Artigo LIMS C. krusei

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ORIGINAL ARTICLE
Photodynamic antimicrobial chemotherapy (PACT) using toluidine blue
inhibits both growth and biofilm formation by Candida krusei
Bruna Graziele Marques da Silva1 & Moisés Lopes Carvalho1 & Isabela Bueno Rosseti2 & Stella Zamuner3 &
Maricilia Silva Costa1
Received: 3 April 2017 /Accepted: 21 December 2017
# Springer-Verlag London Ltd., part of Springer Nature 2018
Abstract
Among non-albicans Candida species, the opportunistic pathogen Candida krusei emerges because of the high mortality related
to infections produced by this yeast. The Candida krusei is an opportunistic pathogen presenting an intrinsic resistance to
fluconazol. In spite of the reduced number of infections produced by C. krusei, its occurrence is increasing in some groups of
patients submitted to the use of fluconazol for prophylaxis. Photodynamic antimicrobial chemotherapy (PACT) is a potential
antimicrobial therapy that combines visible light and a nontoxic dye, known as a photosensitizer, producing reactive oxygen
species (ROS) that can kill the treated cells. The objective of this study was to investigate the effects of PACT, using toluidine
blue, as a photosensitizer on both growth and biofilm formation by Candida krusei. In this work, we studied the effect of the
PACT, using TB on both cell growth and biofilm formation by C. krusei. PACTwas performed using a light source with output
power of 0.068Wand peak wavelength of 630 nm, resulting in a fluence of 20, 30, or 40 J/cm2. In addition, ROS production was
determined after PACT. The number of samples used in this study varied from 6 to 8. Statistical differences were evaluated by
analysis of variance (ANOVA) and post hoc comparison with Tukey-Kramer test. PACT inhibited both growth and biofilm
formation byC. krusei. It was also observed that PACTstimulated ROS production. Comparing to cells not irradiated, irradiation
was able to increase ROS production in 11.43, 6.27, and 4.37 times, in the presence of TB 0.01, 0.02, and 0.05 mg/mL,
respectively. These results suggest that the inhibition observed in the cell growth after PACT could be related to the ROS
production, promoting cellular damage. Taken together, these results demonstrated the ability of PACT reducing both cell growth
and biofilm formation by C. krusei.
Keywords Candida krusei . Photodynamic antimicrobial chemotherapy . PACT . Toluidine blue . Biofilm formation
Introduction
Invasive fungal infections, caused by Candida species, are an
important reason of morbidity and mortality in hospitalized
patients [1–3]. In the USA, Candida species are the fourth
most common cause of nosocomial bloodstream infections
[3, 4]. In spite that Candida albicans is the major cause of
invasive fungal diseases, an increase in the number of blood-
stream infections associated to non-albicans Candida species
has been described in the last years [2, 5–7]. Among non-
albicans Candida species, the opportunistic pathogen
Candida krusei emerges because of the high mortality related
to infections produced by this yeast [8, 9]. Abbas et al. [8]
related that C. krusei fungemia is associated with a high mor-
tality, in neutropenic patients. In addition, an increase in the
frequency of C. krusei fungemia has been related [8]. Pfaller
et al. [10] compared different clinical services reporting the
isolation of C. krusei from patients and demonstrated that
C. krusei was most commonly isolated from patients hospital-
ized in hematology-oncology services. Candida krusei is an
opportunistic pathogen presenting an intrinsic resistance to the
fluconazol [11, 12], probably because of the extensive and re-
petitive use of fluconazol to suppress fungal infections [11, 13].
* Maricilia Silva Costa
mscosta@univap.br
1 Instituto de Pesquisa e Desenvolvimento (IP&D), Universidade do
Vale do Paraíba (UNIVAP), Av. Shishima Hifumi 2911, São José dos
Campos CEP: 12244-000, Brazil
2 Anhanguera Educacional, Av. Doutor João Batista de Souza Soares,
4009—Jardim Morumbi, São José dos Campos, SP, Brazil
3 Posgraduated Program in Medicine, Universidade Nove de Julho
(UNINOVE), São Paulo, SP, Brazil
Lasers in Medical Science
https://doi.org/10.1007/s10103-017-2428-y
Furthermore, different authors have demonstrated that the con-
centration of Amphotericin B necessary to inhibit C. krusei is
always higher than that necessary to inhibitC. albicans [10, 14,
15]. Thus, in spite of the reduced number of infections pro-
duced by C. krusei, its occurrence is significant increasing in
some groups of patients submitted to the use of fluconazol for
prophylaxis. Therefore, the development of effective antifungal
therapies, especially against infections related to the pathogen
Candida krusei, is highly required.
Photodynamic therapy (PDT) is a process involving the use
of a photosensitizer, a visible light source, and oxygen [16,
17]. Since the discovery of PDT, the effects of this therapy in
killing microorganisms have been reported. Currently, the po-
tential of PDT in cancer treatment is already consolidated;
therefore, its application has stimulated the interest in the field
of antimicrobial chemotherapy, because of the several studies
demonstrating diverse advantages, especially the low proba-
bility of resistance by the microorganisms [18, 19]. PDT, also
referred to as photodynamic antimicrobial chemotherapy
(PACT) or antimicrobial photodynamic therapy (aPDT), has
been considered an alternative and innovative therapy [18,
20–22]. PDT and PACT present distinct terminologies; how-
ever, the term PDT is more commonly used in studies
reporting treatment against tumor cells, whereas the term
PACT is highlighted when the antimicrobial action of light
associated with a photosensitizer is considered. Thus, PACT
is a potential antimicrobial therapy that combines visible light
and a nontoxic dye, known as a photosensitizer, producing
reactive oxygen species (ROS) that can kill the treated cells
[21–25]. This technique can promote the photodynamic mi-
crobial damage by producing the highly cytotoxic singlet ox-
ygen and other ROS. ROS can promote damage to DNA,
proteins, and cell membranes, leading to cell death [26]. It
has been demonstrated the antimicrobial effect of a variety
of light-sensitive photosensitizer compounds [21, 27, 28].
The photodynamic effects of different photosensitizer drugs
on the pathogen Candida albicans have been demonstrated
by distinct authors [29–33]. In addition, the fungicide ef-
fect of phenothiazinium salts, such as toluidine blue O
(TBO) and methylene blue (MB), as photosensitizer drugs
on C. albicans has also been demonstrated [31, 34–39].
The phenothiazine salts are the most commonly used pho-
tosensitizers in modern clinical trials, been clinically approved
to use in humans, since they have already shown promising
results in different clinical conditions and presented minimal
damage to human cells at different concentrations [28, 40].
Carvalho et al. [36] reported that PACT, using either MB or
TB, was able to reduce the number ofCandida albicans viable
cells in 80–90%. In addition, they also demonstrated that the
phototoxic effects of MB and TB are different, suggesting that
they have different mechanisms of action against C. albicans.
In addition, Souza et al. [41] also reported the antifungal effect
of PACT, using MB, TB, and malachite green, demonstrating
a pronounced reduction in the number of CFU of C. albicans.
Barbério et al. [42] reported that PACT, using TB, was able to
reduce the number of CFU in approximately 65%, suggesting
that PACT presents a fungicidal effect on C. albicans. In ad-
dition, Giroldo et al. [37] demonstrated that PACT, using MB,
was able to inhibit the viability of C. albicans in ~ 50%, sug-
gesting that the mechanism involved in PACTwas associated
with the increase in the cell permeability, promoting alter-
ations in the plasma membrane integrity andconsequently
producing damage and death of the C. albicans cells. Munin
et al. [35] demonstrated the reduction of germ tube formation
after PACT, using MB, indicating the potential of this therapy
in reducing an essential stage for the virulence by C. albicans.
Recently, it was demonstrated a reduction of both cell growth
and biofilm formation by Candida albicans, after PACT,
using TB [39]. These results demonstrated the potential of
PACT, using either TB or MB as a promising antifungal
therapy.
Different authors have been demonstrated that PACT was
able to interfere with growth of even non-albicans species,
demonstrating its potential as an alternative therapy to current
therapeutic modalities; however, a reduced number of papers
relating the effect of PACTon the pathogen C. krusei is found
in the literature.
Wilson and Mia [43] observed that PACT, using TB, was
able to reduce the number of CFU in different Candida spe-
cies, demonstrating a reduction of 65, 63, and 40% for
Candida tropicalis, Candida stellatoidea, and Candida kefyr,
respectively. Using different Candida spp., either sensitive or
fluconazole resistant, it was demonstrated a reduction in both
the number of viable cells and adhesion of Candida species to
the buccal epithelial cells after photodynamic inactivation
(PDI), using TB [44]. Souza et al. [34] studying the effect of
PACT, using MB on different Candida species (C. albicans,
C. dubliniensis, C. krusei, and C. tropicalis), demonstrated a
great reduction (80%) in growth of both albicans and non-
albicans species. Souza et al. [34] demonstrated that PACT,
using MB, as a photosensitizer drug was able to reduce the
number of colony-forming units in 91.6% for C. krusei.
Furthermore, the effect of PACT, using either MB or TB, as
photosensitizer drugs reducing C. krusei growth was related,
demonstrating that its effect was dependent on the fluence
used [45].
Taken together, these results suggest that PACT could be
used as an alternative therapy to current therapeutic modalities
in different species of the Candida genus. Furthermore,
Rosseti et al. [39] demonstrated that PACT using TB inhibited
both cell growth and biofilm formation by Candida albicans,
by a mechanism evolving ROS production. Therefore, our
hypothesis to initiate this work was that the mechanism by
which PACT, using TB, inhibits Candida krusei could be
similar to that observed in Candida albicans. Thus, the objec-
tive of this study was to investigate the effect of PACT, using
Lasers Med Sci
TB, on both growth and biofilm formation byCandida krusei,
besides ROS formation after PACT.
Material and methods
Organisms and growth conditions
Cultures of Candida krusei (ATCC 6258) were plated on
Sabouraud dextrose agar (Merck, Darmstadt, Hesse,
Germany) and incubated in atmospheric air (37 °C). After
48 h, a sample of colonies was removed from the agar plate
surface (Merck, Darmstadt, Hesse, Germany) and suspended
in sterile physiological solution (0.85%NaCl), at a cell density
of 107 viable cells/mL.
Effect of PACT on Candida krusei growth
Candida suspensions (104 viable cells/mL) were seeded in 96-
well plate and incubated in the dark for 5 min, at room tem-
perature in the presence of different TB concentrations (0.005,
0.01, 0.02, and 0.05 mg/mL) in a final volume of 0.2 mL.
Cells incubated in sterile physiological solution alone were
included as a control. After this period, the cover of the plate
was removed, and the plates were irradiated with the appro-
priated light, at room temperature. The light source used was a
light-emitting diode (LED), with output power of 0.068Wand
peak wavelength of 630 nm. The laser beam illuminated an
area of 0.38 cm2, resulting in a fluence of 20, 30, or 40 J/cm2.
After irradiation, the contents of the wells were properly ho-
mogenized and aliquots of 25 μL were taken and seeded in
24-well plate containing Sabouraud dextrose broth medium
(Merck, Darmstadt, Hesse, Germany) (2 mL). After 18 h of
incubation (37 °C), the medium was homogenized and the
optical density at 570 nm (OD570) was determined using a
Synergy HT Multi-Detection Microplate Reader (Bio-Tek,
Winooski, VT, USA), in order to determine the Candida
growth. The optical density determined in the control group
varied from 0.8 to 1.0 in all experiments. The experiments
were performed under aseptic conditions. The values present-
ed in the figures represent the percentage of growth, calculated
using the control group (cells incubated in the absence of TB
and not irradiated) as 100% of growth.
Effect of PACT on ROS production
Accumulation of ROS was quantified using 2 ′,7 ′-
dichlorodihydrofluorescein diacetate (H2DCF-DA)
(Molecular Probes, Eugene, OR, USA) staining.Candida sus-
pensions (107 viable cells/mL) were seeded in 96-well plate
and incubated in the dark for 5 min, in the presence of different
TB concentrations (0.01, 0.02, and 0.05 mg/mL) in a final
volume of 0.2 mL. After this period, the cover of the plate
was removed, and the plates were irradiated, resulting in an
energy dosage of 40 J/cm2. After irradiation, the contents of
the wells were properly homogenized and maintained in the
rest for 3 h in the dark (37 °C). After this period, aliquots of
50 μL were taken and added to 150 μL PBS in 96-well dark
plate. To each well, 5 μL (H2DCF-DA) (1 mM) was added
and incubated for 1 h in the dark (37 °C). The fluorescence
intensity of suspension was measured directly, in arbitrary
units, using a Synergy HT Multi-Detection Microplate
Reader (Bio-Tek, Winooski, VT, USA) with excitation at
485 nm and emission at 530 nm.
Effect of PACT on biofilm formation
Candida suspensions (107 viable cells/mL) were seeded in 96-
well plate and incubated in the dark (5 min), at room temper-
ature, in the presence of different TB concentrations (0.005,
0.01, and 0.02 mg/mL) in a final volume of 0.2 mL. After this
period, the cover of the plate was removed, and the plates were
irradiated (energy dosage of 40 J/cm2). After irradiation, the
contents of the wells were homogenized and aliquots of 50 μL
were taken and seeded in 96-well plate containing RPMI me-
dium (Sigma, St. Louis, MO, USA), in a final volume of
200 μL. The plates were incubated in order to form biofilm,
during 24 h (37 °C). After this period, the cell suspensions
were aspirated, each well was washed once with 100 μL PBS
to remove the non-adherent cells, and 100 μL PBS was added
to each well. Biofilm formation was monitored by a metabolic
assay based on the reduction of XTT (2,3-bis(2-methoxy-4-
nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium
salt) (Molecular Probes, Eugene, OR, USA) assay. Prior to
each assay, XTT solution (1 mg/mL) was thawed and mixed
with a freshly prepared 0.4 mM menadione solution (a respi-
ratory electron chain-uncoupling agent that accelerates respi-
ration and XTT reduction; Sigma, St. Louis, MO, USA) at a
volume ratio of 4:1. An aliquot of 6 μL from this mixture was
added to each well. After 2 h, the reduced formazan-colored
product was measured at 490 nm (OD490) in a Synergy HT
Multi-Detection Microplate Reader (Bio-Tek, Winooski, VT,
USA).
Statistical analysis
Values were expressed as means ± standard deviation.
Statistical differences were evaluated by analysis of variance
(ANOVA) and post hoc comparison with Tukey-Kramer test.
The statistical analysis was performed by using the software
ORIGIN 7.0 (OriginLab Corporation, Northampton, MA,
USA). A confidence interval of 95% was obtained by using
Shapiro-Wilk computational test. Power analysis > 85% and β
value < 15% were considered for the sample size used in this
study. P values < 0.05 were considered significant.
Lasers Med Sci
Results
Initially, the effect of PACT on Candida krusei growth was
investigated in different fluences (Fig. 1). It was observed that
the growth of C. krusei was inhibited by crescentTB concen-
trations alone (not irradiated). An inhibition of ~ 20 and ~ 35%
was observed in the presence of 0.02 and 0.05 mg/mL TB,
respectively. In a fluence of 20 J/cm2, the effect of PACTwas
modest, promoting an inhibition in the C. krusei growth in the
order of 40% in the presence of TB 0.05 mg/mL (Fig. 1a). An
increase in the fluence improved the inhibition of the growth
by PACT. In the presence of TB 0.05 mg/mL, it was observed
an inhibition of ~ 60% in the growth in fluence of either 30 or
40 J/cm2 (Fig. 1b, c). These results have shown the ability of
PACT, using TB, to inhibit C. krusei growth. It was observed
that, despite that the most significant inhibition was observed
using TB 0.05 mg/mL, the effect of PACT was higher in low
TB concentrations, when TB alone has no effect on growth.
Using TB 0.01 mg/mL, it was observed an inhibition of ~ 20,
45, and 70% in fluences of 20, 30, and 40 J/cm2, respectively
(compare Fig. 1a–c). In this concentration, TB was not able to
inhibit C. krusei growth significantly. This effect can be better
observed when the values were plotted as a ratio of not
irradiated/irradiated cells (Fig. 2). It was observed an increase
in the effect of PACT increasing the fluence used. In the pres-
ence of TB 0.01 mg/mL, the ratio of not irradiated/irradiated
cells was 1.21, 1.66, and 2.74, using 20, 30, and 40 J/cm2,
respectively. However, in the presence of TB 0.05 mg/mL, the
ratio was 1.21, 1.51, and 1.71, using 20, 30, and 40 J/cm2,
respectively. Consequently, the effect of PACT inhibiting
Candida krusei growth was higher in the presence of low
TB concentrations.Morphology of the cells was also observed
by light microscopy. It was observed a great reduction in the
number of cells 18 h after PACT. An important aspect ob-
served was the reduction, in the number of filamentous pro-
duced (data not shown). These results demonstrated the effect
of PACT, using TB, decreasing cell growth and reducing the
number of both yeast and filamentous form in C. krusei. Since
Candida colonization and virulence are related to biofilm for-
mation, the effect of PACT on the ability of the cells to form
biofilm was determined in C. krusei. It was observed that
PACT was able to decrease the ability of the cells to form
biofilm (Fig. 3). Biofilm formation in not irradiated cells
was not significant altered in the presence of TB alone.
However, PACT promoted a great decrease in the cell ability
to form biofilm. After irradiation, it was observed a reduction
of 70, 80, and 90% of biofilm formation in the presence of TB
0.005, 0.01, and 0.02 mg/mL, respectively. The structure of
biofilm produced by C. krusei was observed by light micros-
copy, 24 h after PACT (Fig. 4). It was observed that control
cells presented ability to form biofilm, producing a biofilm
with a great number of cells presenting both yeast and fila-
mentous forms (Fig. 4a). The presence of TB, in different
concentrations, did not change significantly the structure of
biofilm. In addition, only irradiation did not affect the ability
of the cells to form biofilm (Fig. 4b). However, it was ob-
served a significant alteration in the biofilm structure, 24 h
after PACT. The number of the cells decreased, and the form
of the cells modified. After PACT, using TB 0.02 mg/mL,
cells present in biofilm structure are minor and do not present
the typical C. krusei form. It was observed the presence of
some cells presenting a spherical form, and the presence of
filamentous form was not observed. This result indicates that
PACT produced a significant modification in biofilm forma-
tion. In order to understand the mechanism by which PACT
inhibits C. krusei, ROS production was determined after
Fig. 1 Effect of different
toluidine blue concentrations on
Candida albicans growth, in
irradiated (filled circles) and not
irradiated (empty circles) cells.
The experimental conditions are
described under BMaterial and
methods^ section. The cell
growth was determined 18 h after
PACT, using 20 (A), 30 (B), and
40 J/cm2 (C). The data are mean ±
SE (n = 8). *p < 0.05
Lasers Med Sci
PACT. Cells not irradiated, in the presence of TB, shown an
increase in ROS production, comparing to control cells
(Fig. 5). This effect can be related to the inhibition in the
growth by TB in not irradiated cells, observed in Fig. 1.
However, it was observed a pronounced increase in the ROS
production, after PACT. In irradiated cells, it was observed an
increase of 2.89 and 3.92 times, in ROS production in the
presence of TB 0.02 and 0.05 mg/mL, respectively, compar-
ing to the cells incubated in the absence of TB. However,
although the great ROS production was observed in the pres-
ence of TB 0.05 mg/mL, the increase in the ROS production
by irradiation was higher in the presence of low TB concen-
trations. Comparing to the cells not irradiated, irradiation was
able to increase the ROS production in 11.43, 6.27, and 4.37
times, in the presence of TB 0.01, 0.02, and 0.05 mg/mL,
respectively. These results suggest that the inhibition observed
in the cell growth after PACT, using TB, could be related to
the ROS production, promoting cellular damage. Taken to-
gether, these results demonstrated the ability of PACT reduc-
ing both cell growth and biofilm formation by C. krusei.
Discussion
It has been demonstrated that in immunocompromised pa-
tients or those hospitalized with severe diseases, the incidence
and prevalence of invasive fungal infections produced by
yeasts of the Candida genus is a great problem [46–48]. In
addition, an increase in the occurrence of infections which
have become refractory to standard antifungal therapy, due
to the extensive and repetitive use of antifungal azole deriva-
tives, has been observed [49, 50]. Among non-albicans
Candida species, Candida krusei emerges because of the high
mortality related to infections produced by this yeast [7–9].
Thus, the study of news therapies, principally against this
fungus, is critical. PACT is a process that leads to cellular
death by singlet oxygen and other ROS produced through a
variety of photochemical mechanisms, and has been presented
as a potential antimicrobial therapy [21–23, 25]. In this work,
we investigated the effects of PACT, using TB, as a photosen-
sitizer on C. krusei. Curiously, our results demonstrated that
TB alone was able to reduceC. krusei growth. Concentrations
of TB higher than 0.02 mg/mL decreased Candida krusei
growth at least 20%. This effect seems to be specific to
C. krusei, because it was not observed in C. albicans. For this
reason, the effects of PACT, using TB, were more evident in
low TB concentrations. An increase in the fluence increased
the effects of PACT reducing C. krusei growth in a manner
dependent of TB concentrations. These results are according
to literature, showing the effects of PACT, using either MB or
TB on Candida krusei growth [34, 45]. It has been demon-
strated that the transition from budding yeast form to filamen-
tous form, producing biofilms, is a critical step in colonization
and can determine the fugal virulence in Candida albicans
[51]. Moreover, cells detaching from biofilm present an in-
creased pathogenicity, when compared to their planktonic
counterparts [52], and a correlation between antifungal drug
resistance and Candida albicans ability to form hyphae has
been demonstrated [53–55]. In this work, we observed that
PACT, using TB, reduced biofilm formation. This is an im-
portant feature, demonstrating the ability of PACT inhibiting
both growth and biofilm formation by Candida krusei. Bliss
et al. [56] demonstrated that Candida krusei was sensitive to
PACT, using Photofrin, as a photosensitizer drug, suggesting
that photodynamic therapy could be used as a useful adjunct
or alternative to current anti-Candida therapeutic modalities.
Fig. 2 Effect of different TB concentrations on the not irradiated/irradiated ratio of cell growth after PACT, using 20 (squares), 30 (empty
circles), and 40 J/cm2 (filled circles). The values presented in the figure
were obtained by the ration between the values of cell growthmeasured in
not irradiated cells and irradiated cells. The data are mean ± SE (n = 8)
Fig. 3 Effect of different toluidine blue concentrations on biofilm
formation by Candida albicans, in irradiated (filled circles) and not
irradiated (empty circles) cells. The experimental conditions are
described under BMaterial and Methods^ section. The biofilm
formation was determined 24 h after PACT. The data are mean ± SE
(n = 6). *p < 0.05
Lasers Med Sci
The effect of PACT, using MB reducing the number of
colony-forming units for C. krusei, was, also, demonstrat-
ed [34]. In addition, Rodrigues et al. [45] demonstrated the
effect of PACT, using either MB or TB reducing C. krusei
growth. This work demonstrated, for the first time, the
ability of PACT to inhibit biofilm formation by Candida
krusei. Rosseti et al. [39] demonstrated that the effect of
PACT, using TB inhibiting Candida albicans growth,
could be related to ROS production in the medium. In this
work, we demonstrated a significant increase in ROS pro-
duction after PACT. However, this increase was higher in
the presence of low TB concentrations. Thus, we suggest
that the better effect of PACT observed in the presence of
low TB concentrations can be related to this higher ROS
production. Our results demonstrate an important feature,
showing the effect of PACT reducing biofilm formation, an
essential stage to both colonization and virulence. Thus,
we suggest the potential of PACT, using TB, as an effective
fungicidal therapy on Candida krusei.
Fig. 4 Analysis of biofilm
formation by Candida krusei.
Biofilms produced by Candida
krusei were observed at the
presence of not irradiated (a, c, e)
and irradiated (b, d, f) cells.
Biofilm formation was observed
in the absence (a, b) and in the
presence of either 0.005 (c, d) or
0.02 mg/mL of TB (e, f). Bar,
20 μm
Fig. 5 Effect of different toluidine blue concentrations on ROS
production in irradiated (filled circles) and not irradiated (empty circles)
cells. The experimental conditions are described under BMaterial and
methods^ section. The ROS production was determined 3 h after
PACT. The data are mean ± SE (n = 6). *p < 0.05
Lasers Med Sci
Acknowledgements This work was supported by Fundação de Amparo à
Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq).
Funding information The authors would like to thank FAPESP and
CNPq for the financial support.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval In this study, all experiments were performed using
cultures of Candida krusei (ATCC 6258); therefore, there was no need
for approval by local authorities.
Informed consent We have obtained permission from all the authors;
we declare that the material has not been published in whole or in part
elsewhere and the paper is not currently being considered for publication
elsewhere.
References
1. Morgan J, Meltzer MI, Plikaytis BD, Sofair AN, Huie-White S,
Wilcox S, Harrison LH, Seaberg EC, Hajjeh RA, Teutsch SM
(2005) Excess mortality, hospital stay, and cost due to candidemia:
a case control study using data from population-based candidemia
surveillance. Infect Control Hosp Epidemiol 26:540–547
2. Pfaller MA, Diekema DJ (2007) Epidemiology of invasive candi-
diasis: a persistent public health problem. Clin Microbiol Rev 20:
133–163
3. Pfaller MA, Diekema DJ (2010) Epidemiology of invasive myco-
ses in North America. Crit Rev Microbiol 36:1–53
4. Pfaller MA, Jones RN, Messer SA, Edmond MB, Wenzel RP
(1998) National surveillance of nosocomial blood stream infection
due to species of Candida other than Candida albicans: frequency of
occurrence and antifungal susceptibility in the SCOPE Program.
SCOPE Participant Group. Surveillance and Control of Pathogens
of Epidemiologic. Diagn Microbiol Infect Dis 30:121–129
5. Bassetti M, Righi E, Costa A, Fasce R, Molinari MP, Rosso R,
Pallavicini FB, Viscoli C (2006) Epidemiological trends in nosoco-
mial candidemia in intensive care. BMC Infect Dis 6:21
6. Arendrup MC (2010) Epidemiology of invasive candidiasis. Curr
Opin Crit Care 16:445–452
7. Pemán J, Cantón E, Quindós G, Eraso E, Alcoba J, Guinea J,
Merino P, Ruiz-Pérez-de-Pipaon MT, Pérez-del-Molino L,
Linares-Sicilia MJ, Marco F, García J, Roselló EM, Gómez-G-de-
la-Pedrosa E, Borrell N, Porras A, Yagüe G, FUNGEMYCA Study
Group (2012) Epidemiology, species distribution and in vitro anti-
fungal susceptibility of fungaemia in a Spanish multicentre pro-
spective survey. J Antimicrob Chemother 67:1181–1187
8. Abbas J, Bodey GP, Hanna HA, Mardani M, Girgawy E, Abi-Said
D, Whimbey E, Hachem R, Raad I (2000) Candida krusei
fungemia. An escalating serious infection in immunocompromised
patients. Arch Intern Med 160:2659–2664
9. Muñoz P, Saánchez-Somolinos M, Alcalá L, Rodríguez-Créixems
M, Peláez T, Bouza E (2005) Candida krusei fungaemia: antifungal
susceptibility and clinical presentation of an uncommon entity dur-
ing 15 years in a single general hospital. J Antimicrob Chemother
55:188–193
10. Pfaller MA, Diekema DJ, Gibbs DL, Newell VA, Nagy E,
Dobiasova S, Rinaldi M, Barton R, Veselov A, Global Antifungal
Surveillance Group (2008) Candida krusei, a multidrug-resistant
opportunistic fungal pathogen: geographic and temporal trends
from the ARTEMIS DISK Antifungal Surveillance Program,
2001 to 2005. J Clin Microbiol 46:515–521
11. Samaranayake YH, Samaranayake LP (1994) Candida krusei: biol-
ogy, epidemiology, pathogenicity and clinical manifestations of an
emerging pathogen. J Med Microbiol 41:295–310
12. Espinel-Ingroff A, Pfaller MA, Bustamante B, Canton E, Fothergill
A, Fuller J, Gonzalez GM, Lass-Flörl C, Lockhart SR, Martin-
Mazuelos E, Meis JF, Melhem MS, Ostrosky-Zeichner L, Pelaez
T, Szeszs MW, St-Germain G, Bonfietti LX, Guarro J, Turnidge J
(2014) Multilaboratory study of epidemiological cutoff values for
detection of resistance in eight Candida species to fluconazole,
posaconazole, and voriconazole. Antimicrob Agents Chemother
58:2006–2012
13. Wingard JR, MerzWG, Rinaldi MG, Johnson TR, Karp JE, Saral R
(1991) Increase in Candida krusei infection among patients with
bone marrow transplantation and neutropenia treated prophylacti-
cally with fluconazole. N Engl J Med 325:1274–1277
14. Cantón E, Pemán J, Gobernado M, Viudes A, Espinel-Ingroff
A (2004) Patterns of amphotericin B killing kinetics against
seven Candida species. Antimicrob Agents Chemother 48:
2477–2482
15. Scorzoni L, de Lucas MP, Mesa-Arango AC, Fusco-Almeida AM,
Lozano E, Cuenca-Estrella M, Mendes-Giannini MJ, Zaragoza O
(2013) Antifungal efficacy during Candida krusei infection in non-
conventional models correlates with the yeast in vitro susceptibility
profile. PLoS One 8:e60047
16. Dougherty TJ, Gomer CJ, Henderson BW, Jori G, Kessel D,
Korbelik M, Moan J, Peng Q (1998) Photodynamic therapy. J
Natl Cancer Inst 90:889–905
17. Allison RR,Moghissi K (2013) Photodynamic therapy (PDT): PDT
mechanisms. Clin Endoscopy 46:24–29
18. Sharma SK,Mroz P, Dai T, HuangY-Y, St. Denis TG, HamblinMR
(2012) Photodynamic therapy for cancer and for infections: what is
the difference? Isr J Chem 52:691–705
19. Hamblin MR (2016) Antimicrobial photodynamic inactivation: a
bright new technique to kill resistant microbes. Curr Opin
Microbiol 33:67–73
20. Wilson BC, PattersonMS (2008) The physics, biophysics and tech-
nology of photodynamic therapy. Phys Med Biol 53:R61–109
21. Wainwright M (1998) Photodynamic antimicrobial chemotherapy
(PACT). J Antimicrob Chemother 42:13–28
22. Sperandio FF, Sabino CP,Vecchio D, Garcia-Diaz M, Huang L,
Huang Y-Y, Hamblin MR (2015) Antimicrobial photodynamic
therapy in dentistry. In: de Freitas PM, Simões A (eds) Lasers in
Dentistry: Guide for Clinical Practice. John Wiley & Sons, Inc,
Hoboken
23. Maisch T (2007) Anti-microbial photodynamic therapy: useful in
the future? Lasers Med Sci 22:83–91
24. Hamblin MR, Hasan T (2004) Photodynamic therapy: a new anti-
microbial approach to infectious disease? Photochem Photobiol Sci
3:436–450
25. Vera DM, Haynes MH, Ball AR, Dai T, Astrakas C, Kelso MJ,
Hamblin MR, Tegos GP (2012) Strategies to potentiate antimicro-
bial photoinactivation by overcoming resistant phenotypes.
Photochem Photobiol 88:499–511
26. Imlay JA (2003) Pathways of oxidative damage. Annu Rev
Microbiol 57:395–418
27. Huang L, Huang YY, Mroz P, Tegos GP, Zhiyentayev T, Sharma
SK, Lu Z, Balasubramanian T, Krayer M, Ruzié C, Yang E, Kee
HL, Kirmaier C, Diers JR, Bocian DF, Holten D, Lindsey JS,
Hamblin MR (2010) Stable synthetic cationic bacteriochlorins as
selective antimicrobial photosensitizers. Antimicrob Agents
Chemother 54:3834–3841
Lasers Med Sci
28. Calzavara-Pinton P, Rossi MT, Sala R, Venturini M (2012)
Photodynamic antifungal chemotherapy. Photochem Photobiol
88:512–522
29. Chabrier-Roselló Y, Foster TH, Pérez-Nazario N, Mitra S, Haidaris
CG (2005) Sensitivity of Candida albicans germ tubes and biofilms
to photofrin-mediated phototoxicity. Antimicrob Agents
Chemother 49:4288–4295
30. Demidova TN, Hamblin MR (2004) Photodynamic therapy
targeted to pathogens. Int J Immunopathol Pharmacol 17:245–254
31. Demidova TN, Hamblin MR (2005) Effect of cell-photosensitizer
binding and cell density on microbial photoinactivation.
Antimicrob Agents Chemother 49:2329–2335
32. Lambrechts SA, Aalders MC, Van Marle J (2005) Mechanistic
study of the photodynamic inactivation of Candida albicans by a
cationic porphyrin. Antimicrob Agents Chemother 49:2026–2034
33. Lam M, Jou PC, Lattif AA, Lee Y, Malbasa CL, Mukherjee PK,
Oleinick NL, Ghannoum MA, Cooper KD, Baron ED (2011)
Photodynamic therapy with Pc 4 induces apoptosis of Candida
albicans. Photochem Photobiol 87:904–909
34. Souza SC, Junqueira JC, Balducci I, Koga-Ito CY, Munin E, Jorge
AO (2006) Photosensitization of different Candida species by low
power laser light. J Photochem Photobiol B 83:34–38
35. Munin E, Giroldo LM, Alves LP, Costa MS (2007) Study of germ
tube formation by Candida albicans after photodynamic antimicro-
bial chemotherapy (PACT). J Photochem Photobiol B 88:16–20
36. Carvalho GG, Felipe MP, Costa MS (2009) The photodynamic
effect of methylene blue and toluidine blue on Candida albicans is
dependent on medium conditions. J Microbiol 47:619–623
37. Giroldo LM, FelipeMP, deOliveiraMA,Munin E, Alves LP, Costa
MS (2009) Photodynamic antimicrobial chemotherapy (PACT)
with methylene blue increases membrane permeability in Candida
albicans. Lasers Med Sci 24:109–112
38. Prates RA, Kato IT, Ribeiro MS, Tegos GP, Hamblin MR (2011)
Influence of multidrug efflux systems on methylene blue-mediated
photodynamic inactivation of Candida albicans. J Antimicrob
Chemother 66:1525–1532
39. Rosseti IB, Chagas LR, CostaMS (2014) Photodynamic antimicro-
bial chemotherapy (PACT) inhibits biofilm formation by Candida
albicans, increasing both ROS production and membrane perme-
ability. Lasers Med Sci 29:1059–1064
40. Tanaka M, Kinoshita M, Yoshihara Y, Shinomiya N, Seki S,
Nemoto K, Hirayama T, Dai T, Huang L, Hamblin MR,
Morimoto Y (2012) Optimal photosensitizers for photodynamic
therapy of infections should kill bacteria but spare neutrophils.
Photochem Photobiol 88:227–232
41. Souza RC, Junqueira JC, Rossoni RD, Pereira CA, Munin E, Jorge
AO (2010) Comparison of the photodynamic fungicidal efficacy of
methylene blue, toluidine blue, malachite green and low-power
laser irradiation alone against Candida albicans. Lasers Med Sci
25:385–389
42. Barbério GS, da Costa SV, dos Santos Silva M, de Oliveira TM,
Silva TC, de AndradeMoreira MachadoMA (2014) Photodynamic
inactivation of Candida albicans mediated by a low density of light
energy. Lasers Med Sci 29:907–910
43. Wilson M, Mia N (1993) Sensitisation of Candida albicans to kill-
ing by low-power laser light. J Oral Pathol Med 22:354–357
44. Soares BM, da Silva DL, Sousa GR, Amorim JC, de Resende MA,
Pinotti M, Cisalpino OS (2009) In vitro photodynamic inactivation
of Candida spp. growth and adhesion to buccal epithelial cells. J
Photochem Photobiol B 94:65–70
45. Rodrigues GB, Dias-Baruffi M, Holman N, Wainwright M, Braga
GU (2013) In vitro photodynamic inactivation of Candida species
and mouse fibroblasts with phenothiazinium photosensitisers and
red light. Photodiagn Photodyn Ther 10:141–149
46. Espinel-Ingroff A, Canton E, Peman J, Rinaldi MG, Fothergill AW
(2009) Comparison of 24-hour and 48-hour voriconazole MICs as
determined by the Clinical and Laboratory Standards Institute broth
microdilution method (M27–A3 document) in three laboratories:
results obtained with 2,162 clinical isolates of Candida spp. and
other yeasts. J Clin Microbiol 47:2766–2771
47. Ruhnke M, Rickerts V, Cornely OA, Buchheidt D, Glöckner A,
Heinz W, Höhl R, Horré R, Karthaus M, Kujath P, Willinger B,
Presterl E, Rath P, Ritter J, Glasmacher A, Lass-Flörl C, Groll AH
(2011) Diagnosis and therapy of Candida infections: joint recom-
mendations of the German Speaking Mycological Society and the
Paul-Ehrlich-Society for Chemotherapy. Mycoses 54:279–310
48. Van de Veerdonk FL, Netea MG, Joosten LA, van der Meer JW,
Kullberg BJ (2010) Novel strategies for the prevention and treat-
ment of Candida infections: the potential of immunotherapy. FEMS
Microbiol Rev 34:1063–1075
49. White TC, Marr KA, Bowden RA (1998) Clinical cellular and
molecular factors that contribute to antifungal drug resistance.
Clin Microbiol Rev 11:382–402
50. Cannon RD, Lamping E, Holmes AR, Niimi K, Tanabe K, Niimi
M, Monk BC (2007) Candida albicans drug resistance another way
to cope with stress. Microbiology 153:3211–3217
51. Whiteway M, Bachewich C (2007) Morphogenesis in Candida
albicans. Annu Rev Microbiol 61:529–553
52. Uppuluri P, Chaturvedi AK, Srinivasan A, Banerjee M,
Ramasubramaniam AK, Köhler JR, Kadosh D, Lopez-Ribot JL
(2010) Dispersion as an important step in the Candida albicans
biofilm developmental cycle. PLoS Pathog 6:e1000828
53. Ha KC, White TC (1999) Effects of azole antifungal drugs on the
transition from yeast cells to hyphae in susceptible and resistant
isolates of the pathogenic yeast Candida albicans. Antimicrob
Agents Chemother 43:763–768
54. Ramage G, Saville SP, Thomas DP, López-Ribot JL (2005)
Candida biofilms: an update. Eukaryot Cell 4:633–638
55. Chandra J, Mukherjee PK, Ghannoum MA (2012) Candida
biofilms associated with CVC and medical devices. Mycoses 55:
46–57
56. Bliss JM, Bigelow CE, Foster TH, Haidaris CG (2004)
Susceptibility of candida species to photodynamic effects of
photofrin. Antimicrob Agents Chemother 48:2000–2006
Lasers Med Sci
	Photodynamic...
	Abstract
	Introduction
	Material and methods
	Organisms and growth conditions
	Effect of PACT on Candida krusei growth
	Effect of PACT on ROS production
	Effect of PACT on biofilm formation
	Statistical analysis
	Results
	Discussion
	References