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Differentiation between Candida dubliniensis and Candida albicans by fatty acid methyl ester analysis using gas-liquid chromatography.

ABSTRACT: Candida dubliniensis is often found in mixed culture with C. albicans, but its recognition is hampered as the color of its colonies in primary culture on CHROMagar Candida varies. Furthermore, definite identification of C. dubliniensis is difficult to achieve, time-consuming, and expensive. Therefore, a method to discriminate between these two closely related yeast species by fatty acid methyl ester (FAME) analysis using gas-liquid chromatography (Sherlock Microbial Identification System [MIS]; MIDI, Inc., Newark, Del.) was developed. Although the chromatograms of these two species revealed no obvious differences when applying FAME analysis, a new library (CADLIB) was successfully created using Sherlock Library Generation Software (MIDI). The amount and frequency of FAME was analyzed using library training files (n = 10 for each species), preferentially those comprising reference strains. For testing the performance of the CADLIB, clinical isolates genetically assigned to the respective species (C. albicans, n = 32; C. dubliniensis, n = 28) were chromatographically analyzed. For each isolate tested, MIS computed a similarity index (SI) indicating a hierarchy of possible strain fits. When using the newly created library CADLIB, the SIs for C. albicans and C. dubliniensis ranged from 0.11 to 0.96 and 0.53 to 0. 93 (for all but one), respectively. Only three isolates of C. albicans (9.4%) were misidentified as C. dubliniensis, whereas all isolates of C. dubliniensis were correctly identified. Resulting differentiation accuracy was 90.6% for C. albicans and 100% for C. dubliniensis. Cluster analysis and principal component analysis of the resulting FAME profiles showed two clearly distinguishable clusters matching up with two assigned species for the strains tested. Thus, the created library proved to be well suited to discriminate between these two species.

SUBMITTER: Peltroche-Llacsahuanga H

PROVIDER: S-EPMC87459 | biostudies-literature | 2000 Oct

REPOSITORIES: biostudies-literature

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Differentiation between Candida dubliniensis and Candida albicans by fatty acid methyl ester analysis using gas-liquid chromatography.

Peltroche-Llacsahuanga H H Schmidt S S Seibold M M Lütticken R R Haase G G

Journal of clinical microbiology 20001001 10

Candida dubliniensis is often found in mixed culture with C. albicans, but its recognition is hampered as the color of its colonies in primary culture on CHROMagar Candida varies. Furthermore, definite identification of C. dubliniensis is difficult to achieve, time-consuming, and expensive. Therefore, a method to discriminate between these two closely related yeast species by fatty acid methyl ester (FAME) analysis using gas-liquid chromatography (Sherlock Microbial Identification System [MIS]; ...[more]

PMID: 11015386

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Project description:Because Candida dubliniensis is closely related to Candida albicans, we tested whether it underwent white-opaque switching and mating and whether white-opaque switching depended on MTL homozygosity and mating depended on switching, as they do in C. albicans. We also tested whether C. dubliniensis could mate with C. albicans. Sequencing revealed that the MTLalpha locus of C. dubliniensis was highly similar to that of C. albicans. Hybridization with the MTLa1, MTLa2, MTLalpha1, and MTLalpha2 open reading frames of C. albicans further revealed that, as in C. albicans, natural strains of C. dubliniensis exist as a/alpha, a/a, and alpha/alpha, but the proportion of MTL homozygotes is 33%, 10 times the frequency of natural C. albicans strains. C. dubliniensis underwent white-opaque switching, and, as in C. albicans, the switching was dependent on MTL homozygosis. C. dubliniensis a/a and alpha/alpha cells also mated, and, as in C. albicans, mating was dependent on a switch from white to opaque. However, white-opaque switching occurred at unusually high frequencies, opaque cell growth was frequently aberrant, and white-opaque switching in many strains was camouflaged by an additional switching system. Mating of C. dubliniensis was far less frequent in suspension cultures, due to the absence of mating-dependent clumping. Mating did occur, however, at higher frequencies on agar or on the skin of newborn mice. The increases in MTL homozygosity, the increase in switching frequencies, the decrease in the quality of switching, and the decrease in mating efficiency all reflected a general deterioration in the regulation of developmental processes, very probably due to the very high frequency of recombination and genomic reorganization characteristic of C. dubliniensis. Finally, interspecies mating readily occurred between opaque C. dubliniensis and C. albicans strains of opposite mating type in suspension, on agar, and on mouse skin. Remarkably, the efficiency of interspecies mating was higher than intraspecies C. dubliniensis mating, and interspecies karyogamy occurred readily with apparently the same sequence of nuclear migration, fusion, and division steps observed during intraspecies C. albicans and C. dubliniensis mating and Saccharomyces cerevisiae mating.

Project description:There have been increased reports of the isolation of unusual genotypic groups of Candida albicans (groups C and D) based on a well-defined genotypic method; this method uses cellular DNA digested with the EcoRI enzyme and the restriction fragment length polymorphisms (RFLPs) generated by agarose gel electrophoresis. The aim of the present study was to use additional molecular tools to characterize these unusual strains and to compare them with authentic strains of C. dubliniensis, a recently delineated species, and type I C. stellatoidea. The RFLPs of PCR products generated from the intergenic transcribed spacer (ITS) region did not differentiate among C. albicans genotypes A, B, and C and type I C. stellatoidea. However, this method did differentiate the C. albicans genotype D strains, which were identical to C. dubliniensis. The RFLPs generated by HaeIII digestion of the PCR products of the V3 region of the 25S rRNA gene (rDNA) could differentiate the same groups as RFLP analysis of the PCR amplicon of the ITS region. C. albicans genotype B isolates have been shown to have a transposable intron in the 25S rDNA, whereas genotype A isolates do not; C. dubliniensis strains also have an intron that is larger than that in genotype B C. albicans strains but that is in the same location. PCR designed to span this region resulted in a single product for C. albicans genotype A (450 bp), B (840 bp), type 1 C. stellatoidea (840 bp), and C. dubliniensis (1,080 bp), whereas the C. albicans genotype C isolates had two major products (450 and 840 bp). All C. albicans genotype D isolates gave a PCR product identical to that given by C. dubliniensis. These results indicate that those strains previously designated C. albicans genotype D are in fact C. dubliniensis, that no differences were found between type 1 C. stellatoidea and C. albicans genotype B strains, and that the C. albicans genotype C strains appear to have the transposable intron incompletely inserted throughout the ribosomal repeats in their genomes. The results of the antifungal susceptibility testing of 105 of these strains showed that, for fluconazole, strains of C. dubliniensis were significantly more susceptible than strains of each of the C. albicans genotypes (genotypes A, B, and C). The flucytosine susceptibility results indicated that strains of C. albicans genotype A were significantly less susceptible than either C. albicans genotype B or C. albicans genotype C strains. These results indicate that there is a correlation between the Candida groups and antifungal susceptibility.

Dataset Information

Differentiation between Candida dubliniensis and Candida albicans by fatty acid methyl ester analysis using gas-liquid chromatography.

Publications

Differentiation between Candida dubliniensis and Candida albicans by fatty acid methyl ester analysis using gas-liquid chromatography.

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