Mechanisms of antifungal resistance

The many drugs that are available at present to treat fungal infections can be divided into four broad groups on the basis of their mechanism of action. These antifungal agents either inhibit macromolecule synthesis (flucytosine), impair membrane barrier function (polyenes), inhibit ergosterol synthesis (allylamines, thiocarbamates, azole derivatives, morpholines), or interact with microtubules (griseofulvin). Drug resistance has been identified as the major cause of treatment failure among patients treated with flucytosine. A lesion in the UMP-pyrophosphorylase is the most frequent clinical determinant of resistance to 5FC in Candida albicans. Despite extensive use of polyene antibiotics for more than 30 years, emergence of acquired resistance seems not be a significant clinical problem. Polyene-resistant Candida isolates have a marked decrease in their ergosterol content. Acquired resistance to allylamines has not been reported from human pathogens, but, resistant phenotypes have been reported for variants of Saccharomyces cerevisiae and of Ustilago maydis. Tolerance to morpholines is seldom found. Intrinsic resistance to griseofulvin is due to the absence of a prolonged energy-dependent transport system for this antibiotic. Resistance to azole antifungal agents is known to be exceptional, although it does now appear to be increasing in importance in some groups of patients infected with e.g. Candida spp., Histoplasma capsulatum or Cryptococcus neoformans. For example, resistance to fluconazole is emerging in C. albicans, the major agent of oro-pharyngeal candidosis in AIDS patients, after long-term suppressive therapy. In the majority of cases, primary and secondary resistance to fluconazole and cross-resistance to other azole antifungal agents seems to originate from decreased intracellular accumulation of the azoles, which may result from reduced uptake or increased efflux of the molecules. In most C. albicans isolates the decreased intracellular levels can be correlated with enhanced azole efflux, a phenomenon linked to an increase in the amounts of mRNA of a C. albicans ABC transporter gene CDR1 and of a gene (BEN(r) or CaMDR) coding for a transporter belonging to the class of major facilitator multidrug efflux transporters. Not only fluconazole, ketoconazole and itraconazole are substrates for CDR1, terbinafine and amorolfine have also been established as substrates, BEN(r) overexpression only accounts for fluconazole resistance. Other sources of resistance: changes in membrane sterols and phospholipids, altered or overproduced target enzyme(s) and compensatory mutations in the Delta5,6-desaturase.     

Suppression of ATP in Candida albicans by imidazole and derivative antifungal agents

Several antifungal agents, at concentrations of 10 micrograms/ml, were shown to suppress ATP concentrations very rapidly in intact cells and spheroplasts of Candida albicans. The highest ATP-suppressing activity was shown by the highly lipophilic imidazole derivatives difonazole, clotrimazole, econazole, isoconazole, miconazole, oxiconazole and tioconazole, which all caused a reduction of cellular ATP content of more than 50% in 10 min. Relatively hydrophilic imidazole derivatives such as ketoconazole were essentially inactive in the test, as were the triazole derivatives fluconazole, ICI 153066, itraconazole and terconazole, and 5-fluorocytosine. Amphotericin B and terbinafine possessed intermediate ATP-suppressing activity, and the dose-response and pH-response curves for these compounds suggested their mechanism of ATP suppression differed from that of the active imidazole derivatives. ATP suppression by azole antifungals did not involve leakage of ATP from the cells and the effect was entirely abrogated by the presence of serum. Intact cells and spheroplasts of yeast-form and hyphal-form C. albicans were generally equally sensitive to ATP suppression, but stationary-phase cells of both morphological forms were less sensitive than exponential-phase cells. The extent of ATP suppression was significantly reduced in stationary-phase yeast cells of a C. albicans strain with known resistance to azole antifungals, but exponential-phase cells of resistant and susceptible strains were equally sensitive. The effect is tentatively ascribed to membrane damage caused directly by the antifungals.     

Homology model,molecular dynamics simulation and novel pyrazole analogs design of Candida albicans CYP450 lanosterol 14 α-demethylase,a target enzyme for antifungal therapy

Candida albicans infections and their resistance to clinically approved azole drugs are major concerns for human. The azole antifungal drugs inhibit the ergosterol synthesis by targeting lanosterol 14α-demethylase of cytochrome P450 family. The lack of high-resolution structural information of fungal pathogens has been a barrier for the design of modified azole drugs. Thus, a preliminary theoretical molecular dynamic study is carried out to develop and validate a simple homologous model using crystallographic structure of the lanosterol 14α-demethylase of Mycobacterium tuberculosis (PDB ID-1EA1) in which the active site residues are substituted with that of C. albicans (taxid 5476). Further, novel designed pyrazole analogs (SGS1-16) docked on chimeric 1EA1 and revealed that SGS-16 show good binding affinity through non-bonding interaction with the heme, which is different from the leading azole antifungals. The ADME-T results showed these analogs can be further explored in design of more safe and effective antifungal agents.     

2-Hydroxyphenacyl azoles and related azolium derivatives as antifungal agents

2-Hydroxyphenacyl azole and 2-hydroxyphenacyl azolium compounds have been described as a new class of azole antifungals. Most target compounds showed significant in vitro antifungal activities against tested fungi (Candida albicans, Saccharomyces cerevisiae, Aspergillus niger, and Microsporum gypseum) with low MICs values included in the range of 0.25-32 microg/mL comparable to reference drug fluconazole. The most active compounds were also assessed for their cytotoxicity using MTT colorimetric assay on normal mouse fibroblast (NIH/3T3) cells. The results of antifungal activity and toxicity tests indicated that these compounds display antifungal activity at non-cytotoxic concentrations.     

Semisynthetic modifications of hemiaminal function at ornithine unit of mulundocandin, towards chemical stability and antifungal activity

Mulundocandin (1), is an echinocandin class of lipopeptide. It has wide spectrum of antifungal activity against Candida and Aspergillus species. Semisynthetic modification at Ornithine-5-hydroxyl (hemiaminal function) of 1 was carried out to improve solution stability and hence in vivo activity. Synthesis of ether (C-OR), thioether (C-SR) and C-N linkage at hemiaminal function have been described. All synthetic analogues were evaluated for their stability in aqueous solution and found to be more stable than mulundocandin. Antifungal activity of Orn-5 analogues was evaluated both in vitro against Candida albicans and Aspergillus fumigatus by agar well method and in vivo (oral and intraperitoneal) in C. albicans infected Swiss mice. Results of in vivo assays of analogues 2-9 by the oral route suggests that the introduction of either oxygen nucleophiles (-OR) or sulphur nucleophiles (-SR), at either Orn-5 or at both Orn-5 and HTyr-4 positions, results in retaining the activity of the parent compound with improved aqueous stability in most cases. Compound 9 has shown improved antifungal activity in comparison to mulundocandin by oral application in Swiss mice.     

Calcineurin is essential for survival during membrane stress in Candida albicans

The immunosuppressants cyclosporin A (CsA) and FK506 inhibit the protein phosphatase calcineurin and block T-cell activation and transplant rejection. Calcineurin is conserved in microorganisms and plays a general role in stress survival. CsA and FK506 are toxic to several fungi, but the common human fungal pathogen Candida albicans is resistant. However, combination of either CsA or FK506 with the antifungal drug fluconazole that perturbs synthesis of the membrane lipid ergosterol results in potent, synergistic fungicidal activity. Here we show that the C.albicans FK506 binding protein FKBP12 homolog is required for FK506 synergistic action with fluconazole. A mutation in the calcineurin B regulatory subunit that confers dominant FK506 resistance (CNB1-1/CNB1) abolished FK506-fluconazole synergism. Candida albicans mutants lacking calcineurin B (cnb1/cnb1) were found to be viable and markedly hypersensitive to fluconazole or membrane perturbation with SDS. FK506 was synergistic with fluconazole against azole-resistant C.albicans mutants, against other Candida species, or when combined with different azoles. We propose that calcineurin is part of a membrane stress survival pathway that could be targeted for therapy.     

Direct comparison of the pharmacodynamics of four antifungal drugs in a mouse model of disseminated candidiasis using microbiological assays of serum drug concentrations

The aim of this study was to compare the pharmacodynamics of the azole antifungal drugs fluconazole, itraconazole and ketoconazole, and the polyene antifungal amphotericin B, in a mouse model of disseminated Candida albicans infection. In order to directly compare effective serum concentrations of these antifungals, drug concentrations were assayed microbiologically by measuring inhibition of C. albicans mycelial growth (mMIC) in a mouse serum-based assay (serum antifungal titer). Efficacy in the mouse infection model was determined using an organ-based (kidney burden) endpoint. For all four drugs, the serum antifungal titers, 8 hr after administration of single doses of drugs at a range of drug concentrations, correlated closely with C. albicans kidney fungal burden in the mouse model. The results showed that determining serum antifungal titer may be used to accurately represent kidney fungal burden in a mouse model of disseminated candidiasis and allowed direct comparison of the pharmacodynamics of differing classes of antifungal drugs.     

Effects of imidazole- and triazole-derivative antifungal compounds on the growth and morphological development of Candida albicans hyphae

Six azole-derivative antifungal compounds affected several aspects of Candida albicans hyphal development with only a relatively small degree of inhibition of growth rate, measured in terms of ATP concentration, whereas amphotericin B and 5-fluorocytosine affected morphology only when they also substantially inhibited fungal growth rate. At 10(-8) M, all the azoles tested inhibited branch formation by C. albicans hyphae. At 10(-7) M and higher concentrations, clotrimazole and miconazole strongly suppressed emergence of new hyphal outgrowths from parent yeast cells, whereas ICI 153066 and itraconazole had little effect on this phenomenon and ketoconazole and tioconazole had intermediate effects. At the highest concentrations tested (10(-5) M) hyphal development was ultimately arrested by the azole compounds and the fungus grew predominantly in the form of budding yeast cells; however, none of the azole antifungals prevented initial emergence of an apparently normal germ tube. The antifungals only exerted their morphological effects when they were present in the culture medium: removal of the compounds after exposure of C. albicans to them led to reversion to normal growth.     

白色念珠菌的临床分布及Rosco纸片法药敏结果分析

目的了解目前白色念珠菌的临床分布及其耐药现状,以加强抗真菌药物的合理应用。方法采用Rosco纸片扩散法检测2005年2—9月安徽医科大学第一附属医院临床分离的260株白色念珠菌对7种抗真菌药物的药敏结果。结果白色念珠菌的临床分布以痰液标本为主,老年患者居多。白色念珠菌对制霉菌素、两性霉素B和伊曲康唑的敏感率均较高,分别为98.9％、96．9％和94．2％;其次为酮康唑和氟康唑,敏感率分别为84．6％和82．3％;灰黄霉素、益康唑和眯康唑的敏感率较低,分别为73．9％、57．8％和57．6％。结论临床微生物实验室要加强对白色念珠菌的分离培养和药敏试验,指导临床合理用药,有效控制和减少真菌感染。     

Synergistic action of nikkomycin X/Z with azole antifungals on Candida albicans.

Fluconazole, ketoconazole and tioconazole were shown to act synergistically in vitro with the antibiotic nikkomycin X/Z on the pathogenic fungus Candida albicans. The phenomenon was demonstrated using a checkerboard technique and growth inhibition experiments. The azole antifungal agents, even at concentrations not affecting growth, decreased the incorporation of the 14C-label from [14C]glucose into chitin of the candidal cell wall. After 3 h incubation with tioconazole, 1 microgram ml-1, the incorporation of the radiolabelled glucose into chitin of intact cells and regenerating spheroplasts of C. albicans was inhibited by 43% and 30%, respectively. Moreover, the relative chitin content was approximately 45% lower than that of control cells. The chitin content increased after prolonged incubation with azoles, thus confirming the known phenomenon of azole-induced uncoordinated chitin synthesis and deposition. On the other hand, azole derivatives had very little effect on the rate of nikkomycin transport into C. albicans cells. A sequential blockade mechanism of synergism is proposed.     

Fluconazole and itraconazole susceptibility of vaginal yeast isolates from Slovakia

Vulvovaginal candidiasis is a common mucosal infection caused by opportunistic yeasts of the Candida genus. In this study, we isolated and identified the yeast species in the vagina of patients treated in the gynecology clinic and tested in vitro activities of fluconazole and itraconazole against 227 clinical yeast isolates by the NCCLS microdilution method. C. albicans (87.6%) was the most frequently identified species followed by C. glabrata (6.2%) and C. krusei (2.2%). Almost thirteen percent of yeast strains were resistant to fluconazole and 18.5% were resistant to itraconazole. Cross-resistance analyses of C. albicans isolates revealed that fluconazole resistance and itraconazole resistance were also associated with decreased susceptibilities to other azole derivatives mainly to ketoconazole and miconazole. At the same time no cross-resistance to polyene antibiotics amphotericin B and nystatin was observed. These results support the notion that antifungal agents used to treat vaginitis may be contributing to the drug resistance problem by promoting cross-resistance to a range of clinically used antifungals.     

Relationship between respiration deficiency and azole resistance in clinical Candida glabrata

Candida glabrata has become a leading cause of invasive infections around the world and is exhibiting growing resistance to azole antifungals. To study the mechanism of its azole resistance, we analyzed the efflux pumps and found well known increased efflux expression and low metabolic state in all azole-resistant strains. The latter finding led us to further investigate the relationship between respiration status and azole antifungal susceptibility in clinical C.?glabrata by growing them on glycerol-containing agar, measuring the cellular ATP, reactive oxygen species (ROS) levels, oxygen consumption and transmission electron microscopy. All azole-resistant isolates were respiratory-deficient, with reduced generation of ATP and ROS and decreased oxygen consumption; two isolates grew as small colonies and exhibited mitochondrial deficiency. Spot assays and agarose disc diffusion tests were performed to evaluate the effects of respiratory chain inhibitors, sodium azide and salicylhydroxamic acid, on antifungal susceptibility. The results of antifungal susceptibility showed that inhibition of alternative respiration with salicylhydroxamic acid enhanced azole susceptibility of C.?glabrata. In conclusion, clinical azole-resistant C.?glabrata isolates harbor respiratory deficiency exhibiting petite mutant or normal phenotype. The alternative respiratory pathway plays an important role in the decreased susceptibility to azole antifungals.     

SSCP analysis (ITS2, ERG11) in clinical Candida isolates from oral cavity in oncology patients

Antifungal prophylactic therapy in oncology patients has favors the emergence of diverse species of Candida. In the present study 32 clinic isolates of Candida spp., recovered from oral cavity, were evaluated testing their susceptibility to diverse antifungals by means of the microdilution method according with the document M-27A of the National Committee for Clinical Laboratory Standards (NCCLS). The response to antifungals was then compared with SSCP patterns of the gene ERG11, hoping to find mutations in this gene linked to resistance to the azole antifungals from samples of clinical origin. Three SSCP genotypes with diverse response to different antifungals were found. This study suggested that a link of mutations in the ERG11 gene of Candida albicans and antifungal resistance was not supported by this data. In addition, the isolates were also classified by conventional methods and their genetic diversity evaluated by means of SSCP analysis and evaluation of their ITS2 regions, identifying eight SSCP genotypes. This technique has the potential to be a more sensitive method for taxonomic classification.     

Antifungal agents: mechanisms of action

Clinical needs for novel antifungal agents have altered steadily with the rise and fall of AIDS-related mycoses, and the change in spectrum of fatal disseminated fungal infections that has accompanied changes in therapeutic immunosuppressive therapies. The search for new molecular targets for antifungals has generated considerable research using modern genomic approaches, so far without generating new agents for clinical use. Meanwhile, six new antifungal agents have just reached, or are approaching, the clinic. Three are new triazoles, with extremely broad antifungal spectra, and three are echinocandins, which inhibit synthesis of fungal cell wall polysaccharides--a new mode of action. In addition, the sordarins represent a novel class of agents that inhibit fungal protein synthesis. This review describes the targets and mechanisms of action of all classes of antifungal agents in clinical use or with clinical potential.     

The antifungal activity of sulfonylated/carboxylated derivatives of dibenzo-1,4-dioxine-2-acetyloxime may be due to inhibition of lanosterol-14alpha-demethylase

Aryl/alkyl-sulfonyl-, aryl/alkylcarboxyl- and aryl(sulfonyl)carbamyl/thiocarbamyl-derivatives of dibenzo-1,4-dioxine-2-acetyloxime were prepared by reaction of the title compound with sulfonyl halides, sulfonic acid anhydrides, acyl chlorides/carboxylic acids, arylsulfonyl isocyanates, aryl/acyl isocyanates or isothiocyanates. Several of the newly synthesized compounds showed effective in vitro antifungal activity against Aspergillus and Candida spp., some of them showing activities comparable to ketoconazole (with minimum inhibitory concentrations in the range of 1.2-4 microg/mL) against the two Aspergillus strains, but possessing a lower activity as compared to ketoconazole against C. albicans. Of the three investigated strains, best activity was detected against A. flavus. The mechanism of action of these compounds probably involves inhibition of ergosterol biosynthesis by interaction with lanosterol-14-alpha-demethylase (CYP51A1), since reduced amounts of ergosterol were found by means of HPLC, in cultures of the sensitive strain A. flavus treated with some of these inhibitors. Thus, the compounds reported here might possess a similar mechanism of action at molecular level with that of the widely used azole antifungals.     

Activity of anidulafungin against Candida biofilms

Currently, no standardized method to study the in vitro activity of antifungal agents on biofilms is available, thus, the comparison among different authors is difficult. The studies discussed in this review use the XTT reduction to measure the activity of antifungals on biofilms of 24 h of maturation. To date, biofilm anidulafungin MICs of 47 isolates of Candida spp. (25 Candida albicans, 16 Candida tropicalis, 5 Candida dubliniensis and 1 Candida parapsilosis) have been published. The geometric mean MIC of anidulafungin on biofilms of Candida spp. is of 1.18 microg/ml. Against isolates of species with great capacity of biofilm formation, the geometric mean MIC is 0.325 (C. albicans), 2 (C. parapsilosis) and 0.5 microg/ml (C. dubliniensis). No echinocandin has activity on C. tropicalis biofilms. In addition, anidulafungin can be used for lock therapy of catheters since it is the echinocandin with the least in vitro paradoxical effect.     

Calcineurin A of Candida albicans: involvement in antifungal tolerance,cell morphogenesis and virulence

The azole antifungal fluconazole possesses only fungistatic activity in Candida albicans and, therefore, this human pathogen is tolerant to this agent. However, tolerance to fluconazole can be inhibited when C. albicans is exposed to fluconazole combined with the immunosuppressive drug cyclosporin A, which is known to inhibit calcineurin activity in yeast. A mutant lacking both alleles of a gene encoding the calcineurin A subunit (CNA) lost viability in the presence of fluconazole, thus making calcineurin essential for fluconazole tolerance. Consistent with this observation, tolerance to fluconazole was modulated by calcium ions or by the expression of a calcineurin A derivative autoactivated by the removal of its C-terminal inhibitory domain. Interestingly, CNA was also essential for tolerance to other antifungal agents (voriconazole, itraconazole, terbinafine, amorolfine) and to several other metabolic inhibitors (caffeine, brefeldin A, mycophenolic acid, fluphenazine) or cell wall-perturbing agents (SDS, calcofluor white, Congo red), thus indicating that the calcineurin pathway plays an important role in the survival of C. albicans in the presence of external growth inhibitors. Several genes, including PMC1, a vacuolar calcium P-type ATPase, were regulated in a calcineurin- and fluconazole-dependent manner. However, PMC1 did not play a direct role in the survival of C. albicans when exposed to fluconazole. In addition to these different properties, calcineurin was found to affect colony morphology in several media known to modulate the C. albicans dimorphic switch. In particular, calcineurin was found to be essential for C. albicans viability in serum-containing media. Finally, calcineurin was found to be necessary for the virulence of C. albicans in a mice model of infection, thus making calcineurin an important element for adequate adaptation to the conditions of the host environment.     

Multilocus sequence typing confirms synonymy but highlights differences between Candida albicans and Candida stellatoidea

We used multi-locus sequence typing (MLST) to investigate 35 yeast isolates representing the two genome-sequenced strains plus the type strain of Candida albicans, four isolates originally identified as Candida stellatoidea type I and 28 representing type strains of other species now regarded as synonymous with C. albicans. DNA from all 32 C. albicans synonyms readily formed PCR products with the C. albicans MLST primer sets. Their sequences placed all of them within the existing C. albicans clade structure, represented by 1516 isolates. One isolate, originally received as Mycotorula sinensis, was resistant to flucytosine, but no other unusual susceptibilities were found to polyene, azole or echinocandin antifungal agents. The four isolates of C. stellatoidea type I coclustered with two other sucrose-negative isolates, originally identified as examples of Candida africana, in a group of strains highly distinct from the majority of C. albicans. Our results not only confirm the synonymity of all the isolates with C. albicans but also confirm an obvious genotypic difference in the case of C. stellatoidea type I.     

Hsp90 governs dispersion and drug resistance of fungal biofilms

Fungal biofilms are a major cause of human mortality and are recalcitrant to most treatments due to intrinsic drug resistance. These complex communities of multiple cell types form on indwelling medical devices and their eradication often requires surgical removal of infected devices. Here we implicate the molecular chaperone Hsp90 as a key regulator of biofilm dispersion and drug resistance. We previously established that in the leading human fungal pathogen, Candida albicans, Hsp90 enables the emergence and maintenance of drug resistance in planktonic conditions by stabilizing the protein phosphatase calcineurin and MAPK Mkc1. Hsp90 also regulates temperature-dependent C. albicans morphogenesis through repression of cAMP-PKA signalling. Here we demonstrate that genetic depletion of Hsp90 reduced C. albicans biofilm growth and maturation in vitro and impaired dispersal of biofilm cells. Further, compromising Hsp90 function in vitro abrogated resistance of C. albicans biofilms to the most widely deployed class of antifungal drugs, the azoles. Depletion of Hsp90 led to reduction of calcineurin and Mkc1 in planktonic but not biofilm conditions, suggesting that Hsp90 regulates drug resistance through different mechanisms in these distinct cellular states. Reduction of Hsp90 levels led to a marked decrease in matrix glucan levels, providing a compelling mechanism through which Hsp90 might regulate biofilm azole resistance. Impairment of Hsp90 function genetically or pharmacologically transformed fluconazole from ineffectual to highly effective in eradicating biofilms in a rat venous catheter infection model. Finally, inhibition of Hsp90 reduced resistance of biofilms of the most lethal mould, Aspergillus fumigatus, to the newest class of antifungals to reach the clinic, the echinocandins. Thus, we establish a novel mechanism regulating biofilm drug resistance and dispersion and that targeting Hsp90 provides a much-needed strategy for improving clinical outcome in the treatment of biofilm infections.