Molecular epidemiology of global Candida dubliniensis isolates utilizing genomic-wide, co-dominant, PCR-based markers for strain delineation.
ABSTRACT The molecular epidemiology of Candida dubliniensis has been studied using large complex DNA probes for Southern analysis and has revealed the existence of distinct genotypes within this species. The aim of the present study was to utilize a PCR-based analysis of molecular co-dominant markers to assess the relatedness of a global and temporally diverse collection of well characterized isolates of C. dubliniensis. Sixty-two C. dubliniensis strains were collected from the authors of previously published studies. Co-dominant PCR-based markers utilizing five separate PCR fingerprints were obtained in the present investigation. Phylogenetic and statistical analyses utilizing permutation tests were undertaken to assess correlations amongst the isolates. Three distinct PCR-groups were observed and there was evidence that strains isolated since 1990 were genotypically more similar to each other than they were to strains recovered prior to 1990.
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ABSTRACT: 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.Journal of Clinical Microbiology 03/1999; 37(2):417-21. · 4.07 Impact Factor
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ABSTRACT: Infections by Candida spp. have increased in medical importance over the past few decades. Our understanding of species identification, commensalisms, pathogenicity, person-to-person spread, and the development of antifungal resistance within specific strains has been greatly enhanced by the utilization of molecular epidemiological methodology. The aim of the current research was to assess the quantity, species and molecular characterization of oral yeast isolates from well-defined cohorts of immunocompetent patients from a diverse range of clinical settings. Oral rinse samples were assessed for the growth of yeast and degree of colonization. Isolates were defined to the species level by both phenotypic and molecular methods and strains were further genotypically subtyped. Significant variation was shown to exist in the number, species and genotypic subgroups of yeast isolated from the oral cavity in different patient groups. This variation could be attributed to the local oral conditions unique to these patient groups.Oral Microbiology and Immunology 03/2002; 17(1):44-9. · 2.81 Impact Factor
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ABSTRACT: 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.Journal of clinical microbiology 03/2008; 46(2):652-64. · 4.16 Impact Factor