Arch Hellen Med, 18(4), July-August 2001, 354-362
Detection of the KRE9/KNH1 genes in Candida
glabrata of non-killer phenotype
O. KOSTOULA,1 K. KOLLIA,1
M. ARABATZIS,1 D. TOUKAS,1 O. NIKOLATOU,2
OBJECTIVE To determine the distribution and abundance of wild-type Candida glabrata with disrupted ΚRE9/KNH1 genes encoding for cell surface O glycoproteins required for β-1,6-glucan biosynthesis, to determine whether such strains are resistant in vitro to the azole drugs and to develop a PCR system for detecting the presence of these genes in conjunction with screening for their expression using phenotypic tests. An additional aim was to seek evidence for, or against, the phylogenetic relationship between C. glabrata and Clavispora (Candida) lusitaniae based upon identification of identical sequences in their homologous KRE9/KNH1 genes.
METHOD The strains were randomly selected among cases of oral candidiasis from patients receiving radio-chemotherapy for head and neck tumors, and those with candidemia episodes and from bronchopulmonary candidiasis. Primers were selected on studying each gene sequence, as published in the GenBank, and a construction of the amplicon restriction map was computer assisted. All strains were tested by duplex PCR for the simultaneous amplification of sequences from both genes. Expression and partial disruption of the genes was tested by killer tests using the type Saccharomyces cerevisiae killer strain K1V116 and by performing tetracycline resistance tests. Phenotypic resistance tests to antifungal drugs including voriconazole (UK-109,496) were performed by the NCCLS proposed microdilution method.
RESULTS Evidence was produced that one of the mechanisms involved in azole resistance of C. glabrata could be due to the disruption of the KRE9/KNH1 genes. The tested strains also showed azole resistance.
CONCLUSIONS The proposed PCR system is of potential clinical use as a tool for rapid differential diagnosis of C. glabrata and C. lusitaniae. Finally, the results provide evidence of the close phylogenetic relationship of the two yeasts.
Key words: C. glabrata, KRE9/KNH1 genes, Resistance mechanisms.