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Detection of ERG11 point mutations in Iranian fluconazole-resistant Candida albicans isolates.

ABSTRACT: Background and Purpose:Candidiasis is referred to a group of superficial and deep-tissue fungal infections often caused by Candida albicans. The superficial infections affect the oral, oropharynx, esophagus, and vaginal mucosa. The treatment of choice for these infections is the use of azoles, such as fluconazole. However, the increased use of these antifungal agents has led to the emergence of azole-resistant isolates of C. albicans. Different mechanisms have been suggested for the development of drug resistance, such as mutations in the encoding gene ERG11. Mutations in ERG11 result in changes in the ERG11p spatial construction and reduce the affinity between the protein and azole. This study aimed to determine the susceptibility profile of C. albicans clinical isolates to fluconazole using microdilution method. The present research was also targeted toward the detection of mutations that might be related to fluconazole resistance by the amplification and sequencing of ERG11 gene. Materials and Methods:This study was conducted on a total of 216 clinical isolates obtained from Mashhad, Isfahan, and Tehran cities in Iran, during 2016-2018. The clinical isolates were identified using molecular techniques. Furthermore, minimum inhibitory concentration (MICs) was determined according to the clinical and laboratory standards institute M27-A3 and M27-S4 documents. The concentration range for fluconazole was obtained as 0.063-64 ?g/ml. In the resistant strains, ERG11 genes were amplified by specific primers. Subsequently, cycle sequencing reactions were performed on purified polymerase chain reaction (PCR) products in forward and reverse directions. Finally, the results were analyzed by MEGA (version 7) and Gene Runner software (version 6.5.30). Results:Out of 216 strains, 100 (46.3%) species were identified as C. albicans. The MIC values for fluconazole had a range of 0.125-16 ?g/ml with the MIC50 and MIC90 values of 0.5 and 1 ?g/ml, respectively. Totally, 41 nucleotide changes were detected among 4 resistant isolates. In this regard, 4 out of 41 mutations in codons caused changes in ERG11p; however, these mutations did not lead to fluconazole resistance. Conclusion:Fluconazole resistance among clinical isolates is not merely due to the changes in ERG11p. This resistance may be also related to some other mechanisms, such as the prevention of the intracellular accumulation of the antifungal agent and alteration of the target enzyme to diminish drug binding.

SUBMITTER: Sardari A

PROVIDER: S-EPMC6488286 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Project description:Abstract Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Objective To determine the profile of fluconazole-resistant Candida isolates (FRCS) and the proportion of erg11 gene mutation in fluconazole-resistant C. albicans (FRCA) isolates Methods After getting approval from ethics the study was conducted on 150 isolates of FRCS obtained from patients admitted to our tertiary care hospital from September 2019 to December 2021. All the isolates were revived onto Sabouraud dextrose agar (SDA) and identified by Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). Fluconazole-resistance was detected by VITEK 2 automated system. Deoxyribonucleic acid (DNA) of FRCA isolates were extracted by QIAamp DNA Mini kit (Qiagen-51 304, Hilden, Germany) as per manufacturer's instruction. A single primer pair targeting the nucleotide sequence from 1067 to 1576 bp region of erg11 gene was used for amplification as per Xu et al. with minor modification and 500 bp polymerase chain reaction (PCR) products were observed by gel electrophoresis. A positive clinical isolate of FRCA was used as a positive control. The PCR products were purified and subjected to Sanger sequencing with an ABI PRISM DNA analyzer (Applied Biosystems) and mutations were detected by using Mega (version 7) software by comparing with the published GenBank sequence AF153844.1 of C. albicans strain ATCC 28516. Categorical variables were expressed in percentages and continuous variables were expressed in mean (average). Results Out of the total of 150 FRCS, the most common fluconazole-resistant agent of candidiasis was C. auris, which was isolated from 60 (40%) cases followed by 24 (16%) isolates of C. glabrata, 19 (12.7%) isolates of C. krusei, 18 (12%) isolates of C. parapsilosis, 17 (11.3%) isolates of C. albicans, 10 (6.7%) isolates of C. tropicalis, and only two (1.3%) isolates of C. guilliermondii. Among these 17 FRCA isolates, 9 isolates were obtained from genital swabs, 5 from blood, and a single isolate each from the perianal swab, ear swab, and sputum. A total of 12 (70.6%) out of 17 FRCA isolates yielded 500 bp region in erg11 gene. Gel electrophoresis image of PCR products of erg11 gene is depicted in Figure 1. Sequencing of these products detected a single missense mutation A1309G (V437I) in one FRCA isolate. Most of the FRCA isolates had an average of 4 silent mutations (observed in 6 isolates) and a single isolate had 6 silent mutations. A total of 8 different silent mutations were observed among FRCA isolates T1296C (83.3%), C1203T (66.7%), A1440G (66.7%), C1302T (50%), T1470C (50%), T1140C (25%), T1110C (16.7%), and T1284C (8.3%). Conclusion C. auris was the most common fluconazole resistant isolate observed, followed by C. glabrata, C. krusei, C. parapsilosis, C. albicans, C. tropicalis, and C. guilliermondii. There were many silent mutations observed in erg11 gene of FRCA isolates and detected only a single missense mutation (V437I). Fluconazole-resistant in FRCA isolates may be due to mechanisms other than the studied one in our region.

Project description: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.

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Detection of ERG11 point mutations in Iranian fluconazole-resistant Candida albicans isolates.

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