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The photoprocesses involved in hypericin photoinactivation of three different Candida species (C. albicans, C. parapsilosis and C. krusei) have been examined. Production of singlet oxygen from the triplet state and of superoxide from both the triplet state and the semiquinone radical anion are demonstrated. Hydrogen peroxide is formed downstream of these early events. The outcome of the photodynamic treatments is dictated by the intracellular distribution of hypericin, which is different in the three species and affects the ability of hypericin to produce the different reactive oxygen species and trigger cell-death pathways. The results are in line with the previously-observed different susceptibilities of the three Candida species to hypericin photodynamic treatments.

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.)

Superficial mycoses caused by dermatophyte fungi are among the most common infections worldwide, yet treatment is restricted by limited effective drugs available, drug toxicity, and emergence of drug resistance. The stilbene fluorescent brightener calcofluor white (CFW) inhibits fungi by binding chitin in the cell wall, disrupting cell wall integrity, and thus entails a different mechanism of inhibition than currently available antifungal drugs. To identify novel therapeutic options for the treatment of skin infections, we compared the sensitivity of representative strains of the dermatophyte Trichophyton rubrum and Candida albicans to CFW and a panel of fluorescent brighteners and phytoalexin compounds. We identified the structurally related stilbene fluorescent brighteners 71, 85, 113 and 134 as fungicidal to both T. rubrum and C. albicans to a similar degree as CFW, and the stilbene phytoalexins pinosylvan monomethyl ether and pterostilbene inhibited to a lesser degree, allowing us to develop a structure-activity relationship for fungal inhibition. Given the abilities of CFW to absorb UV(365 nm) and bind specifically to fungal cell walls, we tested whether CFW combined with UV(365 nm) irradiation would be synergistic to fungi and provide a novel photodynamic treatment option. However, while both treatments individually were cytocidal, UV(365 nm) irradiation reduced sensitivity to CFW, which we attribute to CFW photoinactivation. We also tested combination treatments of CFW with other fungal inhibitors and identified synergistic interactions between CFW and some ergosterol biosynthesis inhibitors in C. albicans. Therefore, our studies identify novel fungal inhibitors and drug interactions, offering promise for combination topical treatment regimes for superficial mycoses.

The purpose of the present study was to evaluate the mechanism of microbial resistance to oxidative stress induced by photolysis of hydrogen peroxide (H(2)O(2)) in relation to microbial catalase activity. In microbicidal tests, Staphylococcus aureus and Candida albicans were killed and this was accompanied by production of hydroxyl radicals. C. albicans was more resistant to hydroxyl radicals generated by photolysis of H(2)O(2) than was S. aureus. A catalase activity assay demonstrated that C. albicans had stronger catalase activity; accordingly, catalase activity could be one of the reasons for the resistance of the fungus to photolysis of H(2)O(2). Indeed, it was demonstrated that C. albicans with strong catalase activity was more resistant to photolysis of H(2)O(2) than that with weak catalase activity. Kinetic analysis using a modified Lineweaver-Burk plot also demonstrated that the microorganisms reacted directly with hydroxyl radicals and that this was accompanied by decomposition of H(2)O(2). The results of the present study suggest that the microbicidal effects of hydroxyl radicals generated by photolysis of H(2)O(2) can be alleviated by decomposition of H(2)O(2) by catalase in microorganisms., (© 2012 The Societies and Blackwell Publishing Asia Pty Ltd.)

Background: Candida albicans is known as part of human's skin normal flora; but simultaneously, is the most common opportunistic fungal pathogen of human which can cause a variety of infections including cutaneous candidiasis. Because of the importance of superficial infections like cutaneous candidiasis, we tried to use Ultraviolet B light as a method of phototherapy for inducing apoptosis in irradiated colonies of Candida albicans., Materials and Methods: The Ultraviolet B with the wavelength of 302 nanometer was used for irradiating the colonies of Candida albicans. For detecting the eventual apoptotic reactions, the DNA of irradiated colonies as well as control colonies were extracted and then were run in 1% agarose gel electrophoresis containing ethidium bromide to observe luminescent DNA bands., Results: Despite irradiating of Ultraviolet B to yeast cells, no abnormalities including DNA laddering bands or smears were detected in bands formed by total genomic DNA., Discussion: The applied irradiation protocol in this investigation was not successful to induce apoptotic reactions in Ultraviolet-exposed colonies. Maybe, Heat shock proteins as the important part of fungal protein pool, inhibit the inducing pathway of apoptosis in irradiated colonies of Candida albicans.

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.

Objective: The aim of this study was to investigate the effectiveness of two alternatives methods for the disinfection of oral cleaning devices., Methods: One type of toothbrush and two types of tongue scrapers (steel and plastic) were tested in this study. Sixteen specimens of each group were cut with standardized dimensions, contaminated separately with Candida albicans, Streptococcus mutans and Staphylococcus aureus and incubated for 24 h. After this, oral cleaning devices were washed in saline solution to remove non-adhered cells and divided into two groups (n = 8), one irradiated in microwave and other immersed in 3.78% sodium perborate solution, and evaluated for microbial recovery. The values of cfu of each group of microorganism after disinfection were compared by Kruskal-Wallis and Dunn non-parametric test, considering 95% of confidence., Results: The toothbrush harboured a significant larger number of viable organisms than the tongue scrapers. The steel tongue scraper was less susceptible to adhesion of the three oral microorganisms. The time required to inactivate all contaminating microorganisms using microwave oven was 1 min and, for the immersion in 3.78% sodium perborate solution, was 2 and 3 h, respectively, for C. albicans and S. mutans/S. aureus., Conclusion: Microwave irradiation proved to be an effective alternative method to the disinfection of tongue cleaners and toothbrushes., (© 2011 John Wiley & Sons A/S.)

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.)

Unlabelled: Genetic diversity is often generated during adaptation to stress, and in eukaryotes some of this diversity is thought to arise via recombination and reassortment of alleles during meiosis. Candida albicans, the most prevalent pathogen of humans, has no known meiotic cycle, and yet it is a heterozygous diploid that undergoes mitotic recombination during somatic growth. It has been shown that clinical isolates as well as strains passaged once through a mammalian host undergo increased levels of recombination. Here, we tested the hypothesis that stress conditions increase rates of mitotic recombination in C. albicans, which is measured as loss of heterozygosity (LOH) at specific loci. We show that LOH rates are elevated during in vitro exposure to oxidative stress, heat stress, and antifungal drugs. In addition, an increase in stress severity correlated well with increased LOH rates. LOH events can arise through local recombination, through homozygosis of longer tracts of chromosome arms, or by whole-chromosome homozygosis. Chromosome arm homozygosis was most prevalent in cultures grown under conventional lab conditions. Importantly, exposure to different stress conditions affected the levels of different types of LOH events, with oxidative stress causing increased recombination, while fluconazole and high temperature caused increases in events involving whole chromosomes. Thus, C. albicans generates increased amounts and different types of genetic diversity in response to a range of stress conditions, a process that we term "stress-induced LOH" that arises either by elevating rates of recombination and/or by increasing rates of chromosome missegregation., Importance: Stress-induced mutagenesis fuels the evolution of bacterial pathogens and is mainly driven by genetic changes via mitotic recombination. Little is known about this process in other organisms. Candida albicans, an opportunistic fungal pathogen, causes infections that require adaptation to different host environmental niches. We measured the rates of LOH and the types of LOH events that appeared in the absence and in the presence of physiologically relevant stresses and found that stress causes a significant increase in the rates of LOH and that this increase is proportional to the degree of stress. Furthermore, the types of LOH events that arose differed in a stress-dependent manner, indicating that eukaryotic cells generate increased genetic diversity in response to a range of stress conditions. We propose that this "stress-induced LOH" facilitates the rapid adaptation of C. albicans, which does not undergo meiosis, to changing environments within the host.

This study describes the association of curcumin with light emitting diode (LED) for the inactivation of Candida albicans. Suspensions of Candida were treated with nine curcumin concentrations and exposed to LED at different fluences. The protocol that showed the best outcomes for Candida inactivation was selected to evaluate the effect of the preirradiation time (PIT) on photodynamic therapy (PDT) effectiveness, the uptake of curcumin by C. albicans cells and the possible involvement of singlet oxygen in the photodynamic action. Curcumin-mediated PDT was also assessed against biofilms. In addition to the microbiological experiments, similar protocols were tested on a macrophage cell line and the effect was evaluated by Methyltetrazolium assay (MTT) and SEM analysis. The optical properties of curcumin were investigated as a function of illumination fluence. When compared with the control group, a statistically significant reduction in C. albicans viability was observed after PDT (P < 0.05), for both planktonic and biofilm cultures. Photodynamic effect was greatly increased with the presence of curcumin in the surrounding media and the PIT of 20 min improved PDT effectiveness against biofilms. Although PDT was phototoxic to macrophages, the therapy was more effective in inactivating the yeast cell than the defense cell. The spectral changes showed a high photobleaching rate of curcumin., (© 2011 The Authors. Photochemistry and Photobiology © 2011 The American Society of Photobiology.)

The high prevalence of drug resistance necessitates the development of novel antifungal agents against infections caused by opportunistic fungal pathogens, such as Candida albicans. Elucidation of apoptosis in yeast-like fungi may provide a basis for future therapies. In mammalian cells, photodynamic therapy (PDT) has been demonstrated to generate reactive oxygen species, leading to immediate oxidative modifications of biological molecules and resulting in apoptotic cell death. In this report, we assess the in vitro cytotoxicity and mechanism of PDT, using the photosensitizer Pc 4, in planktonic C. albicans. Confocal image analysis confirmed that Pc 4 localizes to cytosolic organelles, including mitochondria. A colony formation assay showed that 1.0 μM Pc 4 followed by light at 2.0 J cm(-2) reduced cell survival by 4 logs. XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide) assay revealed that Pc 4-PDT impaired fungal metabolic activity, which was confirmed using the FUN-1 (2-chloro-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenylquinolinium iodide) fluorescence probe. Furthermore, we observed changes in nuclear morphology characteristic of apoptosis, which were substantiated by increased externalization of phosphatidylserine and DNA fragmentation following Pc 4-PDT. These data indicate that Pc 4-PDT can induce apoptosis in C. albicans. Therefore, a better understanding of the process will be helpful, as PDT may become a useful treatment option for candidiasis., (© 2011 The Authors. Photochemistry and Photobiology © 2011 The American Society of Photobiology.)

Chlorophyll compounds and their derivatives containing metal or phytyl chain can be used as photosensitizer in photodynamic inactivation of microorganisms (PDI). So, the physicochemical properties and antimicrobial effect of chlorophyll derivatives were investigated: Mg-chlorophyll (Mg-Chl), Zn-chlorophyll (Zn-Chl), Zn-chlorophyllide (Zn-Chlde), Cu-chlorophyll (Cu-Chl), pheophytin (Pheo) and pheophorbide (Pheid). The photobleaching experiments showed photostability according to Cu-Chl > Pheo ∼ Pheid ≫ Zn-Chl ∼ Zn-Chlde > Mg-Chl. This order was discussed in terms of metal and the phytyl chain presences. Pheid and Zn-Chl in aqueous Tween 80 solution exhibited highest singlet oxygen yield compared with the other derivatives. Chlorophyll derivatives (CD) with phytyl chain was limited by the self-aggregation phenomenon at high concentrations, even in micellar systems (Tween 80 and P-123). The antimicrobial effect of CD derivatives was investigated against Staphylococcus aureus, Escherichia coli, Candida albicans and Artemia salina. Pheid showed the best results against all organisms tested, Zn-Chlde was an excellent bactericide in the dark and Cu-Chl had no PDI effect. No correlation with CD uptake by microorganisms and darkness cytotoxicity was found. The physicochemical properties allied to bioassays results indicate that Mg-Chl, Pheo, Zn-Chl and Pheid are good candidates for PDI., (© 2011 The Authors. Photochemistry and Photobiology © 2011 The American Society of Photobiology.)

This study describes the use of methylene blue (MB) plus light (photodynamic inactivation, PDI) in the presence of hydrogen peroxide (H(2)O(2)) to kill Staphylococcus aureus, Escherichia coli, and Candida albicans. When H(2)O(2) was added to MB plus light there was an increased antimicrobial effect, which could be due to a change in the type of ROS generated or increased microbial uptake of MB. To clarify the mechanism, the production of ROS was investigated in the presence and absence of H(2)O(2). It was observed that ROS production was almost inhibited by the presence of H(2)O(2) when cells were not present. In addition, experiments using different sequence combinations of MB and H(2)O(2) were performed and MB optical properties inside the cell were analyzed. Spectroscopy experiments suggested that the amount of MB was higher inside the cells when H(2)O(2) was used before or simultaneously with PDI, and ROS formation inside C. albicans cells confirmed that ROS production is higher in the presence of H(2)O(2). Moreover enzymatic reduction of MB by E. coli during photosensitizer uptake to the photochemically inactive leucoMB could be reversed by the oxidative effects of hydrogen peroxide, increasing ROS formation inside the microorganism. Therefore, the combination of a photosensitizer such as MB and H(2)O(2) is an interesting approach to improve PDI efficiency.

This constitutes the first study to report on the relationship between pulsed UV light (PL) irradiation and the simultaneous occurrence of molecular and cellular damage in clinical strains of Candida albicans. Microbial protein leakage and propidium iodide (PI) uptake assays demonstrated significant increases in cell membrane permeability in PL-treated yeast that depended on the amount of UV pulses applied. This finding correlated well with the measurement of increased levels of lipid hydroperoxidation in the cell membrane of PL-treated yeast. PL-treated yeast cells also displayed a specific pattern of intracellular reactive oxygen species (ROS) generation, where ROS were initially localised in the mitochondria after low levels of pulsing (UV dose 0.82 μJ/cm(2)) before more wide-spread cytosolic ROS production occurred with enhanced pulsing. Intracellular ROS levels were measured using the specific mitochondrial peroxide stain dihydrorhodamine 123 and the cytosolic oxidation stain dichloroflurescin diacetate. Use of the dihydroethidium stain also revealed increased levels of intracellular superoxide as a consequence of augmented pulsing. The ROS bursts observed during the initial phases of PL treatment was consistent with the occurrence of apoptotic cells as confirmed by detection of specific apoptotic markers, abnormal chromatin condensation and externalisation of cell membrane lipid phosphatidylserine. Increased amount of PL-irradiation (ca. UV does 1.24-1.65 μJ/cm(2)) also resulted in the occurrence of late apoptotic and necrotic yeast phenotypes, which coincided with the transition from mitochondrial to cytosolic localisation of ROS and with irreversible cell membrane leakage. Use of the comet assay also revealed significant nuclear damage in similarly treated PL samples. Although some level of cellular repair was observed in all test strains during sub-lethal exposure to PL-treatments (≤20 pulses or UV dose 0.55 μJ/cm(2)), this was absent in similar samples exposed to increased amounts of pulsing. This study showed that PL-irradiation inactivates C. albicans test strains through a multi-targeted process with no evidence of microbial ability to support cell growth after ≤20 pulses. Implications of our findings in terms of application of PL for contact-surface disinfection are discussed., (Copyright © 2010 Elsevier B.V. All rights reserved.)

This study examined the antimicrobial/antifungal ability of a tissue conditioner containing a photocatalyst for Escherichia coli, Streptococcus mutans, Staphylococcus aureus and Candida albicans. The photocatalyst was mixed with tissue conditioners powders at concentrations of 0, 10, 15, and 20 wt%. Tissue conditioners powders containing a photocatalyst were mixed with liquid to make test specimens. Test specimens inoculated by each microorganism were irradiated by ultraviolet light for 0-, 2- and 4 hours. The antimicrobial/antifungal effects were evaluated by the CFU technique. The CFU values of each microorganism for tissue conditioners containing a photocatalyst showed significant decrease following UV-irradiation. The improvement in antimicrobial/antifungal effects was concomitant with the increase of the mixing ratio and the irradiation time. Therefore, the results indicated that tissue conditioners containing a photocatalyst might have photocatalytic ability.

The increased resistance of Candida albicans to antibiotic therapy indicates the need for alternative treatments for oral candidiasis. Photodynamic therapy (PDT) has been researched as an alternative tool to inactivate pathogenic microorganisms. It uses a combination of a photosensitizer and a visible light source. This study evaluated the susceptibility of C. albicans to PDT and compared the efficacy of 100 microg/mL methylene blue (MB) and toluidine blue (TB) as photosensitizers. The light source was Indium-Gallium-Aluminum Phosphide (InGaAIP) laser at 53 J/cm2. Suspensions of 108 cells/mL of C. albicans were subject to PDT for 5 minutes in 96-well plates, then decimal dilutions were plated on Sabouraud Dextrose agar After 48h incubation at 37 degreesC, the number of CFU/mL were obtained and submitted to statistical analysis using Kolmogorov-Smirnov, ANOVA (p<0.0001) and Tukey tests. The results showed that MB or laser irradiation alone did not have statistically significant antifungal activity compared to the positive control group (p> 0. 05). Conversely, the number of viable C. albicans cells was reduced significantly after PDT using MB or mainly TB associated to diode laser irradiation. The data proved the efficacy of PDT against C. albicans cells, regardless of the photosensitizer used.

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.)

Riboflavin significantly enhanced the efficacy of simulated solar disinfection (SODIS) at 150 watts per square meter (W m(-2)) against a variety of microorganisms, including Escherichia coli, Fusarium solani, Candida albicans, and Acanthamoeba polyphaga trophozoites (>3 to 4 log(10) after 2 to 6 h; P < 0.001). With A. polyphaga cysts, the kill (3.5 log(10) after 6 h) was obtained only in the presence of riboflavin and 250 W m(-2) irradiance.

Photodynamic inactivation is a rapidly developing antimicrobial treatment that employs a nontoxic photoactivatable dye or photosensitizer in combination with harmless visible light to generate reactive oxygen species that are toxic to cells. Tetrapyrroles (e.g., porphyrins, chlorins, bacteriochlorins) are a class of photosensitizers that exhibit promising characteristics to serve as broad-spectrum antimicrobials. In order to bind to and efficiently penetrate into all classes of microbial cells, tetrapyrroles should have structures that contain (i) one or more cationic charge(s) or (ii) a basic group. In this report, we investigate the use of new stable synthetic bacteriochlorins that have a strong absorption band in the range 720 to 740 nm, which is in the near-infrared spectral region. Four bacteriochlorins with 2, 4, or 6 quaternized ammonium groups or 2 basic amine groups were compared for light-mediated killing against a gram-positive bacterium (Staphylococcus aureus), a gram-negative bacterium (Escherichia coli), and a dimorphic fungal yeast (Candida albicans). Selectivity was assessed by determining phototoxicity against human HeLa cancer cells under the same conditions. All four compounds were highly active (6 logs of killing at 1 microM or less) against S. aureus and showed selectivity for bacteria over human cells. Increasing the cationic charge increased activity against E. coli. Only the compound with basic groups was highly active against C. albicans. Supporting photochemical and theoretical characterization studies indicate that (i) the four bacteriochlorins have comparable photophysical features in homogeneous solution and (ii) the anticipated redox characteristics do not correlate with cell-killing ability. These results support the interpretation that the disparate biological activities observed stem from cellular binding and localization effects rather than intrinsic electronic properties. These findings further establish cationic bacteriochlorins as extremely active and selective near-infrared activated antimicrobial photosensitizers, and the results provide fundamental information on structure-activity relationships for antimicrobial photosensitizers.

The eukaryotic translation initiation factor 4E is an essential and highly conserved protein. As a part of the translational machinery, it plays a key role in the recruitment of mRNA via binding to its m(7)GpppN 5' terminal cap structure. The opportunistic human pathogen Candida albicans is the only known eukaryotic organism with the ability to survive defects in mRNA capping, which suggests unique features of its eIF4E protein. Here, we provide the first experimental evidence of the function of the C. albicans putative gene orf19.7626 as an eIF4E protein. We also show that Ca4E(Leu116) and Ca4E(Ser116) protein variants, both of which occur naturally in C. albicans due to the ambiguous decoding of the CUG(116) codon, display different sensitivities to elevated temperature. Our results clearly point to the importance of the S4-H4 loop for the function of the Ca4E translation initiation factor, and suggest the possible regulatory role of the codon-reading ambiguity within this loop in C. albicans. We proved Saccharomyces cerevisiae as a useful tool organism for studies of C. albicans translation initiation apparatus.

Purpose: To demonstrate the antimicrobial properties of riboflavin/UV-A (365 nm) against fungal pathogens., Methods: The antimicrobial properties of riboflavin/UV-A (365 nm), with or without previous treatment with amphotericin B, were tested on three groups of fungi selected from severe cases of keratomycosis: Candida albicans, Fusarium sp, and Aspergillus fumigatus. They were tested by using Kirby-Bauer discs with empty disc (control), riboflavin 0.1% alone (R), UV-A alone (UV-A), riboflavin 0.1% and additional UV-A exposure (R+UV-A), amphotericin B alone (A), amphotericin B and riboflavin 0.1% (A+R), amphotericin B and UV-A (A+UV-A), amphotericin B and riboflavin 0.1%, and additional UV-A exposure (A+R+UV-A). The mean growth inhibition zone (GIZ) was measured around the discs., Results: C. albicans, Fusarium sp, and A. fumigatus did not show any increased GIZ after treatment without previous amphotericin B medication. However, GIZ was significantly greater after pretreatment with amphotericin B and riboflavin/UV-A (A+R+UV-A) for C. albicans (P = 0.0005), Fusarium sp (P = 0.0023) and A. fumigatus (P = 0.0008) compared with A, A+R, and A+UV-A., Conclusions: Amphotericin B is believed to interact with fungi membrane sterols to produce aggregates that form transmembrane channels. Given that collagen is one of the principal components of the cornea, it is also probable that amphotericin B may diffuse easily after cross-linking. Previous treatment with amphotericin B allowed riboflavin/UV-A effectiveness against C. albicans, Fusarium sp, and A. fumigatus. This schema might be used in the future for the treatment of keratomycosis.

Objective: The purpose of this in vitro study was to investigate the ability of Er,Cr:YSGG laser irradiation at different energy settings to eliminate Candida albicans, either with or without 5.25% sodium hypochlorite (NaOCl) treatment., Methods: In total, 90 extracted human single-rooted teeth were shaped by using a crown-down technique with HERO Shaper rotary instruments and irrigated with 1 mL of 2.5% NaOCl between each instrument. The specimens were autoclaved and incubated with a suspension of C. albicans (ATCC 90028). Then the specimens were divided into six treatment groups (n = 15 per group): group 1, NaOCl + 1-W laser; group 2, 1-W laser; group 3, NaOCl + 0.75-W laser; group 4, 0.75-W laser, group 5, NaOCl; and group 6, no treatment. Aliquots of the experimental teeth were transferred onto Sabouraud's dextrose agar plates with paper points, and colony-forming units (CFUs) were counted as a measure of antifungal activity. One root from each group, which was not submitted to the sampling procedure, was fixed in glutaraldehyde, split into two halves, and evaluated with scanning electron microscopy (SEM). Statistical analysis was performed by using analysis of variance (ANOVA) and the Dunnett t test. Significance was set at alpha = 0.05., Results: All the treatment groups resulted in a significant reduction of C. albicans, but no sterilization, and all the groups were significantly different from each other (p < 0.0001). Group 1 (NaOCl and 1-W laser combination) exhibited the greatest reduction, whereas group 5 (NaOCl) resulted in a minimal reduction in CFUs of C. albicans. The SEM observations were in accordance with the microbiologic analysis., Conclusions: The 5.25% NaOCl and 1-W laser treatment combination may be a valuable cleaning tool for root canals infected with C. albicans.

Rck2p is the Hog1p-MAP kinase-activated protein kinase required for the attenuation of protein synthesis in response to an osmotic challenge in Saccharomyces cerevisiae. Rck2p also regulates rapamycin sensitivity in both S. cerevisiae and Candida albicans. In this study, we demonstrate that the deletion of CaRCK2 renders C. albicans cells sensitive to, and CaRck2p translocates from the cytosol to the nucleus in response to, cell wall stresses caused by Congo red, Calcoflor White, elevated heat and zymolyase. However, the kinase activity of CaRck2p is not required for the cellular response to these cell wall stresses. Furthermore, transcripts of cell wall protein-encoding genes CaBGL2, CaHWP1 and CaXOG1 are reduced in C. albicans cells lacking CaRCK2. The deletion of CaRCK2 also reduces the in vitro filamentation of C. albicans and its virulence in a mouse model of systemic candidasis. The kinase activity of CaRck2p is required for the virulence, but not for the in vitro filamentation, in C. albicans. Therefore, Rck2p regulates cellular responses to cell wall stresses, filamentation and virulence in the human fungal pathogen C. albicans.

Respiratory deficiency increases the sensitivity of the pathogenic fungi Candida albicans and Candida glabrata to oxidative stress induced by photodynamic therapy (PDT) sensitized by the cationic porphyrin meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP-1363). Since disruption of electron transport chain (ETC) function increases intracellular levels of reactive oxygen species in yeast, we determined whether interference with ETC assembly or function increased sensitivity to TMP-1363-PDT in C. albicans, C. glabrata and the non-pathogenic yeast Saccharomyces cerevisiae. Metabolic inhibitor antimycin A and defined genetic mutants were used to identify ETC components that contribute to the sensitivity to PDT. Inhibition of cytochrome bc(1) (Complex III) with antimycin A increases mitochondrial levels of reactive oxygen species. PDT performed following pre-treatment with antimycin A reduced colony forming units (CFU) of C. albicans and C. glabrata by approximately two orders of magnitude relative to PDT alone. A S. cerevisiae mitochondrial glutaredoxin grx5 mutant, defective in assembly of Fe-S clusters critical for Complex III function, displayed increased sensitivity to PDT. Furthermore, C. glabrata and S.cerevisiae mutants in cytochrome c oxidase (Complex IV) synthesis and assembly were also significantly more sensitive to PDT. These included suv3, encoding an ATP-dependent RNA helicase critical for maturation of cytochrome c oxidase subunit transcripts, and pet117, encoding an essential cytochrome c oxidase assembly factor. Following PDT, the reduction in CFU of these mutants was one to two orders of magnitude greater than in their respective parental strains. The data demonstrate that selective inhibition of ETC Complexes III and IV significantly increases the sensitivity of C. albicans, C. glabrata and S. cerevisiae to PDT sensitized with TMP-1363., (2010 Elsevier B.V. All rights reserved.)

This study was to evaluate specific effects of photodynamic therapy (energy density 15.8 J/cm(2), 26.3 J/cm(2) and 39.5 J/cm(2)) using methylene blue, toluidine blue and malachite green as photosensitizers and low-power laser irradiation on the viability of Candida albicans. Suspensions of C. albicans containing 10(6) cells/ml were standardized in a spectrophotometer. For each dye, 120 assays, divided into four groups according to the following experimental conditions, were carried out: laser irradiation in the presence of the photosensitizer; laser irradiation only; treatment with the photosensitizer only; no exposure to laser light or photosensitizer. Next, serial dilutions were prepared and seeded onto Sabouraud dextrose agar for the determination of the number of colony-forming units per milliliter (CFU/ml). The results were subjected to analysis of variance and the Tukey test (P < 0.05). Photodynamic therapy using the photosensitizers tested was effective in reducing the number of C. albicans.. The number of CFU/ml was reduced by between 0.54 log(10) and 3.07 log(10) and depended on the laser energy density used. Toluidine blue, methylene blue and malachite green were effective photosensitizers in antimicrobial photodynamic therapy against C. albicans, as was low-power laser irradiation alone.

We have cloned and characterized the RAD51 and RAD59 orthologs of the pathogenic fungus Candida albicans. CaRad51 exhibited more than 50% identity with several other eukaryotes and the conserved the catalytic domain of a bacterial RecA. As compared to the parental strain, null strains of rad51 exhibited a filamentous morphology, had a decreased grow rate and exhibited a moderate sensitivity to UV light, oxidizing agents, and compounds that cause double-strand breaks (DSB), indicating a role in DNA repair. By comparison, the rad52 null had a higher percentage of filaments, a more severe growth defect and a greater sensitivity to DNA-damaging compounds. Null strains of rad59 showed a UV-sensitive phenotype but behaved similarly to the parental strain in the rest of the assays. As compared to Saccharomyces cerevisiae, C. albicans was much more resistant to bleomycin and the same was true for their respective homologous recombination (HR) mutants. These results indicate that, as described in S. cerevisiae, RAD52 plays a more prominent role than RAD51 in the repair of DSBs in C. albicans and suggest the existence of at least two Rad52-dependent HR pathways, one dependent and one independent of Rad51., (Copyright 2010 Elsevier Inc. All rights reserved.)

Objective: The antimicrobial effect of photoactivated disinfection (PAD) using toluidine blue and an LED lamp was tested on endodontic pathogens in planktonic suspension and after inoculation into extracted teeth. Irradiation time was limited to 30 seconds., Study Design: The effect of PAD on planktonic suspensions of Escherichia coli, Candida albicans, Enterococcus faecalis, Fusobacterium nucleatum, and Streptococcus intermedius was analyzed using Poisson regression. Moreover, cultures of S. intermedius were inoculated into prepared root canals of extracted molars. The effect of PAD performed immediately after inoculation or after overnight bacterial incubation was determined by a 2-sample t test., Results: Photoactivated disinfection yielded significant reductions (P < .001) in the viable counts of all organisms in planktonic suspension. The PAD treatment of S. intermedius in root canals yielded a mean log10 reduction of 2.60 (P < .001) immediately after inoculation and of 1.38 (P < .001) after overnight incubation., Conclusion: Photoactivated disinfection using a conventional light source strongly reduces the number of viable endodontic pathogens in planktonic suspension and in root canals., (Copyright 2010 Mosby, Inc. All rights reserved.)

Objective: In vivo studies of antimicrobial PDT in animal models of oral candidosis are scarce and the association of porphyrin and LED light has not been evaluated for in vivo photoinactivation of Candida. In this study the effectiveness of photodynamic therapy (PDT) on the inactivation of Candida albicans in vivo was evaluated., Study Design: Seventy-one 6-week-old female Swiss mice were immunosuppressed, provided tetracycline to their drinking water, then orally swabbed with a suspension of C. albicans (10(7) CFU/mL). Four days after oral inoculation, PDT was performed on the dorsum of the tongue after topical administration of Photogem at 400, 500, or 1000 mg/L and followed 30 minutes later by illumination with LED light (305 J/cm(2)) at 455 or 630 nm (n = 5 each). After swabbing to recover yeast from the tongue, the number of surviving yeast cells was determined (CFU/mL) and analyzed by ANOVA and Holm-Sidak tests (P < .05). Animals were humanely killed, and the tongues surgically removed and processed for histological evaluation of presence of yeast and inflammatory reaction., Results: PDT resulted in a significant reduction in C. albicans recovered from the tongue (P < .001) when compared with mice from the positive control group. There was no difference between the concentrations of Photogem and LED light wavelengths used. Histological evaluation of the tongue revealed that PDT causes no significant adverse effects to the local mucosa., Conclusion: PDT promoted significant reduction in the viability of C. albicans biofilm without harming the tongue tissue., (Copyright 2010 Mosby, Inc. All rights reserved.)

The possibility of photoeradicating the prokaryotic microorganism Candida albicans by enhancing its endogenous porphyrin production and accumulation was investigated in this study. Induction of porphyrin synthesis was performed by the addition of delta-aminolevulinic acid (ALA), or its hydrophobic derivative ALA methyl ester (m-ALA). Photoinactivation of C. albicans was performed under blue light (407-420 nm) illumination. A decrease in viability of about 1.6 or 2.1 orders of magnitudes was obtained with a light dose of 36 J/cm(2) for an initial concentration of 100-mg/ml ALA or m-ALA, respectively. Endogenous porphyrins extracted from the cells showed that cultures incubated with m-ALA accumulated a relatively higher amount of endogenous porphyrins than ALA, indicating better transport through the yeast cell barriers. When a combination of miconazole and ketoconazole (antifungal agents) is given at a sub-inhibitory concentration (0.5 microg/ml each) with an inducer, a 2.1 or 3.2 orders of magnitude decrease in viability is caused with ALA or with m-ALA, respectively, upon illumination. Fluorescence intensities of the accumulated porphyrins as demonstrated by FACS indicate that the combination of the two azole drugs and an inducer cause a relatively high amount of endogenous porphyrins. Although the additive action of both azole drugs allow better penetration of the inducer, especially m-ALA photoeradication remained limited because of an acidic pH generated in the presence of the inducer. The acidic pH is probably the cause for the inefficiency of the photodynamic treatment. More hydrophobic inducers than m-ALA and less acidic must be investigated to improve the photodynamic treatment by endogenous-induced porphyrins.

Aim of the Study: Phototoxicity can be either harmful or beneficial. Yet the phototoxicity of oriental medicinal plants is an understudied area. The purpose of this study is to fill in this gap., Materials and Methods: The phototoxic potential of oriental medicinal plants was examined in vitro using photohemolysis and the Candida albicans test. Seventeen medicinal plants [Acorus gramineus (ACG), Panax ginseng C.A. (PAG), Platycodon grandiflorum (PLG), Aractylodes japonica (ATJ), Xanthium strumarium (XAS), Dioscorea batatas (DIB), Anemarrhena asphodeloides (ANA), Polygonatum sibiricum Red (PSR), Cocculus trilobus (COT), Ficus carica (FIC), Chelidonium majus var. asiaticum (CMA), Pulsatilla koreana (PUK), Agrimonia pilosa (AGP), Zanthoxylum schinifolium (ZAS), Angelica gigas (ANG), Ledebouriella seseloides (LES), and Cnidium officinale (CNO)] were selected because they showed strong fluorescence in one of our previous studies of 62 plants. We further evaluated in vivo phototoxicity in mice. 0.75 mL/kg of seed oil for Xanthium strumarium (XAS, ), or 1.25 mL/kg of extracted solutions of Atractylodes japonica (ATJ, ), Chelidonium majus var. asiaticum (CMA, ), Zanthoxylum schinifolium (ZAS, ), and Ledebouriella seseloides (LES, ) were given once, and evaluated for sunburn edema, formation of sunburn cell, decrease of epidermal Langerhans cells and local suppression of contact hypersensitivity by UVA irradiation., Results: Sixteen out of the 17 plants tested except COT showed significant photohemolysis, and 5 of those exhibited phototoxic killing of Candida albicans. The phototoxicity of oriental medicines using those 5 plants was then studied in mice. The 5 plants increased sunburn edema and formation of sunburn cell, and suppressed immune responses locally by decreasing epidermal Langerhans cells and contact hypersensitivity by UVA irradiation., Conclusions: More than a quarter of oriental medicinal plants can be phototoxic, and strong fluorescence measured by absorption and fluorescence spectra can be an easier way to screen for phototoxicity. On the other hand, the phototoxicity of the plants may also be used therapeutically. Further studies regarding the phototoxicity of active components extracted from both live and dried oriental medicinal plants are necessary., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

Hsp90 is environmentally contingent molecular chaperone that influences the form and function of diverse signal transducers. Here we discuss our recent findings that Hsp90 regulates the morphogenetic transition from yeast to filamentous forms required for virulence of the most prevalent fungal pathogen of humans, Candida albicans, and does so via cAMP-PKA signalling. This transition is normally regulated by environmental cues that are contingent upon elevated temperature to relieve Hsp90-mediated repression of the morphogenetic program. Intriguingly, Hsp90 inhibition induces filamentation independent of the canonical PKA transcription factor Efg1, in striking similarity to a select set of morphogenetic stimuli. Further investigation will determine the downstream transcription factors through which Hsp90 regulates morphogenesis and the precise mechanism of Hsp90's interaction with the cAMP-PKA pathway. C. albicans is one of many fungal species that undergo a morphological transition in a temperature-dependent manner, thus Hsp90's capacity to govern this key developmental program may provide insight into morphogenesis of diverse organisms.

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 study objective was to evaluate the effects of photodynamic therapy on buccal candidiasis in rats. After experimental candidiasis had been induced on the tongue dorsum, 72 rats were distributed into four groups according to treatment: treated with laser and methylene blue photosensitizer (L+P+); treated only with laser (L+P-); treated only with photosensitizer (L--P+); not treated with laser or photosensitizer (L-P-). The rats were killed immediately, 1 day, or 5 days after treatment, for microscopic analysis of the tongue dorsum. Observation verified that the photodynamic therapy group (L+P+) exhibited fewer epithelial alterations and a lower chronic inflammatory response than the L-P- group. The group L+P- presented more intense epithelial alterations and chronic inflammatory response than the remaining groups. The L-P+ group showed tissue lesions similar to those of the L-P- group. In conclusion, rats treated with photodynamic therapy developed more discrete candidiasis lesions than did the remaining groups.

All organisms have evolved mechanisms that protect them against environmental stress. The major fungal pathogen of humans, Candida albicans, has evolved robust stress responses that protect it against human immune defences and promote its pathogenicity. However, C. albicans is unlikely to be exposed to heat shock as it is obligatorily associated with warm-blooded animals. Therefore, we examined the role of the heat shock transcription factor (Hsf1) in this pathogen. We show that C. albicans expresses an evolutionarily conserved Hsf1 (orf19.4775) that is phosphorylated in response to heat shock, induces transcription via the heat shock element (HSE), contributes to the global transcriptional response to heat shock, and is essential for viability. Why has Hsf1 been conserved in this obligate animal saprophyte? We reasoned that Hsf1 might contribute to medically relevant stress responses. However, this is not the case, as an Hsf1-specific HSE-lacZ reporter is not activated by oxidative, osmotic, weak acid or pH stress. Rather, Hsf1 is required for the expression of essential chaperones in the absence of heat shock (e.g. Hsp104, Hsp90, Hsp70). Furthermore, Hsf1 regulates the expression of HSE-containing genes in response to growth temperature in C. albicans. Therefore, the main role of Hsf1 in this pathogen might be the homeostatic modulation of chaperone levels in response to growth temperature, rather than the activation of acute responses to sudden thermal transitions.

Objective: This study evaluated the clinical effectiveness of two exposure times of microwave irradiation on the disinfection of complete dentures., Methods: Biofilm samples were collected from dentures of 30 patients, who were randomly divided into two experimental groups of 15 subjects each: Group 1-patients had their maxillary denture microwaved for 3 min (650W); Group 2-patients had their maxillary denture microwaved for 2 min (650W). Denture biofilm samples were taken with swabs, before (left side surfaces) and after (right side surfaces) microwave irradiation. All microbial material was plated on selective media for Candida spp., Staphylococcus spp., mutans streptococci and a non-selective media. After incubation (48 h/37 degrees C), the number of colony-forming units (cfu/mL) was counted. Microorganisms which grew on selective media were identified using biochemical methods. The data were statistically analyzed by Kruskal-Wallis test, followed by Dunn's post-test (alpha=0.05)., Results: Microwave irradiation for 3 min (Group 1) resulted in sterilization of all dentures evaluated. After microwave irradiation for 2 min (Group 2), a significant decrease in Candida spp. (P=0.0062), Staphylococcus spp. (P=0.0178), mutans streptococci (P=0.0047) and non-identified species (P<0.0001) was achieved in comparison with the cfu/mL obtained before irradiation. The colonies grown after 2 min of microwave irradiation were identified as Candida albicans, non-aureus Staphylococci and Streptococcus mutans., Conclusion: Microwave irradiation for 3 min may be a potential treatment to prevent cross-contamination.

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.

Purpose: The interference of electric fields (EF) with biological processes is an issue of considerable interest. No studies have as yet been reported on the combined effect of EF plus ionising radiation. Here we report studies on this combined effect using the prokaryote Microcystis panniformis, the eukaryote Candida albicans and human cells., Materials and Methods: Cultures of Microcystis panniformis (Cyanobacteria) in glass tubes were irradiated with doses in the interval 0.5-5 kGy, using a (60)Co gamma source facility. Samples irradiated with 3 kGy were exposed for 2 h to a 20 V . cm(-1) static electric field and viable cells were enumerated. Cultures of Candida albicans were incubated at 36 degrees C for 20 h, gamma-irradiated with doses from 1-4 kGy, and submitted to an electric field of 180 V . cm(-1). Samples were examined under a fluorescence microscope and the number of unviable (red) and viable (apple green fluorescence) cells was determined. For crossing-check purposes, MRC5 strain of lung cells were irradiated with 2 Gy, exposed to an electric field of 1250 V/cm, incubated overnight with the anti-body anti-phospho-histone H2AX and examined under a fluorescence microscope to quantify nuclei with gamma-H2AX foci., Results: In cells exposed to EF, death increased substantially compared to irradiation alone. In C. albicans we observed suppression of the DNA repair shoulder. The effect of EF in growth of M. panniformis was substantial; the number of surviving cells on day-2 after irradiation was 12 times greater than when an EF was applied. By the action of a static electric field on the irradiated MRC5 cells the number of nuclei with gamma-H2AX foci increased 40%, approximately., Conclusions: Application of an EF following irradiation greatly increases cell death. The observation that the DNA repair shoulder in the survival curve of C. albicans is suppressed when cells are exposed to irradiation + EF suggests that EF likely inactivate cellular recovering processes. The result for the number of nuclei with gamma-H2AX foci in MRC5 cells indicates that an EF interferes mostly in the DNA repair mechanisms. A molecular ad-hoc model is proposed.

The photodynamic action of 5-(4-trifluorophenyl)-10,15,20-tris(4-trimethylammoniumphenyl)porphyrin iodide (TFAP(3+)) and 5,10,15,20-tetra(4-N,N,N-trimethylammonium phenyl)porphyrin p-tosylate (TMAP(4+)) has been studied in vitro on Candida albicans. The results of these cationic porphyrins were compared with those of 5,10,15,20-tetra(4-sulphonatophenyl)porphyrin (TPPS(4-)), which characterizes an anionic sensitizer. In vitro investigations show that these cationic porphyrins are rapidly bound to C. albicans cells, reaching a value of approximately 1.4 nmol/10(6) cells, when the cellular suspensions were incubated with 5 microM sensitizer for 30 min. In contrast, TPPS(4-) is poorly uptaken by yeast cells. The fluorescence spectra of these sensitizers into the cells confirm this behaviour. The amount of porphyrin binds to cells is dependent on both sensitizer concentrations (1-5 microM) and cells densities (10(6)-10(8) cells/mL). Photosensitized inactivation of C. albicans cellular suspensions increases with sensitizer concentration, causing a approximately 5 log decrease of cell survival, when the cultures are treated with 5 microM of cationic porphyrin and irradiated for 30 min. However, the photocytotoxicity decreases with an increase in the cell density, according to its low binding to cells. Under these conditions, the photodynamic activity of TFAP(3+) is quite similar to that produced by TMAP(4+), whereas no important inactivation effect was found for TPPS(4)(-). The high photodynamic activity of cationic porphyrins was confirmed by growth delay experiments. Thus, C. albicans cell growth was not detected in the presence of 5 microM TFAP(3+). Photodynamic inactivation capacities of these sensitizers were also evaluated on C. albicans cells growing in colonies on agar surfaces. Cationic porphyrins produce a growth delay of C. albicans colonies and viability of cells was not observed after 3 h irradiation, indicating a complete inactivation of yeast cells. Therefore, these results indicate that these cationic porphyrins are interesting sensitizers for photodynamic inactivation of yeasts in liquid suspensions or in localized foci of infection.

We previously showed that Candida albicans orf19.4590, which we have renamed RFX2, expresses a protein that is reactive with antibodies in persons with candidiasis. In this study, we demonstrate that C. albicans RFX2 shares some functional redundancy with Saccharomyces cerevisiae RFX1. Complementation of an S. cerevisiae rfx1 mutant with C. albicans RFX2 partially restored UV susceptibility and the repression of DNA damage response genes. DNA damage- and UV-induced genes RAD6 and DDR48 were derepressed in a C. albicans rfx2 null mutant strain under basal conditions, and the mutant was significantly more resistant to UV irradiation, heat shock, and ethanol than wild-type strain SC5314. The rfx2 mutant was hyperfilamentous on solid media and constitutively expressed hypha-specific genes HWP1, ALS3, HYR1, ECE1, and CEK1. The mutant also demonstrated increased invasion of solid agar and significantly increased adherence to human buccal epithelial cells. During hematogenously disseminated candidiasis, mice infected with the mutant had a significantly delayed time to death compared to the wild type. During oropharyngeal candidiasis, mice infected with the mutant had significantly lower tissue burdens in the oral cavity and esophagus at 7 days and they were less likely to develop disseminated infections because of mucosal translocation. The data demonstrate that C. albicans Rfx2p regulates DNA damage responses, morphogenesis, and virulence.

The study had to define influence of combined applications of laser radiation and methylene blue (MB) on pathogenic culture of Candida albicans in vitro. The experimental study was done at standard techniques of method of cultivations in a broth. The laser irradiation of cultures was done at once after addition of MB in concentration 0.05%, 0.1% and 0.2%. During studying action of MB in dark, influence of MB to the growth of test-shtam without laser radiation, were prepared fluid Gissa's broth with glucose without Andrede's indicator. Activation of MB was done by laser with wave length 630 nm during 3 or 5 min. All experiments were passed in 4 parallels and 3 repeats. Maximal suppression of growth of microorganisms was noted in group with using 0.1% MB with laser radiation 3 minutes without centrifugation after 24 hours. Maximal suppression of growth was noted in group after centrifugation with 0.05% MB with exposure of laser 3 min. after 48 hours. Sensitivity of pathogenic culture of Candida albicans to application of B and laser raises accordingly to increase of concentration of MB.

Photodynamic antimicrobial chemotherapy (PACT) is a potential antimicrobial therapy that combines light and a photosensitizing drug, promoting a phototoxic effect on the treated cells, in general via oxidative damage. In this work we studied the effect of PACT, using methylene blue (MB), on the permeability of Candida albicans membrane. Our results demonstrated that the combination of MB and laser (684 nm) promoted a decrease in Candida growth. The inhibition was more pronounced in the presence of 0.05 mg/ml MB and with an energy density of 28 J/cm(2). The decrease in Candida growth was associated with an increase in membrane permeabilization. Thus, we suggest that a PACT mechanism using MB can be related to damage in the plasma membranes of the cells.

Purpose: To demonstrate the antimicrobial properties of riboflavin/UVA (365 nm) against common pathogens., Methods: One group of bacteria (Pseudomonas aeruginosa [PA], Staphylococcus aureus [SA], and Staphylococcus epidermidis [SE]) was tested by using Kirby-Bauer discs with (1) empty disc (Control - C); (2) riboflavin 0.1% (B2); (3) riboflavin 0.1% previously activated by UVA (B2'); (4) UVA alone (UVA); (5) group 2+additional UVA exposure (UVA+B2); and (6) group 3+additional UVA exposure (UVA+B2'). In addition, another group of microbes was tested with the same approach: methicillin-resistant S. aureus (MRSA), multidrug-resistant P. aeruginosa (MDRPA), drug-resistant Streptococcus pneumoniae (DRSP), and Candida albicans (CA). The mean growth inhibition zone (GIZ) in square millimeters was measured around the discs. The mean standard deviation (MSD) was calculated to be 3.65 when alpha = 0.01. A mean deviation (MD) > MSD indicates a significant difference., Results: In the first group, the GIZ was significantly greater after UVA (MD = 14.30), UVA+B2 (MD = 39.61), and UVA+B2' (MD = 40.45) when compared with C, B2, and B2'. UVA alone was less effective than UVA+B2 (MD = 25.31) and UVA+B2' (MD = 26.15). The second group demonstrated increased GIZ in UVA (MD = 6.98), UVA+B2 (MD = 17.80), and UVA+B2' (MD = 21.15) when compared with C, B2, and B2'. UVA alone was less effective against the second group of bacteria than was UVA+B2 (MD = 10.82) and UVA+B2' (MD = 14.17). CA did not show any GIZ after treatment., Conclusions: Riboflavin/UVA was effective against SA, SE, PA, MRSA, MDRPA, and DRSP, but was ineffective on CA and has the potential for use in treatment of microbial keratitis in the future.

Candida albicans, clinically the most important human fungal pathogen, rapidly develops resistance to antifungal drugs. The acquisition of resistance has been linked to various types of genome changes. As part of an ongoing study of this problem, we investigated mutation, genome stability and drug resistance acquisition in C. albicans strains with deletions in the base excision repair (BER) genes NTG1, APN1 and OGG1, and in the nucleotide excision repair (NER) genes RAD2 and RAD10. The BER mutants did not exhibit any change in their susceptibility to DNA-damaging agents, but the NER mutants were extremely sensitive to UV-induced DNA damage. We did not observe any significant change in mutation, genome stability and antifungal drug sensitivity in the mutant strains we tested. However, we detected a number of intriguing phenotypic differences between strains bearing deletions in equivalent C. albicans and Saccharomyces cerevisiae BER and NER genes, which may be related to differences in the life cycles of these two fungi.

Purpose: This study evaluated the effectiveness of different exposure times of microwave irradiation on the disinfection of a hard chairside reline resin., Materials and Methods: Sterile specimens were individually inoculated with one of the tested microorganisms (Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Bacillus subtilis) and incubated for 24 hours at 37 degrees C. For each microorganism, 10 specimens were not microwaved (control), and 50 specimens were microwaved. Control specimens were individually immersed in sterile saline, and replicate aliquots of serial dilutions were plated on selective media appropriate for each organism. Irradiated specimens were immersed in water and microwaved at 650 W for 1, 2, 3, 4, or 5 minutes before serial dilutions and platings. After 48 hours of incubation, colonies on plates were counted. Irradiated specimens were also incubated for 7 days. Some specimens were prepared for scanning electron microscopic (SEM) analysis., Results: Specimens irradiated for 3, 4, and 5 minutes showed sterilization. After 2 minutes of irradiation, specimens inoculated with C. albicans were sterilized, whereas those inoculated with bacteria were disinfected. One minute of irradiation resulted in growth of all microorganisms. SEM examination indicated alteration in cell morphology of sterilized specimens. The effectiveness of microwave irradiation was improved as the exposure time increased., Conclusion: This study suggests that 3 minutes of microwave irradiation can be used for acrylic resin sterilization, thus preventing cross-contamination.

The inactivation efficiencies of microorganisms were found to be enhanced by using silver solution together with ultraviolet light (UV-A, 395 nm) irradiation. The inactivation efficiencies were improved remarkably especially in eukaryotic microorganism. To make clear the inactivation mechanism of microorganisms by the combination effect of silver and ultraviolet light irradiation, the resultant solution was characterized by ESR (Electron spin resonance, ESR). Scanning electron microscopy (SEM) and the methnd for measuring enzyme activity of mitochondria for eukarvotic cells were used to conjecture the mechanism, by analysis of the morphological and physiologic changes in eukaryotic cells. It is proposed that silver oxide (Ag20) can be activated by ultraviolet light irradiation and react with water molecules to produce hydroxyl radical (.OH). Hydroxyl radical could damage cell wall of eukaryotic microorganisms, and inactivate the enzyme activity of mitochondria of eukaryotic microorganism cells. Accordingly, eukaryotic microorganism cells would die. In the experiment, Staphylococcus aureus was employed as the representative of prokaryotic microorganisms, and Candida albicans and Trichophyton mentagrophytes as the representative of eukaryotic microorganisms, respectively. Moreover, the results of the technology applied to washing machine were presented and discussed.

The aim of this study was to propose the use of red light-emitting diode (LED) as an alternative light source for methylene blue (MB) photosensitizing effect in photodynamic therapy (PDT). Its effectiveness was tested against Staphylococcus aureus (ATCC 26923), Escherichia coli (ATCC 26922), Candida albicans (ATCC 90028) and Artemia salina. The maximum absorption of the LED lamps was at a wavelength of 663 nm, at intensities of 2,4,6 and 12 J.cm-2 for 10, 20, 30 and 60 min of exposure, respectively. Assays with and without LED exposure were carried out in plates containing MB at concentrations of 7 to 140.8 (micro) M for microorganisms and 13.35 to 668.5 (micro) M for microorganisms or microcrustaceans. The LED exposure induced more than 93.05%, 93.7% and 93.33% of growth inhibition for concentrations of 42.2 (micro)M for S.aureus (D-value=12.05 min) and 35.2 (micro)M for E.coli (D-value=11.51 min) and C.albicans (D-value=12.18 min), respectively after 20 min of exposure. LED exposure for 1 h increased the cytotoxic effect of MB against A.salina from 27% to 75%. Red LED is a promising light device for PDT that can effectively inhibit bacteria, yeast and microcrustacean growth.

Candida albicans is a diploid organism that exhibits high levels of heterozygosity. Although the precise manner by which this heterozygosity provides advantage for the commensal/pathogenic life styles of C. albicans is not known, heterozygous markers are themselves useful for studying genomic rearrangements, which occur frequently in C. albicans. Treatment of CAI-4 with UV light yielded histidine auxotrophs which could be complemented by HIS4, suggesting that strain CAI-4 is heterozygous for HIS4. These auxotrophs appeared to have undergone mitotic recombination and/or chromosome loss. As expected from a heterozygote, disruption of the functional allele of HIS4 resulted in a his4::hisG-URA3-hisG strain that is auxotrophic for histidine. Sequencing of random clones of the HIS4 ORF from CAI-4 and its precursor SC5314 revealed the presence of 11 SNPs, seven synonymous and four non-synonymous. Site-directed mutagenesis indicates that only one of those SNPs, T929G (Gly310Val), is responsible for the non-functionality of the encoded enzyme. HIS4 analysis of five commonly used laboratory strains is reported. This study provides a new, easily measured nutritional marker that can be used in future genetic studies in C. albicans.

Candida albicans is a human fungal pathogen and has been extensively studied because of its clinical importance. Comprehensive gene analyses have, however, made little progress. This is because of the diploid and asexual characteristics of the fungus that hamper gene disruptions. In this study, we found that ultraviolet (UV) irradiation, as well as mutagen treatment, strongly stimulated loss of heterozygosity (LOH) in strains harboring artificially constructed heterozygosity. UV-induced LOH occurred more frequently in cells within the logarithmic phase of growth compared to those within the stationary phase of growth. This was observed at all loci tested on chromosome 7, except for a locus neighboring the centromere. C. albicans RAD52, whose orthologue in Saccharomyces cerevisiae was reported to be involved in DNA repair by homologous recombination, was shown to be required for UV-induced LOH. These results suggest that high efficiency LOH caused by UV irradiation could be a prominent tool for gene analyses in C. albicans.

Due to the augmented number of immunocompromised patients, the infections associated to the pathogen of the genus Candida have increased dramatically in the recent years. In order to proliferate, Candida albicans can produce a germ tube formation extending from the cells. The germ tube formation is a transition state from budding to hyphal cells, and represents an essential stage for virulence. In this work we studied the effect of the photodynamic antimicrobial chemotherapy (PACT), a potential antimicrobial treatment on the germ tube formation by C. albicans. Germ tube formation was induced by goat serum after different treatments with Methylene blue (MB) and Laser (683nm). Our results demonstrated that photodynamic therapy using MB, as a photosensitizing drug; inhibits both the growth and the germ tube formation by C. albicans. Thus, our results suggest the possibility that Methylene blue, combined with light in a specific wavelength, can be used as a promising novel antifungal agent.