Biofilm characterization of Fusarium solani keratitis isolate: increased resistance to antifungals and UV light.

Fusarium solani has drawn phytopathogenic, biotechnological, and medical interest. In humans, it is associated with localized infections, such as onychomycosis and keratomycosis, as well as invasive infections in immunocompromised patients. One pathogenicity factor of filamentous fungi is biofilm formation. There is still only scarce information about the in vitro mechanism of the formation and composition of F. solani biofilm. In this work, we describe the biofilm formed by a clinical keratomycosis isolate in terms of its development, composition and susceptibility to different antifungals and ultraviolet light (UV) at different biofilm formation stages. We found five biofilm formation stages using scanning electron microscopy: adherence, germination, hyphal development, maturation, and cell detachment. Using epifluorescence microscopy with specific fluorochromes, it was elucidated that the extracellular matrix consists of carbohydrates, proteins, and extracellular DNA. Specific inhibitors for these molecules showed significant biofilm reductions. The antifungal susceptibility against natamycin, voriconazole, caspofungin, and amphotericin B was evaluated by metabolic activity and crystal violet assay, with the F. solani biofilm preformation to 24 h increased in resistance to natamycin, voriconazole, and caspofungin, while the biofilm preformation to 48 h increased in resistance to amphotericin B. The preformed biofilm at 24 h protected and reduced UV light mortality. F. solani isolate could produce a highly structured extra biofilm; its cellular matrix consists of carbohydrate polymers, proteins, and eDNA. Biofilm confers antifungal resistance and decreases its susceptibility to UV light. The fungal biofilm functions as a survival strategy against antifungals and environmental factors.

Fungicidal activity of copper-sputtered flexible surfaces under dark and actinic light against azole-resistant Candida albicans and Candida glabrata.

Candida spp. are able to survive on hospital surfaces and causes healthcare-associated infections (HCAIs). Since surface cleaning and disinfecting interventions are not totally effective to eliminate Candida spp., new approaches should be devised. Copper (Cu) has widely recognized antifungal activity and the use of Cu-sputtered surfaces has recently been proposed to curb the spread of HCAIs. Moreover, the activity of Cu under the action of actinic light remains underexplored. We investigated the antifungal activity of Cu-sputtered polyester surfaces (Cu-PES) against azole-resistant Candida albicans and Candida glabrata under dark and low intensity visible light irradiation (4.65mW/cm2). The surface properties of Cu-PES photocatalysts were characterized by diffuse reflectance spectroscopy (DRS) and X-ray fluorescence (XRF). Under dark, Cu-PES showed a fungicidal activity (≥3log10CFU reduction of the initial inoculum) against both C. albicans DSY296 and C. glabrata DSY565 leading to a reduction of the starting inoculum of 3.1 and 3.0log10CFU, respectively, within 60min of exposure. Under low intensity visible light irradiation, Cu-PES exhibited an accelerated fungicidal activity against both strains with a reduction of 3.0 and 3.4log10CFU, respectively, within 30min of exposure. This effect was likely due to the semiconductor Cu2O/CuO charge separation. The decrease in cell viability of the two Candida strains under dark and light conditions correlated with the progressive loss of membrane integrity. These results indicate that Cu-PES represent a promising strategy for decreasing the colonization of surfaces by yeasts and that actinic light can improve its self-disinfecting activity.

Effect of LED Blue Light on Penicillium digitatum and Penicillium italicum Strains.

Studies on the antimicrobial properties of light have considerably increased due in part to the development of resistance to actual control methods. This study investigates the potential of light-emitting diodes (LED) blue light for controlling Penicillium digitatum and Penicillium italicum. These fungi are the most devastating postharvest pathogens of citrus fruit and cause important losses due to contaminations and the development of resistant strains against fungicides. The effect of different periods and quantum fluxes, delaying light application on the growth and morphology of P. digitatum strains resistant and sensitive to fungicides, and P. italicum cultured at 20°C was examined. Results showed that blue light controls the growth of all strains and that its efficacy increases with the quantum flux. Spore germination was always avoided by exposing the cultures to high quantum flux (700 µmol m(-2) s(-1) ) for 18 h. Continuous light had an important impact on the fungus morphology and a fungicidal effect when applied at a lower quantum flux (120 µmol m(-2) s(-1) ) to a growing fungus. Sensitivity to light increased with mycelium age. Results show that blue light may be a tool for P. digitatum and P. italicum infection prevention during handling of citrus fruits.

Stable synthetic mono-substituted cationic bacteriochlorins mediate selective broad-spectrum photoinactivation of drug-resistant pathogens at nanomolar concentrations.

Three stable synthetic mono-substituted cationic bacteriochlorins (BC37, BC38 and BC39) were recently reported to show exceptional activity (low nanomolar) in mediating photodynamic killing of human cancer cells after a 24h incubation upon excitation with near-infrared light (730 nm). The presence of cationic quaternary ammonium groups in each compound suggested likely activity as antimicrobial photosensitizers. Herein this hypothesis was tested against a panel of pathogenic microorganisms that have all recently drawn attention due to increased drug-resistance (Gram-positive bacteria, Staphylococcus aureus and Enterococcus faecalis; Gram-negative bacteria, Escherichia coli and Acinetobacter baumannii; and fungal yeasts, Candida albicans and Cryptococcus neoformans). All three bacteriochlorins were highly effective against both Gram-positive species (>6 logs of eradication at ⩽ 200 nM and 10 J/cm(2)). The dicationic bacteriochlorin (BC38) was best against the Gram-negative species (>6 logs at 1-2 µM) whereas the lipophilic monocationic bacteriochlorin (BC39) was best against the fungi (>6 logs at 1 µM). The bacteriochlorins produced substantial singlet oxygen (and apparently less Type-1 reactive-oxygen species such as hydroxyl radical) as judged by activation of fluorescent probes and comparison with 1H-phenalen-1-one-2-sulfonic acid; the order of activity was BC37 > BC38 > BC39. A short incubation time (30 min) resulted in selectivity for microbial cells over HeLa human cells. The highly active photodynamic inactivation of microbial cells may stem from the amphiphilic and cationic features of the bacteriochlorins.

The two-component sensor kinase TcsC and its role in stress resistance of the human-pathogenic mold Aspergillus fumigatus.

Two-component signaling systems are widespread in bacteria, but also found in fungi. In this study, we have characterized TcsC, the only Group III two-component sensor kinase of Aspergillus fumigatus. TcsC is required for growth under hyperosmotic stress, but dispensable for normal growth, sporulation and conidial viability. A characteristic feature of the ΔtcsC mutant is its resistance to certain fungicides, like fludioxonil. Both hyperosmotic stress and treatment with fludioxonil result in a TcsC-dependent phosphorylation of SakA, the final MAP kinase in the high osmolarity glycerol (HOG) pathway, confirming a role for TcsC in this signaling pathway. In wild type cells fludioxonil induces a TcsC-dependent swelling and a complete, but reversible block of growth and cytokinesis. Several types of stress, such as hypoxia, exposure to farnesol or elevated concentrations of certain divalent cations, trigger a differentiation in A. fumigatus toward a "fluffy" growth phenotype resulting in white, dome-shaped colonies. The ΔtcsC mutant is clearly more susceptible to these morphogenetic changes suggesting that TcsC normally antagonizes this process. Although TcsC plays a role in the adaptation of A. fumigatus to hypoxia, it seems to be dispensable for virulence.

[IXR1 and HMO1 genes jointly control the level of spontaneous mutagenesis in yeast Saccharomyces cerevisiae].

The yeast genes IXR1 and HMO1 encode proteins belonging to the family of chromatin nonhistone proteins, which are able to recognize and bind to irregular DNA structures. The full deletion of gene IXR1 leads to an increase in cell resistance to the lethal action of UV light, gamma-rays, and MMS, increases spontaneous mutagenesis and significantlly decreases the level of UV-induced mutations. It was earlier demonstrated in our works that the hmo 1 mutation renders cells sensitive to the lethal action of cisplatin and virtually does not affect the sensitivity to UV light. Characteristically, the rates of spontaneous and UV-induced mutagenesis in the mutant are increased. Epistatic analysis of the double mutation hmo 1 ixr1 demonstrated that the interaction of these genes in relation to the lethal effect of cisplatin and UV light, as well as UV-induced mutagenesis, is additive. This suggests that the products of genes HMO1 and IXR1 participate in different repair pathways. The ixr1 mutation significantly increases the rate of spontaneous mutagenesis mediated by replication errors, whereas mutation hmo 1 increases the rate of repair mutagenesis. In wild-type cells, the level of spontaneous mutagenesis was nearly one order of magnitude lower than that obtained in cells of the double mutant. Consequently, the combined activity of the Hmo 1 and the Ixr1 proteins provides efficient correction of both repair and replication errors.

Photodynamic inactivation of normal and antifungal resistant Candida species.

BACKGROUND: Susceptibility of bacterial and fungal species to the photodynamic killing effects of various photosensitizing dyes has received increasing attention. In the oral cavity oral candidiasis is primarily caused by Candida albicans. Evidence suggests that Oropharyngeal Candidiasis, found frequently in patients with immunodeficiency, present with mixed Candida organisms and are more difficult to treat than those solely due to C. albicans. In the present study we demonstrate the ability to efficiently kill antifungal resistant isolates of Candida using Photofrin induced PDT. METHODS: Candida strains from the ATCC as well as fluconazole and amphotericin B resistant and sensitive isolates from adults with AIDS were grown cultures and grown under standard conditions. Photofrin was added to appropriate cultures as dictated by experimental design. Light was delivered to assigned cultures using a 630 nm laser source at a power density of 150 mW/cm(2), for appropriate time to deliver 45-135 J/cm(2). Colony forming assays were used to determine survival. RESULTS: After illumination cultures treated with Photofrin had significant reduction in colony forming ability at all light doses examined. Isolates from AIDS patients which had demonstrated antifungal resistance showed equivalent sensitivity to photodynamic killing as did control ATCC cultures of the same strain. CONCLUSION: This study demonstrates Photofrin induced PDT can eliminate Candida species with significant efficiency as revealed by colony forming ability. Further Candida isolates from AIDS patients that had demonstrated fluconazole and amphotericin B resistance were equally susceptible to photodynamic killing.

UVA radiation is highly mutagenic in cells that are unable to repair 7,8-dihydro-8-oxoguanine in Saccharomyces cerevisiae.

UVA (320-400 nm) radiation constitutes >90% of the environmentally relevant solar UV radiation, and it has been proposed to have a role in skin cancer and aging. Because of the popularity of UVA tanning beds and prolonged periods of sunbathing, the potential deleterious effect of UVA has emerged as a source of concern for public health. Although generally accepted, the impact of DNA damage on the cytotoxic, mutagenic, and carcinogenic effect of UVA radiation remains unclear. In the present study, we investigated the sensitivity of a panel of yeast mutants affected in the processing of DNA damage to the lethal and mutagenic effect of UVA radiation. The data show that none of the major DNA repair pathways, such as base excision repair, nucleotide excision repair, homologous recombination, and postreplication repair, efficiently protect yeast from the lethal action of UVA radiation. In contrast, the results show that the Ogg1 DNA glycosylase efficiently prevents UVA-induced mutagenesis, suggesting the formation of oxidized guanine residues. Furthermore, sequence analysis of UVA-induced canavanine-resistant mutations reveals a bias in favor of GC-->TA events when compared with spontaneous or H(2)O(2)-, UVC-, and gamma-ray- induced canavanine-resistant mutations in the WT strain. Taken together, our data point out a major role of oxidative DNA damage, mostly 7,8-dihydro-8-oxoguanine, in the genotoxicity of UVA radiation in the yeast Saccharomyces cerevisiae. Therefore, the capacity of skin cells to repair 7,8-dihydro-8-oxoguanine may be a key parameter in the mutagenic and carcinogenic effect of UVA radiation in humans.