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

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.

Low level of antifungal resistance of Candida glabrata blood isolates in Turkey: Fluconazole minimum inhibitory concentration and FKS mutations can predict therapeutic failure

“…As some isolates were clonal and/or belonged to the same genotype, horizontal transfer could be suggested; however, this hypothesis requires experimental confirmation by performing whole genome sequencing and environmental screening, which are beyond the scope of our study. Nowadays, various next‐generation sequencing platforms have been increasingly employed to assess genotypic diversity as well as to identify mutations responsible for antifungal resistance, which may not be used for a particular gene but rather for numerous genes scattered throughout the genome 59‐61 . In contrast to a previous study, 28 in our study we did not observe the phenomenon of clonal enrichment for FLZ‐resistant C glabrata isolates as evidenced by the lack of statistical association between FLZ MIC values and cluster and MMT patterns (Figure 2).…”

Large-scale genome mining allows identification of neutral polymorphisms and novel resistance mutations in genes involved in Candida albicans resistance to azoles and echinocandins

“…[62][63][64][65] For example, in the study performed by Spettel et al, the amino-acid substitution S937L in Tac1p, identified as a potentially causal resistance mutation, could have been ruled out using the neutral polymorphisms catalog. 62 In our study, two amino-acid substitutions identified in Erg11p (Y64H, P236S) were predicted with high confidence as impacting Erg11p protein structure. Interestingly, these positions were already predicted to be vital for various antifungal interactions with the active site of Erg11p from C. albicans or Saccharomyces cerevisiae.…”

Chromatin Structure and Drug Resistance in Candida spp.

“…This is no more evidenced than when comparing azole resistance mechanisms in C. albicans and C. glabrata. While the majority of C. albicans azole resistant isolates contain a mutation in the binding site of the drug target, Ca_Erg11p, large-scale studies on resistant C. glabrata isolates have revealed that mutations affecting either the expression levels or gene product of Cg_ERG11 seldom occur [ 47 , 48 , 49 , 50 ]. By far the preferred mechanism of azole resistance in C. glabrata is the acquisition of mutations in the Cg_PDR1 transcription factor gene that regulates the expression of the Cg_Cdr1p drug efflux pump [ 47 , 50 , 51 ].…”