Bioactivity and cellular structure of Candida albicans and Candida glabrata biofilms grown in the presence of fluconazole.

OBJECTIVE: The aim of this study was to evaluate whether fluconazole (FLZ) could affect the bioactivity and cellular structure of Candida albicans or Candida glabrata biofilms grown in the presence of FLZ. MATERIALS AND METHODS: Tokens were fabricated using poly(methylmethacrylate) resin (PMMA) in a hot water bath. Salivary pellicles were formed on the PMMA surface, and biofilms of a reference strain and two clinical isolates of C. albicans (ATCC 90028, P01 and P34) and C. glabrata (ATCC 2001, P11 and P31) were developed for a period of 48 h. Control and experimental groups were formed. FLZ at the bioavailable concentration in saliva (2.56 µg/mL) was added to the medium of the experimental group. The culture mediums of the control and experimental groups were changed after 24h. The bioactivities of the biofilms were evaluated using an XTT reduction colorimetric assay. The cellular structure was analysed by confocal scanning laser microscopy and by transmission electron microscopy. The data were analysed by the independent sample Student's t-test, with the significance level set at 5%. RESULTS: The presence of FLZ decreased the bioactivity of all C. albicans biofilms (p<0.001), however, it did not change the cellular structure of C. albicans P34. Regarding the C. glabrata biofilm bioactivity and structure, no statistically significant differences were found between the control and experimental groups. CONCLUSION: FLZ, at the bioavailable concentration present in saliva, interferes with the development of C. albicans biofilms, but does not interfere with the development of C. glabrata biofilms.

Analysis of subtelomeric silencing in Candida glabrata.

Analysis of gene function often involves detailed studies of when a given gene is expressed or silenced. Transposon mutagenesis is a powerful tool to generate insertional mutations that provide with a selectable marker and a reporter gene that can be used to analyze the transcriptional activity of a specific locus in a variety of microorganisms to study gene regulation. Then the reporter gene expression can be easily measured under different conditions to gain insight into the regulation of the particular locus of interest. We have used transposon mutagenesis as a tool to generate insertional mutations with a modified Tn7 transposon containing the reporter gene URA3 (Tn7-URA3) to study subtelomeric silencing in the opportunistic fungal pathogen Candida glabrata. This method consists of two major steps: an in vitro Tn7-URA3 mutagenesis of a plasmid containing the desired subtelomeric region to be analyzed, followed by homologous recombination into the target region of the C. glabrata genome. As an alternative, a fusion PCR protocol can also be used in which the URA3 reporter gene can be "fused" together with the 5' and 3' regions of the desired insertion point by a two step PCR protocol. This fusion product can be introduced into the C. glabrata genome by homologous recombination after transformation in the same way as the Tn7-URA3 mutagenesis products. Once the URA3 reporter gene has been introduced in the desired locus in the C. glabrata genome, a simple plate growth assay is performed to assess the expression of the reporter gene.