Abstract
BackgroundAmino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing.ResultsThe RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96%) isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78%) fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates), G448E (n = 3), G307S (n = 3), K143R (n = 3) and Y123H, S405F and R467K (each n = 1). DNA sequencing revealed a novel substitution, G450V, in one isolate.ConclusionThe sensitive RCA assay described here is a simple, robust and rapid (2 h) method for the detection of ERG11 polymorphisms. It showed excellent concordance with ERG11 sequencing and is a potentially valuable tool to track the emergence and spread of azole-resistant C. albicans and to study the epidemiology of ERG11 mutations. The RCA method is applicable to the study of azole resistance in other fungi.
Highlights
Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans
With increased use of these agents, fluconazole, treatment failures associated with the emergence of azole-resistant strains of C. albicans have occurred [3,4,5,6] This has been most evident in HIV/AIDS patients receiving long-term therapy for oropharyngeal candidiasis (OPC) [3,7]
These include increased expression of the drug efflux pump genes such as MDR1, CDR1 and CDR2 [3,9,10,11], amino acid substitutions in the target enzyme Erg11p due to missense mutations in the ERG11 gene [3,5,10,12,13,14,15] and possibly, overexpression of ERG11 [3,16] Importantly in any one isolate, resistance may be due to a combination of mechanisms [3,4,5,15]
Summary
Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. More than 60 amino acid substitutions have been described in Erg11p with at least 30 of these identified in azole-resistant isolates [5,12,14,15,16,17] The impact of individual substitutions, varies, and may differ between azoles Substitutions such as Y132H, G450E, G464S, R467K and S405F appear to primarily impact on fluconazole and voriconazole, but not posaconazole, susceptibility [12,13,16,17] The effect of substitutions can be additive – strains with G129A and G464S substitutions display higher MICs azoles compared with those with the G129A substitution alone [18]. As most strains of C. albicans are diploid, nucleotide mutations may occur as homozygous (in both alleles) or as heterozygous (in one allele) substitutions; the association of either type of mutation with the resistant (or susceptible) phenotype is not well defined [3,20]
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