Detection of ERG11 point mutations in Iranian fluconazoleresistant 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 μg/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 μg/ml with the MIC50 and MIC90
values of 0.5 and 1 μg/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.