Factors influencing the haematoporphyrin-sensitized photoinactivation of Candida albicans

Photosensitizing activity of haematoporphyrin (Hp) on Candida albicans cells is mainly promoted by unbound dye molecules in the bulk aqueous medium. Moreover, the death of photosensitized cells is dependent on the dye concentration, irradiation time, irradiation temperature, and the composition of the growth media. Morphological and biochemical studies indicate that the photoprocess involves an initial limited alteration of the cytoplasmic membrane, which allows the penetration of the dye into the cell with consequent photodamage of intracellular targets. In this respect, the Hp-sensitized photoinactivation of eukaryotic microbial cells differs from that in prokaryotic cells.     

Optimization of the conditions for RAPD-PCR of Candida spp. and Cryptococcus spp

In this study, we determined the optimal RAPD amplification conditions to obtain genetic molecular markers for the rapid and accurate identification of Cryptococcus spp. and Candida spp. The following parameters are modified: template DNA, DNA polymerase, magnesium cloride and primer concentration; denaturation, annealing and extension time, temperature of annealing and thermal cycles. After the optimization, reliable and reproducible RAPD patterns are obtained.     

Interactions of Candida albicans with other Candida spp. and bacteria in the biofilms

AIMS: To study the interactions between Candida albicans and 12 other species of Candida and bacteria in biofilms. METHODS AND RESULTS: The number of cells within growing biofilms in a polystyrene tube model was measured after adding C. albicans to preformed biofilms of other micro-organisms and vice versa. It was also measured after simultaneous biofilm formation of C. albicans and other micro-organisms. The number of cells of C. albicans within the growing biofilms decreased significantly (P < 0.05) when the fungus was added to preformed biofilms of Candida spp. and bacteria except, with C. parapsilosis, Torulopsis glabrata and the glycocalyx producer Pseudomonas aeruginosa. When C. parapsilosis, Staphylococcus epidermidis (nonglycocalyx producer) or Serratia marcescens was added to preformed biofilms of C. albicans, the number of cells of these micro-organisms increased in the growing biofilms. CONCLUSIONS: Biofilms of C. albicans are capable of holding other micro-organisms and more likely to be heterogeneous with other bacteria and fungi in the environment and on medical devices. SIGNIFICANCE AND IMPACT OF THE STUDY: Recognition of the heterogeneity of biofilm-associated organisms can influence treatment decisions, particularly in patients who do not respond to initial appropriate therapy.     

Ultraviolet photoinactivation of galactosyltransferase. Protection by substrates.

Galactosyltransferase was irreversibly inactivated upon exposure to ultraviolet light and the rate of inactivation followed apparent first-order kinetics. Significant protection against inactivation was observed in the presence of various combinations of substrates. UDPgalactose and Mn2+ together gave the most protection. Amino acid analyses revealed the loss of 1 mol of tryptophan per mol of galactosyltransferase upon ultraviolet photoinactivation. Further evidence for an essential trypotphan was provided by difference spectra and by inactivation with 2-hydroxy-5-nitrobenzyl bromide and protection against this reagent by Mn2+ and UDPgalactose. The protection by UDPgalactose and Mn2+ was greater than that provided by UDPgalactose alone. Since Mn2+ provided no protection by itself, this suggested that the formation of the galactosyltransferase-Mn2+-UDPgalactose complex caused a conformational change which was responsible for the observed protection of the essential tryptophanyl residue.     

Photosensitization of aqueous model systems by hypericin.

Absorption and fluorescence measurements of purified hypericin (HY) were made in various media. Photosensitization of two aqueous systems was investigated: resealed red blood cell membranes (ghosts) and hen lysozyme (Lys). Solubilization of HY by ghost membranes was shown by means of diffuse reflectance spectroscopy. Visible light irradiation of the ghosts incorporating HY led to lipid peroxidation with evidence of singlet oxygen involvement. A binding model applicable for insoluble ligands is indicative of strong HY binding to HSA. The HY-HSA complex photosensitized inactivation of Lys. The pseudo-first-order reaction kinetics with protection by azide ion are consistent with a Type II mechanism mediated by singlet oxygen. The results are discussed in the context of the HY photodynamic and antiretroviral activities.     

Integrin-like proteins in Candida spp. and other microorganisms

The vertebrate integrins provide a paradigm for cell surface proteins involved in adhesion and morphogenesis. However, homologs of integrins have been found in more primitive organisms. This review will discuss the evidence for surface proteins in Candida albicans and Candida tropicalis that contain motifs reminiscent of integrins and will analyze the contributions of one of these proteins, Int1p, to adhesion, morphogenesis, and virulence. Other microorganisms thought to express integrin-like proteins will also be addressed.     

Streptococcus thermophilus and its biosurfactants inhibit adhesion by Candida spp. on silicone rubber.

The adhesion of yeasts, two Candida albicans and two Candida tropicalis strains isolated from naturally colonized voice prostheses, to silicone rubber with and without a salivary conditioning film in the absence and presence of adhering Streptococcus thermophilus B, a biosurfactant-releasing dairy isolate, was studied. Coverage of 1 to 4% of the surface of silicone rubber substrata with adhering S. thermophilus B gave significant reductions in the initial yeast adhesion regardless of the presence of a conditioning film. Mechanistically, this interference in yeast adhesion by S. thermophilus B was not due to direct physical effects but to biosurfactant release by the adhering bacteria, because experiments with S. thermophilus B cells that had released their biosurfactants prior to adhesion to silicone rubber and competition with yeasts did not show interference with initial yeast adhesion. The amounts of biosurfactants released were highest for mid-exponential- and early-stationary-phase bacteria (37 mg.g of cells-1 [dry weight]), but biosurfactants released by stationary-phase bacteria (14 mg.g of cells-1 [dry weight]) were the most surface active. The crude biosurfactants released were mixtures of various components, with a glycolipid-like component being the most surface active. A lipid-enriched biosurfactant fraction reduced the surface tension of an aqueous solution to about 35 mJ.m-2 at a concentration of only 0.5 mg.ml-1. The amount of biosurfactant released per S. thermophilus B cell was estimated to be sufficient to cover approximately 12 times the area of the cross section of the bacterium, making biosurfactant release a powerful defense weapon in the postadhesion competition of the bacterium with microorganisms such as yeasts. Preadsorption of biosurfactants to the silicone rubber prior to allowing yeasts to adhere was as effective against C. albicans GB 1/2 adhesion as covering 1 to 2% of the silicone rubber surface with adhering S. thermophilus B, but a preadsorbed biosurfactant layer was less effective against C. tropicalis GB 9/9.     

Enzymatic differentiation of Candida parapsilosis from other Candida spp. in a membrane filtration test

A previously reported enzyme assay on a membrane filter using 4-methylumbelliferyl (4-MU)-N-acetyl-beta-D-galactosaminide, -phosphate and -pyrophosphate as substrates for the differentiation of four Candida spp. has been extended to Candida parapsilosis. The substrate 4-MU-beta-D-glucoside was hydrolyzed by 28 test strains of this species but to a variable extent by seven other yeasts also. For a full enzymatic differentiation of C. parapsilosis from other medical yeasts, a battery of six reactions was required. Of 71 C. parapsilosis positive clinical samples, 4.2% gave a false negative result due to overgrowth by Candida albicans. The present assay is more rapid than a described spectrofluorometric determination of beta-D-glucosidase in a broth, i.e., 9-11 h versus up to >48 h.     

Potentiation of hypericin and hypocrellin-induced phototoxicity by omeprazole.

Hypericin and hypocrellin are potential antiviral and antineoplastic agents with multiple modes of light-induced biological activity connected with a production of singlet oxygen and/or excited-state proton transfer and consequent pH drop formation in the drugs environment. In present work light-induced cytotoxicity of hypericin (1 x 10(-5) - 10(-9) mol) and hypocrellin (1 x 10(-5) - 10(-9) mol) and potentiating effect of omeprazole on human leukemic cell line HL-60 was studied. Under dark condition cultivation none cytotoxicity was observed. The only one exception was hypocrellin in concentration 1 x 10(-5) mol which displayed full cytotoxic effect. However, illumination increased cytotoxic effect of hypericin and hypocrellin, both. Omeprazole, an inhibitor of H+K+-ATPase, has been used for testing the hypothetical pH decreasing effect of hypericin and hypocrellin in their cytotoxic mechanism of action. The results of our experiments have shown that in HL-60 cell line the effect of hypericin and hypocrellin at 1 x 10(-6) mol (both) was significantly potentiated by omeprazole in concentrations 1 x 10(-6) - 10(-9) mol. Our results support the hypothesis that the excited-state proton transfer and the consequent acidification of hypericin and hypocrellin environment could play a role in the biological activity of both agents.     

Mechanism of photoinactivation in photosynthetic systems I. The dark reaction in photoinactivation

1)General features of photoinactivation of chloroplast reactionsunder moderate light intensity were investigated. 2)The effects of temperature and of intermittent illuminationupon photoinactivation indicated that the process is not a simplephotochemical reaction but includes some dark reactions. 3)The action spectrum of the photoinactivation indicated thatthe energy absorbed by photosynthetic pigments is used for theprocess. 4)The relative sensitivity and different time courses seen inthe inactivation of various photochemical activities suggestedthe presence of different kinds of photoinactivation. ¹This study was aided by grants from the Ministry of Education(407, 160-1965, 91, 402-1966, 1967, 4, 103-1968). Financialsupport from Sanyo Broadcasting Scientific Foundation is alsoacknowledged with cordial thanks (Received July 30, 1969; )     

Susceptibility of Candida spp. clinical isolates to antimycotics and disinfectants

The incidence of candidiasis among immunocompromised patients and emergence of antimycotics resistant strains has increased significantly. The aims of this study were: to examine the in vitro activity of antimycotics and biocides against Candida clinical isolates; to detect cross-resistance of fungi to these preparations and to estimate whether disinfectants applied in hospital areas are active against clinical Candida isolates. In vitro susceptibility of 102 Candida isolates to eight antimycotics was examined by Etest and ATB Fungus. Sensitivity of these strains to four disinfectants and an antiseptic agent was tested according to EN 1275:2005. Amphotericin B, caspofungin and 5-fluorocytosine were the most effective antimycotics against all Candida isolates. Resistance to itraconazole and fluconazole was observed among C. krusei and C. glabrata. The MICs (Minimal Inhibitory Concentrations) for ketoconazole, voriconazole and posaconazole against Candida albicans ranged: 0.003 - >32 μg/ml and one strain was resistant to three agents tested. All analysed Candida strains were sensitive to biocides containing either chlorine, aldehyde, alcohol mixtures, glucoprotamin or chlorhexidine gluconate with isopropanol. Sensitivity to these agents was observed at concentrations lower than those concentrations recommended by manufacturers to achieve proper biocidal activity to those preparations. Our data suggest that these disinfectants can be effectively applied in clinical wards to prevent nosocomial Candida infections.     

Merocyanine-sensitized photoinactivation of enveloped viruses.

A wide range of enveloped viruses, including human herpes simplex virus type 1, human cytomegalovirus, human T cell leukemia/lymphoma virus type I, human immunodeficiency virus type 1, Sindbis virus, and Friend erythroleukemia virus, are highly susceptible to merocyanine 540 (MC 540)-sensitized photoinactivation. By contrast, human pluripotent hematopoietic stem cells, red cells, factor VIII, and von Willebrand factor are much less sensitive. This suggests that MC 540 may be useful for the inactivation of enveloped viruses in blood and blood products. The dye has a low acute systemic toxicity, is rapidly eliminated from the blood stream, and has little or no mutagenic potential. The currently available data support the view that MC 540-sensitized photo-inactivation interferes with early events in the infectious process, notably the ability of the virus to adhere to and penetrate its host cell. The viral envelope is a major target of photodynamic damages which appear to be mediated at least in part by singlet molecular oxygen.     

Three new yeasts from Antarctic soils:Candida nivalis,Candida gelida andCandida frigida spp.n.

During a study of the yeast flora of soil samples from the Ross Dependency of Antarctica three new fermenting yeasts of the genusCandida were isolated. They could all use nitrate as N-source and formed a starch-like substance on non-synthetic media. They were separated on the basis of the sugars they fermented and assimilated.     

Case-control study to evaluate the link between immunosuppression and Candida spp. infection

Candidiasis is one of the fungal infections with the highest incidence in the immunosuppressed host. The evolution of infection and the increase of antifungal medical drugs resistance could both contribute to the mortality attributable to Candida infection in the immunosuppressed host. Even though the data from international studies are well known, few studies have been published in Romania on this subject. In the case-control study we demonstrated the link between the immunosuppression and the presence of Candida infection. Further studies are to be carried out in order to identify more accurately this link and to extend the study to other fungi. There is a need to increase the microbiological diagnosis use at least at the hospital laboratory level in order to better identify the real situation of fungal infections and the link between them and the concrete status of different hosts. Continued surveillance for infections caused by C. albicans and other species of Candida among hospitalized patients is recommended. Control of antimicrobial resistance among nosocomial infections caused by C. albicans and other species of Candida requires rational policies for use of both antifungal and antibacterial agents and appropriate surveillance for the emergence of resistant strains and species.