Detection of ERG۱۱ point mutations in iranian fluconazole-resistant Candida albicans isolates

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 ERG۱۱. Mutations in ERG۱۱ result in changes in the ERG۱۱p 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 ERG۱۱ gene. Materials and Methods: This study was conducted on a total of ۲۱۶ clinical isolates obtained from Mashhad, Isfahan, and Tehran cities in Iran, during ۲۰۱۶-۲۰۱۸. The clinical isolates were identified using molecular techniques. Furthermore, minimum inhibitory concentration (MICs) was determined according to the clinical and laboratory standards institute M۲۷-A۳ and M۲۷-S۴ documents. The concentration range for fluconazole was obtained as ۰.۰۶۳-۶۴ μg/ml. In the resistant strains, ERG۱۱ 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 ۷) and Gene Runner software (version ۶.۵.۳۰). Results: Out of ۲۱۶ strains, ۱۰۰ (۴۶.۳%) species were identified as C. albicans. The MIC values for fluconazole had a range of ۰.۱۲۵-۱۶ μg/ml with the MIC۵۰ and MIC۹۰ values of ۰.۵ and ۱ μg/ml, respectively. Totally, ۴۱ nucleotide changes were detected among ۴ resistant isolates. In this regard, ۴ out of ۴۱ mutations in codons caused changes in ERG۱۱p; however, these mutations did not lead to fluconazole resistance. Conclusion: Fluconazole resistance among clinical isolates is not merely due to the changes in ERG۱۱p. 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.