/12005-1).
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