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

Abstract

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.

FIG. 1.

Modified eBURST “snapshot” for 416 C. albicans isolates from unique sources. The figure is redrawn from the original output of the eBURST analysis to eliminate overlapping lines and numbers. The diameter of each circular spot is proportional to the number of isolates with the same DST (indicated by the number adjacent to the spot). Lines join DSTs that differ in just one of the seven fragments sequenced for MLST and thus indicate a hierarchical relationship between DSTs. Where a putative ancestral DST was determined for a cluster, the DST number has been marked with an asterisk. Each clonal cluster of related isolates is numbered in boldface type. Lengths of lines joining DSTs are of no relevance.

FIG. 2.

UPGMA pairwise-difference dendrogram for 416 C. albicans isolates from separate sources. The dendrogram is broken into three sections, a to c, to fit the printed page. The complete dendrogram is shown at a much reduced vertical scale at the bottom right, with indications of the points at which the tree has been split. For each isolate, its clonal cluster (or singleton status) as determined by eBURST analysis is indicated, as is the result of Ca3 typing for isolates provided by D. R. Soll (Cl is the Soll clade result by UPGMA analysis) or Schmid (Gp is the Schmid group result by neighbor-joining analysis). The assignment of isolates to clades, as indicated in the third data column, was made by consideration of the eBURST and Ca3 data in conjunction with UPGMA bootstrap values as described in the text.

FIG. 2.

UPGMA pairwise-difference dendrogram for 416 C. albicans isolates from separate sources. The dendrogram is broken into three sections, a to c, to fit the printed page. The complete dendrogram is shown at a much reduced vertical scale at the bottom right, with indications of the points at which the tree has been split. For each isolate, its clonal cluster (or singleton status) as determined by eBURST analysis is indicated, as is the result of Ca3 typing for isolates provided by D. R. Soll (Cl is the Soll clade result by UPGMA analysis) or Schmid (Gp is the Schmid group result by neighbor-joining analysis). The assignment of isolates to clades, as indicated in the third data column, was made by consideration of the eBURST and Ca3 data in conjunction with UPGMA bootstrap values as described in the text.

FIG. 3.

Population structure of 416 isolates of C. albicans from separate sources, as determined by p-distance (22) in a neighbor-joining analysis. The tree is displayed in radial format to clarify the relative positions of putative clades of related isolates, which are indicated with gray shading. The clade numbers correspond to those in Fig. 2; note that a minority of isolates in clades 3 and 5 were separated in the neighbor-joining tree labeled 3a and 5a, respectively. Branches to singleton isolates without clade affinities are shown with dashed lines.