Transcriptional regulation of drug-resistance genes in Candida albicans biofilms in response to antifungals.

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.

Effect of filamentation and mode of growth on antifungal susceptibility of Candida albicans.

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.

The influence of single molar crown placement on the smoothness of masticatory movement.

The jerk-cost is an index that can quantify the smoothness of various movements including human body movements. A previous study reported the usefulness of jerk-cost in the evaluation of masticatory movement, and proposed that the masticatory movement of subjects with good occlusion could be explained as a maximum smooth movement. The purpose of this study was to investigate the influence of a single prosthetic molar restoration on the smoothness of masticatory movement. Fourteen adults who visited this hospital seeking a single prosthetic restoration on a molar were selected. Each subject chewed a piece of chewing gum on the molars of the treated side before and after crown placement. Movement trajectory was recorded using the Sirognathograph Analyzing System. Normalized jerk-cost (NJC) was calculated on the closing phase of each chewing cycle and was compared before and after the crown placement. After the prosthetic restoration, NJC significantly decreased (P < 0.05) in seven subjects with a crown placed on a lower molar, whereas significant changes were not observed in seven subjects with a crown placed on an upper molar. These results suggest that restoring a crown on a lower molar could significantly improve the smoothness of masticatory movement on the same side.