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Cited by 511Antifungal resistance and susceptibilityFungal and yeast genetics researchFungal Plant Pathogen Control

Mechanism of Fluconazole Resistance in Candida albicans Biofilms: Phase-Specific Role of Efflux Pumps and Membrane Sterols

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Mukherjee, Pranab K., Chandra, Jyotsna, Kuhn, Duncan M., Ghannoum, Mahmoud A.·2003·Infection and Immunity
DOI 10.1128/iai.71.8.4333-4340.2003PMC PMC165995
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Abstract

Candida albicans biofilms are formed through three distinct developmental phases and are associated with high fluconazole (FLU) resistance. In the present study, we used a set of isogenic Candida strains lacking one or more of the drug efflux pumps Cdr1p, Cdr2p, and Mdr1p to determine their role in FLU resistance of biofilms. Additionally, variation in sterol profile as a possible mechanism of drug resistance was investigated. Our results indicate that parent and mutant strains formed similar biofilms. However, biofilms formed by double and triple mutants were more susceptible to FLU at 6 h (MIC = 64 and 16 μg/ml, respectively) than the wild-type strain (MIC > 256 μg/ml). At later time points (12 and 48 h), all the strains became resistant to this azole (MIC ≥ 256 μg/ml), indicating lack of involvement of efflux pumps in resistance at late stages of biofilm formation. Northern blot analyses revealed that Candida biofilms expressed CDR and MDR1 genes in all the developmental phases, while planktonic cells expressed these genes only at the 12- and 48-h time points. Functionality of efflux pumps was assayed by rhodamine (Rh123) efflux assays, which revealed significant differences in Rh123 retention between biofilm and planktonic cells at the early phase ( P = 0.0006) but not at later stages (12 and 48 h). Sterol analyses showed that ergosterol levels were significantly decreased ( P < 0.001) at intermediate and mature phases, compared to those in early-phase biofilms. These studies suggest that multicomponent, phase-specific mechanisms are operative in antifungal resistance of fungal biofilms.

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  • Ergosterol levels are significantly decreased in both intermediate (12-30h) and mature phases (31-72h) biofilms compared to early-phase (0-11h) biofilms, and this is connected to decreased expression of ERG25, ERG11, and ERG6.

    “Sterol analyses show that ergosterol levels are signi fi cantly decreased in both intermediate (12-30h) and mature phases (31-72h), compared to those in earlyphase (0-11h) bio fi lms, and this is connected to the decreased expression of ergosterol biosynthetic genes ( ERG25 , ERG11 , and ERG6 ).”

    From The synthesis, regulation, and functions of sterols in<i>Candida albicans</i>: Well-known but still lots to learn (2016) · cites this paper as [75]

    The Results report significantly lower ergosterol at 12 and 48 hours than at 6 hours (P < 0.001), supporting the phase comparison at the time points tested. The paper does not connect that decrease to reduced expression of ERG25, ERG11, or ERG6; its gene-expression analysis concerns the efflux-pump genes CDR1, CDR2, and MDR1. It also does not establish the stated reductions across the full phase-hour ranges. (LLM, conf=0.88)

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  • Induction of several ERG genes has been reported during biofilm development, but biofilm cells have also been reported to contain less ergosterol in their cell membranes compared to free-living cells, which possibly reduces the efficiency of azole antifungals.

    “Induction of several ERG genes has been reported during biofilm development38, but contradictory, it was also reported that biofilm cells contain less ergosterol in their cell membranes compared to free-living cells, which possibly reduces the efficiency of azole antifungals affecting biosynthesis of this compound22.”

    From Stimulation of superoxide production increases fungicidal action of miconazole against Candida albicans biofilms (2016) · cites this paper as [22]

    Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

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  • The combination of deletion in the three common ABC transporters does not eliminate drug resistance.

    “This hypothesis is supported by the observation that the combination of deletion in the three common ABC transporters does not eliminate drug resistance [29]”

    From Known Antimicrobials Versus Nortriptyline in Candida albicans: Repositioning an Old Drug for New Targets (2020) · cites this paper as [29]

  • Certain efflux pumps in planktonic cells are up-regulated within the first few hours of surface contact with azole antifungals and remain up-regulated throughout biofilm development.

    “Certain efflux pumps in planktonic cells are often up-regulated within the first few hours of surface contact with azole antifungals and remain up-regulated throughout the biofilm development.”

    From Molecular and genetic basis of azole antifungal resistance in the opportunistic pathogenic fungus<i>Candida albicans</i> (2020) · cites this paper as [171]

  • While under treatment with antifungals, biofilm cells up-regulate these transporters within six hours of surface contact both in vitro and in vivo, even in the absence of drug.

    “While under treatment with antifungals, biofilm cells up-regulate these transporters within six hours of surface contact both in vitro and in vivo, even in the absence of drug [136,137,138,139].”

    From Candida glabrata Biofilms: How Far Have We Come? (2017) · cites this paper as [138]

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Research works printed but not linked: 2

  1. [24]Hitchcock, C. A., N. J. Russell, and K. J. Barrett-Bee. 1987. Sterols in Candida albicans mutants resistant to polyene or azole antifungals, and of a double mutant C. albicans 6.4. Crit. Rev. Microbiol. 15:111-115. [DOI] [PubMed] [Google Scholar]
  2. [31]Larsen, T., and N. E. Fiehn. 1996. Resistance of Streptococcus sanguis biofilms to antimicrobial agents. APMIS 104:280-284. [PubMed] [Google Scholar]

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Authors
Mukherjee, P. K., Chandra, J., Kuhn, D. M., & Ghannoum, M. A.
Affiliations
University Hospitals of Cleveland (US)
ORCID
Venue
Infection and Immunity
Publisher
American Society for Microbiology
Published
2003-08
Volume
71
Issue
8
Pages
4333-4340
ISSN
  • 0019-9567 (ISSN-L)
  • 1098-5522
DOI
10.1128/iai.71.8.4333-4340.2003
PMC
PMC165995
OpenAlex
W2059111198
Cited by
511
References
56
Citations by year
2026: 10 · 2025: 18 · 2024: 14 · 2023: 28 · 2022: 29 · 2021: 23 · 2020: 26 · 2019: 19 · 2018: 25 · 2017: 33 · 2016: 40 · 2015: 33 · 2014: 27 · 2013: 18 · 2012: 23
FWCI
7.38
Type
journal article
Language
en
Open access
Licence
journals.asm.org/non-commercial-tdm-license (tdm)
Locations
  • Infection and Immunity (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • Case Western Reserve University (AI-36219)
  • National Institutes of Health (AI36219)
  • Center for AIDS Research, University of Washington
  • National Institute of Allergy and Infectious Diseases (AI36219, P30AI036219)
  • National Institute of Dental and Craniofacial Research (R01-DE13992, 1R01 DE13932-01A1, R01DE013992, R01DE013932)
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 albicans, biofilm, fluconazole resistance, efflux pumps, Cdr1p, sterols, ergosterol, antifungal resistance, azole resistance
MeSH terms
Antifungal Agents, Base Sequence, Candida albicans, DNA, Fungal, Fluorescent Dyes, Fungal Proteins, Genes, Fungal, Mutation, Sterols, Fluconazole, Gene Expression Regulation, Fungal, Gene Deletion, Biofilms, ATP-Binding Cassette Transporters, Rhodamine 123, ATP Binding Cassette Transporter, Subfamily B, Member 1, Drug Resistance, Fungal, Membrane Transport Proteins

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