Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and DNA sequencing

Abstract

Background: Amino 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.

Results: The 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.

Conclusion: The 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.

Figure 1

Typical design of a circularisable padlock probe. Design and components of a typical padlock probe as exemplified by the Ca-Y132H probe specific for the Y132H amino acid substitution. The probe comprises (i) a 5'-phosphorylated end; (ii) a "backbone" containing binding sites for the RCA primers (RCA primer 1 and 2, respectively) designated by bold upper case letters) as well as the non-specific linker regions (designated by bold lower case letters) and (iii) a 3'-end. The 5'- and 3'-ends of the probe are complementary to the 5' and 3' termini of the target sequence in reverse, in this example, to the C. albicans sequence (GenBank accession no. AF153844). Abbreviations: 5'-P, 5'-phosphorylated binding arm; 3'-, 3' binding arm.

Figure 2

Sensitivity of the RCA assay. RCA was performed on 10-fold serial dilutions of the target template ranging from 10¹¹ to 10⁰ copies of target template (PCR product). The figure illustrates the RCA reaction using the Ca-Y132H-specific probe to detect 10¹¹, 10¹⁰ and 10⁹ copies of the template containing the Y132H mutation (obtained from amplifying DNA from isolate C594). RCA signals are shown as exponential increases in florescence signal above baseline (indicated by the "negative signal" label and defined as the signal obtained when amplifying target template that did not have the mutation of interest). The intensity of the signal weakened with decreasing copy numbers starting at 10¹¹copies and the sensitivity of the assay corresponded to a concentration of 10⁹copies of target template.

Figure 3

Sensitivity of the RCA assay in the presence of DNA mixtures. The accumulation of double-stranded DNA was detected by staining with Sybr green I. RCA signals indicative of the presence of a specific mutation are shown as exponential increases in fluorescence signal above baseline. The figure illustrates the padlock probe-RCA reaction using the Ca-Y257H-specific probe to detect varying concentrations (100%, 50%, 20%, 10% and 5%) of target template (10¹¹copies). The target template was DNA from isolate C594 containing the Y257H mutation; this was diluted with DNA from strain ATCC 10231 (without the Y257H mutation). The intensity of RCA fluorescence signal weakened with decreased template concentration. The sensitivity of the assay corresponded to a concentration of 5% template DNA in the mixture.

Figure 4

Specificity of the RCA assay. RCA results monitored by the RotorGene 6000 real-time PCR machine (Corbett research). The accumulation of double-stranded DNA was detected by staining with Sybr Green I. RCA signals indicating the presence of the mutation of interest ((labeled as "positive signal") are shown as exponential increases in fluorescence. The experiment was conducted using the Ca-Y132H-specific RCA probe and tested on eight C. albicans isolates with known ERG11 mutation sites (Table 1). Ligation-mediated RCA with matched templates (DNA from isolates C527, C594, C507) containing the targeted SNPs produced "positive signals". Other templates showed an absence of signal (labeled as "negative signal").