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Candida bloodstream infection has dramatically increased in the last decade due to the growing number of immunocompromised populations worldwide. In this study, we evaluated the antifungal susceptibility profiles and virulence attributes of Candida bloodstream isolates (CBIs) derived from Hong Kong and Finland, information which are vital for devising empirical clinical strategies. Susceptibility testing of a wide range of antifungals including fluconazole, itraconazole, voriconazole, ketoconazole, 5-fluorocytosine, amphotericin B and caspofungin was performed. Haemolytic activity and secretion of proteinase of CBIs were also examined. All CBIs derived from Hong Kong were susceptible to all the antifungals tested whilst some CBIs from Finland were resistant to azoles and caspofungin. C. albicans, C. glabrata and C. tropicalis showed higher haemolytic activity whereas C. parapsilosis and C. guilliermondii were non-haemolytic in general. Proteinase activity of the Finland C. albicans isolates was significantly higher than the Hong Kong isolates. Our data provide a glimpse of the possible evolutionary changes in pathogenic potential of Candida that may be occurring in different regions of the world. Therefore, continuous surveillance and availability of local data should be taken into consideration when treating candidemia patients.

Biofilm formation is a major virulence attribute of Candida albicans and is directly associated with therapeutic failure. One method by which Candida acquires antifungal resistance is the expression of drug-resistance genes. This study aimed to evaluate the transcriptional regulation of several genes associated with antifungal resistance of C. albicans under planktonic, recently adhered and biofilm growth modes and in C. albicans biofilms in response to antifungal agents. Initially, the antifungal susceptibility of C. albicans cultures in different growth modes was evaluated by standard antifungal susceptibility testing. Next, to assess CDR1, CDR2, MDR1, ERG11, FKS1 and PIL1 expression, RNA was harvested from cells in each growth mode, and from biofilms after drug treatment, and subjected to quantitative real-time RT-PCR (qRT-PCR). Biofilm C. albicans was more resistant to antifungals than recently adhered cells and stationary-phase planktonic cultures. Transcriptional expression of CDR1, CDR2, MDR1, ERG11 and FKS1 was lower in recently adhered C. albicans than in the stationary-phase planktonic cultures. In contrast, PIL1 levels were significantly increased in recently adhered and biofilm modes of growth. The expression of MDR1 in biofilms greatly increased on challenge with amphotericin B but not with the other drugs tested (P<0.01). ERG11 was significantly upregulated by ketoconazole (P<0.01). Caspofungin and amphotericin B significantly upregulated FKS1 expression, whereas they significantly downregulated PIL1 expression (P<0.01). These results indicate that the expression of drug-resistance genes is associated with higher drug resistance of Candida biofilms, and lay a foundation for future large-scale genome-wide expression analysis.

Metergoline, a serotonin receptor antagonist, was evaluated for its antifungal activity against the opportunistic human fungal pathogen Candida krusei by a broth microdilution assay. The minimal inhibitory concentration and minimal fungicidal concentration of metergoline against C. krusei were 4 and 8 μg ml(-1) respectively. Significant synergism was found in combination of metergoline with amphotericin B (fractional inhibitory concentration index: 0.375-0.5) by a chequerboard assay. Metergoline also inhibited extracellular phospholipase secretion in a dose-dependent manner, which may be a possible action mechanism of metergoline on C. krusei., (© 2009 Blackwell Verlag GmbH.)

Photodynamic therapy is a treatment that combines the use of three non-toxic components, viz. photosensitiser, light and oxygen to cause localised oxidative photodamage. In the present study, the antifungal effect of the photosensitiser, BAM-SiPc, an unsymmetrical bisamino phthalocyanine, was investigated. BAM-SiPc was effective in photo-inactivating Candida albicans in a dose-dependent manner. The cell viability as determined by the clonogenic assay was reduced to c. 10% at 0.02 micromol l(-1) BAM-SiPc with a total fluence of 12 J cm(-2) at a cell density of 10(7) cells ml(-1). A short incubation time of 5-15 min was sufficient to allow the photosensitiser to exert its optimal antifungal activity. Microscopical analysis showed that BAM-SiPc was effectively internalised by the fungal cells. Photodynamic treatment led to an increase in the intracellular reactive oxygen species level and disturbed the membrane integrity of the fungal cells.

Biofilm formation involving profuse hyphal growth is a major characteristic of Candida spp. and confers higher antifungal resistance than its planktonic mode of growth. We investigated the antifungal susceptibility of Candida albicans and its hyphal mutants (Delta efg1/efg1, Delta cph1/cph1 and DeltaDelta cph1/cph1 efg1/efg1) to commonly used antifungals during planktonic, adhesion and biofilm modes of growth. The minimum inhibitory concentration (MIC) of each antifungal agent was determined for a lower inoculum (1x10(3) cells/mL) and higher inoculum (1x10(7) cells/mL) of planktonic Candida. Furthermore, MICs of C. albicans biofilms and adhesion modes of growth were determined with a standard XTT assay. Candida albicans in adhesion and biofilm modes of growth, but not in planktonic mode, were resistant to all five antifungal agents tested. Although Delta efg1/efg1 and DeltaDelta cph1/cph1 efg1/efg1 mutants formed less biofilm than wild-type C. albicans SC5314, they were similarly resistant to caspofungin. However, these mutants were more sensitive to amphotericin B and nystatin than the wild-type. Adhesion per se confers increased resistance to antifungal agents, which is further pronounced in the biofilm mode of Candida. Filamentation does not appear to be a major determinant of the antifungal resistance in Candida biofilms.

Unlabelled: Denture related oral candidiasis is a recalcitrant fungal infection not easily resolved by topical antifungals. The antimycotic protein lysozyme, in saliva is an important host defense mechanism although its activity against Candida biofilms on denture acrylic has not been evaluated., Objectives: (i) To establish a clinically relevant denture acrylic assay model to develop standardized Candida albicans biofilms, and (ii) assess the inhibitory effects of lysozyme alone and, the latter combined with antifungals (nystatin, amphotericin B, ketoconazole and 5-fluorocytosine) on sessile Candida cells and, finally (iii) to visualize the accompanying ultrastructural changes., Design: The rotating-disc biofilm reactor was used to develop standardized 48 h Candida biofilms on acrylic discs in YNB/100 mM glucose medium and the biofilm metabolic activity was monitored using a tetrazolium reduction assay., Results: The biofilm metabolic activity was similar in 18 identical denture acrylic discs (p<0.05) thus validating the rotating-disc biofilm model. Very low concentrations of lysozyme (6.25 microg/ml) significantly (p<0.01) inhibited Candida biofilm formation indicating that lysozyme may likely regulate intra-oral Candida biofilm development. Although 100 microg/ml lysozyme killed 45% of sessile Candida cells, further increasing its concentration (up to 240 microg/ml) had no such effect. Nystatin, amphotericin B, and ketoconazole in association with 100 microg/ml lysozyme exhibited effective synergistic killing of biofilm Candida in comparison to drug-free controls. Scanning electron and confocal scanning laser microscopy analysis confirmed the latter trends., Conclusion: Our results indicate that agents found in biological fluids such as lysozyme could be a safe adjunct to antifungals in future treatment strategies for recalcitrant candidal infections.

Biofilm formation is a major virulence attribute of Candida pathogenicity which contributes to higher antifungal resistance. We investigated the roles of cell density and cellular aging on the relative antifungal susceptibility of planktonic, biofilm, and biofilm-derived planktonic modes of Candida. A reference and a wild-type strain of Candida albicans were used to evaluate the MICs of caspofungin (CAS), amphotericin B (AMB), nystatin (NYT), ketoconazole (KTC), and flucytosine (5FC). Standard, NCCLS, and European Committee on Antibiotic Susceptibility Testing methods were used for planktonic MIC determination. Candida biofilms were then developed on polystyrene wells, and MICs were determined with a standard 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide assay. Subsequently, antifungal susceptibility testing was performed for greater inoculum concentrations and 24- and 48-h-old cultures of planktonic Candida. Furthermore, Candida biofilm-derived planktonic cells (BDPC) were also subjected to antifungal susceptibility testing. The MICs for both C. albicans strains in the planktonic mode were low, although on increasing the inoculum concentration (up to 1 x 10(8) cells/ml), a variable MIC was noted. On the contrary, for Candida biofilms, the MICs of antifungals were 15- to >1,000-fold higher. Interestingly, the MICs for BDPC were lower and were similar to those for planktonic-mode cells, particularly those of CAS and AMB. Our data indicate that higher antifungal resistance of Candida biofilms is an intrinsic feature possibly related to the biofilm architecture rather than cellular density or cellular aging.

Anti-candidial activities of eight traditional Chinese medicinal (TCM) herbs were evaluated against six different Candida species. TCM preparations were screened for antifungal activity using a standard agar diffusion assay. Following identification of potential candidate herbs, their minimum inhibitory concentration (MIC) values were determined using the standardised NCCLS M-27A broth microdilution assay. Among TCM herbs, Rhizoma Coptidis had potent antifungal activity against Candida glabrata, Candida krusei and Candida tropicalis, but not against Candida albicans, Candida dubliniensis and Candida parapsolosis. The MIC values of the Rhizoma Coptidis against C. glabrata, C. krusei and C. tropicalis were 50, 50 and 100 microg ml(-1) respectively. We report here, for the first time, the potent antifungal activity of Rhizoma Coptidis and Cortex phellodendri Chinesis on three different non-albicans Candida species, C. glabrata, C. krusei and C. tropicalis and hence their possible use as therapeutic agents.

Photodynamic therapy is a treatment that combines the use of three non-toxic components, viz. photosensitiser, light and oxygen to cause localised oxidative photodamage. In the present study, the antifungal effect of the photosensitiser, BAM-SiPc, an unsymmetrical bisamino phthalocyanine, was investigated. BAM-SiPc was effective in photo-inactivating Candida albicans in a dose-dependent manner. The cell viability as determined by the clonogenic assay was reduced to c. 10% at 0.02 μmol l−1 BAM-SiPc with a total fluence of 12 J cm−2 at a cell density of 107 cells ml−1. A short incubation time of 5–15 min was sufficient to allow the photosensitiser to exert its optimal antifungal activity. Microscopical analysis showed that BAM-SiPc was effectively internalised by the fungal cells. Photodynamic treatment led to an increase in the intracellular reactive oxygen species level and disturbed the membrane integrity of the fungal cells. [ABSTRACT FROM AUTHOR]