Evaluation of phenotypic markers for selection and identification of Candida dubliniensis

Abstract

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.

Figures

FIG. 1

Primary culture of an oral rinse on CHROMagar Candida after 48 h at 37°C showing colonies with three different shades of green (arrowheads), all finally identified as C. dubliniensis.

FIG. 2

(a) Dendrogram of a hierarchical cluster analysis performed on 421 spectra measured from 58 strains of C. albicans and 53 strains of C. dubliniensis. The discriminating information used for clustering is based on five spectral ranges: 822 to 799, 920 to 855, 1,090 to 1,050, 1,235 to 1,180, and 1,385 to 1,355 cm−1. (b) Representative second-derivative spectra of C. albicans and C. dubliniensis in the four most discriminatory spectral windows.

FIG. 2

(a) Dendrogram of a hierarchical cluster analysis performed on 421 spectra measured from 58 strains of C. albicans and 53 strains of C. dubliniensis. The discriminating information used for clustering is based on five spectral ranges: 822 to 799, 920 to 855, 1,090 to 1,050, 1,235 to 1,180, and 1,385 to 1,355 cm−1. (b) Representative second-derivative spectra of C. albicans and C. dubliniensis in the four most discriminatory spectral windows.

FIG. 3

Alignment of the 3′ end of the small-subunit (18S) rDNA sequences of C. albicans JCM 1542 (EMBL accession no. AB013586), C. dubliniensis CD36 (GenBank accession no. X99399), and C. dubliniensis CBS 7987T, CBS 7988, and NCPF 3108 and of ITS1 sequences of C. albicans CBS 562 (EMBL accession no. AB032172), “C. stellatoidea” CBS 1905, and C. dubliniensis CBS 7987T, CBS 7988, and NCPF 3108. Identical nucleotides are indicated by dots, and gaps are indicated by dashes. The ITS1 region begins at position 451 and ends at position 634. Underlining indicates the species-specific signature sequence that could be used to assign a strain unequivocally to C. albicans or C. dubliniensis.

FIG. 3

Alignment of the 3′ end of the small-subunit (18S) rDNA sequences of C. albicans JCM 1542 (EMBL accession no. AB013586), C. dubliniensis CD36 (GenBank accession no. X99399), and C. dubliniensis CBS 7987T, CBS 7988, and NCPF 3108 and of ITS1 sequences of C. albicans CBS 562 (EMBL accession no. AB032172), “C. stellatoidea” CBS 1905, and C. dubliniensis CBS 7987T, CBS 7988, and NCPF 3108. Identical nucleotides are indicated by dots, and gaps are indicated by dashes. The ITS1 region begins at position 451 and ends at position 634. Underlining indicates the species-specific signature sequence that could be used to assign a strain unequivocally to C. albicans or C. dubliniensis.

FIG. 4

Proposed scheme for identification of C. dubliniensis. Some authors claim that a clearer distinction between C. albicans and C. dubliniensis is obtained by subculturing at 45°C (24).