Exponential Clonal Expansion of 5-Fluorocytosine-Resistant Candida tropicalis and New Insights into Underlying Molecular Mechanisms

Abstract

In 2022, we initiated systematic 5-fluorocytosine susceptibility testing of Candida spp. isolates in Denmark; we observed a bimodal MIC distribution in C. tropicalis, with MICs >16 mg/L in half the isolates. This study investigates the epidemiology and molecular mechanisms of 5-fluorocytosine resistance in C. tropicalis. We analyzed 104 C. tropicalis isolates from 3 time periods, alongside 353 C. albicans and 227 C. glabrata isolates from 2022. We determined MICs using EUCAST E.Def 7.3. Sequencing of FCY2 (purine-cytosine permease), FCY1 (cytosine deaminase), FUR1 (uracil phosphoribosyl transferase), and URA3 (orotidine-5'-phosphate decarboxylase) genes revealed FCY2 alterations-E49X (30/32), Q7X (1/32), and K6NfsX10 (1/32)-in resistant C. tropicalis strains. We found a URA3 alteration, K177E, in both susceptible and resistant strains. Microsatellite genotyping showed that all C. tropicalis isolates with E49X were clonally related. The marked increase in resistance, driven by the clonal spread of E49X, necessitates further research into virulence and environmental factors.

Keywords: Candida tropicalis; Denmark; antimicrobial resistance; clonal spread; emerging resistance; flucytosine resistance; fungi.

Figure 1

Minimum spanning tree illustrating the genotypic relationships among Candida tropicalis isolates, Denmark. Each node represents a distinct genotype; node size is proportional to the number of strains sharing the same allelic profile. Lines connecting nodes indicate genetic differences: thick solid black indicates 1 allele difference; thin solid black, 2 alleles; thin solid blue, 3 alleles; thin dashed blue, 4 alleles; orange dashed, 5 alleles; orange dotted, 6 alleles; and grey dotted, >6 alleles. Node colors represent the year of collection and 5-fluorocytosine resistance status. Non–wild-type strains with the E49X alteration form a clonal complex with minimal genetic variation over a 20-year period. In contrast, isolates carrying the Q7X or K6NfsX10 alterations are genetically unrelated to each other and to other strains, suggesting sporadic acquisition of resistance. Note: Line length does not reflect evolutionary distance. Boxed items at top of key indicate MICs for non–wild-type strains. S, susceptible.

Figure 2

FCY2 sequence encoding the purine-cytosine permease derived from contig 14 of the Candida tropicalis strain ATCC 750 genome assembly, published on the ATCC Genome Portal in 2020. The coding sequence spans positions 2152980–2154500. The ORF is depicted at the top left of the figure; long red arrow illustrates the FCY2 forward primer designed by Desnos-Ollivier et al. (12), and the blue arrow indicates the ORF they used. The green arrow represents the tryptophan codon TGG, which is converted to stop codon TGA in the MYA 3404 genome assembly. CDS, coding sequence; ORF, open reading frame.