When examining the drug susceptibility of clinically isolated strains, we have found that 30 clinical isolates of Candida tropicalis were able to produce drug-resistant progeny upon exposures to flucytosine (5FC) on agar media. To determine the molecular mechanism of the resistance, we have collected the 5FC-resistant progeny within the inhibitory ellipses on the agar media and subjected them to sequence analyses on the genes known to be involved in 5FC metabolism. After identifying the mutations on genes potentially responsible for the resistance, we then performed site-directed mutagenesis on the genome of a susceptible strain to assess the effect of those mutations. Among the genes sequenced, 22 of the 30 clinical strains had heterozygous G/T at the 145th position on FCY2, encoding purine-cytosine permease, whereas their progeny recovered from the inhibitory ellipses had homozygous T/T, resulting in null alleles for both copies of the gene and produced only truncated proteins, effecting the 5FC resistance. We further characterized 198 single-nucleotide polymorphisms (SNPs) of 60 loci and found that break-induced replication/allelic recombination is the most likely mechanism contributing to the LOH of all these progeny. In addition, the two major fluconazole-resistant clinical clones, diploid sequence type 98 (DST98) and DST140, had homozygous G/G at the 145th position and neither was able to produce 5FC resistant progeny within the inhibitory ellipses. In conclusion, Candida tropicalis strains containing heterozygous alleles may develop 5FC resistance readily whereas those with homozygous G/G wildtype alleles can be treated with 5FC. Therefore, a combination of 5FC and another antifungal drug is applicable for treating infections of C. tropicalis.
