Population structure and molecular genetic characterization of 5-flucytosine-susceptible and -resistant clinical Candida dubliniensis isolates from Kuwait.

Noura Al-Sweih,Suhail Ahmad,Joy Sturtevant,Mohammad Asadzadeh,Ziauddin Khan

doi:10.1371/journal.pone.0175269

Abstract

Candida dubliniensis and Candida albicans are two closely related species. Although C. dubliniensis is less pathogenic, it has a higher propensity to develop resistance to fluconazole and some strains exhibit intrinsic resistance to 5-flucytosine (5-FC). All 5-FC-resistant isolates from Kuwait were previously shown to belong to one of seven internal transcribed spacer (ITS) region of rDNA-based haplotypes. This study performed fingerprinting of C. dubliniensis isolates by multilocus sequence typing (MLST) to determine population structure of 5-FC-resistant and -susceptible strains and compared the results with data from a global collection of isolates. Fifty-two C. dubliniensis isolates previously analyzed and 58 additional isolates mostly collected during 2010–2013 and characterized by phenotypic and molecular methods were used. ITS-based haplotypes were identified by haplotype-specific PCR and/or by PCR-DNA sequencing of rDNA. Population structure was determined by 8-loci-based MLST. E-test was used to determine susceptibility to 5-FC, fluconazole, voriconazole and amphotericin B. Five ITS haplotypes (ITSH) were detected among 110 C. dubliniensis isolates. The ITSH1 was most common (n = 80 isolates) followed by ITSH4 (n = 25 isolates). Two isolates each belonged to ITSH5 and ITSH8 while one isolate belonged to ITSH7. MLST identified 16 diploid sequence types (DSTs) including six new DSTs. DST11 (n = 52) and DST14 (n = 25) were dominant genotypes and were confined (together with DST21) to Middle-Eastern countries. Other DSTs (excluding some new DSTs) had a wider global distribution as they were identified from various other countries. Only ITSH4 isolates (n = 25) belonged to DST14, were resistant to 5-FC and contained S29L mutation in CdFCA1. ITSH5, ITSH7 and ITSH8 isolates belonged to different DSTs. Thus, clinical C. dubliniensis isolates in Kuwait exhibited limited genotypic heterogeneity and most isolates belonged to region-specific DSTs. All 5-FC-resistant C. dubliniensis isolates belonged to ITSH4 and MLST-based DST14 genotype. Placement of some isolates into additional ITS haplotypes is also supported by MLST data.

Highlights

Candida dubliniensis and Candida albicans are the only two yeast species capable of forming true hyphae which play an important role in adhesion and tissue invasion during infection [1]
The detailed list of all 110 C. dubliniensis isolates with their internal transcribed spacer (ITS) haplotype, diploid sequence type (DST), amino acid at CdFCA1 codon 29 and accession numbers of representative DNA sequences submitted to European Molecular Biology Laboratory (EMBL)/GenBank databases for different ITS haplotypes/new diploid sequence types (DST27-DST33) detected in this study and for CdFCA1 codon 29 region is provided in S1 Table
Based on haplotypespecific PCR amplification and/or DNA sequencing studies, 41, 15, one and one isolate belonged to ITSH1, ITSH4, ITSH5 and ITSH8, respectively, among 58 C. dubliniensis isolates analyzed in this study

Summary

Introduction

Candida dubliniensis and Candida albicans are the only two yeast species capable of forming true hyphae which play an important role in adhesion and tissue invasion during infection [1] Both species share many phenotypic and genotypic characteristics, C. dubliniensis is less pathogenic, likely as a result of significant gene loss since their divergence from the common ancestor [1]. C. dubliniensis exhibits greater karyotype variability than C. albicans, the population structure of a global collection of C. dubliniensis isolates by multilocus sequence typing (MLST) based on 8–10 protein-coding gene fragments was found to be less divergent than C. albicans as it comprised only three distinct clades (C1–C3) and all 5-FCresistant isolates were confined to MLST clade C3, which included some 5-FC susceptible isolates [20]. 7–10 protein-coding gene fragments were initially used in different combinations during development of the MLST scheme, a combination of 8 loci (CdAAT1b, CdACC1, CdADP1, CdMPIb, CdRPN2, CdSYA1, exCdVPS13 and exCdZWF1b) was recommended for the MLST of C. dubliniensis isolates on the basis of the highest number of genotypes per variable base [20]

Methods

Results

Discussion

Conclusion