Heteroresistance to Fluconazole Is a Continuously Distributed Phenotype among Candida glabrata Clinical Strains Associated with In Vivo Persistence
Open accessAbstract
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 FLC HR . 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 ATP-binding 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. IMPORTANCE Heteroresistance refers to variability in the response to a drug within a clonal cell population. This phenomenon may have crucial importance for the way we look at antimicrobial resistance, as heteroresistant strains are not detected by standard laboratory susceptibility testing and may be associated with failure of antimicrobial therapy. We describe for the first time heteroresistance to fluconazole in C. glabrata , a finding that may explain the propensity of this pathogen to acquire resistance following exposure to fluconazole and to persist despite treatment. We found that, rather than being a binary all-or-none trait, heteroresistance was a continuously distributed phenotype associated with increased expression of genes that encode energy-dependent drug efflux transporters. Moreover, we show that heteroresistance is associated with failure of fluconazole to clear infection with C. glabrata . Together, these findings provide an empirical framework for determining and quantifying heteroresistance in C. glabrata .
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Heteroresistance was not present in wild-type C. glabrata strains.
“Further, this mechanism occurred independent of heteroresistance, which was not present in wild-type C. glabrata strains (7).”
From Echinocandin tolerance and persistence <i>in vitro</i> are regulated by calcineurin signaling in <i>Candida glabrata</i> (2026) · cites this paper as [7]
The Abstract reports heteroresistance in 30 of 52 fluconazole-sensitive C. glabrata isolates and says quantitative analysis suggests all isolates exhibit some degree of it. That contradicts the blanket claim that heteroresistance was absent from wild-type strains. The copy does not label those isolates specifically as “wild-type.” (LLM, conf=0.88)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measuredThe production of long-lived persister-like cells in C. glabrata occurred independent of heteroresistance.
“Further, this mechanism occurred independent of heteroresistance, which was not present in wild-type C. glabrata strains (7).”
From Echinocandin tolerance and persistence <i>in vitro</i> are regulated by calcineurin signaling in <i>Candida glabrata</i> (2026) · cites this paper as [7]
The Results section ('HR is associated with persistence of viable C. glabrata cells in mouse kidney tissue'; page unavailable) reports persistent infection in both groups, but significantly more often with a heteroresistant strain (45.8% versus 15%). The Discussion (page unavailable) also reports heteroresistance in 30 of 52 fluconazole-susceptible clinical isolates, contrary to the citing sentence’s claim that it was absent. The paper does not establish production of long-lived persister-like cells independently of heteroresistance. (LLM, conf=0.87)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measuredBy altering gene dosage or expression levels, aneuploidy allows fungi to quickly adjust to adverse conditions without permanent genetic mutations, promoting survival in fluctuating environments.
“By altering gene dosage or expression levels, aneuploidy allows fungi to quickly adjust to adverse conditions without permanent genetic mutations, promoting survival in fluctuating environments [99,129,145,152,154].”
From Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance (2025) · cites this paper as [99]
The Results report increased expression of efflux-transporter genes in heteroresistant Candida glabrata strains, and the Discussion links heteroresistance to persistence during fluconazole treatment. Neither section attributes these effects to aneuploidy or shows that altered gene dosage enables rapid adaptation without permanent mutations; the claim assigns that mechanism to the source without support. (LLM, conf=0.85)
Experimental — accuracy not yet measuredAntifungal heteroresistance was first found in C. neoformans and Cryptococcus gattii and is considered an intrinsic property of these species, associated with disomic chromosomes.
“Antifungal heteroresistance was first been found in C. neoformans and Cryptococcus gattii, and was considered as an intrinsic property of these species, associating with disomic chromosomes [24–27].”
From Antifungal persistence: Clinical relevance and mechanisms (2025) · cites this paper as [24]
The Introduction (page not provided) says heteroresistance has been described in Cryptococcus neoformans. It does not say it was first found there or in C. gattii, or that it is intrinsic to those species and associated with disomic chromosomes. Searches for C. gattii and disomy found no supporting passage in our copy. (LLM, conf=0.85)
Experimental — accuracy not yet measuredBy increasing the copy number of chromosomes containing drug resistance genes, aneuploidy leads to resistance against multiple antifungals including azoles and echinocandins.
“By increasing the copy number of chromosomes containing drug resistance genes, such as those encoding transcription factors, efflux pumps or involved in ergosterol biosynthesis, aneuploidy leads to resistance against multiple antifungals. including azoles and echinocandins [99,129,154].”
From Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance (2025) · cites this paper as [99]
The Introduction (page not provided) links azole resistance in Candida glabrata to upregulated drug transporters and notes emerging echinocandin resistance. It does not attribute resistance to aneuploidy or increased chromosome copy number. The Results (page not provided) also found no significant echinocandin heteroresistance in the isolates studied. (LLM, conf=0.84)
Experimental — accuracy not yet measured
Heteroresistant N. glabrata isolates can lead to higher fungal burden in mice kidneys.
“Heteroresistant N. glabrata isolates can lead to higher fungal burden in mice kidneys, making it harder to thoroughly clear the pathogen and resulting in persistent infection [99].”
From Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance (2025) · cites this paper as [99]
The Results section reports that high kidney fungal burdens and persistent infection occurred more often in mice infected with a highly fluconazole-heteroresistant C. glabrata strain than in mice infected with a nonheteroresistant strain. The finding is specifically for mice receiving seven days of fluconazole treatment; the citing sentence omits that condition and describes higher burden rather than a higher frequency of persistent high burdens. (LLM, conf=0.83)
Experimental — accuracy not yet measuredAntifungal heteroresistance refers to the coexistence of a small population of resistant cells, usually much less than 1%, and a large population of susceptible fungal cells.
“Antifungal heteroresistance refers to the coexistence of a small population of resistant cells (usually <<1%) and a large population of susceptible fungal cells (Fig 1).”
From Antifungal persistence: Clinical relevance and mechanisms (2025) · cites this paper as [24]
Heteroresistance is more continuous than binary (on/off).
“As a variant, it is more of continuous than binary (on/off).”
From Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance (2025) · cites this paper as [99]
Heteroresistant N. glabrata isolates make it harder to thoroughly clear the pathogen and result in persistent infection.
“Heteroresistant N. glabrata isolates can lead to higher fungal burden in mice kidneys, making it harder to thoroughly clear the pathogen and resulting in persistent infection [99].”
From Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance (2025) · cites this paper as [99]
Misclassification of heteroresistant infections as susceptible can lead to selection of antifungal therapies ineffective against resistant subpopulations, resulting in treatment failure, persistent infections, or emergence of fully resistant strains.
“This misclassification can lead to the selection of antifungal therapies that are ineffective against resistant subpopulations, resulting in treatment failure, persistent infections, or even the emergence of fully resistant strains [99,165].”
From Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance (2025) · cites this paper as [99]
Heteroresistance might cause false outcomes in susceptibility tests, which may result in the misidentification of potentially resistant isolates as susceptible and even in fatal treatment failure.
“To complicate things further, heteroresistance might cause false outcomes in susceptibility tests, which may result in the misidentification of potentially resistant isolates as susceptible and even in fatal treatment failure [137].”
From Evolutionary Emergence of Drug Resistance in Candida Opportunistic Pathogens (2018) · cites this paper as [137]
Heteroresistance to fluconazole is highly frequent among clinical C. glabrata strains, and is associated with failure of fluconazole to clear C. glabrata from tissues of experimentally infected immunocompetent mice.
“Heteroresistance, specifically to fluconazole, was also shown to be highly frequent among clinical C. glabrata strains, and is associated with failure of fluconazole to clear C. glabrata from tissues of experimentally infected immunocompetent mice [68].”
From Treatment of Invasive Candidiasis: A Narrative Review (2018) · cites this paper as [68]
Heteroresistance to fluconazole is associated with up-regulation of ABC-type drug transporters, and may be detected and quantified using population analysis profiling.
“Heteroresistance to fluconazole is associated with up-regulation of ABC-type drug transporters, and may be detected and quantified using population analysis profiling [68], a method not routinely used by clinical microbiological laboratories.”
From Treatment of Invasive Candidiasis: A Narrative Review (2018) · cites this paper as [68]
C. glabrata heteroresistance was not observed in response to the echinocandins.
“C. glabrata heteroresistance was not observed in response to the echinocandins”
From Genetic Drivers of Multidrug Resistance in Candida glabrata (2016) · cites this paper as Ben-Ami et al., 2016
C. glabrata subpopulations originating from a clonal population can develop heteroresistance to fluconazole through transient upregulation of efflux pumps.
“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).”
From Genetic Drivers of Multidrug Resistance in Candida glabrata (2016) · cites this paper as Ben-Ami et al., 2016
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References 36 · Cited by 88
- Authors
- Ben-Ami, R., Zimmerman, O., Finn, T., Amit, S., Novikov, A., Wertheimer, N., Lurie-Weinberger, M., & Berman, J.
- Affiliations
- Infectious Diseases Unit, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel
- Tel Aviv Sourasky Medical Center (IL)
- Tel Aviv University (IL)
- Corresponding author
- Berman, Judith
- Venue
- mBio
- Publisher
- American Society for Microbiology
- Published
- 2016-09-07
- Volume
- 7
- Issue
- 4
- ISSN
- 2150-7511 (ISSN-L)
- 2161-2129
- PMC
- PMC4981708
- OpenAlex
- W2495234190
- Cited by
- 88
- References
- 36
- Citations by year
- 2026: 10 · 2025: 13 · 2024: 7 · 2023: 6 · 2022: 5 · 2021: 9 · 2020: 12 · 2019: 5 · 2018: 12 · 2017: 7 · 2016: 2
- FWCI
- 8.00
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- mBio (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Ben Dov Physician Researcher Grant
- European Union's 7th Framework Programme (303635)
- EC | European Research Council (340087)
- Topics
- Health Sciences › Medicine › Infectious Diseases › Antifungal resistance and susceptibility
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
- Life Sciences › Agricultural and Biological Sciences › Ecology, Evolution, Behavior and Systematics › Fungal Plant Pathogen Control
- Keywords
- Candida glabrata, population analysis profiling, drug efflux, ABC transporters, CDR1, hematogenous infection, immunocompetent mice, clonal variation, antifungal susceptibility
- MeSH terms
- Animals, Antifungal Agents, Biological Transport, Candidiasis, Disease Models, Animal, Genetics, Population, Humans, Phenotype, Genetic Variation, Fluconazole, ATP-Binding Cassette Transporters, Drug Resistance, Fungal, Candida glabrata, Epigenesis, Genetic, Mice
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