Two new species, Candida orthopsilosis and C. metapsilosis, are proposed to replace the existing designations of C. parapsilosis groups II and III, respectively. The species C. parapsilosis is retained for group I isolates. Attempts to construct a multilocus sequence typing scheme to differentiate individual strains of C. parapsilosis instead revealed fixed DNA sequence differences between pairs of subgroups in four genes: COX3, L1A1, SADH, and SYA1. PCR amplicons for sequencing were obtained for these four plus a further seven genes from 21 group I isolates. For nine group II isolates, PCR products were obtained from only 5 of the 11 genes, and for two group III isolates PCR products were obtained from a different set of 5 genes. Three of the PCR products from group II and III isolates differed in size from the group I products. Cluster analysis of sequence polymorphisms from COX3, SADH, and SYA1, which were common to the three groups, consistently separated the isolates into three distinct sets. All of these differences, together with DNA sequence similarities <90% in the ITS1 sequence, suggest the subgroups should be afforded species status. The near absence of DNA sequence variability among isolates of C. parapsilosis and relatively high levels of sequence variability among isolates of C. orthopsilosis suggest that the former species may have evolved very recently from the latter.

We analyzed data on multilocus sequence typing (MLST), ABC typing, mating type-like locus (MAT) status, and antifungal susceptibility for a panel of 1,391 Candida albicans isolates. Almost all (96.7%) of the isolates could be assigned by MLST to one of 17 clades. eBURST analysis revealed 53 clonal clusters. Diploid sequence type 69 was the most common MLST strain type and the founder of the largest clonal cluster, and examples were found among isolates from all parts of the world. ABC types and geographical origins showed statistically significant variations among clades by univariate analysis of variance, but anatomical source and antifungal susceptibility data were not significantly associated. A separate analysis limited to European isolates, thereby minimizing geographical effects, showed significant differences in the proportions of isolates from blood, commensal carriage, and superficial infections among the five most populous clades. The proportion of isolates with low antifungal susceptibility was highest for MAT homozygous a/a types and then ␣/␣ types and was lowest for heterozygous a/␣ types. The tree of clades defined by MLST was not congruent with trees generated from the individual gene fragments sequenced, implying a separate evolutionary history for each fragment. Analysis of nucleic acid variation among loci and within loci supported recombination. Computational haplotype analysis showed a high frequency of recombination events, suggesting that isolates had mixed evolutionary histories resembling those of a sexually reproducing species.

We submitted a panel of 416 isolates of Candida albicans from separate sources to multilocus sequence typing (MLST). The data generated determined a population structure in which four major clades of closely related isolates were delineated, together with eight minor clades comprising five or more isolates. By Fisher's exact test, a statistically significant association was found between particular clades and the anatomical source, geographical source, ABC genotype, decade of isolation, and homozygosity versus heterozygosity at the mating type-like locus (MTL) of the isolates in the clade. However, these associations may have been influenced by confounding variables, since in a univariate analysis of variance, only the clade associations with ABC type and anatomical source emerged as statistically significant, providing the first indication of possible differences between C. albicans strain type clades and their propensity to infect or colonize different anatomical locations. There were no significant differences between clades with respect to distributions of isolates resistant to fluconazole, itraconazole, or flucytosine. However, the majority of flucytosine-resistant isolates belonged to clade 1, and these isolates, but not flucytosine-resistant isolates in other clades, bore a unique mutation in the FUR1 gene that probably accounts for their resistance. A significantly higher proportion of isolates resistant to fluconazole, itraconazole, and flucytosine were homozygous at the MTL, suggesting that antifungal pressure may trigger a common mechanism that leads both to resistance and to MTL homozygosity. The utility of MLST for determining clade assignments of clinical isolates will form the basis for strain selection for future research into C. albicans virulence.Differentiation of microbial isolates by sequencing a small sample of unrelated housekeeping genes has become established as a reliable and effective method for typing strains of many bacteria (9, 50). Such multilocus sequence typing (MLST) is highly reproducible, and data can be archived in Web-based databases accessible to all users. For the fungal pathogen Candida albicans, an MLST system based on seven DNA fragments was developed as an optimal consensus for typing strains within the species (6) following two earlier proposed systems (5, 48). Because C. albicans is a diploid organism, sequence data contain heterozygous as well as homozygous sites, adding an extra discriminatory feature to MLST for this species.Among other approaches to strain typing of C. albicans, DNA fingerprinting based on the moderately repetitive sequence Ca3 has been widely used. By this method, C. albicans populations were shown to comprise five major clades of closely related strain types (46), including clades enriched in isolates from Europe (36) and South Africa (2, 3). Resistance of C. albicans to flucytosine in vitro was found to be a property restricted almost entirely to isolates from a single C. albicans clade as determined by Ca3 fingerprinting (37), and the sole me...

A panel of 86 different Candida albicans isolates was subjected to multilocus sequence typing (MLST) in two laboratories to obtain sequence data for 10 published housekeeping gene fragments. Analysis of data for all possible combinations of five, six, seven, eight, and nine of the fragments showed that a set comprising the fragments AAT1a, ACC1, ADP1, MPIb, SYA1, VPS13, and ZWF1b was the smallest that yielded 86 unique diploid sequence types for the 86 isolates. This set is recommended for future MLST with C. albicans.Multilocus sequence typing (MLST) is becoming a widely used approach to microbial isolate differentiation for epidemiological purposes (6). For Candida albicans, the species most often involved in deep-organ fungal diseases, MLST was introduced in 2002 (1), and a central internet database has been set up for deposition and analysis of C. albicans MLST data from any global source (http://calbicans.mlst.net). This MLST system is based on fragments of six C. albicans genes (in alphabetical order): ACC1, ADP1, GLN4, RPN2, SYA1, and VPS13.A second study of C. albicans MLST involved four of these genes plus four other gene fragments, AAT1a, AAT1b, MPIb, and ZWF1b (5). Both sets of gene fragments provided highly discriminatory typing systems that gave stable, reproducible results and could distinguish even closely related strains. While in principle the use of as many gene sequences as possible should enhance the discriminatory power of an MLST scheme, in practical and technical terms a compromise is required to provide the maximum level of isolate differentiation with the minimum set of fragments. Our two groups therefore agreed to collaborate by exchanging C. albicans isolates that had already been described in the published MLST papers and compiling a data set of sequences based on all 10 fragments that have been used for MLST. Analysis of these data allows us to propose an optimized gene set for routine use in C. albicans MLST research.A total of 92 C. albicans isolates (1, 5) were shared between the laboratories. These included duplicate cultures of isolate SC5314 and one culture of CAF2, derived from SC5314. As expected, identical MLST results were obtained for these three isolates, so two of the three data sets were excluded from analysis. Among the remaining 90 unique isolates, incomplete sequence data were obtained for 4, so the results for 86 isolates were analyzed to determine the optimal set of gene fragments for MLST.The method used for MLST was as previously described (1, 5). Both DNA strands in this diploid fungus were sequenced for each of 10 fragments (Table 1), and the sequences were recorded by the one-letter code for nucleotides from the International Union of Pure and Applied Chemistry nomenclature. For each fragment, each different genotype was assigned a unique number. Diploid sequence types (DSTs) are the numbers assigned to each unique combination of genotypes. Table 1 summarizes the characteristics of the 10 DNA fragments used for MLST with the 86 C. albicans isolates. The sizes ...