Curcuminoid-Mediated Antimicrobial Photodynamic Therapy on a Murine Model of Oral Candidiasis.

Antimicrobial Photodynamic Therapy (aPDT) has been extensively investigated in vitro, and preclinical animal models of infections are suitable for evaluating alternative treatments prior to clinical trials. This study describes the efficacy of aPDT in a murine model of oral candidiasis. Forty mice were immunosuppressed with subcutaneous injections of prednisolone, and their tongues were inoculated using an oral swab previously soaked in a C. albicans cell suspension. Tetracycline was administered via drinking water during the course of the experiment. Five days after fungal inoculation, mice were randomly distributed into eight groups; a ninth group of untreated uninfected mice was included as a negative control (n = 5). Three concentrations (20 µM, 40 µM, and 80 µM) of a mixture of curcuminoids were tested with a blue LED light (89.2 mW/cm2; ~455 nm) and without light (C+L+ and C+L- groups, respectively). Light alone (C-L+), no treatment (C-L-), and animals without infection were evaluated as controls. Data were analyzed using Welch's ANOVA and Games-Howell tests (α = 0.05). Oral candidiasis was established in all infected animals and visualized macroscopically through the presence of characteristic white patches or pseudomembranes on the dorsum of the tongues. Histopathological sections confirmed a large presence of yeast and filaments limited to the keratinized layer of the epithelium in the C-L- group, and the presence of fungal cells was visually decreased in the images obtained from mice subjected to aPDT with either 40 µM or 80 µM curcuminoids. aPDT mediated by 80 µM curcuminoids promoted a 2.47 log10 reduction in colony counts in comparison to those in the C-L- group (p = 0.008). All other groups showed no statistically significant reduction in the number of colonies, including photosensitizer (C+L-) or light alone (C-L+) groups. Curcuminoid-mediated aPDT reduced the fungal load from the tongues of mice.

Zerumbone Disturbs the Extracellular Matrix of Fluconazole-Resistant Candida albicans Biofilms.

This study assessed the effect of zerumbone (ZER) against fluconazole-resistant (CaR) and -susceptible Candida albicans (CaS) biofilms and verified the influence of ZER on extracellular matrix components. Initially, to determine the treatment conditions, the minimum inhibitory concentration (MIC), the minimum fungicidal concentration (MFC) and the survival curve were evaluated. Biofilms were formed for 48 h and exposed to ZER at concentrations of 128 and 256 µg/mL for 5, 10 and 20 min (n = 12). One group of biofilms did not receive the treatment in order to monitor the effects. The biofilms were evaluated to determine the microbial population (CFU/mL), and the extracellular matrix components (water-soluble polysaccharides (WSP), alkali-soluble polysaccharides (ASPs), proteins and extracellular DNA (eDNA), as well as the biomass (total and insoluble) were quantified. The MIC value of ZER for CaS was 256 µg/mL, and for CaR, it was 64 µg/mL. The survival curve and the MFC value coincided for CaS (256 µg/mL) and CaR (128 µg/mL). ZER reduced the cellular viability by 38.51% for CaS and by 36.99% for CaR. ZER at 256 µg/mL also reduced the total biomass (57%), insoluble biomass (45%), WSP (65%), proteins (18%) and eDNA (78%) of CaS biofilms. In addition, a reduction in insoluble biomass (13%), proteins (18%), WSP (65%), ASP (10%) and eDNA (23%) was also observed in the CaR biofilms. ZER was effective against fluconazole-resistant and -susceptible C. albicans biofilms and disturbed the extracellular matrix.

The Effect of Sub-Lethal Successive Applications of Photodynamic Therapy on Candida albicans Biofilm Depends on the Photosensitizer.

This study aimed to evaluate the potential of successive applications of sub-lethal doses of the antimicrobial photodynamic therapy (aPDT) mediated by Photodithazine® (PDZ) and curcumin (CUR) associated with LED in the viability, reactive oxygen species (ROS) production, and gene expression of Candida albicans. The microbial assays were performed using planktonic cultures and biofilms. Ten successive applications (Apl#) were performed: aPDT (P+L+; C+L+), photosensitizer (P+L-; C+L-), and LED (P-L+; C-L+). Control groups were used (P-L-; C-L-). The viability of C. albicans was determined by cultivating treated cultures on agar plates with or without fluconazole (FLU). In addition, the ROS detection and expression of SOD1, CAP1, and ERG11 genes were determined. For planktonic cultures, no viable colonies were observed after Apl#3 (without FLU) and Apl#2 (with FLU) for either photosensitizer. Biofilm treated with P+L+ resulted in the absence of cell viability after Apl#7, while C+L+ showed ~1.40 log10 increase in cell viability after Apl#2, regardless of FLU. For both photosensitizers, after the last application with viable colonies, the production of ROS was higher in the biofilms than in the planktonic cultures, and SOD1 expression was the highest in P+L+. A reduction of CAP1 and ERG11 expression occurred after P+L+, regardless of FLU. C+L+ had a higher level of ROS, and the treatments were non-significant for gene expression. Sub-lethal doses of aPDT mediated by CUR could induce C. albicans resistance in biofilms, while C. albicans cells in biofilms were susceptible to aPDT mediated by PDZ.

DNase enhances photodynamic therapy against fluconazole-resistant Candida albicans biofilms.

OBJECTIVE: This study evaluated the effectiveness of DNase I combined with antimicrobial photodynamic therapy, mediated by Photodithazine® and light-emitting diode light, against biofilms formed by a fluconazole-resistant Candida albicans strain (ATCC 96901) and two clinical isolates (R14 and R70). MATERIALS AND METHODS: Biofilms were grown for 48 h and exposed to DNase for 5 min, followed by application of a photosensitizer (P) and light (L), either singly or combined (P+L+, P-L+, P+L-, P-L-, P-L-DNase, P+L+DNase, P+L-DNase, and P-L+DNase; n = 12). Biofilm analysis included quantification of extracellular matrix components (water-soluble and insoluble polysaccharides, proteins and extracellular DNA), and biomass (total and insoluble), as well as the enumeration of colony-forming units. The data were analyzed using three-way analysis of variance with Bonferroni's post hoc test. RESULTS: The DNase treatment combined with aPDT showed a reduction of 1.92, 1.65, and 1.29 log10 of cell viability compared with untreated controls for ATCC 96901, R14, and R70 strains, respectively. It also reduced extracellular matrix contents of water-soluble polysaccharides (36.3%) and extracellular DNA (72.3%), as well as insoluble biomass content (43.3%). CONCLUSION: The three strains showed similar behavior when treated with DNase, and the extracellular matrix components were affected, improving the effectiveness of antimicrobial photodynamic therapy.

DNase improves the efficacy of antimicrobial photodynamic therapy in the treatment of candidiasis induced with Candida albicans.

The study evaluated the association of DNase I enzyme with antimicrobial photodynamic therapy (aPDT) in the treatment of oral candidiasis in mice infected with fluconazole-susceptible (CaS) and -resistant (CaR) Candida albicans strains. Mice were inoculated with C. albicans, and after the infection had been established, the tongues were exposed to DNase for 5 min, followed by photosensitizer [Photodithazine®(PDZ)] and light (LED), either singly or combined. The treatments were performed for 5 consecutive days. Treatment efficacy was evaluated by assessing the tongues via fungal viable population, clinical evaluation, histopathological and fluorescence microscopy methods immediately after finishing treatments, and 7 days of follow-up. The combination of DNase with PDZ-aPDT reduced the fungal viability in mice tongues immediately after the treatments by around 4.26 and 2.89 log10 for CaS and CaR, respectively (versus animals only inoculated). In the fluorescence microscopy, the polysaccharides produced by C. albicans and fungal cells were less labeled in animals treated with the combination of DNase with PDZ-aPDT, similar to the healthy animals. After 7 days of the treatment, DNase associated with PDZ-aPDT maintained a lower count, but not as pronounced as immediately after the intervention. For both strains, mice treated with the combination of DNase with PDZ-aPDT showed remission of oral lesions and mild inflammatory infiltrate in both periods assessed, while animals treated only with PDZ-aPDT presented partial remission of oral lesions. DNase I enzyme improved the efficacy of photodynamic treatment.

The biological effects of different LED wavelengths in the health field. A review / Os efeitos biológicos de diferentes comprimentos de onda de LED na área da saúde. Uma revisão

Introduction: the use of light emitting diodes (LED) in domestic and public vias have increased in the last 20 years. In addition, the LED light has been used as a light source for medical applications. Objective: since humans are increasingly exposed to LEDs, there is an urgency to investigate the possible biological effects on tissues caused by this exposure. So, researchers have been focused their investigations in the application of this light in the health field. Material and method: in this review, a search in important databases was performed on the biological effects caused after application of different LED light protocols in in vitro and in vivo studies. Result: although most published papers have shown positive results, some of them reported negative biological effects of light LEDs technology on humans' cells/tissues. Conclusion: therefore, the comprehension of the biological effects caused by light LEDs will provide a better assessment of the risks involved using this technology.

Introdução: o uso de diodos emissores de luz ("LED") em vias domésticas e públicas tem aumentado nos últimos 20 anos. Além disso, a luz LED tem sido usada para aplicações médicas. Objetivo: pelo fato de seres humanos estarem cada vez mais expostos aos LEDs, há urgência em investigar os possíveis efeitos biológicos nos tecidos causados por esta exposição. Assim, pesquisadores têm focado suas investigações no uso desta luz na área da saúde. Material e método: nesta revisão foi realizada uma pesquisa em bancos de dados conceituados sobre os efeitos biológicos causados após aplicação de diferentes protocolos de luz LED em estudos in vitro e in vivo. Resultado: embora a maioria dos artigos publicados tenham mostrado resultados positivos, alguns deles relataram efeitos biológicos negativos da tecnologia de LEDs nas células/tecidos humanos. Conclusão: portanto, a compreensão dos efeitos biológicos causados pela luz LED proporcionará uma melhor avaliação dos riscos envolvidos no uso desta tecnologia.

Efficacy of Antimicrobial Photodynamic Therapy Mediated by Photosensitizers Conjugated with Inorganic Nanoparticles: Systematic Review and Meta-Analysis.

Antimicrobial photodynamic therapy (aPDT) is a method that does not seem to promote antimicrobial resistance. Photosensitizers (PS) conjugated with inorganic nanoparticles for the drug-delivery system have the purpose of enhancing the efficacy of aPDT. The present study was to perform a systematic review and meta-analysis of the efficacy of aPDT mediated by PS conjugated with inorganic nanoparticles. The PubMed, Scopus, Web of Science, Science Direct, Cochrane Library, SciELO, and Lilacs databases were searched. OHAT Rob toll was used to assess the risk of bias. A random effect model with an odds ratio (OR) and effect measure was used. Fourteen articles were able to be included in the present review. The most frequent microorganisms evaluated were Staphylococcus aureus and Escherichia coli, and metallic and silica nanoparticles were the most common drug-delivery systems associated with PS. Articles showed biases related to blinding. Significant results were found in aPDT mediated by PS conjugated with inorganic nanoparticles for overall reduction of microorganism cultured in suspension (OR = 0.19 [0.07; 0.67]/p-value = 0.0019), E. coli (OR = 0.08 [0.01; 0.52]/p-value = 0.0081), and for Gram-negative bacteria (OR = 0.12 [0.02; 0.56/p-value = 0.0071). This association approach significantly improved the efficacy in the reduction of microbial cells. However, additional blinding studies evaluating the efficacy of this therapy over microorganisms cultured in biofilm are required.

Efficacy of curcumin-mediated antibacterial photodynamic therapy for oral antisepsis: A systematic review and network meta-analysis of randomized clinical trials.

BACKGROUND: The presence of oral microorganisms resistant to traditional treatment is increasing, thus a search for new therapies is needed. In this context, antimicrobial photodynamic therapy (aPDT) is an approach for the treatment of antibiotic resistant andnon resistant microorganisms. Therefore, the aim of the present study was to conduct a systematic review and meta-analysis of randomized clinical trials of aPDT for oral antisepsis against oral polymicrobial biofilms. METHODS: PubMed, Science Direct, Scopus, SciELO, Lilacs, Cochrane Library and Embase databases were searched. In total, five articles were included for qualitative analysis and four articles were used for quantitative analyses. Bias assessment of the eligible articles was made using the RoB 2 criteria. Network meta-analysis was performed using the random-effect model. Subgroup's analysis was also conducted. The groups evaluated were aPDT, exposure to light only and no treatment at all (control group). The quality of evidence was assessed by CINeMA approach. RESULTS: aPDT mediated by curcumin had significant results in the reducing bacterial load (0.31-0.49 log10 UFC/ I2=0%) when compared with the control group. The included articles were classified as low risk of bias, despite biases detected by allocation and blinding. Moreover, quantitative analysis between aPDT and control group and between light and control group were classified with low risk of confidence rating, while the results from aPDT versus light were classified as moderate risk of confidence rating. CONCLUSION: aPDT has significant efficacy for oral antisepsis, however more randomized clinical trials will be needed to validate the present results.

Gene expression of Candida albicans strains isolates from patients with denture stomatitis submitted to treatments with photodynamic therapy and nystatin.

The study evaluated the effect of antimicrobial photodynamic therapy (aPDT) and nystatin (NYS) in the expression of genes (ACT1, ALS1, CAP1, CAT1, EFG1, HWP1, LIP3, PLB1, SAP1, and SOD1) involved in the virulence of Candida albicans strains recovered from patients with denture stomatitis (DS). These strains were isolated from the patients before (initial) and after treatment (final), and 45 days after the treatments (follow-up). For gene expression analyses, RNA was isolated from the clinical strains, followed by cDNA synthesis and qPCR using specific primers for each target gene. The samples that present integrity were pooled to increase the RNA yield. In the end, four patients treated with aPDT and five patients treated with NYS had the clinical isolates of C. albicans submitted to gene expression evaluation. The data demonstrated a statistical difference in the expression of PLB1 and ACT1 for the different therapies (aPDT versus NYS). Also, there was a statistical difference in the expression of CAT1, SOD1, and LIP3 at the time intervals assessed (initial, final, and follow-up). In contrast, no statistical difference was found in the expression of ALS1, HWP1, EFG1, CAP1, CAT1, SOD1, LIP3, and SAP1 between the therapies, while no significant difference was detected at the time intervals evaluated for ALS1, HWP1, EFG1, CAP1, and SAP1. Therefore, the topical treatments for DS with aPDT or NYS did not effect the expression of most C. albicans virulence genes evaluated.

Consecutive treatments with photodynamic therapy and nystatin altered the expression of virulence and ergosterol biosynthesis genes of a fluconazole-resistant Candida albicans in vivo.

This investigation assessed the effect of five consecutive daily topical treatments of antimicrobial photodynamic therapy (aPDT), nystatin (NYS), and an association of treatments on a fluconazole-resistant strain of Candida albicans colonizing the tongues of mice. After the last treatments application, colonies of C. albicans were recovered from the tongues and used to determine their fluconazole susceptibility. After 24 hours of the last treatment, the mice tongues were processed to evaluate the expression of C. albicans genes related to the virulence and ergosterol production. The fluconazole susceptibility test yielded a resistance profile similar for all treatment groups and the control group (no treatment). The treatments aPDT, NYS, NYS+aPDT, and aPDT+NYS promoted a reduction in ALS1, EFG1, CAP1, SOD1, SAP1, and LIP3 expression. The expression of HWP1 was higher in the three groups containing nystatin. In contrast, the treatments produced a significative increase in CAT1 gene expression, mainly in the groups in which aPDT was performed. The expression of genes related to ergosterol production was significantly reduced by the treatments evaluated (aPDT, NYS, NYS+aPDT, and aPDT+NYS). Thus, the consecutive topical treatments performed on mice tongues promoted a reduction in the expression of virulence and ergosterol biosynthesis genes of a fluconazole-resistant C. albicans.

Successive applications of Antimicrobial Photodynamic Therapy effects the susceptibility of Candida albicans grown in medium with or without fluconazole.

Antimicrobial Photodynamic Therapy (aPDT) was introduced as a therapy due to resistance that microorganisms have developed to conventional drugs. The study aimed to evaluate the potential of successive applications of aPDT in effecting Candida albicans susceptibility and also whether the presence of fluconazole effected the recovery of the fungi in the culture medium. Planktonic cultures and biofilm were subjected to successive applications of Photodithazine-mediated (25 mg/L) LED-associated aPDT (660 nm, 34 mW/cm2). Plating was performed on Sabouraud Dextrose Agar supplemented or not with fluconazole to recover colony-forming units per milliliter (CFU/mL). Surviving cells were recovered, recultivated, and again exposed to the treatment. The treatments were performed until not enough colonies were available for recultivation and continuation of the protocol. The complete inactivation of the fungus was obtained after three and five applications for planktonic culture and biofilm, respectively. A reduction of 6.3 log10 was observed after third applications in the planktonic cultures grown on medium without fluconazole, while there was a 7 log10 reduction of these cultures grown on fluconazole medium. However, a reduction of 6.1 log10 occurred for biofilms after fifth applications for cultures grown on medium without fluconazole, while a reduction of 6.7 log10 was observed for cultures grown on medium with the antifungal. Thus, aPDT was potentiated by fluconazole. C. albicans in planktonic and biofilm cultures are susceptible to successive applications of PDZ-mediated aPDT, and tolerance to aPDT is higher in the biofilm.

Antimicrobial photodynamic therapy reduces gene expression of Candida albicans in biofilms.

The present study evaluated whether the oxidative stress caused by antimicrobial photodynamic therapy (aPDT) affects the expression of C. albicans genes related to adhesion and biofilm formation (ALS1 and HPW1) and oxidative stress response (CAP1, CAT1, and SOD1). The aPDT was mediated by two photosensitizing agents (PSs) Photodithazine® (PDZ at 100 and 200 mg/L) or Curcumin (CUR at 40 and 80 µM) and LED (37.5 J/cm2 or 50 J/cm2). The quantification of the expression was performed by Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) using specific primers for the target genes. The data were analyzed by Analysis of Variance (α = 0.05), followed by Tukey's post-test. It was observed reduction in the expression of ALS1, HWP1, CAP1, CAT1, and SOD1 when aPDT was performed using 200 mg/L PDZ and 80 µM CUR associated to LED (37.7 and 50 J/cm2, respectively) and using 100 mg/L PDZ and 40 µM CUR with LED of 50 J/cm2 (versus control). Also, the expression of CAP1 and SOD1 genes was reduced after aPDT using 100 mg/L PDZ and LED of 37.5 J/cm2. There was a significant reduction in the expression of genes HWP1, CAP1, and SOD1 after aPDT using 40 µM CUR and 37.5 J/cm2 (versus the control group). The application of LED only at 37.5 and 50 J/cm2 promoted down-regulation of ALS1, CAP1, CAT1, and SOD1 genes (versus the control group). Therefore, aPDT mediated by LED -associated PSs PDZ and CUR promoted a reduction in the expression of the five C. albicans genes evaluated.