Nitrogen concentration affects amphotericin B and fluconazole tolerance of pathogenic cryptococci
Open accessAbstract
Environmental stress often causes phenotypic changes among pathogenic cryptococci, such as altered antifungal susceptibility, changes in capsule and melanin formation, as well as altered levels of the membrane sterol and antifungal target, ergosterol. We therefore hypothesised that nitrogen limitation, a prevalent environmental stress in the natural habitat of these yeasts, might affect virulence and antifungal susceptibility. We tested the effect of different nitrogen concentrations on capsule, melanin and ergosterol biosynthesis, as well as amphotericin B (AmB) and fluconazole (FLU) susceptibility. This was achieved by culturing cryptococcal strains representing Cryptococcus neoformans and Cryptococcus gattii in media with high (0.53 g/l), control (0.42 g/l) and low (0.21 g/l) NH4Cl concentrations. India ink staining was used to determine capsule thickness microscopically, while melanin and ergosterol content were determined spectrophotometrically. We found that lower nitrogen concentrations enhanced both ergosterol and capsule biosynthesis, while a variable effect was observed on melanisation. Evaluation of drug tolerance using time-kill methodology, as well as tests for FLU heteroresistance, revealed that the low nitrogen cultures had the highest survival percentages in the presence of both AmB and FLU, and showed the highest frequency of FLU heteroresistance, suggesting that nitrogen concentration may indeed influence drug tolerance.
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Environmental stress, such as nitrogen limitation, increases frequency of heterogeneous resistance to fluconazole in C. neoformans.
“Bosch et al. demonstrated that environmental stress, such as the nitrogen limitation commonly encountered in its natural habitats, increases the resistance of C. neoformans to AmB and fluconazole, and increases the frequency of heterogeneous resistance to fluconazole [57].”
From Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance (2025) · cites this paper as [57]
The ‘Frequency of fluconazole heteroresistance/tolerance’ section reports a higher combined frequency under low nitrogen, but identifies only C. gattii CAB 761 as heteroresistant. It classifies the tested C. neoformans strains as fluconazole-tolerant, not heteroresistant. No page number is available in our copy. (LLM, conf=0.90)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measuredHeteroresistance may be induced by pharmacological factors or non-pharmacological agents.
“Heteroresistance may be induced by pharmacological factors or non-pharmacological agents.”
From Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance (2025) · cites this paper as [57]
The Frequency of fluconazole heteroresistance/tolerance section reports more heteroresistance/tolerance after growth under low nitrogen, a non-pharmacological condition, although only one of four strains produced heteroresistant colonies. Fluconazole was used to identify those colonies; the passages do not establish that a pharmacological factor induced heteroresistance. (LLM, conf=0.83)
Experimental — accuracy not yet measuredEnvironmental stress, such as nitrogen limitation, increases resistance of C. neoformans to fluconazole.
“Bosch et al. demonstrated that environmental stress, such as the nitrogen limitation commonly encountered in its natural habitats, increases the resistance of C. neoformans to AmB and fluconazole, and increases the frequency of heterogeneous resistance to fluconazole [57].”
From Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance (2025) · cites this paper as [57]
The Time-kill assays section reports that low-nitrogen cultures, including C. neoformans, survived amphotericin B and fluconazole exposure better than high-nitrogen cultures. But the Frequency of fluconazole heteroresistance/tolerance section classifies the tested C. neoformans strains as fluconazole-tolerant, not heteroresistant; increased heteroresistance was observed in one C. gattii strain. The claim overstates tolerance as resistance and extends the heteroresistance finding to C. neoformans. (LLM, conf=0.90)
Experimental — accuracy not yet measuredEnvironmental stress, such as nitrogen limitation commonly encountered in natural habitats, increases resistance of C. neoformans to AmB.
“Bosch et al. demonstrated that environmental stress, such as the nitrogen limitation commonly encountered in its natural habitats, increases the resistance of C. neoformans to AmB and fluconazole, and increases the frequency of heterogeneous resistance to fluconazole [57].”
From Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance (2025) · cites this paper as [57]
The Time-kill assays section reports that low-nitrogen cultures, including C. neoformans, survived AmB exposure better than cultures grown with more nitrogen. The Discussion describes this as increased drug tolerance and says low-nitrogen cultures showed no change in minimum inhibitory concentration (MIC), so the paper does not establish increased AmB resistance in the stricter sense. (LLM, conf=0.85)
Experimental — accuracy not yet measuredIn C. neoformans, time kill assays have revealed that subpopulations of cells can survive for prolonged periods despite presence of high drug concentrations.
“In C. neoformans, time kill assays have been used to study azole and AmB heteroresistance, revealing that subpopulations of cells can survive for prolonged periods despite the presence of high drug concentrations [57].”
From Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance (2025) · cites this paper as [57]
The Time-kill assays section reports survival of C. neoformans strains after exposure to fluconazole and amphotericin B, with the highest survival in low-nitrogen cultures even after 24 hours. But the paper identifies heteroresistant or tolerant subpopulations in a separate fluconazole-plating assay, not through the time-kill assays; its amphotericin B time-kill concentrations also did not exceed the MIC. Page numbers are unavailable in our copy. (LLM, conf=0.85)
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References 70 · Cited by 12
Research works printed but not linked: 6
- Breivik ON, Owades JL. Spectrophotometric semi- microdetermination of ergosterol in yeast. J Agr Food Chem 1957;5:360-3.
- Emmons CW. Saprophytic sof Cryptococcus neoformans associ- ated with the pigeon (Columba livia). Am J Hyg 1955;62: 227-32.
- Ene IV, Brunke S, Brown AJ et al. Metabolism in fungal pathogen- esis. Cold Spring Harb Perspect Med 2014;4:a019695.
- Kwon-Chung KJ, Fraser JA, Doering TL et al. Cryptococcus neofor- mans and Cryptococcus gattii, the etiologic agents of crypto- coccosis. Cold Spring Harb Perspect Med 2014;4:a019760.
- Mondon P, Petter R, Amalfitano G et al. Heteroresistance to flu- conazole and voriconazole in Cryptococcus neoformans. Antimi- crob Agents Chemother 1999;43:1856-61.
- Velagapudi R, Hsueh YP, Geunes-Boyer S et al. Spores as infec- tious propagules of Cryptococcus neoformans. Infect Immun 2009;77:4345-55.
- Authors
- Bosch, C., Toplis, B., Vreulink, J.-M., Volschenk, H., & Botha, A.
- Affiliations
- Stellenbosch University (ZA)
- Corresponding author
- Botha, Alfred
- Venue
- FEMS Yeast Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2020-02-19
- Volume
- 20
- Issue
- 2
- ISSN
- 1567-1356 (ISSN-L)
- 1567-1364
- OpenAlex
- W3006763031
- Cited by
- 12
- References
- 70
- Citations by year
- 2026: 1 · 2025: 2 · 2024: 1 · 2022: 4 · 2021: 4
- FWCI
- 1.43
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model (vor)
- Locations
- FEMS Yeast Research (publishedVersion)
- PubMed (publishedVersion)
- Funders
- South African National Research foundation
- Harry Crossley Foundation
- Topics
- Health Sciences › Medicine › Epidemiology › Fungal Infections and Studies
- Health Sciences › Medicine › Infectious Diseases › Antifungal resistance and susceptibility
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
- Keywords
- Cryptococcus neoformans, Cryptococcus gattii, nitrogen limitation, antifungal resistance, capsule biosynthesis, melanogenesis, ergosterol biosynthesis, virulence, environmental stress
- MeSH terms
- Ammonium Chloride, Amphotericin B, Antifungal Agents, Cryptococcus, Cryptococcus neoformans, Culture Media, Ergosterol, Melanins, Microbial Sensitivity Tests, Nitrogen, Fluconazole, Biosynthetic Pathways, Cryptococcus gattii
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