Analysis of antifungal resistance genes in Candida albicans and Candida glabrata using next generation sequencing.

Kathrin Spettel,Wolfgang Barousch,Brigitte Selitsch,Iris Zeller,Athanasios Makristathis,Peter-Michael Rath,Michaela Lackner,Marion Nehr,Joerg Steinmann,Birgit Willinger,Joy Sturtevant

doi:10.1371/journal.pone.0210397

Abstract

Introduction/ObjectivesAn increase in antifungal resistant Candida strains has been reported in recent years. The aim of this study was to detect mutations in resistance genes of azole-resistant, echinocandin-resistant or multi-resistant strains using next generation sequencing technology, which allows the analysis of multiple resistance mechanisms in a high throughput setting.MethodsForty clinical Candida isolates (16 C. albicans and 24 C. glabrata strains) with MICs for azoles and echinocandins above the clinical EUCAST breakpoint were examined. The genes ERG11, ERG3, TAC1 and GSC1 (FKS1) in C. albicans, as well as ERG11, CgPDR1, FKS1 and FKS2 in C. glabrata were sequenced.ResultsFifty-four different missense mutations were identified, 13 of which have not been reported before. All nine echinocandin-resistant Candida isolates showed mutations in the hot spot (HS) regions of FKS1, FKS2 or GSC1. In ERG3 two homozygous premature stop codons were identified in two highly azole-resistant and moderately echinocandin-resistant C. albicans strains. Seven point mutations in ERG11 were determined in azole-resistant C. albicans whereas in azole-resistant C. glabrata, no ERG11 mutations were detected. In 10 out of 13 azole-resistant C. glabrata, 12 different potential gain-of-function mutations in the transcription factor CgPDR1 were verified, which are associated with an overexpression of the efflux pumps CDR1/2.ConclusionThis study showed that next generation sequencing allows the thorough investigation of a large number of isolates more cost efficient and faster than conventional Sanger sequencing. Targeting different resistance genes and a large sample size of highly resistant strains allows a better determination of the relevance of the different mutations, and to differentiate between causal mutations and polymorphisms.

Highlights

Candida spp. has emerged as an important pathogen causing bloodstream infections associated with a high mortality
Seven point mutations in ERG11 were determined in azole-resistant C. albicans whereas in azole-resistant C. glabrata, no ERG11 mutations were detected
In 10 out of 13 azole-resistant C. glabrata, 12 different potential gain-of-function mutations in the transcription factor CgPDR1 were verified, which are associated with an overexpression of the efflux pumps CDR1/2

Summary

Introduction

Candida spp. has emerged as an important pathogen causing bloodstream infections associated with a high mortality. Echinocandins and azoles play an important role in the therapeutic management of invasive candidiasis. Echinocandins inhibit the glycosyltransferase 1,3-β-D-glucan synthase (FKS) non-competitively. This enzyme is responsible for the biosynthesis of the oligosaccharide 1,3-β-D-glucan, an important structural component of the fungal cell wall [5]. Decreased susceptibility to echinocandins is associated with target mutations in the hot spot (HS) regions of Fks proteins, which represent the putative binding domain of the echinocandins. Point mutations in these regions can reduce the affinity of the echinocandins to 1,3-β-D-glucan synthase [3, 6, 7]

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