Azole-resistant Isolates Research Articles

Involvement of calcium inhibitable binding to the cell wall in the fungicidal activity of CAN-296.

CAN-296 is a heat stable, complex carbohydrate (molecular mass 4300 Da) isolated from the cell wall of the filamentous fungus Mucor rouxii. It possesses potent in-vitro fungicidal activity against a wide spectrum of pathogenic yeasts, including azole-resistant isolates of Candida albicans and Candida glabrata. As a preliminary step in the study of the mode of action of this novel antifungal agent, we investigated the effect of various cations on the antifungal activity as well as the binding of CAN-296 to intact cells and cell-wall fractions of C. albicans. The antifungal activity of CAN-296 was inhibited by low concentrations of calcium, magnesium and lithium and by high concentrations of barium, cobalt and manganese, but not by potassium and copper. The calcium-mediated inhibition of the antifungal activity of CAN-296 was readily reversible by the removal of calcium by dialysis, and the fungicidal activity of the inhibited compound was fully restored. The uptake/binding of CAN-296 to intact cells and to the cell-wall fraction of C. albicans was time and concentration dependent. Maximum uptake/binding was obtained at 5 mg/L within 60 min and was associated with the aggregation of intact cells. Washing intact cells and the cell-wall fraction preincubated with radiolabelled CAN-296 with 150-fold excess of unlabelled compound failed to remove CAN-296 associated with the intact cells and the cell-wall fraction, suggesting that the binding of CAN-296 to C. albicans is tight. The uptake/binding of CAN-296 and the drug-mediated aggregation of intact cells were inhibited by calcium in a concentration-dependent manner. The fact that CAN-296 is a fungicidal agent that binds to intact cells and the cell-wall fraction of C. albicans very tightly, together with the observation that calcium was able to inhibit the fungicidal activity as well as the uptake/binding of CAN-296, suggests that the mode of action of this novel antifungal agent may involve interaction with the cell wall of C. albicans.

Identification of polymorphic mutant alleles of CaMDR1, a major facilitator of Candida albicans which confers multidrug resistance, and its in vitro transcriptional activation.

CaMDR1 (Candida albicans Multi Drug Resistance) encodes a major facilitator whose expression in Saccharomyces cerevisiae confers resistance to several unrelated drugs. We describe here the identification and molecular characterization of seven mutant alleles of CaMDR1 (CaMDR1-1 to 1-7). The complete sequencing of CaMDR1 alleles revealed several in-frame point mutations leading to a change in amino-acid residues where insertion/replacement of an aspartate residue in a serine-asparagine-aspartate-rich domain was most noteworthy. Interestingly, these alleles showed a distinct drug resistance profile. The expression of CaMDR1, or of its alleles, in C. albicans cells was enhanced by benomyl, methotrexate and several other unrelated drugs, and was more pronounced in at least one of the azole-resistant clinical isolates.

Expression of CDR1, a multidrug resistance gene of Candida albicans: transcriptional activation by heat shock, drugs and human steroid hormones

We have examined the expression of CDR1 ( Candida drug resistance gene) in different stress conditions. There was a significant but transient enhancement of CDR1 expression associated with elevated temperatures. Most noteworthy transcriptional activation was observed with miconazole and vinblastine. Interestingly, β-estradiol and progesterone were also able to enhance CDR1 expression. Elevated levels of CDR1 and CDR2 (a homologue of CDR1) mRNA were found in some azole-resistant clinical isolates of C. albicans. CaMDR1 (benomyl-resistant) expression, however, did not differ among all the resistant isolates. Our results confirm the existence of multiple mechanisms of azole resistance in C. albicans.

Expression ofCDR1, a multidrug resistance gene ofCandida albicans: transcriptional activation by heat shock, drugs and human steroid hormones

We have examined the expression of CDR1 (Candida drug resistance gene) in different stress conditions. There was a significant but transient enhancement of CDR1 expression associated with elevated temperatures. Most noteworthy transcriptional activation was observed with miconazole and vinblastine. Interestingly, beta-estradiol and progesterone were also able to enhance CDR1 expression. Elevated levels of CDR1 and CDR2 (a homologue of CDR1) mRNA were found in some azole-resistant clinical isolates of C. albicans. CaMDR1 (benomyl-resistant) expression, however, did not differ among all the resistant isolates. Our results confirm the existence of multiple mechanisms of azole resistance in C. albicans.

Activity of fluconazole againstCandida albicansisolates from HIV + patients in a digestive candidosis turkey model

Fluconazole activity against four strains of Candida albicans (three isolates from AIDS patients and one azole-resistant isolate, NCPF 3363) was studied in a turkey crop infection model. Isolate NCPF 3363 showed confirmed azole resistance in vitro and in vivo. Two isolates from AIDS patients were susceptible to fluconazole in vitro and in vivo. The third isolate was resistant to fluconazole in vitro (MIC = 100 micrograms ml-1), insensitive to 2.5 mg kg-1 but sensitive in vivo to a 5 mg kg-1 daily dose.

Defective sterol C5-6 desaturation and azole resistance: A new hypothesis for the mode of action of azole antifungals

Two azole resistant isolates of Saccharomyces cerevisiae carried mutations allelic to erg 3 and were blocked to differing degrees at the C5-6 desaturation step of ergosterol biosynthesis. When treated with the sterol 14α-demethylation inhibitor fluconazole the wild-type sensitive strain accumulated lanosterol and 14α-methyl-ergosta-8,24(28)-dien-3β,6α-diol (14-methyl-3,6 diol). The stringent desaturase mutant, A2, accumulated 14α-methyl-8,24(28)-dien-3β-ol (14-methyl fecosterol) and lanosterol as the major sterol components when treated with fluconazole. Resistant isolate A3 accumulated 14-methyl-3,6-diol, 14-methyl fecosterol, and lanosterol and was only partially blocked at sterol C5-6 desaturation. We conclude that functional sterol C5-6 desaturase is required for the synthesis of 14-methyl-3,6-diol under conditions of azole inhibition. We present a new hypothesis for the mode of action of azole antifungals based on the inability of 14-methyl-3,6-diol to support growth, and suggest that growth can occur through utilisation of 14-methyl fecosterol, produced by a combination of azole inhibition and defective sterol C5-6 desaturation.

Azole resistance inCandida albicans

An isolate of Candida albicans from a patient with chronic mucocutaneous candidosis who relapsed during ketoconazole treatment was compared with a number of other azole-sensitive and azole-resistant isolates by tests in vitro and in three animal models of vaginal or disseminated infection. In-vitro tests indicated that the isolate was cross-resistant to all imidazole and triazole antifungals tested. In the animal models, treatment with miconazole, ketoconazole, itraconazole or fluconazole failed to influence the infection.