Lasers Med Sci https://doi.org/10.1007/s10103-017-2344-1
ORIGINAL ARTICLE
Biofilm formation by Candida albicans is inhibited by photodynamic antimicrobial chemotherapy (PACT), using chlorin e6: increase in both ROS production and membrane permeability Moisés Lopes Carvalho 1 & Ana Paula Pinto 1 & Leandro José Raniero 1 & Maricilia Silva Costa 1
Received: 1 June 2017 / Accepted: 29 September 2017 # Springer-Verlag London Ltd. 2017
Abstract Candida albicans is an opportunistic fungal producing both superficial and systemic infections in immunocompromised patients. Furthermore, it has been described an increase in the frequency of infections which have become refractory to standard antifungal therapy. 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 chlorin e6, as a photosensitizer on C. albicans. In this work, we studied the effect of PACT on both cell growth and biofilm formation by C. albicans. In addition, both ROS production and cell permeability were determined after PACT. PACT inhibited both growth and biofilm formation by C. albicans. We have also observed that PACT increased both ROS production (six times) and cell membrane permeability (five times) in C. albicans. PACT decreased both cell growth and biofilm development. The effect of PACT using chlorin e6 on C. albicans could be associated with an increase in ROS production, which could increase cell permeability, producing permanent damage to the cell membranes, leading to the cell death.
Keywords Photodynamic antimicrobial chemotherapy . PACT . Candida albicans . Chlorin e6
* Maricilia Silva Costa
[email protected] 1
Instituto de Pesquisa e Desenvolvimento—IP&D. Universidade do Vale do Paraíba—UNIVAP, Av. Shishima Hifumi, 2911, São José dos Campos, SP 12244-000, Brazil
Introduction The increase in both invasive and mucocutaneous fungal infections, mainly in immunocompromised individuals with severe underlying diseases, has been described by different authors [1–6]. Candida albicans has been described as one of the main opportunistic fungi found in a variety of clinical scenario producing infections ranging from superficial to systemic and invasive [7, 8]. In the USA, infections caused by C. albicans still represent one of the most prevalent problems in the hospital environment and are described as the fourth most common cause of bloodstream infections [3, 5]. Furthermore, an increase in the incidence of localized and invasive fungal infections that has become refractory to current antifungal therapies has been reported. It has been described the resistance of Candida species for a variety of conventional antifungal agents [6, 9–13]. Therefore, the development of more effective antifungal therapies is of great relevance. Photodynamic antimicrobial chemotherapy (PACT) is a potential antimicrobial therapy that combines a visible light and a photosensitizer (nontoxic), which in the presence of molecular oxygen produces reactive oxygen species (ROS), including singlet oxygen, which are highly reactive to biological components [14, 15]. ROS can promote damage to several vital components of the microorganism, such as DNA, proteins, and lipids, resulting in cell death [16, 17]. The antimicrobial effect of a variety of photosensitizers on different pathogenic microorganisms including Candida albicans has been demonstrated by different authors [18–25]. Park et al. [26], using chlorin e6 (chl-e6) as a photosensitizer, demonstrated the ability of photodynamic therapy (PDT) to inhibit different pathogenic bacteria, suggesting that chl-e6-mediated PDT could be an effective alternative for antimicrobial treatment. Moreover, the effect of chl-e6-mediated PDT inhibiting
Lasers Med Sci
Staphylococcus aureus Xen29 growth was demonstrated both in vitro and in vivo [27]. Winkler et al. [28] demonstrated the effect of PDT, using chl-e6 in reducing the viability of multidrug-resistant Staphylococcus aureus. Furthermore, the fungicidal effect of chl-e6 on C. albicans was also demonstrated. Uliana et al. [29] demonstrated the ability of PDT, using chl-e6 to inhibit Candida albicans growth, showing that this effect was dependent on the fluence used. Ryu et al. [24] demonstrated that chl-e6-mediated PDT was able to inhibit different microorganisms, including Candida albicans. These results suggest the potential of PACT, using chl-e6 as an antimicrobial therapy. Thus, the objective of this work was to study the effect of PACT, using chlorin e6 as a photosensitizer on both growth and biofilm formation by C. albicans.
Material and methods Organisms and growth conditions Cultures of C. albicans (ATCC 10231) were plated on Sabouraud dextrose agar (Merck) and incubated in atmospheric air (37 °C). After 48 h of incubation, a sample of the colonies was removed from the agar plate surface and suspended in sterile physiological solution (0.9% NaCl), at a cell density of 108 viable cells/ml. Effect of PACT on Candida albicans growth Candida suspensions (106 and 107 viable cells/ml) were seeded in a 96-well plate and incubated in the dark for 20 min, at room temperature in the presence of different chl-e6 concentrations (1, 2, 5, 7, 10, and 20 μg/ml) in a final volume of 150 μl. Cells incubated in sterile physiological solution alone were included as a control. After this period, the cover of the plate was removed, and the content of each well was irradiated with the appropriated light, at room temperature. The light source used was one light-emitting diode (LED), with output power of 0.118 W and peak wavelength of 660 nm. The light source irradiated an area of 0.38 cm2, resulting in a fluence of 40 J/cm2. After irradiation, the contents of the wells were properly homogenized, and aliquots of 25 μl were taken and seeded in a 24-well plate containing Sabouraud dextrose broth medium (Merck) (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 presented 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 biofilm formation After take of content of the 96-well plate to determine cell growth, aliquots of 25 μl were taken and seeded in a 96-well plate containing RPMI medium (Sigma, St. Louis, MO, USA), in a final volume of 200 μl. The plates were incubated to form biofilm, during 24 h (37 °C). After this period, the cell suspensions were aspirated, each well was washed three times with 200 μl PBS to remove the non-adherent cells, and 200 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-5carboxanilide 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 respiratory electron chain-uncoupling agent that accelerates respiration and XTT reduction; Sigma, St. Louis, MO, USA) at a volume ratio of 9:1. 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. 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 suspensions (108 viable cells/ml) were seeded in a 96-well plate and incubated in the dark for 20 min, in the presence of chl-e6 (20 μg/ml) in a final volume of 150 μl. 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, 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, the contents of the wells were homogenized and aliquots of 50 μl were taken to determine both ROS production and SYTOX Green uptake. Aliquots of 50 μl were taken and added to 50 μl PBS in a 96-well dark plate. To each well, 20 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 with excitation at 485 nm and emission at 530 nm. Effect of PACT on cell permeability Cell permeability was determined using the high-affinity nucleic acid stain SYTOX Green (Molecular Probes, Eugene, OR, USA), which does not cross the intact cell
Lasers Med Sci
Initially, the effect of PACT, using chl-e6 as a photosensitizer, was evaluated on C. albicans growth. It was observed a significant reduction in cell growth after PACT in a dependent manner on chl-e6 concentration (Fig. 1). Curiously, the effect of PACT reducing cell growth was decreased when cell density in the medium increased. It was observed that the concentration of chl-e6 necessary to produce an inhibition of 50% in the cell growth increased from < 10 to ~ 20 μM, when the cell densities increased from 106 to 107 viable cells/ml (compare Fig. 1a, b). At a cell density of 106 cells/ml, 20 μM chl-e6 inhibited C. albicans growth by 90%; however, it was only
about 50%, at cell density of 107 cells/ml. This inhibitory effect by chl-e6 was not observed in cells not irradiated, showing that chl-e6 alone does not affect Candida albicans growth. These results demonstrated the ability of PACT, using chl-e6 to inhibit C. albicans growth; however, this effect was dependent of the number of cells in the medium. Since Candida albicans colonization and virulence are related to biofilm formation, the effect of PACT, using chl-e6, was determined on the ability of the cells to form biofilm. Figure 2 shows the effect of different concentrations of chl-e6 on biofilm formation using C. albicans suspensions at different cell densities. It was observed that PACT decreased the ability of the cells to form biofilm, also in a dependent manner on chl-e6 concentrations. The inhibitory profile produced by chl-e6 on biofilm formation was similar in biofilms forming using either 106 or 107 cells/ml. The concentration of chl-e6 necessary to inhibit 50% of biofilm formation was ~ 7 μM, in the presence of either 106 or 107 cells/ml. At a cell density of 106 cells/ml, 10 μM chl-e6 inhibited C. albicans growth by 80%; however, it was about 55%, at a cell density of 107 cells/ml (compare Fig. 2a, b). It was observed that PACT, using 20 μM chl-e6, was able to reduce biofilm formation at > 90% in the presence of either 106 or 107 cells/ml. The effect of PACT, using chl-e6 on the morphology of the biofilm formation, was observed by light microscopy (Fig. 3). It was observed a mixture of yeasts and filaments in the structure of the biofilm formed, especially in the control group. The structure of the biofilm was not modified by only irradiation (compare Fig. 3a, b). In addition, chl-e6 had no effect on biofilm morphology in cells not irradiated (compare Fig. 3a, c, e, and g). However, it was observed a reduction in both yeast cells and hyphae formation after PACT. A significant reduction in the number of hyphae
Fig. 1 Effect of different chl-e6 concentrations on Candida albicans growth, in irradiated (•) and not irradiated (ο) cells. The experimental conditions are described under Materials and Methods. The cell growth was determined using suspensions at cell density of 106 (a) and 107 (b) viable cells/ml. Values are expressed as a percentage of the optical density determined at 570 nm (OD570) in treated cells from the optical density measured in the control group (cells incubated in the absence of TB and not irradiated). Data are means ± SD (n = 8). Asterisks indicate p < 0.05
Fig. 2 Effect of different chl-e6 concentrations on biofilm formation by Candida albicans, in irradiated (•) and not irradiated (ο) cells. The biofilm formation was determined using suspensions at cell density of 106 (a) and 107 (b) viable cells/ml. Values are expressed as a percentage of the optical density determined at 490 nm (OD490) in treated cells from the optical density measured in the control group. The data are mean ± SE (n = 8). Asterisks indicate p < 0.05
membranes, however, penetrates into cells with damaged plasma membranes. Aliquots of 50 μl were taken 3 h after irradiation and added to 50 μl phosphate-buffered saline (PBS) in a 96-well dark plate. To each well, 20 μl SYTOX Green (0.1 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 the Synergy HT multidetection microplate reader with excitation at 480 nm and emission at 530 nm. Statistical analysis Values were expressed as means ± standard deviation (SD). Statistical differences were evaluated by analysis of variance (ANOVA) and post hoc comparison with the Tukey–Kramer test. p values of < 0.05 were considered significant.
Results
Lasers Med Sci Fig. 3 Analysis of biofilm formation by Candida albicans. Biofilms produced by Candida albicans were observed in not irradiated (a, c, e, and g) and irradiated (b, d, f, and h) cells. Biofilm formation was observed in the absence (a and b) and in the presence of 5 μM (c and d), 10 μM (e and f) or 20 μM chl-e6 (g and h). Bars indicate 20 μm
form was observed by increasing the concentrations of chl-e6 in the medium (compare Fig. 3b, d, f, and h). In fact, the cells treated with PACT, using 20 μM chl-e6, presented a great decrease in both yeast cells and hyphae formation, showing complete absence of the filamentous form in the biofilm structure. This result demonstrated that the inhibition in biofilm formation by PACT was related, also, to reduce the transition from budding yeast form to filamentous form, an essential stage to both colonization and virulence, demonstrating the potential of PACT, using chl-e6 as an antifungal therapy. In addition, 24-h-old biofilms produced by C. albicans were inhibited at ~ 50% in the presence of 20 μM chl-e6 during irradiation (data not shown). Taken together, these results indicate that PACT, using chl-e6, could be a potential antifungal
therapy reducing both cell growth and biofilm formation by C. albicans. In order to study the mechanism by which PACT, using chl-e6, inhibits C. albicans, ROS production was determined. It was observed a significant increase in the ROS production after PACT, using 20 μM chl-e6 (Fig. 4). Comparing to control cells, PACT was able to increase ROS production by six times. In cells not irradiated, 20 μM chl-e6 did not modify ROS accumulation. The same effect was observed in cells only irradiated, in the absence of chl-e6. This result indicates that the mechanism by which PACT with chl-e6 inhibits C. albicans can be related to the production of oxidative damage to the cells, caused by the ROS production. The potential of PACT,
Lasers Med Sci
Discussion
Fig. 4 Effect of PACT on ROS production. The ROS production was determined 3 h after PACT, using 20 μg/ml chl-e6. Values are expressed as a percentage of the fluorescence intensity determined, in arbitrary units, in treated cells from the fluorescence intensity measured in the control group (cells incubated in the absence of TB and not irradiated). The data are mean ± SE (n = 8). Asterisk indicates p < 0.05
using chl-e6 to damage cell membranes, was demonstrated using the high-affinity nucleic acid stain SYTOX Green. This probe does not cross the membranes of intact cells; however, it enters into cells with damaged plasma membranes. It was observed an increase in the cell permeability only in cells submitted to PACT (Fig. 5). Comparing to control, cells treated with PACT presented an increase in cell permeability by ~ 5 times, 3 h after irradiation. Taken together, these results suggest that the mechanism by which PACT, with chl-e6 inhibits both growth and biofilm formation by C. albicans, could be related to the ROS production, which, consequently, could increase the cell permeability, producing cell damages.
Fig. 5 Effect of PACT on cellular permeability. Cell permeability was determined 3 h after PACT, using 20 μg/ml chl-e6. Values are expressed as a percentage of the fluorescence intensity determined, in arbitrary units, in treated cells from the fluorescence intensity measured in the control group (cells incubated in the absence of TB and not irradiated). The data are mean ± SE (n = 8). Asterisk indicates p < 0.05
It has been demonstrated that the incidence of both mucocutaneous and invasive fungal infections has increased in recent years, particularly in immunocompromised individuals [1, 2, 4, 6, 17]. Therefore, it has been described the increase in the cases of Candida species presenting resistance to conventional antifungal agents [6, 9–13]. Thus, the development of more effective antifungal therapies is crucial. Photodynamic antimicrobial chemotherapy (PACT) has been presented as a potential antimicrobial therapy, presenting as main advantages a wide spectrum of action and low probability to develop resistance by microorganisms [18, 30]. In spite of the antimicrobial effects of a variety of photosensitizers has been demonstrated against different pathogenic microorganisms [18, 20, 21, 23, 31–35], few studies demonstrate the potential fungicidal of chl-e6 on C. albicans. Our results demonstrated the potential of PACT, using chl-e6, to inhibit both growth and biofilm formation by C. albicans. It has been shown that C. albicans present different factors contributing to its virulence and colonization, including the transition from yeast to filamentous form, a crucial step to biofilm formation in medical devices and host tissue [36–38]. Our results demonstrate, for the first time, that PACT with chl-e6 is able to decrease both formation and viability of biofilms produced, suggesting its potential to reduce cell proliferation and colonization by C. albicans. This is a very interesting feature, since the formation of biofilm in medical devices is characterized as a major cause of morbidity and mortality in hospitalized individuals [37–41]. It has been demonstrated that 70–80% of invasive candidemia are associated with central venous catheters [42]. It has been described that the effect of PACT is related to a variety of photochemical mechanisms, which lead to the formation of reactive oxygen species (ROS), including singlet oxygen, a highly toxic specie to different cellular components [30, 31, 43, 44]. Our results demonstrated the increase in the ROS production after PACT. We suggest that this increase in ROS production could produce damage to the cell membranes, increasing cell permeability after PACT. ROS production associated to the increase in the membrane permeability was also demonstrated after PACT, using toluidine blue, as a photosensitizer [21]. In this work, we demonstrate that PACT, using chl-e6, leads to the ROS production, which, probably, increases the cellular permeability, exposing nuclear contents, causing irreversible damage and, consequently, cell death. The correlation between ROS production and cell permeability suggests that the mechanism by which PACT, using chl-e6, inhibits both cell proliferation and biofilm formation is associated with ROS production, inducing damage to the cell membrane, leading to cell death.
Lasers Med Sci
Conclusion We concluded that PACT, using chl-e6 as a photosensitizer, could inhibit both cell growth and biofilm formation by C. albicans, probably by a mechanism evolving ROS production, which, consequently, produces irreversible damages to the cell membranes, leading to the cell death. We suggest that the ability of Candida albicans to form biofilm could be decreased by PACT, corroborating the potential of PACT, using chl-e6 as an effective fungicidal therapy.
9. 10.
11.
12.
13.
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).
14.
Role of funding source The authors would like to thank FAPESP for the financial support.
15. 16.
Compliance with ethical standards Conflict of interest The authors have no financial, personal, or other conflicts of interest related to this work. Ethical approval In this study, all experiments were performed using cultures of Candida albicans (ATCC 10231), therefore, it is not necessarily approved by local authorities.
17.
18.
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; the paper is not currently being considered for publication elsewhere.
19.
References
21.
Wisplinghoff H, Bischoff T, Tallent SM, Seifert H, Wenzel RP, Edmond MB (2004) Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis 39:309–317 2. Pfaller MA, Diekema DJ (2010) Epidemiology of invasive mycoses in North America. Crit Rev Microbiol 36:1–53 3. Lockhart SR, Iqbal N, Cleveland AA, Farley MM, Harrison LH, Bolden CB, Baughman W, Stein B, Hollick R, Park BJ, Chiller T (2012) Species identification and antifungal susceptibility testing of Candida bloodstream isolates from population-based surveillance studies in two U.S. cities from 2008 to 2011. J Clin Microbiol 50: 3435–3442 4. Groll AH, Lumb J (2012) New developments in invasive fungal disease. Future Microbiol 7:179–184 5. Wisplinghoff H, Ebbers J, Geurtz L, Stefanik D, Major Y, Edmond MB, Wenzel RP, Seifert H (2014) Nosocomial bloodstream infections due to Candida spp. in the USA: species distribution, clinical features and antifungal susceptibilities. Int J Antimicrob Agents 43: 78–81 6. Tsui C, Kong EF, Jabra-Rizk MA (2016) Pathogenesis of Candida albicans biofilm. Pathog Dis 74:ftw018 7. Ganguly S, Mitchell AP (2011) Mucosal biofilms of Candida albicans. Curr Opin Microbiol 14:380–385 8. Nobile CJ, Johnson AD (2015) Candida albicans biofilms and human disease. Annu Rev Microbiol 69:71–92
20.
1.
22.
23.
24.
25.
26.
27.
28.
Seneviratne CJ, Jin L, Samaranayake LP (2008) Biofilm lifestyle of Candida: a mini review. Oral Dis 14:582–590 Sardi JC, Almeida AM, Mendes Giannini MJ (2011) New antimicrobial therapies used against fungi present in subgingival sites—a brief review. Arch Oral Biol 56:951–959 Lewis RE, Viale P, Kontoyiannis DP (2012) The potential impact of antifungal drug resistance mechanisms on the host immune response to Candida. Virulence 3:368–376 Tobudic S, Kratzer C, Lassnigg A, Presterl E (2012) Antifungal susceptibility of Candida albicans in biofilms. Mycoses 55: 199–204 Gonçalves SS, Souza AC, Chowdhary A, Meis JF, Colombo AL (2016) Epidemiology and molecular mechanisms of antifungal resistance in Candida and Aspergillus. Mycoses 59:198–219 Dai T, Fuchs BB, Coleman JJ, Prates RA, Astrakas C, St Denis TG, Ribeiro MS, Mylonakis E, Hamblin MR, Tegos GP (2012) Concepts and principles of photodynamic therapy as an alternative antifungal discovery platform. Front Microbiol 3:1–16 Allison RR, Moghissi K (2013) Photodynamic therapy (PDT): PDT mechanisms. Clin Endosc 46:24–29 Alves E, Faustino MA, Neves MG, Cunha A, Tome J, Almeida A (2014) An insight on bacterial cellular targets of photodynamic inactivation. Future Med Chem 6:141–164 Taraszkiewicz A, Szewczyk G, Sarna T, Bielawski KP, Nakonieczna J (2015) Photodynamic inactivation of Candida albicans with imidazoacridinones: influence of irradiance, photosensitizer uptake and reactive oxygen species generation. PLoS One 10:e0129301 Calzavara-Pinton P, Rossi MT, Sala R, Venturini M (2012) Photodynamic antifungal chemotherapy. Photochem Photobiol 88:512–522 Yang YT, Chien HF, Chang PH, Chen YC, Jay M, Tsai T, Chen CT (2013) Photodynamic inactivation of chlorin e6-loaded CTAB-liposomes against Candida albicans. Lasers Surg Med 45:175–185 Prates RA, Fuchs BB, Mizuno K, Naqvi Q, Kato IT, Ribeiro MS, Mylonakis E, Tegos GP, Hamblin MR (2013) Effect of virulence factors on the photodynamic inactivation of Cryptococcus neoformans. PLoS One 8:e54387 Rosseti IB, Chagas LR, Costa MS (2014) Photodynamic antimicrobial chemotherapy (PACT) inhibits biofilm formation by Candida albicans, increasing both ROS production and membrane permeability. Lasers Med Sci 29:1059–1064 Rosseti IB, Costa MS (2014) The viability of biofilm produced by Candida albicans is decreased by photodynamic antimicrobial chemotherapy (PACT) with toluidine blue. Trends in Photochemistry & Photobiology 6:19–24 Dovigo LN, Carmello JC, Carvalho MT, Mima EG, Vergani CE, Bagnato VS, Pavarina AC (2013) Photodynamic inactivation of clinical isolates of Candida using Photodithazine®. Biofouling 29:1057–1067 Ryu AR, Han CS, Oh HK, Lee MY (2015) Chlorin e6-mediated photodynamic inactivation with halogen light against microbes and fungus. Toxicol Environ Health Sci 7:231–238 Quishida CC, Carmello JC, Mima EG, Bagnato VS, Machado AL, Pavarina AC (2015) Susceptibility of multispecies biofilm to photodynamic therapy using Photodithazine®. Lasers Med Sci 30: 685–694 Park JH, Moon YH, Bang IS, Kim YC, Kim SA, Ahn SG, Yoon JH (2010) Antimicrobial effect of photodynamic therapy using a highly pure chlorin e6. Lasers Med Sci 25:705–710 Park JH, Ahn MY, Kim YC, Kim SA, Moon YH, Ahn SG, Yoon JH (2012) In vitro and in vivo antimicrobial effect of photodynamic therapy using a highly pure chlorin e6 against Staphylococcus aureus Xen29. Biol Pharm Bull 35:509–514 Winkler K, Simon C, Finke M, Bleses K, Birke M, Szentmáry N, Hüttenberger D, Eppig T, Stachon T, Langenbucher A, Foth HJ,
Lasers Med Sci Herrmann M, Seitz B, Bischoff M (2016) Photodynamic inactivation of multidrug-resistant Staphylococcus aureus by chlorin e6 and red light (λ=670nm). J Photochem Photobiol B 162:340–347 29. Uliana MP, Pires L, Pratavieira S, Brocksom TJ, de Oliveira KT, Bagnato VS, Kurachi C (2014) Photobiological characteristics of chlorophyll a derivatives as microbial PDT agents. Photochem Photobiol Sci 13:1137–1145 30. Donnelly RF, McCarron PA, Tunney MM (2008) Antifungal photodynamic therapy. Microbiol Res 163:1–12 31. Wainwright M (1998) Photodynamic antimicrobial chemotherapy (PACT). J Antimicrob Chemother 42:13–28 32. Harris F, Chatfield LK, Phoenix DA (2005) Phenothiazinium based photosensitisers—photodynamic agents with a multiplicity of cellular targets and clinical applications. Curr Drug Targets 6:615–627 33. Fuchs BB, Tegos GP, Hamblin MR, Mylonakis E (2007) Susceptibility of Cryptococcus neoformans to photodynamic inactivation is associated with cell wall integrity. Agents and Chemotherapy 51:2929–2936 34. Giroldo LM, Felipe MP, Oliveira MA, 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 35. Huang L, Dai T, Hamblin MR (2010) Antimicrobial photodynamic inactivation and photodynamic therapy for infections. Methods Mol Biol 635:155–173
36. 37. 38.
39. 40. 41.
42.
43.
44.
Cullen PJ, Sprague GF Jr (2012) The regulation of filamentous growth in yeast. Genetics 190:23–49 Mathé L, Van Dijck P (2013) Recent insights into Candida albicans biofilm resistance mechanisms. Curr Genet 59:251–264 Sardi JC, Pitangui Nde S, Rodríguez-Arellanes G, Taylor ML, Fusco-Almeida AM, Mendes-Giannini MJ (2014) Highlights in pathogenic fungal biofilms. Rev Iberoam Micol 31:22–29 Miceli MH, Díaz JA, Lee AS (2011) Emerging opportunistic yeast infections. Lancet Infect Dis 11:142–151 Finkel JS, Mitchell AP (2011) Genetic control of Candida albicans biofilm development. Nat Rev Microbiol 9:109–118 DiDone L, Oga D, Krysan DJ (2011) A novel assay of biofilm antifungal activity reveals that amphotericin B and caspofungin lyse Candida albicans cells in biofilms. Yeast 28:561–568 Sardi JC, Scorzoni L, Bernardi T, Fusco-Almeida AM, Mendes Giannini MJ (2013) Candida species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options. J Med Microbiol 62:10–24 Hamblin MR, Hasan T (2004) Photodynamic therapy: a new antimicrobial approach to infectious disease? Photochem Photobiol Sci 3:436–450 Bacellar IO, Tsubone TM, Pavani C, Baptista MS (2015) Photodynamic efficiency: from molecular photochemistry to cell death. Int J Mol Sci 16:20523–20559
