Lasers Med Sci

DOI 10.1007/s10103-015-1826-2

ORIGINAL ARTICLE

In vitro effectiveness of 455-nm blue LED to reduce the load

of Staphylococcus aureus and Candida albicans biofilms
in compact bone tissue
Luciano Pereira Rosa 1,2 & Francine Cristina da Silva 1 & Magda Souza Viana 1 &
Giselle Andrade Meira 1

Received: 26 February 2015 / Accepted: 19 October 2015

# Springer-Verlag London 2015

Abstract The aim of this study was to evaluate the effective-
ness of a 455-nm blue light-emitting diode (LED), at different
application times, to reduce the load of Staphylococcus aureus
and Candida albicans biofilms applied to compact bone tis-
sue. The microorganisms S. aureus (ATCC 25923) and
C. albicans (ATCC 18804) were used to form biofilms on
160 specimens of compact bones that had been divided into
eight experimental groups (n=10) for each microorganism,
according to the times of application of the 455-nm blue
LED (1, 2, 3, 4, 5, 7, and 10 min) with an irradiance of
75 mW/cm2. After LED application, decimal dilutions of mi-
croorganisms were performed, plated on BHI or Sabouraud
agar and incubated for 24 h/35 °C to obtain CFU/mL counts.
The findings were statistically analyzed using a ANOVA 5 %.
For the group of S. aureus biofilms, all groups of 455-nm LED
application differ compared with the control group (p<0.05),
in which no treatment was given. The largest reduction was
obtained in the group receiving LED for 10 min (p=0.00);
within this group, a 3.2 log reduction was observed. For the
C. albicans biofilms, only those samples receiving 3, 7, and
* Luciano Pereira Rosa
drlucianorosa@yahoo.com.br
Francine Cristina da Silva
drfransilva@yahoo.com.br
Magda Souza Viana
magda—viana@hotmail.com
Giselle Andrade Meira
gih.andradem@gmail.com
1
Multidisciplinary Health Institute, UFBA, Vitória da Conquista, BA,
Brazil
2
Present address: Instituto Multidisciplinar em Saúde, Universidade
Federal da Bahia, Rua Rio de Contas, 58. Candeias, Vitória da
Conquista, BA CEP: 45029-094, Brazil
10 min of LED application presented a significant difference
compared with the control group (p<0.00), indicating that
longer application times are required to achieve efficacy.
The results of this study show that 455-nm LED light was
effective to reduce the load of S. aureus and C. albicans
biofilms, especially during 10 min of application.
Keywords Bone . Blue light . Phototherapy . Staphylococcus
aureus . Candida albicans
Introduction
The main microbial agent isolated from bone infections is
Staphylococcus aureus, which is responsible for 60 to 90 %
of such infections in children and more than 95 % in adults
[1–4]. Garcia et al. [5] reported that S. aureus accounted for
50 % of the microorganisms isolated from bone infections in
their study. In another study, Lima and Zumiotti [3] reported
an incidence of 48.5 to 70 % of the studied cases related to
S. aureus. With the increasing occurrence of immunocompro-
mised individuals, diagnoses of candidemia and invasive can-
didiasis due to Candida albicans and other fungi have in-
creased dramatically in recent years [6, 7]. Arias et al. [6]
reported clinical cases of severe bone infection due to
C. albicans; using optical microscopy, the researchers ob-
served bone and fibrous tissue with microabscesses and the
presence of yeast within the tissues. In a systematic review,
Figueiredo et al. [8] found 783 cases of fungal osteomyelitis
reported in the literature after 1990, with Candida sp. being
the most common etiologic agent.
These infections, which are called osteomyelitis, are pri-
marily related to fractures but can also occur due to internal
dissemination via the bloodstream, thus facilitating the micro-
organism’s access to bone tissues. Osteomyelitis, which is

considered a true emergency, jeopardizes the reconstitution of
the bone tissue, which is essential for a quick and accurate
diagnosis and the subsequent appropriate treatment. Currently,
treatment is based on fracture reduction and antibiotic therapy
for prolonged periods, which may promote the emergence of
microbial resistance, either by treatment neglect or by chro-
nicity of the disease due to symptoms decrease [7, 9].
The increased resistance of S. aureus and C. albicans to
antibiotic therapy indicates the need for alternative treatments
for infections with these microorganisms. However, new ther-
apeutic approaches that employ light sources such as lasers
and light-emitting diodes (LEDs) have recently been identi-
fied as potential treatments for infections because they have
phototoxic effects on microorganisms, thereby eliminating the
possible expression of resistance to conventional drugs [7].
Blue light (405–470 nm) without the addition of exogenous
photosensitizers (PS) has intrinsic antimicrobial effects and
shows fewer deleterious effects to mammalian cells than does
ultraviolet irradiation [10, 11].
. The aim of this study was to evaluate the effectiveness of
455-nm blue LED, at different application times, to reduce the
load of S. aureus and C. albicans biofilms applied to compact
bone tissue specimens.
Materials and methods
Bone specimens
One hundred sixty specimens of compact bone were obtained
from the diaphysis regions of bovine tibias using carborun-
dum discs (Carborundum Abrasives SA, Recife, Pernambuco,
Brazil); the resulting specimens had dimensions of 5×2×
2 mm. The specimens were rinsed in saline (0.85 % NaCl)
and sterilized at 121 °C for 15 min.
This study was approved by the Animal Research Ethics
Committee (UNESP Dental School, São José dos Campos-
SP) under protocol number 05/2008-PA/CEP. The study strict-
ly followed all of the ethical principles set forth by the Decla-
ration of Helsinki (2000).
Microorganisms
Reference strains [American Type Culture Collection
(ATCC)] of S. aureus (ATCC 25923) and C. albicans (ATCC
18804) were used to generate single-species biofilms in bone
tissues for the evaluation of the proposed treatments. Micro-
bial suspensions of S. aureus and C. albicans containing 10×6
cells/mL were prepared by seeding 24-h cultures onto Manni-
tol agar (Difco, Detroit, MI, USA) and Sabouraud agar (Difco,
Detroit, MI, USA) using a spectrophotometer, for the respec-
tive microorganisms.
Lasers Med Sci
In vitro formation of single-species biofilms
Biofilm formation was performed according to the procedure
described in the previous works of Rosa et al. [12]. Compact
bones were used as substrates for growing in vitro S. aureus
and C. albicans single-species biofilms. To perform the exper-
iment, the specimens were distributed according to microor-
ganisms (80 S. aureus and 80 C. albicans) into 160 24-well
flat-bottom microtiter plates. The plates were organized ac-
cording to the experimental groups (eight plates with ten com-
pact bone specimens for S. aureus and eight plates with ten
compact bone specimens for C. albicans). In each plate, ten
wells were filled with 2 mL of sterile TSB (Tryptic Soy Broth,
Difco, Detroit, MI, USA) or Sabouraud broth (Difco, Detroit,
MI, USA) and inoculated with 0.1 mL of the S. aureus or
C. albicans standard suspension. The 24-well flat-bottom mi-
crotiter plates were incubated for 14 days at 35 °C. The hu-
midity and nutrition conditions were evaluated daily.
Light source
A Twin Flex Evolution ® device (MMOptics, São Carlos-SP,
Brazil) fitted with a 455-nm light probe was used to irradiate
the biofilms. This device delivers light through an acrylic-
polished rectangular guide containing three LEDs, spot of
9.3 cm2, bandwidth of 435–475 nm, 700 mW average power,
and 75 mW/cm2 irradiance. The probe was positioned perpen-
dicular to the plate, maintaining a distance of 2 cm from each
specimen. For the application times mentioned, the energy
densities were obtained as follows: 4.51, 9.03, 13.54, 18.06,
22.58, 31.61, and 45.16 J/cm2.
Experimental conditions
After the incubation period, each specimen was transferred to
a sterile 2.5 mL polypropylene microtube containing 1 mL of
sterile saline and then agitated in a shaker apparatus for 1 min,
obtaining an initial suspension. From the initial suspension,
decimal dilutions of 10–1, 10–2, and 10–3 were made. The
initial suspension and the other dilutions were plated in dupli-
cate on Petri dishes containing TSA (Tryptic Soy Agar, Difco,
Detroit, MI, USA) culture medium for S. aureus or Sabouraud
agar (Difco, Detroit, MI, USA) culture medium for
C. albicans. The plates were incubated at 35 °C for 24 h. After
the incubation period, those plates containing from 30 to 300
colonies were used to calculate the colony forming units per
milliliter (CFU/mL) using a colony counter device (Spencer,
Santo André, São Paulo, Brazil).
Statistical analysis
The logarithm of colony-forming unit per milliliter (CFU/mL)
(log10 CFU/mL) was calculated for each specimen.
Lasers Med Sci
Adherence to the assumptions of normality and homoscedas-
ticity was verified using the Kolmogorov-Smirnov normality
test, and the data exhibited a normal distribution. Using an
ANOVA with an alpha value of 5 % followed by the Tukey
test, comparisons were made between experimental treat-
ments; comparisons of the mean CFU/mL of the experimental
treatments were also evaluated.
Results
Figure 1 shows the mean and standard deviation of the S. aureus
and C. albicans groups. All groups of S. aureus biofilms re-
ceiving the 455-nm LED application differed compared with
the control group (p<0.05), in which no treatment was given.
The largest reduction was obtained in the group of application
of LED for 10 min (p=0.00), in which a 3.2 log reduction was
observed. There was no significant difference when comparing
the groups receiving 1-, 2-, 3-, 4-, and 5-min LED application,
as they indicated the same reduction in log CFU/mL of
S. aureus biofilms. Furthermore, LED application for 7 min
did not differ statistically from 2, 3, and 4 min of application.
For the group of C. albicans biofilms, only those treated
with 3, 7, and 10 min of LED application exhibited a signif-
icant difference compared with the control group (p<0.00),
indicating that for the microorganism in question, longer ap-
plication times are required to achieve efficacy. Additionally,
for this group, the greater reduction in log CFU/mL was ob-
tained for the group of 10-min LED application (2.3 log re-
duction). The group that demonstrated less effectiveness was
the group receiving 2 min of LED application; in this group,
an increase of 0.1 log CFU/ml was observed compared with
the control group. There was no significant difference between
Fig. 1 The means, expressed in units of log10 CFU/mL, and standard
deviations (error bars) were obtained using an ANOVA with an alpha
value of 5 % for the LED treatment groups for the specimens infected
with S. aureus and C. albicans biofilms
the groups receiving 1, 3, 4, 5, and 7 min of 455-nm LED
application on C. albicans biofilms.
In comparing the groups of 455-nm LED application on the
different microorganisms, the effectiveness in the reduction of
log CFU/ml was similar for the times of 1, 3, 4, 5, 7, and
10 min. Only the application time of 2 min presented a signif-
icant difference (p=0.006) in obtaining a greater reduction in
log CFU/mL among the group of S. aureus biofilms.
Discussion
The inappropriate usage of antimicrobials has made the treat-
ment of infections challenging due to the capability of micro-
organisms to develop resistance to these agents [13–17].
One of the microorganisms that are most commonly cited
as being able to express this resistance is S. aureus, which is
often associated with severe infections [3, 9, 13, 16]. It is the
most important representative of the staphylococcal group,
which causes clinically significant infections within immuno-
compromised patients. It lives commensally on human skin,
nares, and mucosal surfaces as an opportunistic pathogen that
possesses the ability to infect, invade, persist, and replicate in
many human tissues [3, 9].
For these reasons, the search for new therapeutic modalities
has been undertaken to reduce the production of antimicrobial
resistance [13–17]. The use of lights, whether associated or
not with photosensitizing dyes, has been studied to fill this gap
because the literature does not indicate that microorganisms
can develop resistance to the phototoxic effects of light [18].
Many types of lights have been studied with antimicrobial
purpose in in vitro studies or clinical trials [19, 20]; among
them, the most frequently used are lasers and LEDs of various
wavelengths. Studies in the literature have shown the effec-
tiveness of visible blue light against Propionibacterium acnes
[21], Enterococcus faecalis [22], Porphyromonas gingivalis
[23], Fusobacterium nucleatum [23], S. aureus [13, 14], and
Gram-negative microorganisms. [23] In this study, blue LED
with a wavelength of 455 nm was used to inactivate the
S. aureus and C. albicans biofilms produced in vitro in com-
pact bone specimens. The choice of this machine model with
this specific wavelength was based on availability because it is
easily found and routinely used in dental offices.
Lights are able to produce the death of microorganisms
through the formation of reactive oxygen species (ROS) that
result from light absorption by endogenous cellular photosen-
sitizers [24]. Low fluxes of ROS serve as cellular signaling
messengers that induce processes such as transcription factor
activation, gene expression, muscle contraction, and cell
growth [25]. To be influenced by light, the cells must possess
suitable photosensitizers. Potential endogenous photosensi-
tizers that absorb light in the visible range include porphyrins
[26], cytochromes [27], pyridine cofactors, nicotinamide

adenine dinucleotide (NADH) and nicotinamide adenine di-
nucleotide phosphate (NADPH) [28], Fe-S clusters, and fla-
vins [29]. The plasma membrane contains the NADPH oxi-
dase system (a transmembrane complex containing both heme
molecules and flavins), which is also a potential photosensi-
tizer because it produces superoxide radicals that are capable
of destroying pathogens [30].
Several studies in the literature have evaluated the effects of
antimicrobial agents on biofilms, and lasers or LEDs have
been included in these studies [31, 32]. In these previous ex-
periments, the biofilms were formed on solid artifacts and,
most commonly, acrylic discs. In our present work, we chose
to use bone tissue specimens to promote the formation of
biofilms on a biological tissue, thus simulating the challenges
that could be encountered in vivo due to the anatomical and
structural aspects of these tissues. The organization of micro-
bial cells within the different layers of the biofilm may provide
some protection to the microorganisms within the inner layers,
ultimately contributing to the bacterial resistance to conven-
tional drugs [33]. In vitro susceptibility tests in model biofilms
have revealed significant microorganism survival after treat-
ment with antibiotics. Drug resistance can include
target alterations, overexpressed efflux capabilities, and drug
inactivation [34].
In addition to the expression of antimicrobial resistance,
another factor that can lead to greater difficulty in treating
infections is the organization of microorganisms in biofilms.
Biofilms are communities that are embedded in a matrix,
which reduces the ability of antimicrobials to penetrate and
represents a significant therapeutic barrier for many antibi-
otics. In addition, it has the ability to produce extracellular
enzymes, which have shown very aggressive behavior [31].
Clinical conditions that are often associated with S. aureus
biofilms include urinary tract infections, catheter infections,
middle-ear infections, sinusitis, formation of dental plaque
periodontitis, gingivitis, endodontics, osteomyelitis, infected
contact lenses, endocarditis, infections in cystic fibrosis, and
infections of permanent indwelling devices such as joint pros-
theses, heart valves, and implants. In the head and neck area,
biofilms are a major etiologic factor in periodontitis, wound
infections, oral candidiasis, and sinus and ear infections [16].
Lipovsky et al. [35] showed a bacterial reduction of
S. aureus and E. coli with blue light (415 and 455 nm) alone,
with fluence of 120 J/cm2. In addition, Feuerstein et al. [19]
showed the bacterial reduction of P. gingivalis and
F. nucleatum, with a 450-nm blue light at fluences of 62, 78,
and 94 J/cm2. Considering blue LED applications, several
clinical trials for the treatment of acne showed positive effects
of this wavelength [21, 36].
Gois et al. [37] also reported a reduction in the number of
S. aureus cells of 0.97, 0.97, and 1.05 log10 when irradiated with
red LED (20 J/cm2) and diode laser light (20 and 40 J/cm2),
respectively, in the absence of a photosensitizer.
Lasers Med Sci
Our results showed a significant reduction in the number of
CFU/mL at almost all time/doses used compared with the
control, in which no treatment was given. Special attention
should be paid to the group in which LED was applied for
10 min because there was a significant reduction of 3.2 log10
compared with the control group. Similar results were obtain-
ed by Enwemeka et al. [13, 14], who used LED at 470 and
405 nm, both in the visible blue color, to inhibit the growth of
methicillin-resistant S. aureus (MRSA) in planktonic cultures.
The authors obtained up to 90 % efficacy; however, the results
indicated dose-dependent features. Similar results were found
in in vitro studies by Guffey and Wilborn [38], who used 405
and 470 nm laser light on S. aureus and Pseudomonas
aeruginosa.
Andrade et al. [39] evaluated the effects of pre-irradiation
time on curcumin-mediated photodynamic therapy, using a
blue LED light of 455 nm against planktonic and biofilm
cultures of reference strains of Candida albicans, Candida
glabrata, and Candida dubliniensis. Their findings indicated
no reduction on the tested yeast biofilms with the application
of LED blue light alone. In other work, Dovigo et al. [33]
investigated the efficacy of Photogem with a blue LED light
for the photoinactivation of fluconazole-resistant strains of
C. albicans and C. glabrata. Exposure to LED light alone or
Photogem alone had no effect on the viability of the strains
evaluated.
Costa et al. [40] evaluated the effect of erythrosine and
green LED-mediated photodynamic therapy on planktonic
cultures and biofilms of C. albicans and C. dubliniensis. The
LED used in this study did not exhibit cytotoxic effects when
used alone against either planktonic cultures or biofilms of
both species, as shown previously for red and blue LEDs used
in association with erythrosine against microbial cells and
fibroblasts [41, 42].
Different results were found in our work. Irradiation with
blue LED of 455 nm for 3, 7, and 10 min showed a significant
reduction in the log of CFU/mL of C. albicans biofilms com-
pared with the control group. These findings show that longer
application times of blue light are required to achieve a rele-
vant clinical reduction of C. albicans compared with S. aureus,
which demonstrated positive results in shorter irradiation
times. When comparing the reduction in log CFU/mL be-
tween the tested microorganisms, a greater effectiveness in
the 10-min application groups was observed, though the best
results emerged from the group of specimens infected with
S. aureus compared with C. albicans (decreases of 3.2 and
2.3 logs, respectively). This fact can be explained by the fact
that fungi present much more complex targets than bacteria.
For example, yeasts, which constitute a large group of rather
disparate eukaryotic organisms, are enveloped by a thick ex-
ternal wall that is composed of a mixture of glucan, mannan,
chitin, and lipoproteins and is separated from the plasma
membrane by a periplasmic space. However, the available
Lasers Med Sci
evidence suggests that the response of such cells to photody-
namic processes is less strictly controlled by structural factors
compared with bacteria [43]. It has been widely noted that
C. albicans, similar to other yeasts, is slightly more difficult
to kill by PACT than are Gram-positive bacteria cells, neces-
sitating higher drug and light doses [44]. This has been attrib-
uted to the presence of a nuclear membrane in the yeasts, the
greater cell size and the reduced number of targets for singlet
oxygen per unit volume of cell [9, 45].
Studies in the literature employing LED to inhibit the
growth of S. aureus and C. albicans planktonic culture proved
the efficacy and potential of this technique. However, works
using LED in biofilms attached to bone tissue are scarce,
showing that more studies on this topic should be conducted
to establish protocols/parameters to circumvent the difficulties
encountered. In this work, according to the method and pro-
tocol used, 455 nm LED light was effective to reduce the load
of S. aureus and C. albicans biofilms, especially for 10 min of
application.
Acknowledgments The authors thank the Institutional Program of Sci-
entific Initiation of Federal University of Bahia for the Scientific Initiation
scholarships awarded for the development of this work.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
Ethics approval This study was approved by the Animal Research
Ethics Committee (UNESP Dental School, São José dos Campos-SP)
under protocol number 05/2008-PA/CEP. The study strictly followed all
of the ethical principles set forth by the Declaration of Helsinki (2000).
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