Efflux Pump Inhibitor Potentiates the Antimicrobial Photodynamic Inactivation of Multidrug-Resistant Acinetobacter baumannii.

Background: Acinetobacter baumannii, a nosocomial pathogen, poses a major public health problem due to generating resistance to several antimicrobial agents. Antimicrobial photodynamic inactivation (APDI) employs a nontoxic dye as a photosensitizer (PS) and light to produce reactive oxygen species that destroy bacterial cells. The intracellular concentration of PS could be affected by factors such as the function of efflux pumps to emit PS from the cytosol. Objective: To evaluate the augmentation effect of an efflux pump inhibitor, verapamil, three multidrug-resistant A. baumannii were subjected to APDI by erythrosine B (EB). Methods and results: The combination of EB and verapamil along with irradiation at 530 nm induced a lethal effect and more than 3 log colony-forming unit reduction to all A. baumannii strains in planktonic state. In contrast, EB and irradiation alone could produce only a sublethal effect on two of the strains. Conclusions: These data suggest that verapamil increases the intracellular concentration of EB, which potentiates the lethal efficacy of APDI. Verapamil could be applied with EB and green light to improve their antimicrobial efficacy against A. baumannii-localized infections.

Optimization of the Erythrosine-mediated photodynamic therapy against Escherichia coli using response surface methodology.

BACKGROUND: The efficacy of photodynamic therapy (PDT) depends on the combination of light and a photosensitizer for inactivation of microorganisms. However, finding the ideal conditions for the factors involved in this technique is time and cost-consuming. The rotational composite central design (RCCD) is a tool that can be allied with PDT to achieve precise results within a shorter working time. METHODS: This study used the response surface methodology to optimize the parameters of PDT mediated by Erythrosine (ERY) and green light-emitting diodes (LED) in different Escherichia coli strains by applying RCCD. RESULTS: The RCCD predicted optimum values of ERY and light exposure on PDT. According to the experimental results, the light exposure time showed the most significant influence on the inactivation of the evaluated bacteria. The optimized operating conditions were validated in laboratory tests, and no viable cells were recovered with ERY at 116 µmol L-1 and 30 min of light (33.34 J cm2) for E. coli ATCC 25922, 108 µmol L-1 and 40 min (44.38 J cm2) for E. coli ATCC 35218, and 108 µmol L-1 and 29.3 min (32.5 J cm2) for E. coli O157:H7 EDL 933. CONCLUSION: The adjusted polynomial models provided accurate information on the combined effects of ERY and lighting time with green LED on PDT. The application of the RCCD, in addition to reducing the number of experiments, also allows for increased quantity and quality of the results. Therefore, surface response methodology combined with PDT is a promising approach to inactivate E. coli.

Morphological changes and viability of Streptococcus mutans biofilm treated with erythrosine: A confocal laser scanning microscopy analysis.

Antimicrobial photodynamic therapy (a-PDT) is a modality that aims to induce microorganisms through visible light, a photosensitizer, and molecular oxygen. This therapy has shown promising results in controlling cariogenic biofilm in vitro and in vivo counterparts. This study investigated bacterial viability and morphological characterization of Streptococcus mutans mature biofilms after combination of erythrosine and a high potency dental curing light. Biofilms were formed on saliva-coated hydroxyapatite disks in batch culture. The samples were performed in triplicates. Fresh medium was replaced daily for five days and treated using 40 µM of E activated by HL 288 J/cm2 and total dose of 226 J at 1200 mW/cm2. Phosphate buffer saline and 0.12% of chlorhexidine were used as negative and positive control, respectively. After treatment, biofilms were assessed for microbial viability and morphological characterization by means of bio-volume and thickness. COMSTAT software was used for image analysis. Data were analyzed using two-way ANOVA followed by Tukey test with significance level 5%. The application of a-PDT and CHX treatments decreased S. mutans bacterial viability. The image analysis showed more red cells on biofilms when compared to other groups, demonstrating photobacterial killing. Erythrosine irradiated with a high potency curing light can potentially act as an antimicrobial tool in the treatment of cariogenic biofilms. The morphology and viability of microorganisms were impacted after treatment. Treatment with photodynamic therapy may be able to reduce the bio-volume and viability of bacteria present in biofilms. CLINICAL RELEVANCE AND RESEARCH HIGHLIGHTS: The use of the a-PDT technique has been applied in dentistry with satisfactory results. Some applications of this technique are in stomatology and endodontics. In the present study, we sought to understand the use of photodynamic therapy in the control of biofilm and the results found are compatible with the objective of microbiological control proposed by this technique, thus raising the alert for future studies in vivo using the combination of a-PDT with erythrosine, since they are easily accessible materials for the dental surgeon and can be applied in clinical practice.

Antimicrobial photodynamic therapy against Lactobacillus casei using curcumin, nano-curcumin, or erythrosine and a dental LED curing device.

This study aimed to investigate the photodynamic effects of curcumin, nanomicelle curcumin, and erythrosine on Lactobacillus casei (L. casei). Various concentrations of curcumin (1.5 g/L, 3 g/L), nano-curcumin (3 g/L), and erythrosine (100 µM/L, 250 µM/L) were tested either alone or combined with light irradiation (PDT effect) against L. casei in planktonic and biofilm cultures. The light was emitted from a light-emitting diode (LED) with a central wavelength of 450 nm. A 0.12% chlorhexidine digluconate (CHX) solution served as the positive control, and a solution containing neither photosensitizer nor light was the negative control group. The number of viable microorganisms was determined using serial dilution. There was a significant difference in the viability of L. casei in both planktonic and biofilm forms (P < 0.05). In the planktonic culture, the antibacterial effects of CHX and PDT groups with curcumin 3 g/L and erythrosine 250 µM/L were significantly greater than the other groups (P < 0.05). For L. casei biofilms, the greatest toxic effects were observed in CHX and PDT groups with curcumin 3 g/L, erythrosine 250 µmol/L, erythrosine 100 µmol/L, and nanomicelle curcumin 3 g/L, with a significant difference to other groups (P < 0.05). The antibacterial effects of all photosensitizers (except erythrosine 250 µmol/L at planktonic culture) enhanced significantly when combined with light irradiation (P < 0.05). PDT with curcumin 3 g/L or erythrosine 250 µmol/L produced comparable results to CHX against L. casei at both planktonic and biofilm cultures. Alternatively, PDT with erythrosine 100 µmol/L or nanomicelle curcumin 3 g/L could be suggested to kill L. casei biofilms.

Efficient Degradation of Intracellular Antibiotic Resistance Genes by Photosensitized Erythrosine-Produced 1O2.

Intracellular antibiotic resistance genes (iARGs) constitute the important part of wastewater ARGs and need to be efficiently removed. However, due to the dual protection of intracellular DNA by bacterial membranes and the cytoplasm, present disinfection technologies are largely inefficient in iARG degradation. Herein, we for the first time found that erythrosine (ERY, an edible dye) could efficiently degrade iARGs by producing abundant 1O2 under visible light. Seven log antibiotic-resistant bacteria were inactivated within only 1.5 min, and 6 log iARGs were completely degraded within 40 min by photosensitized ERY (5.0 mg/L). A linear relationship was established between ARG degradation rate constants and 1O2 concentrations in the ERY photosensitizing system. Surprisingly, a 3.2-fold faster degradation of iARGs than extracellular ARGs was observed, which was attributed to the unique indirect oxidation of iARGs induced by 1O2. Furthermore, ERY photosensitizing was effective for iARG degradation in real wastewater and other photosensitizers (including Rose Bengal and Phloxine B) of high 1O2 yields could also achieve efficient iARG degradation. The findings increase our knowledge of the iARG degradation preference by 1O2 and provide a new strategy of developing technologies with high 1O2 yield, like ERY photosensitizing, for efficient iARG removal.

Boosting the photodynamic activity of erythrosine B by using thermoresponsive and adhesive systems containing cellulose derivatives for topical delivery.

Erythrosine displays potential photodynamic activity against microorganisms and unhealthy cells. However, erythrosine has high hydrophilicity, negatively impacting on permeation through biological membranes. Combining biological macromolecules and thermoresponsive polymers may overcome these erythrosine-related issues, enhancing retention of topically applied drugs. The aim of this work was to investigate the performance of adhesive and thermoresponsive micellar polymeric systems, containing erythrosine in neutral (ERI) or disodium salt (ERIs) states. Optimized combinations of poloxamer 407 (polox407) and sodium carboxymethylcellulose (NaCMC) or hydroxypropyl methylcellulose (HPMC) were used as platforms for ERI/ERIs delivery. The rheological and mechanical properties of the systems was explored. Most of the formulations were plastic, thixotropic and viscoelastic at 37 °C, with suitable gelation temperature for in situ gelation. Mechanical parameters were reduced in the presence of the photosensitizer, improving the softness index. Bioadhesion was efficient for all hydrogels, with improved parameters for mucosa in contrast to skin. Formulations composed of 17.5 % polox407 and 3 % HPMC or 1 % NaCMC with 1 % (w/w) ERI/ERIs could release the photosensitizer, reaching different layers of the skin/mucosa, ensuring enough production of cytotoxic species for photodynamic therapy. Functional micelles could boost the photodynamic activity of ERI and ERIs, improving their delivery and contact time with the cells.

The physiochemical and photodynamic inactivation properties of corn starch/erythrosine B composite film and its application on pork preservation.

This study explored the effect of erythrosine B (EB) as a photosensitizer in corn starch (CS) film and its physicochemical properties and photodynamic bacteriostatic ability against Staphylococcus aureus, Escherichia coli, and Salmonella both in vitro and inoculated on pork under the irradiation of D65 light-emitting diode (LED) (400-800 nm). The study revealed that the physiochemical properties of CS films: moisture content, water solubility, and water vapor transmission were improved with the addition of EB. In addition, the elasticity and the thermal stability of the film were enhanced. The results showed that the CS-EB films stimulated a maximum of 26.36 µg/mL hydrogen peroxide and 74.5 µg/g hydroxyl radical under irradiation. The CS composite films with a 5 % concentration of EB inhibited the bacterial growth by 4.7 Log CFU/mL in vitro after 30 min of illumination, and 2.4 Log CFU/mL on the pork samples under the same experimental condition. Moreover, the antibacterial ability was enhanced with the increase in EB concentration. Overall, the CS-EB composite films can inhibit the growth of bacteria through photodynamic inactivation and has the potential to become a new type of environmentally friendly packaging material.

Plasma membrane permeabilization to explain erythrosine B phototoxicity on in vitro breast cancer cell models.

Lipid oxidation is ubiquitous in cell life under oxygen and essential for photodynamic therapy (PDT) of carcinomas. However, the mechanisms underlying lipid oxidation in rather complex systems such as plasma membranes remain elusive. Herein, Langmuir monolayers were assembled with the lipid extract of glandular breast cancer (MCF7) cells and used to probe the molecular interactions allowing adsorption of the photosensitizer (PS) erythrosine B and subsequent photooxidation outcomes. Surface pressure (π) versus area (cm2/mL) isotherms of MCF7 lipid extract shifted to larger areas upon erythrosine incorporation, driven by secondary interactions that affected the orientation of the carbonyl groups and lipid chain organization. Light-irradiation increased the surface area of the MCF7 lipid extract monolayer containing erythrosine owing to the lipid hydroperoxidation, which may further undergo decomposition, resulting in the chain cleavage of phospholipids and membrane permeabilization. Incorporation of erythrosine by MCF7 cells induced slight toxic effects on in vitro assays, differently of the severe phototoxicity caused by light-irradiation, which significantly decreased cell viability by more than 75% at 2.5 × 10-6 mol/L of erythrosine incubated for 3 and 24 h, reaching nearly 90% at 48 h of incubation. The origin of the phototoxic effects is in the rupture of the plasma membrane shown by the frontal (FSC) and side (SSC) light scattering of flow cytometry. Consistent with hydroperoxide decomposition, membrane permeabilization was also confirmed by cleaved lipids detected in mass spectrometry and subsidizes the necrotic pathway of cell death.

Erythrosine as a photosensitizer for antimicrobial photodynamic therapy with blue light-emitting diodes - An in vitro study.

BACKGROUND: This study aims to test the absorbance of a new composition of erythrosine, its pH, cell viability and potential as a photo sensitizer against Candida albicans when irratiaded with blue light emitting-diode (LED). METHODS: For pH and absorbance tests, erythrosine was prepared at a concentration of 0.03/ml. The cells of the L929 strain were cultured and the alamarBlue® assay was performed on samples to assess cell viability. For the microbiological essay, the strain of Candida albicans ATCC 90028 was selected. Yeast suspensions were divided into the following groups: control without irradiation or photosensitizer (C), irradiated group without photosensitizer (L), photosensitizer group without irradiation (0), and groups that received photosensitizer and irradiation, called aPDT groups. RESULTS: Erythrosine had no significant changes in pH and its absorbance was also consistent (≅400 nm). When it came to cell viability, on the first day, the group that was in contact with the dye and irradiated with the LED in minimun power was found to have the higher cell proliferation. On day 3, both irradiated groups (maximum and minimum) showed the highest cell proliferation. In the microbiological essay with C. albicans, aPDT groups started to show microbial reduction after 60 and 90 s of irradiation and when irradiated for 120 s, 6 microbial reduction logs were found. CONCLUSIONS: The erythrosine in question is a PS, with pH stability, blue light absorbance, cell viability and efficacy against C. albicans. More studies with this PS should be encouraged in order to verify its performance in aPDT.

Eradication of Acinetobacter baumannii Planktonic and Biofilm Cells Through Erythrosine-Mediated Photodynamic Inactivation Augmented by Acetic Acid and Chitosan.

Photodynamic inactivation (PDI) is an attractive treatment modality for multidrug-resistant bacterial infections. The effectiveness of photosensitization by anionic photosensitizers such as erythrosine B can be further enhanced by the addition of biological or chemical molecules. This study aimed to investigate of the enhancement effect of acetic acid and chitosan on erythrosine-mediated PDI of Acinetobacter baumannii in planktonic and biofilm forms. The planktonic cell growth of three A. baumannii strains was subjected to PDI by using erythrosine B (50 µM) in 0.01% acetic acid and green laser light (530 nm) at fluence of 40 J/cm2. The phototoxic effect of erythrosine B (100 µM) in combination with chitosan (12.5 mg/ml) (in a solution of acetic acid) at fluence of 80 J/cm2 on biofilms was also evaluated. Finally, the cytotoxicity and phototoxicity of the mentioned mixture were assessed on human fibroblasts. Planktonic cells of all three studied A. baumannii strains were almost eradicated by erythrosine B-mediated PDI in the presence of acetic acid. Also, PDI combined with chitosan resulted in a marked decrease in the number of viable biofilm cells (> 3 log10 CFU). At the same experimental conditions, only 15% of the fibroblasts were photoinactivated. The results showed that PDI by using erythrosine B in acetic acid is very effective against A. baumannii planktonic cells and could eliminate them significantly. Also, chitosan enhanced the anti-biofilm efficacy of erythrosine B-mediated PDI against A. baumannii, suggesting that combination therapy may be useful in targeting biofilms.

Photodynamic optimization by combination of xanthene dyes on different forms of Streptococcus mutans: An in vitro study.

OBJECTIVE: The photokilling rate in antimicrobial photodynamic therapy (a-PDT) is highly related to interaction of reactive oxygen species (ROS) produced, ability of photosensitizers (PS) in incorporating into microorgansims and light devices/microorganism type. Since xanthene dyes (Rose Bengal and Erythrosine) are present in the dental practice as PS, have high quantum yield of singlet oxygen and are efficiently incorporated into bacterial cells, the additive bactericidal ability of a combination of xanthene dyes was tested on planktonic cultures and biofilms of Streptococcus mutans when irradiated by a hand-held LED photopolymerizer unit. METHODS: Planktonic cultures of S. mutans (UA 159 ATCC 700610) were grown in BHI broth with 1 % sucrose. This culture was exposed to a concentrations of Rose Bengal (RB) and Erythrosine (ER) at 1.5, 3.5 µM, in combination (RB + ER + L+) / alone (RB + L+/ ER + L+) and irradiated with a blue LED high light intensity (L). Accordingly, concentrations of dyes and time irradiation were increased in 10 times and applied on 120 h - biofilms of S. mutans and compared with a 0.12 % Chlorhexidine solution (0.12 % - CHX). For statistical analysis, parametrical procedures were applied (n = 6; ANOVA test and Tukey post hoc test; α = 0.05) and data transformed into log 10. RESULTS: Substantial antimicrobial reduction was verified in planktonic cultures (∼ 7 log reduction) and biofilm (∼ 1 log reduction) for combined a-PDT group (RB + ER + L+) presenting a significant statistical difference to control group (p < 0.05) with similar effect to CHX group (p > 0.05). CONCLUSION: Different forms of S. mutans can be effectively controlled by photodynamic therapy and optimized when in combination of xanthene dyes.

Photo-Induced Necrosis on Oropharyngeal Carcinoma (HEp-2) Cells Mediated by the Xanthene Erythrosine.

The photodynamic therapy (PDT) has been outstanding as a promising alternative for treating different carcinomas. However, the lack of detailed knowledge on the mechanisms of action prevents exploitation of the therapy full potential. Herein we shall evaluate not only the photodynamic efficiency but the mechanism of cell death triggered by the photoactivated erythrosine in oropharyngeal cancer cells (HEp-2). Cytotoxic assays were performed by MTT at distinct concentrations (10-3 to 10-6 mol/L) and incubation time (3, 24 and 48 h) of erythrosine in HEp-2 in vitro culture. In addition to the cytotoxic effect, the mechanisms of cell death were evaluated by flow cytometry following the annexin V/propidium iodide double staining protocol. Erythrosine was incorporated by HEp-2 cells in a dose- and time-dependent pathway. The incubation of erythrosine in dark has not shown any significant effect over the culture until 24 h and 1.25×10-6 mol/L concentration, from which a small portion (<25% and statistically significant) of the cell population have undergone apoptosis. On the other hand, 50% of cell viability is reduced mainly by necrosis when 10, 3.75 and 1.9×10-6 mol/L of erythrosine concentrations at 3, 24 and 48 h of incubation are photoactivated, respectively. Bioinspired models of tumor membrane based on Langmuir monolayers of 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) mixture reveled that electrostatic interactions with the lipid head groups are the main driving forces allowing the erythrosine adsorption. Furthermore, light-induced hydroperoxidation significantly increased the surface area of the monolayers, which might be the origin of the necrotic pathway triggered in HEp-2 cells.

Effects of Erythrosine on Neural Tube Development in Early Chicken Embryos.

OBJECTIVE: Erythrosine (E127), a synthetic food dye containing iodine and sodium, has often been used inside packaged foods and beverages in Turkey and many other countries. We evaluated the effects of erythrosine on neural tube development in early-stage chicken embryos. METHODS: The study included 4 groups, with a total of 80 embryos: a control group, a normal saline group, a half-dose group, and a high-dose group. After 30 hours of incubation, saline and erythrosine solution was injected under the embryonic discs. At the end of 72 hours, the embryos were excised and evaluated macroscopically and histopathologically. RESULTS: Neural tube defects were detected in the erythrosine-administered groups with statistically significant differences. In contrast, the embryos in the control and saline groups displayed normal development. CONCLUSIONS: Erythrosine increased the risk of neural tube defects in early-stage chicken embryos, even at half of the approved dose.

"Biotin-targeted mixed liposomes: A smart strategy for selective release of a photosensitizer agent in cancer cells".

The high incidence of cancer, necessity of treatment, and prognosis times are urgent issues that need to be addressed. In this work, we present DPPC liposomes coated with F127 triblock copolymers as a promising alternative in drug delivery systems for cancer therapy. The proposed mixed liposomes exhibit adequate size, high stability, and passive targeting that result from the EPR effect. An interesting strategy to obtain both passive and active targeting is the vectorization with a covalent bond between F127 and Biotin (a vitamin). Cancer cells can overexpress Biotin receptors, such as Avidin. Here, we evaluate the cytotoxic effects of the erythrosine-decyl ester (ERYDEC). This is a photosensitizer that can be utilized in photodynamic therapy (PDT) and incorporated in DPPC liposomes coated with F127 (F127/DPPC) and the biotinylated-F127 (F127-B/DPPC). The results showed that DPPC liposomes were efficiently mixed with common F127 or F127B, exhibiting adequate physical properties with simple and low-cost preparation. An HABA/Avidin assay showed the amount of Biotin available at the liposome surface. In addition, ERYDEC interaction with lipid vesicles showed high encapsulating efficiency and slow release kinetics. The ERYDEC monomeric species are represented by high light absorption and high singlet oxygen generation (1O2), which confirm the presence of the drug in its monomeric state, as required for PDT. The ERYDEC/liposome system showed high stability and absence of significant cytotoxic effects (absence of light) in fibroblasts of the Mus musculus cell line. In addition, phototoxicity studies showed that ERYDEC/liposomes were able to inhibit cancer cells. However, in the biotinylated system, the effect was much greater than the common F127 coating. This dramatically decreased the inhibitory concentration of CC50 and CC90. In addition, cellular uptake studies based on fluorescence properties of ERYDEC showed that a two-hour incubation period was enough for the uptake by the cell. Therefore, the new vectorized-coated liposome is a potential system for use in cancer treatments, considering that it is a theranostic platform.

PEG-coated vesicles from Pluronic/lipid mixtures for the carrying of photoactive erythrosine derivatives.

Liposomes are very attractive membrane models and excellent drug delivery systems. Concerning their drug delivery aspects, the mixing liposomes with biocompatible copolymers allows for stability and the incorporation of several drugs. We developed PEG coated vesicles from the mixture of DPPC and F127 Pluronic copolymer to obtain long-circulating nanoparticles (mixed vesicles). We employed an innovative process previously developed by us: a small amount of F127 mixed in DPPC, thin film preparation, followed by hydration (lipids plus F127) using a bath sonicator cleaner type, forming unilamellar spherical vesicles with diameter ∼100 nm. The formed PEG coated vesicles were incorporated with the xanthene dye Erythrosine B (ERY), and its ester derivatives as photosensitizers (PS) for photodynamic proposes. The F127/DPPC mixed vesicles promoted a higher PS incorporation, and with better thermal and kinetic stability, at least 60 days, when compared to conventional DPPC liposome. The binding constant and quenching analysis revealed that with a higher PS hydrophobicity, PS affinity increases toward the nanoparticle and results in a deeper PS location inside the lipid bilayer. An increment in the fluorescence quantum yield was observed, while the PS singlet oxygen generations remained high. Dialysis studies demonstrated that PS were released based on their hydrophobicity. Permeation analysis showed that all PS can reach the deeper regions of the skin. The Decyl Ester derivative/nanoparticle exhibited high photoactivity against Caco-2 cancer cells (in vitro studies). The PEG coated from F127/DPPC mixed vesicles are very promising nanocarriers for erythrosine and its derivatives.

Antimicrobial Photodynamic Inactivation Mediated by Rose Bengal and Erythrosine Is Effective in the Control of Food-Related Bacteria in Planktonic and Biofilm States.

The thermal and chemical-based methods applied for microbial control in the food industry are not always environmentally friendly and may change the nutritional and organoleptic characteristics of the final products. Moreover, the efficacy of sanitizing agents may be reduced when microbial cells are enclosed in biofilms. The objective of this study was to investigate the effect of photodynamic inactivation, using two xanthene dyes (rose bengal and erythrosine) as photosensitizing agents and green LED as a light source, against Staphylococcus aureus, Listeria innocua, Enterococcus hirae and Escherichia coli in both planktonic and biofilm states. Both photosensitizing agents were able to control planktonic cells of all bacteria tested. The treatments altered the physicochemical properties of cells surface and also induced potassium leakage, indicating damage of cell membranes. Although higher concentrations of the photosensitizing agents (ranging from 0.01 to 50.0 µmol/L) were needed to be applied, the culturability of biofilm cells was reduced to undetectable levels. This finding was confirmed by the live/dead staining, where propidium iodide-labeled bacteria numbers reached up to 100%. The overall results demonstrated that photoinactivation by rose bengal and erythrosine may be a powerful candidate for the control of planktonic cells and biofilms in the food sector.

Biofilms of Candida albicans and Streptococcus sanguinis and their susceptibility to antimicrobial effects of photodynamic inactivation.

This study evaluated the effects of photodynamic inactivation (PDI) on single and multi-species biofilms, compounds by Candida albicans and Streptococcus sanguinis. Biofilms were formed, on microplate of 96 wells, by suspensions of C. albicans (ATCC 18804) and S. sanguinis (ATCC 7073) adjusted in 107 cells/mL, followed by incubation of 48 h (with 5% CO2). The effects of the photosensitizer erythrosine (ER) at 400 µM for 5 min and green light-emitting diode (LED - 532 ±â€¯10 nm) for 3 min, alone and conjugated, were evaluated. After normality test, results was analysed by Tukey´s test (P < 0.05). PDI group promoted reductions of 1.07 and 0.39 log10, respectively, in biofilms of C. albicans alone and in association with S. sanguinis. Biofilms of S. sanguinis alone were more sensitive, with reduction of 4.48 log10. When in association with the yeast, S. sanguinis have a decrease of 2.67 log10. SEM analysis revealed a decrease in bacterial and fungal structures of biofilms treated with PDI. In conclusion PDI promoted significant microbial reductions in both species of microorganisms grown on mixed biofilms. This study is one of the pioneers to evaluate the antimicrobial action of PDI on biofilms of S. sanguinis and C. albicans, demonstrating a way to control these microorganisms of clinical importance.

Antimicrobial effect of photodynamic therapy using erythrosine/methylene blue combination on Streptococcus mutans biofilm.

BACKGROUND: Photodynamic therapy (PDT) has demonstrated promising results in the treatment of several clinical pathologies through the photochemical reaction caused by the combination of a photosensitizer and a light source. The objective of this study was to evaluate the antimicrobial effect of the combination of the photosensitizers (PSs) erythrosine/methylene blue activated by a white halogen light device on Streptococcus mutans biofilm. METHODS: Two separate experiments were conducted, the first using the PSs at the concentration of 100 µM, and the second 250 µM. The PSs were tested on S. mutans biofilms cultured for 24 h in isolation, in combination, with and without light activation for 2 min fractionated in 4 periods of 30 s. After treatment, biofilms were diluted and plated on BHI medium and incubated for 24 h for colony forming units (CFU) counting. The results (log10) were analyzed with ANOVA followed by Tukey test (p < 0.05). RESULTS: The erythrosine/methylene blue combination activated by white halogen light at 100 and 250 µM, and erythrosine at 250 µM, methylene blue at 250 µM presented significantly reduced cell counts (3.2 log10, 5.3 log10, 4.5 log10, 4.3 log10, respectively) when compared to controls (p < 0.05). CONCLUSION: PDT with the combination of erythrosine/methylene blue demonstrated better results that the PSs in isolation regardless of the concentration. The use of this combination at the concentration of 250 µM shows promise as an antibacterial treatment for carious lesions and should be further assessed.

Photodynamic inactivation in the expression of the Candida albicans genes ALS3, HWP1, BCR1, TEC1, CPH1, and EFG1 in biofilms.

The objective of this study was to evaluate the effects of photodynamic inactivation (PDI) on Candida albicans biofilms, evaluating its effects on gene expression of ALS3, HWP1, BCR1, TEC1, CPH1, and EFG1 by yeast. Three samples of C. albicans were used in this study: a clinical sample from a patient with HIV (39S), a clinical sample from a patient with denture stomatitis lesion (Ca30), and a standard strain ATCC 18804. The quantification of gene expression was related to the production of those genes in the samples referred above using quantitative polymerase chain reaction (qPCR) assay in real time. The photosensitizer methylene blue at 300 uM and erythrosine at 400 uM, sensitized with low-power laser (visible red, 660 nm) and green LED (532 nm), respectively, were used for PDI. Four groups of each sample and PDI protocol were evaluated: (a) P+L+: sensitization with the photosensitizer and irradiation with light, (b) P+L-: only treatment with the photosensitizer, (c) P-L+: only irradiation with light, and (d) P-L-: without sensitization with the dye and absence of light. The results were analyzed by t test, with a significance level of 5%. The photodynamic inactivation was able to reduce the expression of all genes for both treatments, laser and LED. The fold-decrease for the genes ALS3, HWP1, BCR1, TEC1, CPH1, and EFG1 were 0.73, 0.39, 0.77, 0.71, 0.67, and 0.60 for laser, respectively, and 0.66, 0.61, .050, 0.43, 0.54, and 0.66 for LED, respectively. It could be concluded that PDI showed a reduction in the expression of C. albicans genes, suggesting its virulence decrease.

Erythrosin B is a potent and broad-spectrum orthosteric inhibitor of the flavivirus NS2B-NS3 protease.

Many flaviviruses, such as Zika virus (ZIKV), Dengue virus (DENV1-4) and yellow fever virus (YFV), are significant human pathogens. Infection with ZIKV, an emerging mosquito-borne flavivirus, is associated with increased risk of microcephaly in newborns and Guillain-Barré syndrome and other complications in adults. Currently, specific therapy does not exist for any flavivirus infections. In this study, we found that erythrosin B, an FDA-approved food additive, is a potent inhibitor for flaviviruses, including ZIKV and DENV2. Erythrosin B was found to inhibit the DENV2 and ZIKV NS2B-NS3 proteases with IC50 in low micromolar range, via a non-competitive mechanism. Erythrosin B can significantly reduce titers of representative flaviviruses, DENV2, ZIKV, YFV, JEV, and WNV, with micromolar potency and with excellent cytotoxicity profile. Erythrosin B can also inhibit ZIKV replication in ZIKV-relevant human placental and neural progenitor cells. As a pregnancy category B food additive, erythrosin B may represent a promising and easily developed therapy for management of infections by ZIKV and other flaviviruses.