Molecular phylogenetic analysis of Candida tropicalis isolates by multi-locus sequence typing.

Multi-locus sequencing data for 242 different isolates of Candida tropicalis generated a dendrogram showing 235 strains assigned to a single large recently evolved group which contained several small clonal clusters. Haplotype analysis of a representative strain subset revealed a high level of recombination events in an otherwise clonal population. Pairs of isolates from single sources showed non-identity attributable to loss of heterozygosity in some genes in a manner similar to that established for C. albicans.

Comparison of Candida albicans strain types among isolates from three countries.

Multi-locus sequence typing data for 217 Candida albicans isolates cultured since 1990 from blood and vaginal samples in Japan, England/Wales and the USA were analysed for geographically related variations. While no significant differences were found between distributions of diploid sequence types (DSTs) in blood vs. vaginal isolates, there were highly significant differences in the clade distributions of isolates from the three geographical sources. Clade 2 strains were predominantly isolates from England/Wales, while clade 3 strains came mainly from the USA. The isolates from Japan were highly prevalent among strains in clades 5-17, and provided the first example seen so far in C. albicans of an amino acid encoded by three separate codons. Within clade 1, the most commonly encountered clade for isolates from all three regions, 15 Japanese isolates and 1 English isolate formed a separate clonal cluster in eBURST analysis. A similarly well demarcated clonal cluster rich in isolates from Japan was also found among the clade 4 strains. The data suggest C. albicans undergoes localized evolution, but human movements and person-to-person spread considerably blur the boundaries of such evolution.

One year prospective survey of Candida bloodstream infections in Scotland.

A 12 month survey of candidaemia in Scotland, UK, in which every Scottish hospital laboratory submitted all blood isolates of yeasts for identification, strain typing and susceptibility testing, provided 300 isolates from 242 patients, generating incidence data of 4.8 cases per 100,000 population per year and 5.9 cases per 100,000 acute occupied bed days; 27.9 % of cases occurred in intensive care units. More than half the patients with candidaemia had an underlying disease involving the abdomen, 78 % had an indwelling intravenous catheter, 62 % had suffered a bacterial infection within the 2 weeks prior to candidaemia and 37 % had undergone a laparotomy. Candida albicans was the infecting species in 50 % of cases, followed by Candida glabrata (21 %) and Candida parapsilosis (12 %). Seven cases of candidaemia were caused by Candida dubliniensis, which was more prevalent even than Candida lusitaniae and Candida tropicalis (six cases each). Among C. glabrata isolates, 55 % showed reduced susceptibility to fluconazole, but azole resistance among other species was extremely low. Multilocus sequence typing showed isolates with high similarity came from different hospitals across the country, and many different types came from the hospitals that submitted the most isolates, indicating no tendency towards hospital-specific endemic strains. Multiple isolates of C. albicans and C. glabrata from individual patients were of the same strain type with single exceptions for each species. The high prevalence of candidaemia in Scotland, relative to other population-based European studies, and the high level of reduced fluconazole susceptibility of Scottish C. glabrata isolates warrant continued future surveillance of invasive Candida infections.

Molecular phylogenetics of Candida albicans.

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 alpha/alpha types and was lowest for heterozygous a/alpha 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.

Multilocus sequence typing for differentiation of strains of Candida tropicalis.

A system is described for typing isolates of the pathogenic fungus Candida tropicalis, based on sequence polymorphisms in fragments of six genes: ICL1, MDR1, SAPT2, SAPT4, XYR1, and ZWF1a. The system differentiated 87 diploid sequence types (DSTs) among a total of 106 isolates tested or 80 DSTs among 88 isolates from unique sources. Replicate isolates from the same source clustered together with high statistical similarity, with the exception of one isolate. However, a clade of very closely related isolates included replicate isolates from three different patients, as well as single isolates from eight other patients. This clade, provisionally designated clade 1, was one of three clusters of isolates with high statistical similarity. Five of six isolates in one cluster that may acquire clade status were resistant to flucytosine. This study adds C. tropicalis to Candida albicans and Candida glabrata as Candida species for which a multilocus sequence typing (MLST) system has been set up. The C. tropicalis MLST database can be accessed at http://pubmlst.org/ctropicalis/.

Population structure and properties of Candida albicans, as determined by multilocus sequence typing.

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.

Candida orthopsilosis and Candida metapsilosis spp. nov. to replace Candida parapsilosis groups II and III.

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.