Segmentation of hyphae and yeast in fungi-infected tissue slice images and its application in analyzing antifungal blue light therapy.

Candida albicans is a pathogenic fungus that undergoes morphological transitions between hyphal and yeast forms, adapting to diverse environmental stimuli and exhibiting distinct virulence. Existing research works on antifungal blue light (ABL) therapy have either focused solely on hyphae or neglected to differentiate between morphologies, obscuring potential differential effects. To address this gap, we established a novel dataset of 150 C. albicans-infected mouse skin tissue slice images with meticulously annotated hyphae and yeast. Eleven representative convolutional neural networks were trained and evaluated on this dataset using seven metrics to identify the optimal model for segmenting hyphae and yeast in original high pixel size images. Leveraging the segmentation results, we analyzed the differential impact of blue light on the invasion depth and density of both morphologies within the skin tissue. U-Net-BN outperformed other models in segmentation accuracy, achieving the best overall performance. While both hyphae and yeast exhibited significant reductions in invasion depth and density at the highest ABL dose (180 J/cm2), only yeast was significantly inhibited at the lower dose (135 J/cm2). This novel finding emphasizes the importance of developing more effective treatment strategies for both morphologies.

We studied the effects of blue light therapy on hyphal and yeast forms of Candida albicans. Through image segmentation techniques, we discovered that the changes in invasion depth and density differed between these two forms after exposure to blue light.

Assessment of multispecies biofilm growth on root canal dentin under different radiation therapy regimens.

OBJECTIVES: To assess the growth of a multispecies biofilm on root canal dentin under different radiotherapy regimens. MATERIALS AND METHODS: Sixty-three human root dentin cylinders were distributed into six groups. In three groups, no biofilm was formed (n = 3): NoRT) non-irradiated dentin; RT55) 55 Gy; and RT70) 70 Gy. In the other three groups (n = 18), a 21-day multispecies biofilm (Enterococcus faecalis, Streptococcus mutans, and Candida albicans) was formed in the canal: NoRT + Bio) non-irradiated + biofilm; RT55 + Bio) 55 Gy + biofilm; and RT70 + Bio) 70 Gy + biofilm. The biofilm was quantified (CFUs/mL). Biofilm microstructure was assessed under SEM. Microbial penetration into dentinal tubules was assessed under CLSM. For the biofilm biomass and dentin microhardness pre- and after biofilm growth assessments, 45 bovine dentin specimens were distributed into three groups (n = 15): NoRT) non-irradiated + biofilm; RT55 + Bio) 55 Gy + biofilm; and RT70 + Bio) 70 Gy + biofilm. RESULTS: Irradiated specimens (70 Gy) had higher quantity of microorganisms than non-irradiated (p = .010). There was gradual increase in biofilm biomass from non-irradiated to 55 Gy and 70 Gy (p < .001). Irradiated specimens had greater reduction in microhardness after biofilm growth. Irradiated dentin led to the growth of a more complex and irregular biofilm. There was microbial penetration into the dentinal tubules, regardless of the radiation regimen. CONCLUSION: Radiotherapy increased the number of microorganisms and biofilm biomass and reduced dentin microhardness. Microbial penetration into dentinal tubules was noticeable. CLINICAL RELEVANCE: Cumulative and potentially irreversible side effects of radiotherapy affect biofilm growth on root dentin. These changes could compromise the success of endodontic treatment in oncological patients undergoing head and neck radiotherapy.

Latex membranes with methylene blue dye for antimicrobial photodynamic therapy.

The search for new materials that can be applied in the treatment of injured human tissues has led to the development of new dressings. Membranes have potential as dressing materials because they can be fitted to and interact with the tissue surface. In this study, we analyze the morphological properties and wettability of latex membranes, along with the incorporation of the photosensitizer methylene blue, in the context of the utility of the membranes in curative applications involving photodynamic therapy (PDT). It was observed that deposition of the photosensitizer into latex membranes increased both the surface roughness and wettability. Antifungal testing indicated that antimicrobial PDT assisted by the latex membranes incorporating methylene blue effectively inactivated Candida albicans.

Antimicrobial Blue Light Inactivation of Microbial Isolates in Biofilms.

BACKGROUND AND OBJECTIVES: Biofilms cause more than 80% of infections in humans, including more than 90% of all chronic wound infections and are extremely resistant to antimicrobials and the immune system. The situation is exacerbated by the fast spreading of antimicrobial resistance, which has become one of the biggest threats to current public health. There is consequently a critical need for the development of alternative therapeutics. Antimicrobial blue light (aBL) is a light-based approach that exhibits intrinsic antimicrobial effect without the involvement of exogenous photosensitizers. In this study, we investigated the antimicrobial effect of this non-antibiotic approach against biofilms formed by microbial isolates of multidrug-resistant bacteria. STUDY DESIGN/MATERIALS AND METHODS: Microbial isolates of Acinetobacter baumannii, Candida albicans, Escherichia coli, Enterococcus faecalis, MRSA, Neisseria gonorrhoeae, Pseudomonas aeruginosa, and Proteus mirabilis were studied. Biofilms were grown in microtiter plates for 24 or 48 hours or in the CDC biofilm reactor for 48 hours and exposed to aBL at 405 nm (60 mW/cm2 , 60 or 30 minutes). The anti-biofilm activity of aBL was measured by viable counts. RESULTS: The biofilms of A. baumannii, N. gonorrhoeae, and P. aeruginosa were the most susceptible to aBL with between 4 and 8 log10 inactivation after 108 J/cm2 (60 mW/cm2 , 30 minutes) or 216 J/cm2 (60 mW/cm2 , 60 minutes) aBL were delivered in the microplates. On the contrary, the biofilms of C. albicans, E. coli, E. faecalis, and P. mirabilis were the least susceptible to aBL inactivation (-0.30, -0.24, -0.84, and -0.68 log10 inactivation, respectively). The same aBL treatment in biofilms developed in the CDC biofilm reactor, caused -1.68 log10 inactivation in A. baumannii and -1.74 and -1.65 log10 inactivation in two different strains of P. aeruginosa. CONCLUSIONS: aBL exhibits potential against pathogenic microorganisms and could help with the significant need for new antimicrobials in clinical practice to manage multidrug-resistant infections. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Quinine Improves the Fungicidal Effects of Antimicrobial Blue Light: Implications for the Treatment of Cutaneous Candidiasis.

BACKGROUND AND OBJECTIVE: Candida albicans is an opportunistic fungal pathogen of clinical importance and is the primary cause of fungal-associated wound infections, sepsis, or pneumonia in immunocompromised individuals. With the rise in antimicrobial resistance, it is becoming increasingly difficult to successfully treat fungal infections using traditional antifungals, signifying that alternative non-traditional approaches must be explored for their efficacy. STUDY DESIGN/MATERIALS AND METHODS: We investigated the combination of antimicrobial blue light (aBL) and quinine hydrochloride (Q-HCL) for improved inactivation of C. albicans, in vitro and in vivo, relative to either monotherapy. In addition, we evaluated the safety of this combination therapy in vivo using the TUNEL assay. RESULTS: The combination of aBL (108 J/cm2 ) with Q-HCL (1 mg/mL) resulted in a significant improvement in the inactivation of C. albicans planktonic cells in vitro, where a 7.04 log10 colony forming units (CFU) reduction was achieved, compared with aBL alone that only inactivated 3.06 log10 CFU (P < 0.001) or Q-HCL alone which did not result in a loss of viability. aBL + Q-HCL was also effective at inactivating 48-hour biofilms, with an inactivation 1.73 log10 CFU at the dose of 108 J/cm2 aBL and 1 mg/mL Q-HCL, compared with only a 0.73 or 0.66 log10 CFU by aBL and Q-HCL alone, respectively (P < 0.001). Transmission electron microscopy revealed that aBL + Q-HCL induced morphological and ultrastructural changes consistent with cell wall and cytoplasmic damage. In addition, aBL + Q-HCL was effective at eliminating C. albicans within mouse abrasion wounds, with a 2.47 log10 relative luminescence unit (RLU) reduction at the dose of 324 J/cm2 aBL and 0.4 mg/cm2 Q-HCL, compared with a 1.44 log10 RLU reduction by aBL alone. Q-HCL or nystatin alone did not significantly reduce the RLU. The TUNEL assay revealed some apoptotic cells before and 24 hours following treatment with aBL + Q-HCL. CONCLUSION: The combination of aBL + Q-HCL was effective at eliminating C. albicans both in vitro and in vivo. A comprehensive assessment of toxicity (cytotoxicity and genotoxicity) is required to fully determine the safety of aBL + Q-HCL therapy at different doses. In conclusion, the combination of aBL and Q-HCL may be a viable option for the treatment of cutaneous candidiasis. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Deletion of GLX3 in Candida albicans affects temperature tolerance, biofilm formation and virulence.

Candida albicans is a predominant cause of fungal infections in mucosal tissues as well as life-threatening bloodstream infections in immunocompromised patients. Within the human body, C. albicans is mostly embedded in biofilms, which provides increased resistance to antifungal drugs. The glyoxalase Glx3 is an abundant proteomic component of the biofilm extracellular matrix. Here, we document phenotypic studies of a glx3Δ null mutant concerning its role in biofilm formation, filamentation, antifungal drug resistance, cell wall integrity and virulence. First, consistent with its function as glyoxalase, the glx3 null mutant showed impaired growth on media containing glycerol as the carbon source and in the presence of low concentrations of hydrogen peroxide. Importantly, the glx3Δ mutant showed decreased fitness at 37°C and formed less biofilm as compared to wild type and a reintegrant strain. At the permissive temperature of 28°C, the glx3Δ mutant showed impaired filamentation as well as increased sensitivity to Calcofluor white, Congo red, sodium dodecyl sulfate and zymolyase, indicating subtle alterations in wall architecture even though gross quantitative compositional changes were not detected. Interestingly, and consistent with its impaired filamentation, biofilm formation and growth at 37°C, the glx3Δ mutant is avirulent. Our results underline the role of Glx3 in fungal pathogenesis and the involvement of the fungal wall in this process.

Role of homologous recombination genes RAD51, RAD52, and RAD59 in the repair of lesions caused by γ-radiation to cycling and G2/M-arrested cells of Candida albicans.

We have analysed the role of homologous recombination (HR) genes on the repair of double-strand breaks induced by γ-ionising radiation in Candida albicans. Depletion of either CaRad51 or CaRad52 caused a dramatic drop in the number of survivors compared with wild type, whereas depletion of CaRad59 caused a moderate decrease. Besides, compared with Saccharomyces cerevisiae, C. albicans relies more on HR proteins for repair of ionising radiation lesions. Pulse-field electrokaryotypes of survivors identified genetic alterations mainly in the form of aneuploidy in HR mutants and chromosome length polymorphism and ectopic translocation in wild type. Increasing irradiation (4 to 80 krad) of both cycling and nocodazole-treated (G2/M-arrested) cells revealed a gradual loss of chromosomes, larger chromosomes being more affected than smaller ones. For cycling wild-type cells, shattered chromosomes were progressively restored following incubation in yeast extract, peptone, dextrose medium, but not in phosphate-buffered saline, and this accompanied by a moderate increase in colony-forming units, suggesting that repair was followed by replication of survivors. Irradiated G2/M arrested cells from wild type but not from HR mutants partially restored the chromosome ladder following incubation (4-8 hr) in yeast peptone dextrose-nocodazole. However, HR mutants showed a chromosome shattering pattern similar to wild type, an indication that lesions other than double-strand breaks, likely single-strand break, are responsible for their drastically reduced survivability.

Characterizing the Antimicrobial Properties of 405 nm Light and the Corning® Light-Diffusing Fiber Delivery System.

BACKGROUND AND OBJECTIVES: Hospital-acquired infections (HAIs) and multidrug resistant bacteria pose a significant threat to the U.S. healthcare system. With a dearth of new antibiotic approvals, novel antimicrobial strategies are required to help solve this problem. Violet-blue visible light (400-470 nm) has been shown to elicit strong antimicrobial effects toward many pathogens, including representatives of the ESKAPE bacterial pathogens, which have a high propensity to cause HAIs. However, phototherapeutic solutions to prevention or treating infections are currently limited by efficient and nonobtrusive light-delivery mechanisms. STUDY DESIGN/MATERIALS AND METHODS: Here, we investigate the in vitro antimicrobial properties of flexible Corning® light-diffusing fiber (LDF) toward members of the ESKAPE pathogens in a variety of growth states and in the context of biological materials. Bacteria were grown on agar surfaces, in liquid culture and on abiotic surfaces. We also explored the effects of 405 nm light within the presence of lung surfactant, human serum, and on eukaryotic cells. Pathogens tested include Enterococcus spp, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., Staphylococcus epidermidis, Streptococcus pyogenes, Candida albicans, and Escherichia coli. RESULTS: Overall, the LDF delivery of 405 nm violet-blue light exerted a significant degree of microbicidal activity against a wide range of pathogens under diverse experimental conditions. CONCLUSIONS: The results exemplify the fiber's promise as a non-traditional approach for the prevention and/or therapeutic intervention of HAIs. Lasers Surg. Med. © 2019 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.

Twice-daily red and blue light treatment for Candida albicans biofilm matrix development control.

Phototherapy has been proposed as a direct means of affecting local bacterial infections. However, the use of phototherapy to prevent fungal biofilm development has received comparatively less attention. This study aimed to determine the effects of red light treatment and blue light treatment, without a photosensitizer, on the development of Candida albicans biofilm. During the development of 48-h biofilms of C. albicans SN 425 (n = 10), the biofilms were exposed twice-daily to noncoherent blue and red light (LumaCare; 420 nm and 635 nm). The energy density applied was 72 J cm-2 for blue light and 43.8 J cm2, 87.6 J cm2, and 175.5 J cm2 for red light. Positive control (PC) and negative control (NC) groups were treated twice-daily for 1 min with 0.12% chlorhexidine (CHX) and 0.89% NaCl respectively. Biofilms were analyzed for colony forming units (CFU), dry-weight, and exopolysaccharides (EPS-soluble and EPS-insoluble). Data was analyzed by one-way ANOVA and Tukey post hoc test (α = 0.05). Dry-weight was lower than NC (p < 0.001) and approached PC levels with both red and blue light treatments. CFU were also lower in groups exposed to blue light and higher durations of red light (p < 0.05). EPS-soluble and EPS-insoluble measures were variably reduced by these light exposures. In conclusion, twice-daily exposure to both blue and red lights affect the biofilm development and physiology of polysaccharide production and are potential mechanisms for the control of C. albicans biofilm matrix development.

Effects of Nd:YAG laser irradiation on the growth of Candida albicans and Streptococcus mutans: in vitro study.

The purpose of this study was to evaluate the effects of Nd:YAG laser with flat-top handpiece on the in vitro growth of Candida albicans and Streptococcus mutans. The incidence of C. albicans (opportunistic commensal) and S. mutans (facultatively anaerobic) infections is increasing, despite available treatments. Cultures of Streptococcus mutans and Candida albicans were irradiated using Nd:YAG laser (LightWalker, Fotona) with flat-top handpiece (Genova, LightWalker, Fotona) at the following parameters: group G1: 0.25 W, 10 Hz, 15 s, 3 J and group G2: 1 W, 10 Hz, 60s, 59 J. The results were evaluated directly and 24 h after irradiation using a quantitative culture method (estimation of colony-forming units in 1 ml of suspension, cfu/ml), and microscopic analysis with Janus green stain and compared with control group in which laser was not applied. C. albicans was reduced by 20 up to 54% for G1, and for G2 by 10 up to 60% directly after the application. The cfu/ml values for S. mutans decreased by 13% (p = 0.1771) for G1 and 89% (p < 0.0001) for G2. In both test groups 24 h after the application, the number of colony-forming units decreased by 15-46% for G1 and by 15-64% for G2. The arrested cell division, increasing the surface area and increasing the number of metabolically inactive cells, were observed in morphometric analysis. Macroscopic and microscopic analyses revealed a reduction in cell number and a significant decrease of cell metabolism after laser application for both C. albicans and S. mutans.

The antifungal agent of silver nanoparticles activated by diode laser as light source to reduce C. albicans biofilms: an in vitro study.

Candida albicans is a normal flora caused fungal infections and has the ability to form biofilms. The aim of this study was to improve the antifungal effect of silver nanoparticles (AgNPs) and the light source for reducing the biofilm survival of C. albicans. AgNPs were prepared by silver nitrate (AgNO3) and trisodium citrate (Na3C6H5O7). To determine the antifungal effect of treatments on C. albicans biofilm, samples were distributed into four groups; L + P+ was treatment with laser irradiation and AgNPs; L + P- was treatment with laser irradiation only; L - P+ was treatment with AgNPs only (control positive); L - P- was no treatment with laser irradiation or AgNPs (control negative). The growth of fungi had been monitored by measuring the optical density at 405 nm with ELISA reader. The particle size of AgNPs was measured by using (particle size analyzer) and the zeta potential of AgNPs was measured by using Malvern zetasizer. The PSA test showed that the particle size of AgNPs was distributed between 7.531-5559.644 nm. The zeta potentials were found lower than - 30 mV with pH of 7, 9 or 11. The reduction percentage was analyzed by ANOVA test. The highest reduction difference was given at a lower level irradiation because irradiation with a density energy of 6.13 ± 0.002 J/cm2 resulted in the biofilm reduction of 7.07 ± 0.23% for the sample without AgNPs compared to the sample with AgNPs that increased the biofilm reduction of 64.48 ± 0.07%. The irradiation with a 450-nm light source had a significant fungicidal effect on C. albicans biofilm. The combination of light source and AgNPs provides an increase of biofilm reduction compared to the light source itself.

Bactericidal effects of deep ultraviolet light-emitting diode for solutions during intravenous infusion.

Background: Ultraviolet irradiation is effectively used as a disinfection method for inactivating microorganisms. Methods: We investigated the bactericidal effects by irradiation with a deep-ultraviolet light-emitting diode (DUV-LED) on the causative microorganisms of catheter related blood stream infection contaminating the solution for intravenous infusion. For irradiation, prototype modules for water disinfection with a DUV-LED were used. Experiments were conducted on five kinds of microorganisms. We examined the dependence of bactericidal action on eleven solutions. Administration sets were carried out three types. Results: When the administration set JY-PB343L containing the infusion tube made of polybutadiene was used, the bactericidal action of the DUV-LED against all tested microorganisms in the physiological saline solutions was considered to be effective. We confirmed that the number of viable bacteria decreased in 5% glucose solution and electrolyte infusions with DUV-LED irradiation. Conclusions: These results indicate that the DUV-LED irradiation has bactericidal effects in glucose infusion and electrolyte infusions by irradiating via a plasticizer-free polybutadiene administration set. We consider DUV-LED irradiation to be clinically applicable.

Effect of 1064-nm Q-switched Nd:YAG laser on invasiveness and innate immune response in keratinocytes infected with Candida albicans.

Candida albicans is an opportunistic pathogen commensal in the oral cavity, vagina, and healthy skin. Common therapeutic options for fungal infections are topical or systemic antifungal drugs. Recently, in cutaneous pathologies, lasers and light-based treatments have emerged showing few contraindications and minimal side effects. The Q-switched (Nd-YAG) laser at a wavelength of 1064 nm has been shown to be useful in dermatology, dentistry, and some other medical specialties. It is used to treat onychomycoses, warts, and wounds and in some other treatments. We analyzed the effect of Q-switched (Nd-YAG) laser 1064 nm on human keratinocytes infected with C. albicans. In particular, we evaluated the effect of laser on invasiveness of C. albicans and on inflammatory and protective response of HaCaT cells infected. The results obtained did not show inhibitory, fungicidal, or fungistatic effects of laser on yeast; in addition, laser did not affect HaCaT vitality. HaCaT cells infected with C. albicans and irradiated with laser showed a reduction of invasiveness of TNF-α and IL8 gene expression and an increase of immunomodulatory cytokines such as TGFß. Furthermore, laser induces a significant over-expression of HSP70B (heat shock protein) and of HBD-2 (Human ß defensin-2) in HaCaT infected with C. albicans, compared to the untreated control. The use of Q-switched Nd:YAG laser in skin mycosis caused by C. albicans reduces yeast invasiveness in keratinocytes, downregulates inflammatory activities, and facilitates cytoprotection and antimicrobial defense. Our results offer a promising therapeutic strategy in the management of skin candidiasis, also in combination with conventional therapies.

Influence of pre-irradiation time employed in antimicrobial photodynamic therapy with diode laser.

The aim of the present study was to evaluate, in vitro, the effect of different pre-irradiation times of the photosensitizer in photodynamic therapy in biofilms formed by Streptococcus mutans and Candida albicans, through the evaluation of the microbial load. The factors under study were as follows: times of pre-irradiation of the photosensitizer in three levels (1, 2, or 5 min). For the control of the cariogenic dental biofilm with antimicrobial photodynamic therapy (aPDT), methylene blue (0.01%) was used in association with the diode laser (InGaAlP) with a wavelength of 660 nm. Chlorhexidine digluconate (0.12% CHX) and saline were used as positive and negative controls, respectively. The study design was carried out in complete and randomized blocks. The sample consisted of 15 S. mutans biofilms cultures, randomly divided into five groups and 15 C. albicans cultures, also divided into five groups. The experiment was performed in triplicate (n = 3) and the response variables were obtained through quantitative analysis of bacterial viability, expressed in colony-forming units (CFU) per square millimeter of the specimen area. The data were analyzed with the aid of the ANOVA one-way test and Tukey's post-test. All analyses were performed using the Graph Pad Prism 4.0 program, with a significance level of 5%. For the S. mutans group, only the saline solution presented a statistically significant difference when compared to the other treatments (p < 0.05), that is, the treatment with aPDT, irrespective of the irradiation time applied, was similar to the treatment with CHX and both were more effective in reducing cariogenic biofilm compared to saline. For the group of C. albicans, there was no statistical difference between the groups (p > 0.05). Therefore, it can be concluded that the treatment with aPDT reduced the number of CFUs of S. mutans in a similar way to CHX, independently of the pre-irradiation time applied. No effect of this therapy or of the different pre-irradiation times on the C. albicans biofilm could be observed. In this way, the pre-irradiation time of 1 min can be used to reduce the microbial load of S. mutans.

Photodynamic Inactivation Potentiates the Susceptibility of Antifungal Agents against the Planktonic and Biofilm Cells of Candida albicans.

Photodynamic inactivation (PDI) has been shown to be a potential treatment modality against Candida infection. However, limited light penetration might leave some cells alive and undergoing regrowth. In this study, we explored the possibility of combining PDI and antifungal agents to enhance the therapeutic efficacy of Candida albicans and drug-resistant clinical isolates. We found that planktonic cells that had survived toluidine blue O (TBO)-mediated PDI were significantly susceptible to fluconazole within the first 2 h post PDI. Following PDI, the killing efficacy of antifungal agents relates to the PDI dose in wild-type and drug-resistant clinical isolates. However, only a 3-log reduction was found in the biofilm cells, suggesting limited therapeutic efficacy under the combined treatment of PDI and azole antifungal drugs. Using confocal microscopic analysis, we showed that TBO-mediated PDI could partially remove the extracellular polymeric substance (EPS) of biofilm. Finally, we showed that a combination of PDI with caspofungin could result in the complete killing of biofilms compared to those treated with caspofungin or PDI alone. These results clearly indicate that the combination of PDI and antifungal agents could be a promising treatment against C. albicans infections.

An in Vitro Study on the Effect of Combined Treatment with Photodynamic and Chemical Therapies on Candida albicans.

Candida albicans is the most commonly encountered human fungal pathogen, and it is traditionally treated with antimicrobial chemical agents. The antimicrobial effect of these agents is largely weakened by drug resistance and biofilm-associated virulence. Enhancement of the antimicrobial activity of existing agents is needed for effective candidiasis treatment. Our aim was to develop a therapy that combined biofilm disruption with existing antimicrobial agents. Photodynamic therapy (PDT) utilizing curcumin and blue light was tested as an independent therapy and in combination with fluconazole treatment. Viability assays and morphology analysis were used to assess the effectiveness of C. albicans treatment. Results showed that fluconazole treatment decreased the viability of planktonic C. albicans, but the decrease was not as pronounced in adherent C. albicans because its biofilm form was markedly more resistant to the antimicrobiotic. PDT effectively eradicated C. albicans biofilms, and when combined with fluconazole, PDT significantly inhibited C. albicans to a greater extent. This study suggests that the addition of PDT to fluconazole to treat C. albicans infection enhances its effectiveness and can potentially be used clinically.

Antimicrobial Photodynamic Therapy Mediated by Curcumin-Loaded Polymeric Nanoparticles in a Murine Model of Oral Candidiasis.

Antimicrobial photodynamic therapy (aPDT) has been proposed as an alternative method for oral candidiasis (OC), while nanocarriers have been used to improve the water solubility of curcumin (CUR). The aim of this study is to encapsulate CUR in polymeric nanoparticles (NPs) and to evaluate its photodynamic effects on a murine model of OC. Anionic and cationic CUR-NP is synthesized using poly-lactic acid and dextran sulfate and then characterized. Female mice are immunosuppressed and inoculated with Candida albicans (Ca) to induce OC. aPDT is performed by applying CUR-NP or free CUR on the dorsum of the tongue, followed by blue light irradiation for five consecutive days. Nystatin is used as positive control. Afterward, Ca are recovered and cultivated. Animals are euthanized for histological, immunohistochemical, and DNA damage evaluation. Encapsulation in NP improves the water solubility of CUR. Nystatin shows the highest reduction of Ca, followed by aPDT mediated by free CUR, which results in immunolabelling of cytokeratins closer to those observed for healthy animals. Anionic CUR-NP does not show antifungal effect, and cationic CUR-NP reduces Ca even in the absence of light. DNA damage is associated with Ca infection. Consecutive aPDT application is a safe treatment for OC.

Employment of methylene blue irradiated with laser light source in photodynamic inactivation of biofilm formed by Candida albicans strain resistant to fluconazole.

A promising approach for the eradication of biofilm formed by the yeast Candida albicans seems to be photodynamic inactivation (PDI). This work presents a use of methylene blue (MB, 1 mM) irradiated with a red laser (output power 190 mW/cm2, wavelength 660 nm) for the eradication of a biofilm formed by the fluconazole-resistant (FLC-resistant) strain C. albicans CY 1123 compared to the standard strain C. albicans SC5314. The periods of irradiation corresponded to the fluence of 15, 23 and 57 J/cm2. Effectiveness of PDI was evident with following percentage of survived biofilm cells: 24.57, 23.46, and 22.29% for SC5314 and 40.28, 17.91, and 5.89% for CY 1123, respectively, compared to the samples without irradiation. Light and confocal laser scanning microscopy confirmed the effectiveness of PDI. However, the morphological form of C. albicans seems to play an important role as well, since prolonged duration of irradiation did not increase efficiency of PDI on C. albicans SC5314. An experiment with the yeast-to-hyphae transition revealed that the FLC-resistant strain expressed a markedly reduced capacity to form hyphae compared to SC5314. We summarized that PDI was effective on biofilm formed by the FLC-resistant strain, but resistance most likely did not play significant role in PDI. Additionally, we observed differences in susceptibility to PDI between biofilms composed of the mycelia and only of the yeasts, and finally, the employment of a laser in PDI enabled a decreasing period of irradiation while maintaining the high effectiveness of PDI.

Preclinical study of a cost-effective photodynamic therapy protocol for treating oral candidoses.

Photodynamic therapy (PDT) is a promising treatment for oral candidoses. Its use as an alternative to antifungals prevents several adverse effects, including microbial resistance. However, most PDT protocols do not employ devices and consumables commonly available in dental practice, thus influencing treatment affordability. This study aimed to determine the efficacy of a PDT method based on light curing units' blue LEDs combined to a plaque-disclosing composition (5% erythrosine) against C. albicans in culture and in a murine model of oral candidosis. Standard and resistant fungal strains were tested in vitro in planktonic and biofilm forms. PDT (pre-irradiation time periods: 30 and 60 s; irradiation time: 3 min) was compared to control conditions without light and/or erythrosine. Mice with induced oral candidosis (n = 40) randomly received PDT or similar control conditions with subsequent C. albicans count. These mice underwent histological analysis, as well as 12 healthy mice submitted to experimental treatments. PDT completely inactivated C. albicans planktonic cells and biofilm. Control conditions presented minor differences (ANOVA, p < 0.05), with mean values ranging from 5.2 to 6.8 log10 (UFC/mL). Infected mice presented no significant difference in C. albicans counts consequent to treatments (ANOVA, p = 0.721), although the PDT protocol was able to enhance the inflammatory infiltrate in healthy mice. It can be concluded that the tested PDT protocol can inactivate C. albicans but still needs further investigation in order to achieve efficacy and safety.

Microwave radiation is effective at disinfecting dental stone surfaces without changing their physical properties.

The aims of this study were to evaluate the effectiveness of different microwave radiation regimens for disinfection of type IV dental stone surfaces and to assess the influence of these regimens on surface roughness and dimensional change following disinfection. Three hundred cylindrical (20 × 2-mm) test specimens were made in type IV stone and divided into subgroups of 20 according to the microorganisms tested (Staphylococcus aureus, Escherichia coli, or Candida albicans) and the 900-W microwave radiation protocol (cycles of 3, 5, or 7 minutes; a positive control; or a negative control). To test physical changes, 80 test specimens were made with the same dimensions except that they had 2 parallel and symmetrical indentations measuring 8 × 4 mm. These specimens were divided into 4 subgroups of 20 each (a subgroup for each radiation time and a negative control). The mean dimensional change and roughness data were analyzed by mixed models for repeated measures and Tukey-Kramer tests. Disinfection was analyzed with descriptive statistics. For E coli and C albicans, all radiation times proved effective at sterilizing the test specimens. For S aureus, sterilization was achieved with 5 and 7 minutes of exposure; however, colonies were observed in 10 Petri dishes (50%) exposed to 3 minutes of microwave radiation. No statistically significant difference in dimensional change or surface roughness was observed for any radiation regimen (P > 0.05).