Heteroresistance to fluconazole is a continuously distributed phenotype among candida glabrata clinical strains associated with in vivo persistence

Candida glabrata causes persistent infections in patients treated with fluconazole and often acquires resistance following exposure to the drug. Here we found that clinical strains of C. glabrata exhibit cell-to-cell variation in drug response (heteroresistance). We used population analysis profiling (PAP) to assess fluconazole heteroresistance (FLC^HR) and to ask if it is a binary trait or a continuous phenotype. Thirty (57.6%) of 52 fluconazole-sensitive clinical C. glabrata isolates met accepted dichotomous criteria for FLCHR. However, quantitative grading of FLC^HR by using the area under the PAP curve (AUC) revealed a continuous distribution across a wide range of values, suggesting that all isolates exhibit some degree of heteroresistance. The AUC correlated with rhodamine 6G efflux and was associated with upregulation of the CDR1 and PDH1 genes, encoding ATPbinding cassette (ABC) transmembrane transporters, implying that HetR populations exhibit higher levels of drug efflux. Highly FLC^HR C. glabrata was recovered more frequently than nonheteroresistant C. glabrata from hematogenously infected immunocompetent mice following treatment with high-dose fluconazole (45.8% versus 15%, P=0.029). Phylogenetic analysis revealed some phenotypic clustering but also variations in FLC^HR within clonal groups, suggesting both genetic and epigenetic determinants of heteroresistance. Collectively, these results establish heteroresistance to fluconazole as a graded phenotype associated with ABC transporter upregulation and fluconazole efflux. Heteroresistance may explain the propensity of C. glabrata for persistent infection and the emergence of breakthrough resistance to fluconazole.