Investigation of mutations which cause alterations in cytochrome p450 14?-demethylase enzyme of fluconazole resistant candida albicans isolates

Increase in frequency of life-threatening fungal infections has contributed to an important problem nowadays. The increase in the number of the transplantation and intensive care patients who are at risk for fungal infections and empirical use of antifungals has elevated the importance of the problem. Especially widespread and repeated use of fluconazole resulted in resistance problem correlating with clinical failure. Our study was planned to investigate resistance mechanisms in fluconazole resistant C. albicans isolates. In our study, investigation of Y132H and other mutations in ERG11 gene that encodes lanosterole 14?-demethylase enzyme which is one of the resistance mechanisms in fluconazole resistant/dose dependent susceptible C. albicans isolates and determination of the role of efflux pumps in this resistance were aimed. In this study three fluconazole resistant and ten fluconazole susceptible isolates chosen from 650 C. albicans strains isolated from Dokuz Eylül University Hospital Mycology Laboratory and four fluconazole resistant/dose dependent susceptible C. albicans isolates obtained from other universities were included. DSY 289, DSY 347 and DSY 292 numbered C. albicans isolates were used as control strains. The susceptibility of C. albicans isolates were determined by broth microdilution method according to the Clinical and Laboratory Standards Institute M27-A2 standards. All of the seven fluconazole resistant/dose dependent susceptible C. albicans isolates were found to be resistant to flucytosine except one, all isolates were susceptible to amphotericine B and all isolates except one were resistant to itraconazole and ketoconazole and all of them except one were susceptible to clotrimazole. Six fluconazole susceptible C. albicans isolates were found to be resistant to flucytosine and the others were intermediate. All of the ten isolates were susceptible to amphotericine B, ketoconazole, clotrimazole. Restriction enzyme analysis was performed to all isolates for Y132H mutation and sequence analysis was done for other mutations in ERG11 gene which encodes lanosterole 14?-demethylase enzyme. DNA extraction was performed with Nucleospin Tissue Kit (Macherey-Nagel, Germany) according to the manufacturer?s instructions by adding EDTA, lyticase, sorbitol buffer. After determining a 673 bp polymerase chain reaction product, digestion with PagI enzyme was performed following purification. 315 and 358 bp fragments were determined in all fluconazole resistant/dose dependent susceptible and susceptible C. albicans isolates and for this reason it is concluded that Y132H mutation does not exist in our strains. Sequence analysis was performed to all strains after amplication of 1600 bp fragment of ERG11 gene by polymerase chain reaction and the results were evaluated with Bio-edit version 7.0 program. When the sequence analysis results were interpreted, it was found that Y132H mutation does not exist in all isolates as determined by restriction enzyme analysis. No mutation was detected in one of the fluconazole resistant/dose dependent susceptible, two of the fluconazole susceptible C. albicans isolates. D116E, K128T, E266D, L280Y, L280F, L281M, L281Y, I282D, S284I, T285H, V437I mutations were determined in fluconazole susceptible C. albicans isolates and D116E, K143R, D153E, E266D, S412T, G464S, G465S, R469K, V488I were found in resistant/dose dependent susceptible C. albicans isolates. K143R, G464S, G465S and V488I mutations which has been reported as important for fluconazole resistance previously were determined in three of the fluconazole resistant/dose dependent susceptible isolates. S412T and R469K mutations determined only in this group of strains by sequence analysis were thought to play a role in resistance but they should be evaluated with advenced studies. Efflux pumps which is another mechanism for resistance were investigated by susceptibility testing performed by microdilution method according to Clinical and Laboratory Standards Institute M27-A2 standards. When the study performed by using substrates and inhibitors was evaluated, possible overexpression of efflux pumps was determined especially in three fluconazole resistant C. albicans strains isolated in Dokuz Eylül University Hospital. In conclusion, mutations such as K143R, G464S, G465S and V488I in ERG11 gene which encodes lanosterole 14?-demethylase enzyme and overexpression of efflux pumps were determined to be effective mechanisms in our fluconazole resistant/dose dependent susceptible C. albicans isolates, however other mechanisms of resistance such as overexpression of ERG11, mutations in ERG3 gene shoud also be investigated.