Review
Resistance of human fungal pathogens to antifungal drugs

https://doi.org/10.1016/S1369-5274(02)00344-2Get rights and content

Abstract

Resistance mechanisms can be engaged in clinically relevant fungal pathogens under different conditions when exposed to antifungal drugs. Over past years, active research was undertaken in the understanding of the molecular basis of antifungal drug resistance in these pathogens, and especially against the class of azole antifungals. The isolation of various alleles of the gene encoding the target of azoles has enabled correlation of the appearance of resistance with distinct mutations. Resistance mechanisms to azoles also converge to the upregulation of multidrug transporter genes, whose products have the capacity to extrude from cells several chemically unrelated antifungal agents and toxic compounds. Genome-wide studies of azole-resistant isolates are now permitting a more comprehensive analysis of the impact of resistance on gene expression, and may deliver new clues to their mechanisms. Several laboratories are also exploring, as well as possible alternative resistance pathways, the role of biofilm formation by several fungal species in the development of resistance to various antifungals, including azoles.

Introduction

When faced with antifungal drugs, fungal pathogens have, in principle, the capacity to overcome their inhibitory action through specific resistance mechanisms. This biological response, reflected in vitro by the ability to select mutants resistant to antifungal drugs, has even been used on several occasions to identify the cellular targets of antifungal drugs 1., 2.. In a clinical context, whenever antifungal agents are used to combat fungal infections, the exposure of fungal pathogens to these agents is therefore expected to give rise to resistant isolates. The increasing number of fungal infections documented in several hospital sites around the world could potentially favor the occurrence of this phenomenon, as the number of antifungal treatments will be higher [3]. The occurrence of resistance will be, of course, dependent on the type of fungal pathogen to be treated and the type of antifungal agents applied. An outlook on the different classes of antifungals is given in Table 1, which summarizes their use against major fungal pathogens and the occurrence of resistance among clinical isolates. The facts show that resistance to antifungal drugs, which is measured as an increase in minimum inhibitory concentration (MIC) as compared to values measured in susceptible reference organisms, has been reported in clinical use for three classes of antifungal drugs up to now: the polyenes, pyrimidine analogues (5-fluorocytosine) and the azoles (Table 1) [4•].

Resistance to azoles has a leading position in published reports. The repeated use of azoles (especially fluconazole) in treatments of HIV-positive patients with mucosal fungal infections in the period preceding the introduction of highly active antiretroviral therapy has favored the acquisition of azole resistance in several fungal pathogens. These were mostly Candida species, including (with decreasing importance) C. albicans, C. glabrata, C. dubliniensis and C. tropicalis, and (less frequently) Cryptococcus species [5]. Azole resistance in systemic fungal infections of severely immunocompromised patients is much less frequent and has been described mainly for C. albicans [6] and A. fumigatus species [7]. Mechanisms of azole resistance have been most extensively investigated in recent years, as a large number of yeast isolates were available to research laboratories. Several reviews are available that describe in detail the different mechanisms resulting in resistance to the azoles 8., 9., 10.. In this review, I summarize the most recent findings that deal with the molecular basis of the mechanisms of azole resistance in yeast pathogens, including Candida, Cryptococcus and Aspergillus species.

Section snippets

Resistance mechanisms involving target alterations

The major cellular target of azole compounds in yeast and fungi is a cytochrome P450 (Erg11p) involved in the demethylation of the lanosterol molecule in position 14α. This step is necessary for the biosynthesis of ergosterol, a fungal-specific sterol that maintains membrane functions. Inhibiting the activity of Erg11p by azoles leads to ergosterol depletion and accumulation of 14α-methylated sterols (lanosterol and 14α-methyl-3-6-diol) [11]. Both effects result in growth arrest and not in cell

Resistance mechanisms affecting antifungal transport

A prerequisite for the inhibitory action of azoles is to reach intracellular concentration levels that are able to block the function of every Erg11p molecule present in the membrane of the endoplasmic reticulum. The transport of azoles into the fungal cells is still not totally understood. Current models support the idea of passive transport through cell wall and cell membrane barriers, given that: first, modification of cell wall structures by altering the glycosylation of surface proteins

Molecular basis of multidrug transporter upregulation

The molecular basis of the upregulation of multidrug transporters belonging to the ABC and major facilitator families is being actively investigated in yeast pathogens. Several questions must be addressed. Are mutations responsible for upregulation in cis or in trans? Which are the regulatory elements present in the promoters of the genes? Which are the transcription factors responsible for gene upregulation? What are the pathways (CDR- and CaMDR1-specific) that start from drug exposure to

Resistance mechanisms and their combinations in clinical isolates

In some studies investigating resistance mechanisms to azoles in clinical isolates, it was possible to recover from patients treated with these compounds sequential isolates showing stepwise increase in azole resistance, as measured by susceptibility testing. The stepwise increase in azole resistance indicated that different resistance mechanisms probably operate and, through their sequential addition, explains the increase in azole MIC values. Several examples have been reported that document

Alternative mechanisms of azole resistance

Besides the two main resistance mechanisms described above, alternative pathways for azole resistance can be used by fungi. One of these alternative pathways targets specific steps in ergosterol biosynthesis. For example, mutation in the gene ERG3, which encodes the enzyme Δ5,6 desaturase (Erg3p) is linked to azole resistance in C. albicans clinical isolates. Azole resistance is thought to be caused by the inability of cells to produce a sterol metabolite (3,6-diol) from 14α-methylfecosterol, a

Genome-wide studies with azole-resistant isolates

As mentioned above, the ABC-transporter genes CDR1 and CDR2 and the major facilitator CaMDR1 respond to the presence of distinct drugs. Azole resistance is also mostly correlated with the upregulation of genes of a single family in individual isolates, meaning that it is only on rare occasions that genes of both families are co-regulated in the same azole-resistant isolate. Microarray experiments, with their ability to deliver collections of genes differentially expressed in a genome, represent

Conclusions

Studies on resistance mechanisms to the azoles have delivered the many different resources utilized by simple microorganisms to circumvent the effect of growth inhibitory substances. Although antifungal resistance is now less of a problem than it was several years ago, before the introduction of highly active antiretroviral therapy to combat HIV, several basic biological processes that emerged from these studies will continue to be investigated and can be used when screening new antifungal

Acknowledgements

DS is supported by Grant Number 3100-055901 from the Swiss National Foundation and from the Howard Hughes Medical Institute.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

References (50)

  • C.M. Beck-Sague et al.

    Secular trends in the epidiemology of nosocomial fungal infections in the United States. National Nosocomial Infections Surveillance System

    J Infect Dis

    (1993)
  • K.A. Marr et al.

    Development of fluconazole resistance in Candida albicans causing disseminated infection in a patient undergoing marrow transplantation

    Clin Infect Dis

    (1997)
  • T.C. White et al.

    Clinical, cellular, and molecular factors that contribute to antifungal drug resistance

    Clin Microbiol Rev

    (1998)
  • M.A. Ghannoum et al.

    Antifungal agents: mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance

    Clin Microbiol Rev

    (1999)
  • M.E. Klepser et al.

    Antifungal pharmacodynamic characteristics of fluconazole and amphotericin B against Cryptococcus neoformans

    J Antimicrob Chemother

    (1998)
  • C. Lass-Florl et al.

    Studies of in vitro activities of voriconazole and itraconazole against Aspergillus hyphae using viability staining

    Antimicrob Agents Chemother

    (2001)
  • E.K. Manavathu et al.

    Comparative study of susceptibilities of germinated and ungerminated conidia of Aspergillus fumigatus to various antifungal agents

    J Clin Microbiol

    (1999)
  • P. Marichal et al.

    Molecular biological characterization of an azole-resistant Candida glabrata isolate

    Antimicrob Agents Chemother

    (1997)
  • T.C. White et al.

    Resistance mechanisms in clinical isolates of Candida albicans

    Antimicrob Agents Chemother

    (2002)
  • A.S. Orozco et al.

    Mechanism of fluconazole resistance in Candida krusei

    Antimicrob Agents Chemother

    (1998)
  • T.D. Edlind et al.

    Aspergillus fumigatus CYP51 sequence: potential basis for fluconazole resistance

    Med Mycol

    (2001)
  • E. Mellado et al.

    Identification of two different 14-alpha sterol demethylase-related genes (cyp51A and cyp51B) in Aspergillus fumigatus and other Aspergillus

    J Clin Microbiol

    (2001)
  • D. Sanglard et al.

    Amino acid substitutions in the cytochrome P-450 lanosterol 14α-demethylase (CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents

    Antimicrob Agents Chemother

    (1998)
  • L.M. Podust et al.

    Crystal structure of cytochrome P450 14α-sterol demethylase (CYP51) from Mycobacterium tuberculosis in complex with azole inhibitors

    Proc Natl Acad Sci USA

    (2001)
  • K.L. Jensen-Pergakes et al.

    Sequencing, disruption, and characterization of the Candida albicans sterol methyltransferase (ERG6) gene: drug susceptibility studies in erg6 mutants

    Antimicrob Agents Chemother

    (1998)
  • Cited by (148)

    • Evaluation of synergistic anticandidal and apoptotic effects of ferulic acid and caspofungin against Candida albicans

      2018, Journal of Food and Drug Analysis
      Citation Excerpt :

      The synergistic activity of the combination of caspofungin and ferulic acid caused a pronounced reduction in the MIC of C. albicans and C. glabrata. Only a few classes of antifungals such as polyenes, azoles, echinocandins, allylamines, and flucytosine are available for the treatment of Candida infections [18]. However, studies on their mechanism of action alone or in synergism with known antifungals are still scarce [19].

    View all citing articles on Scopus
    View full text