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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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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/cm 2 , 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 log 10 inactivation after 108 J/cm 2 (60 mW/cm 2 , 30 minutes) or 216 J/cm 2 (60 mW/cm 2 , 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 log 10 inactivation, respectively). The same aBL treatment in biofilms developed in the CDC biofilm reactor, caused -1.68 log 10 inactivation in A. baumannii and -1.74 and -1.65 log 10 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., (© 2019 Wiley Periodicals, Inc.)

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 (AgNO 3 ) and trisodium citrate (Na 3 C 6 H 5 O 7 ). 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/cm 2 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.

Aims: 1) Evaluation of the photo-thermal (PT) and photo-activated (PAD) antibacterial effect of the 445/970 nm diode laser on E. faecalis, S. aureus and C. albicans mixed biofilms grown together inside root canals of human teeth. 2) Defining a potentially efficient clinical protocol for safe and predictable usage in endodontic procedures., Methodology: The root canals of 100 extracted human teeth with single straight canals were prepared with ProTaper NEXT files, sterilized, contaminated with a combination of three cultures (E. faecalis, S. aureus, C. albicans) and incubated for 15 days. The samples were randomly distributed into three groups (n = 20) and treated as follows: Group 1 (G1) - the 445 nm photo-thermal (PT) effect, Group 2 (G2) - a combination of the 445 nm and 970 nm PT effect, Group 3 (G3) - the 445 nm photo-activated (PAD) effect with 0.1% riboflavin, Group 4 (G4) - a combination of 3% sodium hypochlorite (NaOCl) and the 445 nm PAD effect. Four samples were used as positive control (non-treated) and four as a negative control. 12 aditional samples were used as a control for the G4 (3% NaOCl rinse without the laser). The number of viable microbes in each canal was determined by the colony forming unit (CFU) count., Results: A statistically significant reduction in the microbial population after all treatments was observed (P < 0.001). Groups 2 and 3 showed similar results, both better than Group 1. Group 4 produced the best results., Conclusions: The 445 nm PAD protocol has a stronger antimicrobial effect than the 445 nm PT protocol. Prolonged exposure time to laser light and a combination of wavelengths (445/970 PT protocol) helps in the reduction of microbes. C. albicans appears to be more sensitive to laser irradiation than the other bacteria tested in this study. Following current results, tested laser protocols could be recommended for clinical usage but only as an adjunct to "classic" NaOCl rinse since alone they are not able to completely eradicate all microorganisms., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: The aim of this study was to evaluate the effect of photodynamic therapy (PDT) on multispecies oral caries biofilms composed of Streptococcus mutans , Lactobacillus casei , and Candida albicans . Background: The abovementioned microorganisms largely cause dental caries, especially early childhood caries (ECC), by synthesizing of acids in the presence of sugar. PDT is considered an effective process to remove oral biofilms, and erythrosine, an oral bacterial disclosing agent, is an ideal dye that can be used as a photosensitizer in PDT. However, until now, there are no studies that have reported the effect of erythrosine-mediated PDT on biofilms, including the three microorganisms. Methods: The biofilms were formed on hydroxyapatite discs, and erythrosine was used as the photosensitizer, diluted to a concentration of 40 μM for 3 min. Light was irradiated for 10 and 20 sec using a blue light-emitting diode dental curing light. After the experiment, the colony-forming units of each microbial group cultured on blood agar plates were counted, and a confocal laser-scanning microscope was used to evaluate the effect of PDT. Results: The counts of all three microorganisms significantly decreased in the PDT group compared with those in the control group. For S. mutans and L. casei , there was a larger decrease proportional to the amount of energy irradiated. Conclusions: Overall, PDT showed a significant antimicrobial effect against oral biofilms composed of the three microorganisms, suggesting its potential clinical application for infants with ECC.

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J·cm -2 ). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

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 cm 2 , 87.6 J cm 2 , and 175.5 J cm 2 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.

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.

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., Competing Interests: The authors declare no conflict of interest.

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., (© The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: In this study, the photoinactivation of 5-aminolevulinic acid (ALA) has been investigated on Candida albicans biofilms in vitro., Methods: After culture and proliferation of Candida albicans biofilms in vitro, the metabolic activity was confirmed using XTT reduction assay. Then, the suitable incubation time and concentration of ALA were determined by measuring PpIX accumulation quantities. Photosensitivity of the biofilms treated with ALA solution was studied in optical doses of 50, 100, 200 and 300J/cm(2) while light irradiation was applied by a red light semiconductor. Finally, rapid immunofluorescence staining method using the LIVE/DEAD FungaLight Yeast Viability Kit and XTT assay were conducted to visualize and quantify the antifungal effect of ALA-PDT on Candida albicans biofilms., Results: A 5h incubation time and 15mM ALA concentration were determined for this study. Photoinactivation of ALA-PDT on Candida albicans biofilms showed a significant increase of protoporphyrin IX (PpIX) in the biofilms. The metabolic activity of Candida albicans biofilms tread with ALA-PDT confirmed the inhibition efficacy compared with control groups. Upon radiation at 300J/cm(2), cells in Candida albicans biofilms were 74.45% inhibited., Conclusions: PpIX can be absorbed in biofilm-grown Candida albicans in vitro and under appropriate parameters, photochemistry can be triggered by light in combination with ALA and inhibits Candida albicans biofilms effectively., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: The aim of this study is to investigate the photoinactivation of C. albicans biofilm on acrylic resin discs (the standard material for dental prosthesis) using the photosensitizers Methylene Blue and a Protoporphyrin IX., Methods: Eighteen thermally activated Methyl Methacrylate Polymers were used for the biofilm growth of Candida albicans ATCC 10231. Two photosensitizers were tested: methylene blue (50μM) and protoporphyrin IX (10μM). Two custom-made LEDs emitting at 660nm and 630nm with approximately 800mW each were used for the irradiation, with duration ranging from 2 to 10min., Results: This study demonstrates that MB decreased the aPDT CFUs by approximately two orders of magnitude, but the protoporphyrin was ineffective., Conclusion: The aPDT with MB significantly reduces (but does not sterilize) the amount of CFU after 10min of irradiation, and it is not dose-dependent. The lack of effect of the protoporphyrin is likely because the negative charges of the proteoglycans present in the extracellular matrix repel the negative charges of the PS, thus preventing its diffusion in the cells., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The aim of this study was to evaluate the effectiveness of a 455-nm blue light-emitting diode (LED), at different application times, to reduce the load of Staphylococcus aureus and Candida albicans biofilms applied to compact bone tissue. The microorganisms S. aureus (ATCC 25923) and C. albicans (ATCC 18804) were used to form biofilms on 160 specimens of compact bones that had been divided into eight experimental groups (n = 10) for each microorganism, according to the times of application of the 455-nm blue LED (1, 2, 3, 4, 5, 7, and 10 min) with an irradiance of 75 mW/cm2. After LED application, decimal dilutions of microorganisms were performed, plated on BHI or Sabouraud agar and incubated for 24 h/35 °C to obtain CFU/mL counts. The findings were statistically analyzed using a ANOVA 5 %. For the group of S. aureus biofilms, all groups of 455-nm LED application differ compared with the control group (p < 0.05), in which no treatment was given. The largest reduction was obtained in the group receiving LED for 10 min (p = 0.00); within this group, a 3.2 log reduction was observed. For the C. albicans biofilms, only those samples receiving 3, 7, and 10 min of LED application presented a significant difference compared with the control group (p < 0.00), indicating that longer application times are required to achieve efficacy. The results of this study show that 455-nm LED light was effective to reduce the load of S. aureus and C. albicans biofilms, especially during 10 min of application.

In this investigation, the effectiveness of successive applications of antimicrobial photodynamic inactivation (API) mediated by Photodithazine(®) (PDZ) and LED light was evaluated against a multispecies biofilm formed by Candida albicans, Candida glabrata, and Streptococcus mutans on denture base acrylic resin. Standard cell suspensions (bacteria and yeast) were inoculated on acrylic resin samples, and the biofilm was grown for 48 h (37 °C/75 rpm). API was performed by the administration of PDZ (175 and 200 mg/L) and exposure to 37.5 J/cm(2) of LED light (660 nm). Additional samples were treated with PDZ or LED light only. Untreated control samples were not submitted to light or PDZ. The conditions described were applied once or in three consecutive applications for all groups. Cell viability was determined by colony counts (CFU/mL), metabolic activity, total biomass, and confocal laser scanning microscopy (CLSM). Data were analyzed by a nonparametric two-way ANOVA and Tukey tests (α = 0.05). The results obtained demonstrated a significant effect (p < 0.05) of number of applications and treatment groups for CFU/mL, and S. mutans showed the highest susceptibility to API. The metabolic activity of the multispecies biofilm was significantly reduced (p < 0.05) after API for both numbers of applications, which were also significantly different (p < 0.05) between them. The total biomass of the biofilm was significantly different (p < 0.05) only between groups submitted to one and three API applications. CLSM showed a visual increase of dead cells after API. API-mediated PDZ was effective in reducing the cell viability of multispecies biofilm. Three consecutive applications of API were more effective for reducing the cell viability and the total biomass of multispecies biofilm.

Antimicrobial photodynamic therapy (APDT) combined with endodontic treatment has been recognized as an alternative approach to complement conventional root canal disinfection methods on bacterial biofilms. We developed an in  vitro model of bioluminescent Candida albicans biofilm inside curved dental root canals and investigated the microbial reduction produced when different light delivery methods are employed. Each light delivery method was evaluated in respect to the light distribution provided inside curved root canals. After conventional endodontic preparation, teeth were sterilized before canals were contaminated by a bioluminescent strain of C. albicans (CEC789). Methylene blue (90 μM) was introduced into the canals and then irradiated (λ = 660 nm, P = 100 mW, beam diameter = 2 mm) with laser tip either in contact with pulp chamber or within the canal using an optical diffuser fiber. Light distribution was evaluated by CCD camera, and microbial reduction was monitored through bioluminescence imaging. Our findings demonstrated that the bioluminescent C. albicans biofilm model had good reproducibility and uniformity. Light distribution in dental tissue was markedly dependent on the light delivery system, and this strategy was directly related to microbial destruction. Both light delivery systems performed significant fungal inactivation. However, when irradiation was performed with optical diffuser fiber, microbial burden reduction was nearly 100 times more effective. Bioluminescence is an interesting real-time analysis to endodontic C. albicans biofilm inactivation. APDT showed to be an effective way to inactivate C. albicans biofilms. Diffuser fibers provided optimized light distribution inside curved root canals and significantly increased APDT efficiency.

Onychomycoses represent approximately 50 % of all nail diseases worldwide. In warmer and more humid countries like Brazil, the incidence of onychomycoses caused by non-dermatophyte molds (NDM, including Fusarium spp.) or yeasts (including Candida albicans) has been increasing. Traditional antifungal treatments used for the dermatophyte-borne disease are less effective against onychomycoses caused by NDM. Although some laser and light treatments have demonstrated clinical efficacy against onychomycosis, their US Food and Drug Administration (FDA) approval as "first-line" therapy is pending, partly due to the lack of well-demonstrated fungicidal activity in a reliable in vitro model. Here, we describe a reliable new in vitro model to determine the fungicidal activity of laser and light therapies against onychomycosis caused by Fusarium oxysporum and C. albicans. Biofilms formed in vitro on sterile human nail fragments were treated with 1064 nm neodymium-doped yttrium aluminum garnet laser (Nd:YAG), 420 nm intense pulsed light (IPL) IPL 420, followed by Nd:YAG, or near-infrared light ((NIR) 700-1400 nm). Light and laser antibiofilm effects were evaluated using cell viability assay and scanning electron microscopy (SEM). All treatments were highly effective against C. albicans and F. oxysporum biofilms, resulting in decreases in cell viability of 45-60 % for C. albicans and 92-100 % for F. oxysporum. The model described here yielded fungicidal activities that matched more closely to those observed in the clinic, when compared to published in vitro models for laser and light therapies. Thus, our model might represent an important tool for the initial testing, validation, and "fine-tuning" of laser and light therapies against onychomycosis.

Statement of Problem: Proper sterilization or disinfection of removable prostheses and surgical guides has been problematic in dental practice because of the absence of simple and low-cost techniques that do not cause damage to acrylic resins., Purpose: The purpose of this study was to study the effect of photodynamic therapy against Streptococcus mutans, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans biofilms formed on acrylic resin specimens., Material and Methods: The specimens were sterilized in ethylene oxide gas and submitted to in vitro biofilm growth. The photodynamic therapy consisted of the application of 0.05% methylene blue (P+) conjugated to irradiation with a light-emitting-diode of 630 nm and 150 mW (L+). The specimens were randomly divided into groups (n=5): negative control (P-L-); stained and irradiated at 10 J/cm(2) (P+L+ 10); stained and irradiated at 30 J/cm(2) (P+L+ 30); stained and not irradiated (P+L-); not stained and irradiated at 10 J/cm(2) (P-L+ 10); not stained and irradiated at 30 J/cm(2) (P-L+ 30); and gold standard (GS), sterilized. Afterward, the specimens were submitted to contact with culture medium agar for 10 minutes in petri plates, which were incubated for 48 hours at 37°C. The number of colony-forming units was obtained, and the data were expressed according to scores (1=0; 2=1-10; 3=11-100; 4=101-1000) and analyzed by the Friedman and Dunn tests (α=.05)., Results: Streptococcus mutans was sensitized by (P+L-); P aeruginosa and C albicans were also sensitized by the dye but showed a slight microbial reduction with (P+L+ 30), as did S aureus (P>.05); E coli presented an initial score of 3 and achieved a bacterial reduction to score 2 with (P+L+ 30) (P=.039)., Conclusions: Photodynamic therapy was effective in reducing E coli counts on biofilms formed on acrylic resin specimens. The inhibition of microorganism growth tended to be directly proportional to the amount of energy provided by the light-emitting diode., (Copyright © 2014 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.)

The opportunistic fungal Candida albicans is able to produce both superficial and systemic infections in immunocompromised patients. Photodynamic antimicrobial chemotherapy (PACT) is a process that combines visible light and a photosensitizer, producing reactive oxygen species (ROS) that can kill the treated cells and has been presented as a potential antimicrobial therapy. In this work, we study the effects of PACT, using toluidine blue (TB) as a photosensitizer drug, on ROS production and cell damage and the ability of C. albicans to form biofilm. A significant decrease was observed in the cell growth after PACT in a TB concentration-dependent manner. This effect was dependent on the incubation time after PACT. In addition, an increase in both the ROS production and cell permeability, after PACT, in a TB concentration-dependent manner was observed. PACT, using 0.1 mg/ml TB was able to reduce biofilm formation in 30, 50, and 62%, in cells submitted to incubation times of 1, 2, and 3 h, respectively. These results suggested that PACT, using TB, is able to decrease both growth and biofilm formation by C. albicans, possibly by a mechanism evolving both ROS production and the increase in the cell permeability.

New drug delivery systems, such as nanoemulsions (NE), have been developed to allow the use of hydrophobic drugs on the antimicrobial photodynamic therapy. This study evaluated the photodynamic potential of aluminum-chloride-phthalocyanine (ClAlPc) entrapped in cationic and anionic NE to inactivate Candida albicans planktonic cultures and biofilm compared with free ClAlPc. Fungal suspensions were treated with different delivery systems containing ClAlPc and light emitting diode. For planktonic suspensions, colonies were counted and cell metabolism was evaluated by XTT assay. Flow cytometry evaluated cell membrane damage. For biofilms, the metabolic activity was evaluated by XTT and ClAlPc distribution through biofilms was analyzed by confocal laser scanning microscopy (CLSM). Fungal viability was dependent on the delivery system, superficial charge and light dose. Free ClAlPc caused photokilling of the yeast when combined with 100 J cm(-2). Cationic NE-ClAlPc reduced significantly both colony counts and cell metabolism (P < 0.05). In addition, cationic NE-ClAlPc and free ClAlPc caused significant damage to the cell membrane (P < 0.05). For the biofilms, cationic NE-ClAlPc reduced cell metabolism by 70%. Anionic NE-ClAlPc did not present antifungal activity. CLSM showed different accumulation on biofilms between the delivery systems. Although NE system showed a lower activity for planktonic culture, cationic NE-ClAlPc showed better results for Candida biofilms., (© 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.)

Objective: This study evaluated the influence of the area of Candida albicans biofilm on denture disinfection by microwave energy., Materials and Methods: Candida albicans biofilm was allowed to form for 72 h on resin discs, and three small coverage or seven large coverage discs were placed onto the palatal surface of sterile maxillary dentures. Each denture was immersed in 200 ml distilled water and individually irradiated at a power of 450, 630 or 900 W for different time intervals (1, 2 or 3 min) (n = 6). The effectiveness of disinfection was evaluated by counting the residual cells. The data were analysed by anova and Tukey's HSD test (α = 0.05). Pearson's correlation test was performed to determine the correlation between effectiveness of sterilisation and temperature., Results: Dentures with a larger area of biofilm demanded a longer irradiation exposure to achieve disinfection (p < 0.001), irrespective of power setting, and in this time no yeast growth was detected. Dentures with small areas of biofilm were disinfected after 1 min at 900 W and 2 min at 450 or 630 W. A positive correlation was found between water temperature and effectiveness of disinfection (r = 0.6170; p < 0.001)., Conclusion: The C. albicans biofilm area influenced disinfection by microwave energy; therefore dentures with larger biofilm areas required longer irradiation exposure to be disinfected., (© 2010 The Gerodontology Society and John Wiley & Sons A/S.)

Effective calculus and biofilm removal is essential to treat periodontitis. Sonic and ultrasonic technologies are used in several scaler applications. This was the first feasibility study to assess the potential of a shock wave device to remove calculus and biofilms and to kill bacteria. Ten extracted teeth with visible subgingival calculus were treated with either shock waves for 1 min at an energy output of 0.4 mJ/mm(2) at 3 Hz or a magnetostrictive ultrasonic scaler at medium power setting for 1 min, which served as a control. Calculus was determined before and after treatment planimetrically using a custom-made software using a grey scale threshold. In a second experiment, multispecies biofilms were formed on saliva-preconditioned bovine enamel discs during 64.5 h. They were subsequently treated with shock waves or the ultrasonic scaler (N = 6/group) using identical settings. Biofilm detachment and bactericidal effects were then assessed. Limited efficiency of the shock wave therapy in terms of calculus removal was observed: only 5% of the calculus was removed as compared to 100% when ultrasound was used (P ≤ 0.0001). However, shock waves were able to significantly reduce adherent bacteria by three orders of magnitude (P ≤ 0.0001). The extent of biofilm removal by the ultrasonic device was statistically similar. Only limited bactericidal effects were observed using both methods. Within the limitations of this preliminary study, the shock wave device was not able to reliably remove calculus but had the potential to remove biofilms by three log steps. To increase the efficacy, technical improvements are still required. This novel noninvasive intervention, however, merits further investigation.

Bacterial colonization of central venous catheters (CVCs) causes severe complications in patients. As a result, developing methods to remove and prevent bacterial and fungal colonization of CVCs is imperative. Recently, we have demonstrated that disinfection by radiation of polymer tubes with UVC light is possible. In this paper we present dose-response results using a newly developed UVC disinfection device, which can be connected to a Luer catheter hub. The device was tested on soft polymer tubes contaminated with a pallet of microorganisms, including Candida albicans, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa (ca 10(3) CFU mL(-1)). The tubes were equipped with a modified catheter hub and interfaced to the disinfection device via a middle piece separating the disinfection device from the hub. The contamination lasted for 3 h prior to treatment to simulate an aseptic breach. Our results show UVC killing in a dose and time dependent manner, with no viable counts after 2 min of radiation for bacteria. Killing of C. albicans was obtained at >20 min in an UVC absorbing suspension. We believe our results to be transferable directly to the clinic, and we are currently working on a setup for clinical trial., (© 2011 The Authors. Photochemistry and Photobiology © 2011 The American Society of Photobiology.)

The aim of this study was to evaluate the effect of specific parameters of low-level laser therapy (LLLT) on biofilms formed by Streptococcus mutans, Candida albicans or an association of both species. Single and dual-species biofilms--SSB and DSB--were exposed to laser doses of 5, 10 or 20 J/cm(2) from a near infrared InGaAsP diode laser prototype (LASERTable; 780 ± 3 nm, 0.04 W). After irradiation, the analysis of biobilm viability (MTT assay), biofilm growth (cfu/mL) and cell morphology (SEM) showed that LLLT reduced cell viability as well as the growth of biofilms. The response of S. mutans (SSB) to irradiation was similar for all laser doses and the biofilm growth was dose dependent. However, when associated with C. albicans (DSB), S. mutans was resistant to LLLT. For C. albicans, the association with S. mutans (DSB) caused a significant decrease in biofilm growth in a dose-dependent fashion. The morphology of the microorganisms in the SSB was not altered by LLLT, while the association of microbial species (DSB) promoted a reduction in the formation of C. albicans hyphae. LLLT had an inhibitory effect on the microorganisms, and this capacity can be altered according to the interactions between different microbial species.

Aims: The major objective of the study was to evaluate the enhanced germicidal effects of low-frequency pulsed ultraviolet A (UVA)-light-emitting diode (LED) on biofilms., Methods and Results: The germicidal effects of UVA-LED irradiation (365 nm, 0·28 mW cm(-2) , in pulsed or continuous mode) on Candida albicans or Escherichia coli biofilms were evaluated by determining colony-forming units. The morphological change of microbial cells in biofilms was observed using scanning electron microscopy. After 5-min irradiation, over 90% of viable micro-organisms in biofilms had been killed, and pulsed irradiation (1-1000 Hz) had significantly greater germicidal ability than continuous irradiation. Pulsed irradiation (100 Hz, 60 min) almost completely killed micro-organisms in biofilm (>99·9%), and 20-min irradiation greatly damaged both microbial species. Interestingly, few hyphae were found in irradiated Candida biofilms. Moreover, mannitol treatment, a scavenger of hydroxyl radicals (OH(•) ), significantly protected viable micro-organisms in biofilms from UVA-LED irradiation., Conclusions: The study demonstrated that pulsed UVA-LED irradiation has a strong germicidal effect (maximum at 100 Hz, over 5-min irradiation) and causes the disappearance of hyphal forms of Candida., Significance and Impact of the Study: This study can assist in developing a low-frequency pulsed UVA-LED system to be applied to pathogenic biofilms for disinfection., (© 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.)

Biofilms have been found to be involved in a wide variety of microbial infections in the body, by one estimate 80% of all infections. Infectious processes in which biofilms have been implicated include common problems such as urinary tract infections, catheter infections, middle-ear infections, sinusitis, formation of dental plaque, gingivitis, coating contact lenses, endocarditis, infections in cystic fibrosis, and infections of permanent indwelling devices such as joint prostheses and heart valves. Bacteria living in a biofilm usually have significantly different properties from free-floating bacteria of the same species, as the dense and protected environment of the film allows them to cooperate and interact in various ways. One benefit of this environment is increased resistance to detergents and antibiotics, as the dense extracellular matrix and the outer layer of cells protect the interior of the community. In some cases antibiotic resistance can be increased 1000-fold. Also, the biofilm bacteria excrete toxins that reversibly block important processes such as translation and protecting the cell from bactericidal antibiotics that are ineffective against inactive targets. In the head and neck area, biofilms are a major etiologic factor in periodontitis, wound infections, oral candidiasis, and sinus and ear infections. For the past several decades, photodynamic treatment has been reported in the literature to be effective in eradicating various microorganisms using different photosensitizers, different wavelengths of light, and different light sources. PDT has been further studied to demonstrate its effectiveness for the eradication of both Gram-negative and Gram-positive antibiotic-resistant bacteria. This chapter will focus on the use of PDT in the treatment of antibiotic-resistant biofilms, antibiotic-resistant wound infections, and azole-resistant oral candidiasis using methylene blue-based photodynamic therapy.

The different forms of superficial and systemic candidiasis are often associated with biofilm formation on surfaces of host tissues or medical devices. The biofilm formation of Candida spp., in general, necessitates significantly increased amounts of antifungal agents for therapy. Often the therapeutic effect is doubtful. A 5-day biofilm model with oral Candida isolates was established according to Chandra et al. (J Dent Res 80:903-908, 2001) on glass and titanium surfaces and was modified by Sennhenn-Kirchner et al. (Z Zahnärztl Implantol 3:45-51, 2007) to investigate different aspects unanswered in the field of dentistry. In this model, the efficacy of erbium:yttrium-aluminium-garnet (Er:YAG) light (2940 nm, 100 mJ, 10 Hz, 300 micros pulsed mode applied for 80 s) and diode laser light (810 nm, 1 W, continuous wave mode applied for 20 s with four repetitions after 30 s pauses each) was evaluated and compared to untreated controls. The photometric evaluation of the samples was completed by observations on morphological changes of yeast cells grown in the biofilm. Compared to the untreated controls Candida cells grown in mature in vitro biofilms were significantly reduced by both wavelengths investigated. Comparison between the different methods of laser treatment additionally revealed a significantly greater effect of the Er:YAG over the diode laser. Scanning electron microscopy findings proved that the diode laser light was effective in direct contact mode. In contrast, in the areas without direct contact, the fungal cells were left almost unchanged. The Er:YAG laser damaged the fungal cells to a great extent wherever it was applied.

Treatment of mucocutaneous and cutaneous Candida albicans infections with photosensitizing agents and light, termed photodynamic therapy (PDT), offers an alternative to conventional treatments. Initial studies using the clinically approved photosensitizer Photofrin demonstrated the susceptibility of C. albicans to its photodynamic effects. In the present study, we have further refined parameters for Photofrin-mediated photodynamic action against C. albicans and examined whether mechanisms commonly used by microorganisms to subvert either antimicrobial oxidative defenses or antimicrobial therapy, including biofilm formation, were operative. In buffer and defined medium, germ tubes preloaded with Photofrin retained their photosensitivity for up to 2 hours, indicating the absence of degradation or export of Photofrin by the organism. The addition of serum resulted in a gradual loss of photosensitivity over 2 hours. In contrast to an adaptive response by germ tubes to oxidative stress by hydrogen peroxide, there was no adaptive response to singlet oxygen-mediated stress by photodynamic action. C. albicans biofilms were sensitive to Photofrin-mediated phototoxicity in a dose-dependent manner. Finally, the metabolic activity of C. albicans biofilms following photodynamic insult was significantly lower than that of biofilms treated with amphotericin B for the same time period. These results demonstrate that several of the mechanisms microorganisms use to subvert either antimicrobial oxidative defenses or antimicrobial therapy are apparently not operative during Photofrin-mediated photodynamic treatment of C. albicans. These observations provide support and rationale for the continued investigation of PDT as an adjunctive, or possibly alternative, mode of therapy against cutaneous and mucocutaneous candidiasis.