Nucleotide differences in internal transcribed spacer regions of rDNA and 5-flucytosine resistance among clinical Candida dubliniensis isolates in Kuwait

Abstract number: P726

Ahmad S., Mokaddas E., Khan Z.

Objective: Clinical Candida dubliniensis isolates are classified into four genotypes based on nucleotide sequences of internal transcribed spacer (ITS)1 and ITS2 regions of rDNA. Some of the isolates from the Middle Eastern region are resistant to 5-flucytosine. This study was carried out to determine nucleotide differences in ITS region (including ITS1–5.8S rRNA-ITS2) of rDNA that define 5-flucytosine resistance among clinical C. dubliniensis isolates in Kuwait.

Methods: A total of 76 C. dubliniensis isolates recovered from various clinical specimens in Kuwait were assigned to one of four genotypes based on amplification of rDNA with genotype-specific primers. The susceptibility of the isolates to commonly used antifungal agents including 5-flucytosine was performed by Etest. DNA sequencing was performed to correlate specific nucleotides in ITS region of rDNA with the four genotypes and to define signature residues associated with resistance of C. dubliniensis isolates to 5-flucytosine.

Results: Majority (49 of 76, 64%) of C. dubliniensis isolates belonged to genotype 1, DNA sequences of ITS region of rDNA from 12 randomly selected isolates were identical and matched completely with the prototype strain CD36. A total of 21 isolates (21 of 76, 28%) yielded an amplicon with genotype 4 specific primers only and their ITS region sequences matched completely with the prototype strain of genotype 4. Only 1 isolate belonged to genotype 3. None of the C. dubliniensis strains belonged to genotype 2. Five isolates yielded amplicons with more than one genotype-specific primer pair and DNA sequences of the ITS region from these isolates identified them as hybrid strains. A total of 21 C. dubliniensis isolates were resistant to 5-flucytosine, all 21 belonged to genotype 4 and were characterised by the presence of a T residue at nucleotide position 82 in the ITS region.

Conclusions: Only three of four genotypes based on ITS region sequences were detected among clinical C. dubliniensis isolates in Kuwait with most (64%) of the isolates belonging to genotype 1. All C. dubliniensis isolates resistant to 5-flucytosine belonged to genotype 4 and were defined by a signature nucleotide in the ITS region of rDNA. The C. dubliniensis hybrid strains closely related to genotype 4 but not containing this signature nucleotide were not resistant to 5-flucytosine.