Multilocus sequence typing of Candida glabrata reveals geographically enriched clades

Abstract

The haploid pathogenic yeast Candida glabrata is the second most common Candida species isolated from cases of bloodstream infection. The clinical relevance of C. glabrata is enhanced by its reduced susceptibility to fluconazole. Despite this, little is known of the epidemiology or population structure of this species. We developed a multilocus sequence typing (MLST) scheme for C. glabrata and used it to fingerprint a geographically diverse collection of 107 clinical isolates and 2 reference strains. Appropriate loci were identified by amplifying and sequencing fragments of the coding regions of 11 C. glabrata genes in 10 unrelated isolates. The 6 most variable loci (FKS, LEU2, NMT1, TRP1, UGP1, and URA3) were sequenced in the collection of 109 isolates. From the 3,345 bp sequenced in each isolate, 81 nucleotide sites were found to be variable. These defined 30 STs among the 109 strains. The technique was validated by comparison with random amplified polymorphic DNA and the complex DNA fingerprinting probes Cg6 and Cg12. MLST identified 5 major clades among the isolates studied. Three of the clades exhibited significant geographical bias. Our data demonstrate for the first time, with such a large geographically diverse strain collection, that distinct genetic clades of C. glabrata prevail in different geographical regions.

FIG. 1.

Polymorphic sites of the 6 loci used in the MLST scheme. The polymorphisms comprising each of the 30 STs found in the 109 isolates tested are shown. The STs are arranged in the 5 groups defined in the study, followed by the pairs of STs differing at only one allele, followed finally by the STs not fitting these criteria. The allelic profiles and groups of the STs are also shown. The allelic profiles are given in the order FKS, LEU2, NMT1, TRP1, UGP1, and URA3. Synaptomorphic alleles are indicated in boldface type. The letters under the figure indicate whether the polymorphism is synonymous (S) or nonsynonymous (N). The position of the polymorphism in the coding sequence is given by reading the numbers vertically.

FIG. 2.

Dendrogram showing the relationships of all 109 isolates typed by MLST. The dendrogram was generated by using the UPGMA method from the concatenated sequence obtained from all 6 loci used in the MLST scheme. Bootstrap values of >60% are shown. The STs to which the isolates belong are shown. Groups defined by a sequence similarity of >99.6% (shown by a dashed line) are also shown. Isolates from Europe are shown in blue, those from Japan are shown in red, and those from the United States are shown in green. All other isolates are shown in black. The 8 isolates known to be fluconazole resistant (MIC ≥ 64 μg/ml) are underlined.

FIG. 3.

Dendrograms based on the computed S_ABs between the 26 isolates typed by MLST (a), RAPD (b), and combined data obtained by Southern blotting with the DNA probes Cg6 and Cg12 (c). The arbitrary S_AB thresholds used to define clusters are shown by dashed lines.