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


CONCLUSIONS
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.
Wang
Huiping
H
Department of Dermatology, Tianjin Medical University General Hospital, Tianjin Medical University, Tianjin, PR China. huiping1208@163.com
Kong
Fanrong
F
Sorrell
Tania C
TC
Wang
Bin
B
McNicholas
Paul
P
Pantarat
Namfon
N
Ellis
David
D
Xiao
Meng
M
Widmer
Fred
F
Chen
Sharon Ca
SC
D019943:Amino Acid Substitution; D000935:Antifungal Agents / Q000494:pharmacology*; D001483:Base Sequence; D002176:Candida albicans / Q000235:genetics* / Q000302:isolation & purification; D004252:DNA Mutational Analysis; D004271:DNA, Fungal / Q000235:genetics; D025141:Drug Resistance, Fungal / Q000235:genetics*; D015725:Fluconazole / Q000494:pharmacology*; D005656:Fungal Proteins / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity
D000935:Antifungal Agents; D004271:DNA, Fungal; D005656:Fungal Proteins; D015725:Fluconazole
J Clin Microbiol. 2008 Apr;46(4):1200-6
18234874
Trends Biotechnol. 2006 Feb;24(2):83-8
16378651
Antimicrob Agents Chemother. 2002 Jun;46(6):1704-13
12019079
Microbiology (Reading). 1999 Oct;145 ( Pt 10):2715-25
10537193
Med Mycol. 2005 Jun;43(4):285-318
16110776
Lancet Infect Dis. 2002 Feb;2(2):73-85
11901654
J Antimicrob Chemother. 2008 Apr;61(4):798-804
18218640
Antimicrob Agents Chemother. 1999 May;43(5):1163-9
10223930
Antimicrob Agents Chemother. 1995 Jan;39(1):1-8
7695288
Lancet Infect Dis. 2003 Nov;3(11):685-702
14592598
J Clin Microbiol. 2008 Apr;46(4):1192-9
18234865
Clin Chim Acta. 2006 Jan;363(1-2):32-47
16182268
Histochem Cell Biol. 2006 Aug;126(2):159-64
16807721
Antimicrob Agents Chemother. 2004 Feb;48(2):568-74
14742211
J Antimicrob Chemother. 2004 Feb;53(2):217-24
14688046
Antimicrob Agents Chemother. 1997 Jul;41(7):1482-7
9210670
Biochim Biophys Acta. 2002 Jul 18;1587(2-3):240-8
12084466
Antimicrob Agents Chemother. 2005 May;49(5):1745-52
15855491
J Clin Microbiol. 2006 Mar;44(3):876-80
16517870
J Clin Microbiol. 2006 Oct;44(10):3826-9
17021120
J Clin Microbiol. 2005 May;43(5):2339-44
15872263
J Clin Microbiol. 2008 Jul;46(7):2423-7
18495860
Antimicrob Agents Chemother. 1998 Nov;42(11):2932-7
9797228
Antimicrob Agents Chemother. 2004 Jun;48(6):2124-31
15155210
Antimicrob Agents Chemother. 1997 Jul;41(7):1488-94
9210671
Genetics. 2006 Apr;172(4):2139-56
16452151
Microbiology (Reading). 1999 Oct;145 ( Pt 10):2701-2713
10537192
Antimicrob Agents Chemother. 2007 Jun;51(6):1897-904
17371828
Clin Microbiol Rev. 1998 Apr;11(2):382-402
9564569
Antimicrob Agents Chemother. 1998 Feb;42(2):241-53
9527767
J Biol Chem. 1997 Feb 28;272(9):5682-8
9038178
Antimicrob Agents Chemother. 2001 Oct;45(10):2676-84
11557454
J Med Vet Mycol. 1988;26(5):277-83
3236147
Biochem Soc Trans. 1993 Nov;21(4):1034-8
8131893
Antimicrob Agents Chemother. 1998 Dec;42(12):3065-72
9835492