Project description:Candida dubliniensis is often associated with C. albicans in cultures. Easy-to-perform selective isolation procedures for these closely related species do not exist. Therefore, we evaluated previously described discriminatory phenotypic markers for C. dubliniensis. A total of 150 oral rinses from human immunodeficiency virus (HIV)-infected patients were cultured on CHROMagar Candida. Dark green colonies described as being indicative of C. dubliniensis and other green colonies, 170 in total, were isolated. Chlamydospore formation, intracellular beta-D-glucosidase activity, ability to grow at 42 degrees C, carbohydrate assimilation pattern obtained by the API ID 32C, and Fourier transform infrared (FT-IR) spectroscopy were used for phenotypic characterization. Sequencing of the 5' end of the nuclear large-subunit (26S) ribosomal DNA gene was used for definitive species identification for C. dubliniensis. C. dubliniensis was found in 34% of yeast-colonized HIV-infected patients. The color of the colonies on CHROMagar Candida proved to be insufficient for selecting C. dubliniensis, since only 30 of 53 proven C. dubliniensis isolates showed a dark green color in primary cultures. The described typical chlamydospore formation can give only some indication of C. dubliniensis. The assimilation pattern proved to be insufficient to discriminate C. dubliniensis from C. albicans. All C. dubliniensis strains showed no or highly restricted growth at 42 degrees C and a lack of beta-D-glucosidase activity. Unfortunately, atypical C. albicans strains can also exhibit these phenotypic traits. FT-IR spectroscopy combined with hierarchical clustering proved to be as reliable as genotyping for discriminating the two species.
Project description:We isolated Candida dubliniensis from a nonhuman source, namely, tick samples from an Irish seabird colony. The species was unambiguously identifi ed by phenotypic and genotypic means. Analysis of the 5.8S rRNA gene showed that the environmental isolates belong to C. dubliniensis genotype 1.
Project description:Candida dubliniensis is a recently described Candida species associated with oral candidosis in human immunodeficiency virus (HIV)-infected and AIDS patients, from whom fluconazole-resistant clinical isolates have been previously recovered. Furthermore, derivatives exhibiting a stable fluconazole-resistant phenotype have been readily generated in vitro from fluconazole-susceptible isolates following exposure to the drug. In this study, fluconazole-resistant isolates accumulated up to 80% less [3H] fluconazole than susceptible isolates and also exhibited reduced susceptibility to the metabolic inhibitors 4-nitroquinoline-N-oxide and methotrexate. These findings suggested that C. dubliniensis may encode multidrug transporters similar to those encoded by the C. albicans MDR1, CDR1, and CDR2 genes (CaMDR1, CaCDR1, and CaCDR2, respectively). A C. dubliniensis homolog of CaMDR1, termed CdMDR1, was cloned; its nucleotide sequence was found to be 92% identical to the corresponding CaMDR1 sequence, while the predicted CdMDR1 protein was found to be 96% identical to the corresponding CaMDR1 protein. By PCR, C. dubliniensis was also found to encode homologs of CDR1 and CDR2, termed CdCDR1 and CdCDR2, respectively. Expression of CdMDR1 in a fluconazole-susceptible delta pdr5 null mutant of Saccharomyces cerevisiae conferred a fluconazole-resistant phenotype and resulted in a 75% decrease in accumulation of [3H]fluconazole. Northern analysis of fluconazole-susceptible and -resistant isolates of C. dubliniensis revealed that fluconazole resistance was associated with increased expression of CdMDR1 mRNA. In contrast, most studies showed that overexpression of CaCDR1 was associated with fluconazole resistance in C. albicans. Increased levels of the CdMdr1p protein were also detected in fluconazole-resistant isolates. Similar results were obtained with fluconazole-resistant derivatives of C. dubliniensis generated in vitro, some of which also exhibited increased levels of CdCDR1 mRNA and CdCdr1p protein. These results demonstrate that C. dubliniensis encodes multidrug transporters which mediate fluconazole resistance in clinical isolates and which can be rapidly mobilized, at least in vitro, on exposure to fluconazole.
Project description:Telomeric ORFs (TLOs) in pathogenic Candida spp. encode Mediator subunits that regulate the transcription of distinct subsets of genes
Project description:Candida dubliniensis is a recently described opportunistic fungal pathogen that is closely related to Candida albicans. Candida dubliniensis readily develops resistance to the azole antifungal agent fluconazole, both in vitro and in infected patients, and this resistance is usually associated with upregulation of the CdMDR1 gene, encoding a multidrug efflux pump of the major facilitator superfamily. To determine the role of CdMDR1 in drug resistance in C. dubliniensis, we constructed an mdr1 null mutant from the fluconazole-resistant clinical isolate CM2, which overexpressed the CdMDR1 gene. Sequential deletion of both CdMDR1 alleles was performed by the MPA(R)-flipping method, which is based on the repeated use of a dominant mycophenolic acid resistance marker for selection of integrative transformants and its subsequent deletion from the genome by FLP-mediated, site-specific recombination. In comparison with its parental strain, the mdr1 mutant showed decreased resistance to fluconazole but not to the related drug ketoconazole. In addition, we found that CdMDR1 confers resistance to the structurally unrelated drugs 4-nitroquinoline-N-oxide, cerulenin, and brefeldin A, since the enhanced resistance to these compounds of the parent strain CM2 compared with the matched susceptible isolate CM1 was abolished in the mdr1 mutant. In contrast, CdMDR1 inactivation did not cause increased susceptibility to amorolfine, terbinafine, fluphenazine, and benomyl, although overexpression of CdMDR1 in a hypersusceptible Saccharomyces cerevisiae strain had previously been shown to confer resistance to these compounds. The effect of CdMDR1 inactivation was identical to that seen in two similarly constructed C. albicans mdr1 mutants. Therefore, despite species-specific differences in the amino acid sequences of the Mdr1 proteins, overexpression of CaMDR1 and CdMDR1 in clinical C. albicans and C. dubliniensis strains seems to confer the same drug resistance profile in both species.