The pathogenic yeast Candida dubliniensis is phylogenetically very closely related to Candida albicans, and both species share many phenotypic and genetic characteristics. DNA fingerprinting using the species-specific probe Cd25 and sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal gene cluster previously showed that C. dubliniensis is comprised of three major clades comprising four distinct ITS genotypes. Multilocus sequence typing (MLST) has been shown to be very useful for investigating the epidemiology and population biology of C. albicans and has identified many distinct major and minor clades. In the present study, we used MLST to investigate the population structure of C. dubliniensis for the first time. Combinations of 10 loci previously tested for MLST analysis of C. albicans were assessed for their discriminatory ability with 50 epidemiologically unrelated C. dubliniensis isolates from diverse geographic locations, including representative isolates from the previously identified three Cd25-defined major clades and the four ITS genotypes. Dendrograms created by using the unweighted pair group method with arithmetic averages that were generated using the data from all 10 loci revealed a population structure which supports that previously suggested by DNA fingerprinting and ITS genotyping. The MLST data revealed significantly less divergence within the C. dubliniensis population examined than within the C. albicans population. These findings show that MLST can be used as an informative alternative strategy for investigating the population structure of C. dubliniensis. On the basis of the highest number of genotypes per variable base, we recommend the following eight loci for MLST analysis of C. dubliniensis: CdAAT1b, CdACC1, CdADP1, CdMPIb, CdRPN2, CdSYA1, exCdVPS13, and exCdZWF1b, where "Cd" indicates C. dubliniensis and "ex" indicates extended sequence.Candida dubliniensis is a pathogenic yeast species that is phenotypically, genetically, and phylogenetically very closely related to Candida albicans, the yeast species most commonly associated with infection in humans (49, 51). Despite their close relationship, C. albicans is significantly more pathogenic (49, 50). C. dubliniensis is most commonly associated with oral carriage and infection in human immunodeficiency virus (HIV)-infected and diabetic patients, although it has been identified as a minor constituent of the commensal floras in the oral cavities of healthy individuals (40,49,50). Although C. dubliniensis has also been recovered from patients with systemic infections, its incidence is far lower than that of C. albicans. While the latter is responsible for 40 to 60% of cases of candidemia, C. dubliniensis has been identified in only 1 to 2% of blood culture yeast samples (11,15,27,(29)(30)(31). These epidemiological data are reflected in the results of animal infection model studies that demonstrated that C. albicans is significantly more pathogenic than C. dubliniensis (22,47,56). The reasons for the lower viru...

SummaryThe expression of the ERG1, ERG3, ERG7, ERG9, ERG11 and ERG25 genes in response to incubation with fluconazole and biofilm formation was investigated using reversetranscription PCR and real-time PCR in Candida albicans and Candida dubliniensis clinical isolates. The viability of biofilm was measured using an 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay and confocal scanning laser microscopy (CSLM). Expression of the ERG11 gene was found to be low or moderate and it was regulated by fluconazole addition more so than by biofilm formation. Very low or non-detectable expression of ERG1, ERG7 and ERG25 genes was detected in C. albicans. The expression of the ERG9 increased in the presence of fluconazole in some isolates. Following incubation with fluconazole, formation of biofilm by C. dubliniensis was coupled with up-regulation of the ERG3 and ERG25 genes as have been observed previously in C. albicans. Planktonic cells of both Candida species released from biofilm displayed similar resistance mechanisms to fluconazole like attached cells. The XTT reduction assay and CSLM revealed that although incubation with fluconazole decreased the biofilm thickness, these were still comprised metabolically active cells able to disseminate and produce biofilm. Our data indicate that biofilm represents a highly adapted community reflecting the individuality of clinical isolates.

Candida infections are frequently associated with formation of biofilms on artificial medical devices. This work studied variation of cell surface hydrophobicity (CSH) and formation of biofilm in relation to Candida albicans and Candida dubliniensis genotypes and an effect of some conventional antifungal agents on both CSH and biofilm. The 50 isolates of C. albicans and C. dubliniensis were classified into genotypes A, B, C, and D, genotype D being exclusively represented by C. dubliniensis. No significant differences between CSH of genotypes A and B and B and C were observed with respect to cultivation temperature 25 or 37 degrees C. Candida dubliniensis showed increased CSH in comparison with other C. albicans genotypes (p < 0.001) regardless of temperature used. Using XTT reduction assay and dry masses, genotypes B and C showed reduced ability to form biofilm in comparison with genotype A (p < 0.05) and C. dubliniensis (p < 0.001). Fluconazole reduced biofilm in C. albicans genotypes A, B, and C (p < 0.05) but not CSH. The opposite effect was observed in C. dubliniensis. Voriconazole effectively reduced both biofilm formation and CSH in all tested genotypes of C. albicans and C. dubliniensis (p < 0.05).

The Candida antigen CR3-RP (complement receptor 3-related protein) is supposed to be a 'mimicry' protein because of its ability to bind antibody directed against the a subunit of the mammalian CR3 (CD11b/CD18). This study aimed to (i) investigate the specific humoral isotypic response to immunization with CR3-RP in vivo in a rabbit animal model, and (ii) determine the role of CR3-RP in the adherence of Candida albicans in vitro using the model systems of buccal epithelial cells (BECs) and biofilm formation. The synthetic C. albicans peptide DINGGGATLPQ corresponding to 11 amino-acids of the CR3-RP sequence DINGGGATLPQALXQITGVIT, determined by N-terminal sequencing, was used for immunization of rabbits to obtain polyclonal anti-CR3-PR serum and for subsequent characterization of the humoral isotypic response of rabbits. A significant increase of IgG, IgA and IgM anti-CR3-RP specific antibodies was observed after the third (P,0.01) and the fourth (P,0.001) immunization doses. The elevation of IgA levels suggested peptide immunomodulation of the IgA1 subclass, presumably in coincidence with Candida epithelial adherence. Blocking CR3-RP with polyclonal anti-CR3-RP serum reduced the ability of Candida to adhere to BECs, in comparison with the control, by up to 35 % (P,0.001), and reduced biofilm formation by 28 % (P,0.001), including changes in biofilm thickness and integrity detected by confocal laser scanning microscopy. These properties of CR3-RP suggest that it has potential for future vaccine development. INTRODUCTIONCandida albicans is the most frequently isolated fungal pathogen associated with infection of immunocompromised patients in hospital settings. The high propensity to cause disease is due to the expression of many virulence factors including highly immunogenic cell surface proteins (Alberti-Segui et al., 2004;Jeng et al., 2005;Pietrella et al., 2006) able to trigger cellular and humoral response (Viudes et al., 2001;Ló pez-Ribot et al., 2004;Omaetxebarria et al., 2005;Fukuizumi et al., 2006). Additionally, a variety of cell surface molecules expressed by Candida help it to evade the host immune response by mimicry of host receptors (Gustafson et al., 1991;Phan et al., 2007). The receptor MAC-1 (CD11b/CD18) on lymphocytes is the surface b 2 integrin that mediates lymphocyte adhesion to C. albicans (Forsyth & Mathews, 2002). However, Candida is not only the target for MAC-1, but also CR3-RP identified on the yeast surface might be classified as a member of the integrin family due to its antigenic, structural and functional relation to the a subunit of the mammalian neutrophil receptor CD11b/CD18 (Gilmore et al., 1988;Abbreviations: BEC, buccal epithelial cell; CLSM, confocal laser scanning microscopy; RT, room temperature; XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.The GenBank/EMBL/DDBJ accession number for the N-terminal sequence fragment of CR3-RP is P85437. Hostetter et al., 1990;Hostetter, 1996). Binding the human complement fragment iC3b to this receptor results i...