Heteroresistance to Fluconazole in Cryptococcus neoformans Is Intrinsic and Associated with Virulence
Open accessAbstract
In 1999, heteroresistance to triazoles was reported in Cryptococcus neoformans strains isolated from an azole therapy failure case of cryptococcosis in an AIDS patient and in a diagnostic strain from a non-AIDS patient. In this study, we analyzed 130 strains of C. neoformans isolated from clinical and environmental sources before 1979, prior to the advent of triazoles, and 16 fluconazole (FLC)-resistant strains isolated from AIDS patients undergoing FLC maintenance therapy during 1990 to 2000. All strains isolated prior to 1979 manifested heteroresistance (subset of a population that grows in the presence of FLC) at concentrations between 4 and 64 μg/ml, and all 16 FLC-resistant AIDS isolates manifested heteroresistance at concentrations between 16 and 128 μg/ml. Upon exposure to stepwise increases in the concentration of FLC, subpopulations that could grow at higher concentrations emerged. Repeated transfer on drug-free media caused the highly resistant subpopulations to revert to the original level of heteroresistance. The reversion pattern fell into four categories based on the number of transfers required. The strains heteroresistant at ≥32 μg/ml were significantly more resistant to other xenobiotics and were also more virulent in mice than were those heteroresistant at ≤8 μg/ml. During FLC treatment of mice infected by strains with low levels of heteroresistance, subpopulations exhibiting higher levels of heteroresistance emerged after a certain period of time. The ABC transporter AFR1 , known to efflux FLC, was unrelated to the heteroresistance mechanism. Our study showed that heteroresistance to azole is universal and suggests that heteroresistance contributes to relapse of cryptococcosis during azole maintenance therapy.
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For C. neoformans and C. gattii, heteroresistance to fluconazole is intrinsic and increased resistance can be induced.
“For C. neoformans and C. gattii, the heteroresistance to fluconazole is intrinsic and increased resistance can be induced [19,102].”
From Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance (2025) · cites this paper as [102]
The Abstract and Results (page not provided) show that fluconazole heteroresistance is intrinsic in C. neoformans and that exposure to increasing fluconazole concentrations can produce subpopulations with higher resistance. The strains described are C. neoformans; our copy does not establish either point for C. gattii. (LLM, conf=0.88)
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 [25]
The Abstract defines heteroresistance as a resistant subpopulation growing at a drug concentration that inhibits the majority of cells. The Results report a representative strain with 0.3–0.6% resistant cells, but also report a strain with a 10% resistant subpopulation; the paper does not establish that the resistant fraction is usually much less than 1%. (LLM, conf=0.86)
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 [25]
The Abstract and Discussion (pages not provided) report early observations of heteroresistance in C. neoformans and conclude that fluconazole heteroresistance is intrinsic in the strains tested. They do not establish the claim for C. gattii. The Discussion says whether a chromosome-based mechanism explains heteroresistance in C. neoformans remains to be determined, so it does not support the asserted association with disomic chromosomes. (LLM, conf=0.89)
Experimental — accuracy not yet measuredIn a mouse model, heteroresistant C. cryptococcus isolates increase virulence.
“For fungi, a mouse model showed that heteroresistant C. cryptococcus isolates suppress the host immune response to enhance evasion, achieve higher fungal burden in different organs, and increase virulence [48,102].”
From Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance (2025) · cites this paper as [102]
The Results report that three high-level fluconazole-heteroresistant C. neoformans strains caused greater mouse mortality than low-level heteroresistant strains; the Discussion describes an association between heteroresistance level and virulence, not an increase for every isolate. The paper does not establish the citing sentence’s claims about suppression of the host immune response or higher fungal burdens in different organs. (LLM, conf=0.87)
Experimental — accuracy not yet measuredThe presence on chr1 of ERG11, the drug target of fluconazole, and AFR1, which encodes the drug efflux pump, explains why chr1 disomy is associated with fluconazole heteroresistance; however, AFR1 does not directly lead to heteroresistance.
“This is due to the presence on chr1 of ERG11, which is the drug target of fluconazole, and AFR1, which encodes the drug efflux pump. However, the AFR1 gene does not directly lead to heteroresistance [102].”
From Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance (2025) · cites this paper as [102]
The Results and Discussion state that AFR1 contributes to fluconazole resistance but is not involved in the mechanism of heteroresistance, supporting the sentence’s final point. The paper does not establish that ERG11 and AFR1 on chromosome 1 explain an association between chromosome 1 disomy and heteroresistance; the Discussion says whether a similar aneuploidy mechanism operates in C. neoformans remains to be elucidated. Page numbers were not available in our copy. (LLM, conf=0.88)
Experimental — accuracy not yet measured
In a mouse model, heteroresistant C. cryptococcus isolates achieve higher fungal burden in different organs.
“For fungi, a mouse model showed that heteroresistant C. cryptococcus isolates suppress the host immune response to enhance evasion, achieve higher fungal burden in different organs, and increase virulence [48,102].”
From Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance (2025) · cites this paper as [102]
The Methods describe a mouse study that measured fungal burden in the brain, and the Results report brain-burden differences associated with strains’ heteroresistance levels during fluconazole treatment. They do not report higher fungal burden across different organs; the separate virulence comparison measured mouse survival, not organ burdens. No page numbers were supplied for these passages. (LLM, conf=0.83)
Experimental — accuracy not yet measuredIn a mouse model, heteroresistant C. cryptococcus isolates suppress host immune response to enhance evasion.
“For fungi, a mouse model showed that heteroresistant C. cryptococcus isolates suppress the host immune response to enhance evasion, achieve higher fungal burden in different organs, and increase virulence [48,102].”
From Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance (2025) · cites this paper as [102]
The Results report that three high-level fluconazole-heteroresistant C. neoformans strains caused greater mortality in mice than low-level heteroresistant strains. The paper does not report suppression of the host immune response or immune evasion; its fungal-burden measurements concern the brain, not multiple organs. (LLM, conf=0.86)
Experimental — accuracy not yet measuredThe majority subpopulation in heteroresistance can never be purified, meaning a minority remains phenotypically resistant.
“Interestingly, this majority subpopulation can never be purified, meaning that a minority remains phenotypically resistant [102].”
From Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance (2025) · cites this paper as [102]
There are studies showing existence of intrinsic heteroresistance.
“However, there are also studies showing the existence of intrinsic heteroresistance [48,102]”
From Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance (2025) · cites this paper as [102]
The traits of genomic plasticity and physiological adaptability can be inherited and are often transient, such that they are lost when the stress is removed.
“These traits can be inherited, and are often transient, such that they are lost when the stress is removed17,18.”
From Treatment strategies for cryptococcal infection: challenges, advances and future outlook (2021) · cites this paper as [18]
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References 58 · Cited by 179
Research works printed but not linked: 1
- [11]Casadevall, A., and J. R. Perfect. 1998. Cryptococcus neoformans. ASM Press, Washington, DC.
- Authors
- Sionov, E., Chang, Y. C., Garraffo, H. M., & Kwon-Chung, K. J.
- Affiliations
- National Institutes of Health (US)
- National Institute of Allergy and Infectious Diseases (US)
- Corresponding author
- Kwon-Chung, Kyung J.
- Venue
- Antimicrobial Agents and Chemotherapy
- Publisher
- American Society for Microbiology
- Published
- 2009-07
- Volume
- 53
- Issue
- 7
- Pages
- 2804-2815
- ISSN
- 0066-4804 (ISSN-L)
- 1098-6596
- PMC
- PMC2704677
- OpenAlex
- W2052931063
- Cited by
- 179
- References
- 58
- Citations by year
- 2026: 10 · 2025: 14 · 2024: 14 · 2023: 10 · 2022: 12 · 2021: 10 · 2020: 6 · 2019: 14 · 2018: 11 · 2017: 13 · 2016: 10 · 2015: 8 · 2014: 11 · 2013: 9 · 2012: 10
- FWCI
- 5.85
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Antimicrobial Agents and Chemotherapy (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health
- National Institute of Allergy and Infectious Diseases
- Topics
- Health Sciences › Medicine › Epidemiology › Fungal Infections and Studies
- Health Sciences › Medicine › Infectious Diseases › Antifungal resistance and susceptibility
- Life Sciences › Agricultural and Biological Sciences › Ecology, Evolution, Behavior and Systematics › Fungal Plant Pathogen Control
- Keywords
- Cryptococcus neoformans, intrinsic drug resistance, triazole resistance, cryptococcosis, AIDS, virulence, xenobiotic resistance, relapse, clinical and environmental isolates
- MeSH terms
- Animals, Antifungal Agents, Azoles, Cryptococcosis, Cryptococcus neoformans, Female, Fungal Proteins, Humans, Mice, Inbred BALB C, Microbial Sensitivity Tests, Virulence, Fluconazole, Reverse Transcriptase Polymerase Chain Reaction, Drug Resistance, Fungal, Mice
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