Genetic diversity of medically important and emerging Candida species causing invasive infection

Abstract

Background: Genetic variation in the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region has been studied among fungi. However, the numbers of ITS sequence polymorphisms in the various Candida species and their associations with sources of invasive fungal infections remain poorly investigated. Here, we characterized the intraspecific and interspecific ITS diversity of Candida spp. strains collected from patients with bloodstream or oroesophageal candidiasis.

Methods: We selected cultures of representative medically important species of Candida as well as some rare and emerging pathogens. Identification was performed by micromorphology and by biochemical testing using an ID32C system, as well as by the sequencing of rDNA ITS. The presence of intraspecific ITS polymorphisms was characterized based on haplotype networks, and interspecific diversity was characterized based on Bayesian phylogenetic analysis.

Results: Among 300 Candida strains, we identified 76 C. albicans, 14 C. dubliniensis, 40 C. tropicalis, 47 C. glabrata, 34 C. parapsilosis (sensu stricto), 31 C. orthopsilosis, 3 C. metapsilosis, 21 Meyerozyma guilliermondii (C. guilliermondii), 12 Pichia kudriavzevii (C. krusei), 6 Clavispora lusitaniae (C. lusitaniae), 3 C. intermedia, 6 Wickerhamomyces anomalus (C. pelliculosa), and 2 C. haemulonii strains, and 1 C. duobushaemulonii, 1 Kluyveromyces marxianus (C. kefyr), 1 Meyerozyma caribbica (C. fermentati), 1 Pichia norvegensis (C. norvegensis), and 1 Lodderomyces elongisporus strain. Out of a total of seven isolates with inconsistent ID32C profiles, ITS sequencing identified one C. lusitaniae strain, three C. intermedia strains, two C. haemulonii strains and one C. duobushaemulonii strain. Analysis of ITS variability revealed a greater number of haplotypes among C. albicans, C. tropicalis, C. glabrata and C. lusitaniae, which are predominantly related to endogenous sources of acquisition. Bayesian analysis confirmed the major phylogenetic relationships among the isolates and the molecular identification of the different Candida spp.

Conclusions: Molecular studies based on ITS sequencing are necessary to identify closely related and emerging species. Polymorphism analysis of the ITS rDNA region demonstrated its utility as a genetic marker for species identification and phylogenetic relationships as well as for drawing inferences concerning the natural history of hematogenous infections caused by medically important and emerging Candida species.

Figure 1

Analysis of nucleotide sequence variation of rDNA ITS in medically important Candida spp. Polymorphic sites in ITS1-5.8S-ITS2 rDNA were evaluated along the total sequence lengths for the following species for the assessment of the percentage of nucleotide variation per site, for which the sequences of the clinical strains were compared with the sequence of the reference or type strain for each species: (A) C. albicans (447 bp, 78 sequences), (B) C. tropicalis (437 bp, 41 sequences), (C) C. glabrata (803 bp, 48 sequences), (D) C. parapsilosis (sensu stricto) (430 bp, 35 sequences), (E) C. orthopsilosis (426 bp, 32 sequences), and (F) M. guilliermondii (516 bp, 22 sequences).

Figure 2

Genetic diversity of medically important and emerging Candida spp. based on rDNA ITS sequences. (A) The median-joining haplotype network of 319 DNA sequences of clinical (n = 300) and reference/type strains (n = 19) belonging to 17 Candida species and 1 non-Candida species (Lodderomyces elongisporus). Each circle represents one haplotype (H1-H67), and the circle circumference is proportional to the haplotype frequency of the dataset. The black dots (median vectors, m) represent unsampled or extinct haplotypes in the population. Mutational steps are represented by lines between haplotypes and, in cases of long branches, by values. (B) The haplotypes are color-coded, and each color represents the predominantly associated source of acquired infection for the haplotype (endogenous versus exogenous).

Figure 3

Unrooted Bayesian consensus phylogenetic tree of rDNA ITS haplotype representatives of Candida spp. Species names were identified according to anamorphic nomenclature. Posterior probabilities and bootstrap values are depicted in the main nodes (pp/bootstrap). The Bayesian tree was inferred from 2 million generations with a 50% burn-in, and runs were saved every 100 generations. The average standard deviation of the split frequencies was 0.008203. The selected model was fA = 0.274572, fC = 0.203973, fG = 0.223583, and fT = 0.297972 with the rate matrix [A–C] = 0.151397, [A–G] = 0.142740, [A–T] = 0.199583, [C–G] = 0.112413, [C–T] = 0.269359 and [G–T] = 0.124502. The shape parameter of the gamma distribution was alpha = 1.517708, and the proportion of invariant sites was I = 0.196504.