Heteroresistance to Fluconazole Is a Continuously Distributed Phenotype among Candida glabrata Clinical Strains Associated with In Vivo Persistence

Abstract

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 (FLCHR) 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 FLCHR 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 ATP-binding cassette (ABC) transmembrane transporters, implying that HetR populations exhibit higher levels of drug efflux. Highly FLCHR 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 FLCHR 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.

Genetic Drivers of Multidrug Resistance in Candida glabrata

“…As stated earlier, specific pdr1 mutations can also enhance the fitness of C. glabrata and increase virulence through adhesion upregulation (Vale-Silva et al, 2016). C. glabrata heteroresistance was not observed in response to the echinocandins, which may reflect its fungicidal mechanism of action (at least in vitro ) (Ben-Ami et al, 2016) or potential associated fitness costs. However, yeast are exposed to additional pressures in vivo that may induce some kind of transient heterotolerance to the echinocandins that is not necessarily measurable in vitro .…”

“…Although acquisition of a pdr1 mutation is required for stable triazole resistance, studies have demonstrated the ability of C. glabrata subpopulations originating from a clonal population to develop heteroresistance to fluconazole through transient upregulation of efflux pumps (Claudino et al, 2009; Ben-Ami et al, 2016). Development of tolerance to fluconazole treatment at the population level may be one consequence of heteroresistance, which in turn, may allow the yeast to adapt and “buy time” until beneficial, stable mutations develop.…”

Rapid detection of <i>Candida albicans</i> in oral exfoliative cytology samples by loop-mediated isothermal amplification

“…In vitro studies have shown that the adhesion of non-albicans species to buccal epithelial and vascular endothelial cells is weaker than that of C. albicans (26). C. glabrata, which is the second most frequently isolated fungus causing single infections or combined infections with C. albicans, causes persistent infections in patients treated with fluconazole and often acquires resistance to antifungal drugs (27). Because the sensitivity to antifungal drugs varies, identification of Candida species is important for treatment of candidiasis, allowing physicians to prescribe the appropriate antifungal drug.…”

Heteroresistance and fungi

“…For fungi, some studies show that heteroresistance to azoles seems to be intrinsic and others indicate that it is acquired. Several studies focused on Cryptococcus heteroresistance showed that all strains tested in repeated exposure to fluconazole or itraconazole led to a subset of resistant subpopulations . However, different data were found for Candida glabrata, Aspergillus fumigatus and even for Cryptococcus neoformans (Table ).…”

“…Several studies focused on Cryptococcus heteroresistance showed that all strains tested in repeated exposure to fluconazole or itraconazole led to a subset of resistant subpopulations . However, different data were found for Candida glabrata, Aspergillus fumigatus and even for Cryptococcus neoformans (Table ). These difference in the results may be attributed to the subset of strains tested and also to the different methodologies used, demonstrating the need for well‐established standards to study heteroresistance.…”