Acquisition Of Azole Resistance Research Articles

The Effect of Posaconazole, Itraconazole and Voriconazole in the Culture Medium on Aspergillus fumigatus Triazole Resistance

Triazoles are the only compounds used as antibiotics in both medicine and agriculture. The presence of triazoles in the environment can contribute to the acquisition of azole resistance among isolates of Aspergillus fumigatus. The objective of this study was to investigate the effect of A. fumigatus exposure to triazoles on susceptibility to these compounds. Seventeen triazole-resistant and 21 triazole-sensitive A. fumigatus isolates were examined. The isolates were transferred 20 times on the Sabouraud medium supplemented with posaconazole, itraconazole or voriconazole, followed by five times on the medium not supplemented. The minimum inhibitory concentrations of antimycotics were examined according to the EUCAST broth microdilution method after the 20th transfer and also the 25th transfer. In addition, the expression levels of genes mdr1, mdr2, mdr3, atrF, cyp51A and cyp51B were determined. Cultivation of A. fumigatus on media supplemented with posaconazole, itraconazole and voriconazole resulted in the acquisition of resistance to the tested triazoles of all examined isolates. After recultivation on Sabouraud without azoles, most of the isolates lost their acquired resistance. The long-term use of triazole compounds in agriculture may result in the occurrence of triazole resistant A. fumigatus isolates in the environment, not only by induction or selection of mutations in the cyp51A gene, but also by contribution to changes in the gene expression.

Resistance in the Environmental Pathogenic Fungus Aspergillus fumigatus.

Aspergillus fumigatus, an opportunistic pathogenic fungus, is common in the environment. Azole-resistant strains of A. fumigatus have recently been discovered in the environment. Acquisition of azole resistance has been considered to occur through unexpected selection due to fungicide use in agriculture. This review discusses the relationships of humans with A. fumigatus in the environment and the importance of anthropogenic activities in the spread of A. fumigatus worldwide.

Environmental azole fungicide, prochloraz, can induce cross-resistance to medical triazoles in Candida glabrata.

Acquisition of azole resistance by clinically relevant yeasts in nature may result in a significant, yet undetermined, impact in human health. The main goal of this study was to assess the development of cross-resistance between agricultural and clinical azoles by Candida spp. An in vitro induction assay was performed, for a period of 90 days, with prochloraz (PCZ) - an agricultural antifungal. Afterward, the induced molecular resistance mechanisms were unveiled. MIC value of PCZ increased significantly in all Candida spp. isolates. However, only C.glabrata developed cross-resistance to fluconazole and posaconazole. The increased MIC values were stable. Candida glabrata azole resistance acquisition triggered by PCZ exposure involved the upregulation of the ATP binding cassette multidrug transporter genes and the transcription factor, PDR1. Single mutation previously implicated in azole resistance was found in PDR1 while ERG11 showed several synonymous single nucleotide polymorphisms. These results might explain why C.glabrata is so commonly less susceptible to clinical azoles, suggesting that its exposure to agricultural azole antifungals may be associated to the emergence of cross-resistance. Such studies forward potential explanations for the worldwide increasing clinical prevalence of C.glabrata and the associated worse prognosis of an infection by this species.

Development of Azole Resistance in Aspergillus fumigatus during Azole Therapy Associated with Change in Virulence

Four sequential Aspergillus fumigatus isolates from a patient with chronic granulomatous disease (CGD) eventually failing azole-echinocandin combination therapy were investigated. The first two isolates (1 and 2) were susceptible to antifungal azoles, but increased itraconazole, voriconazole and posaconazole MICs were found for the last two isolates (3 and 4). Microsatellite typing showed that the 4 isolates were isogenic, suggesting that resistance had been acquired during azole treatment of the patient. An immunocompromised mouse model confirmed that the in vitro resistance corresponded with treatment failure. Mice challenged with the resistant isolate 4 failed to respond to posaconazole therapy, while those infected by susceptible isolate 2 responded. Posaconazole-anidulafungin combination therapy was effective in mice challenged with isolate 4. No mutations were found in the Cyp51A gene of the four isolates. However, expression experiments of the Cyp51A showed that the expression was increased in the resistant isolates, compared to the azole-susceptible isolates. The microscopic morphology of the four isolates was similar, but a clear alteration in radial growth and a significantly reduced growth rate of the resistant isolates on solid and in broth medium was observed compared to isolates 1 and 2 and to unrelated wild-type controls. In the mouse model the virulence of isolates 3 and 4 was reduced compared to the susceptible ones and to wild-type controls. For the first time, the acquisition of azole resistance despite azole-echinocandin combination therapy is described in a CGD patient and the resistance demonstrated to be directly associated with significant change of virulence.

DNA microarray analysis of fluconazole resistance in a laboratory <italic>Candida albicans</italic> strain

Several mechanisms are responsible for the acquired fluconazole (FLC) resistance in Candida albicans. In this study, we developed a FLC-resistant C. albicans strain through serial cultures of a FLC-susceptible C. albicans strain with inhibitory concentrations of FLC. Complimentary DNA microarray analysis and real-time reverse transcription-polymerase chain reaction were used to investigate gene expression changes during the acquisition of azole resistance in the susceptible parental strain and the resistant daughter strain. The differentially expressed genes represented functions as diverse as transporters (e.g. CDR1, PDR17), ergosterol biosynthesis (e.g. ERG2, ERG9), sterol metabolism (e.g. ARE2, IPF6464), energy metabolism (e.g. ADH3, AOX2) and transcription factors (e.g. FCR1, ECM22). Functional analysis revealed that energy-dependent efflux activity of membrane transporters increased and that ergosterol content decreased with the accumulation of sterol intermediates in the resistant strain as compared with the susceptible strain. We found that a point mutation (N977K) in transcription factor TAC1 that resulted in hyperactivity of Tac1 could be the reason for overexpression of CDR1, CDR2, and PDR17 in the resistant strain. Furthermore, a single amino acid difference (D19E) in ERG3 that led to the inactivation of Erg3 could account for both sterol precursor accumulation and the changes in the expression of ergosterol biosynthesis genes in this resistant strain. These findings expand the understanding of potential novel molecular targets of FLC resistance in clinical C. albicans isolates.

Effects of Y132H and F145L Substitutions on the Activity, Azole Resistance and Spectral Properties of Candida albicans Sterol 14-Demethylase P450 (CYP51): A Live Example Showing the Selection of Altered P450 through Interaction with Environmental Compounds

Three variants of Candida albicans CYP51 (sterol 14-demethylase P450) having Y132H and/or F145L substitutions were purified and characterized to reveal the effects of these amino acid substitutions on the enzymatic properties and azole resistance of the enzyme. Y132H and F145L substitutions modified the spectral properties of the enzyme, suggesting that they caused some structural change modifying the heme environments of CYP51. Y132H and F145L substitutions increased the resistance of the enzyme to azole compounds but considerably decreased the catalytic activity. This fact represents a trade-off between acquisition of azole resistance and maintenance of high activity in the CYP51 having Y132H and F145L substitutions. A fluconazole-resistant C. albicans strain DUMC136 isolated from patients receiving long-term azole treatment was a homozygote of the altered CYP51 having Y132H and F145L substitutions. However, neither of these substitutions was found in CYP51 of wild-type C. albicans so far studied. These facts suggest that the azole-resistant variant having Y132H and/or F145L substitutions might be selected only under azole-rich environments because of its azole resistance and impaired catalytic activity. This may be a live example showing one of the important processes of P450 diversification, the selection of altered P450 through the interaction with environmental compounds.

The pfr1 gene from the human pathogenic fungus Paracoccidioides brasiliensis encodes a half-ABC transporter that is transcribed in response to treatment with fluconazole.

We have isolated a gene that encodes a half-ABC-transporter, designated Pfr1, from the dimorphic human pathogenic fungus Paracoccidioides brasiliensis, which has high identity with members of the ABC-superfamily involved in multidrug resistance. The pfr1 gene is predicted to encode a 827 amino acid protein that, in common with mammalian Mdr1, has a TM-NBD topology. The transcription of the pfr1 gene is induced by the triazole drug fluconazole but not by amphotericin B, suggesting a role in transport-mediated azole resistance. However, Pfr1 has greatest identity to the mitochondrial ABC transporters Mdl1 and Mdl2 from Saccharomyces cerevisiae and mammalian ABC-me, with identities of 47.2%, 40.6% and 39.5%, respectively, over the length of these proteins. Furthermore, the N-terminus of Pfr1 is rich in positively charged residues, a feature of mitochondrial targeting sequences. Considering these features, it seems likely that Pfr1 is a mitochondrial protein. Previous studies have revealed that the acquisition of azole resistance in S. cerevisiae is linked to mitochondrial loss and, conversely, that mitochondrial dysfunction can lead to the upregulation of PDR transporters mediated by the transcription factor Pdr3. Our studies suggest that a mitochondrial ABC transporter is induced as part of the cellular response to drug treatment. The promoter region of pfr1 contains a PDRE-like consensus sequence to which Pdr3 binds, which may be the element responsible for the upregulation of Pfr1 in response to fluconazole. The nucleotide binding domain of Pfr1 was expressed and purified from Escherichia coli and shown to retain ATPase activity, consistent with Pfr1 functioning as a homodimeric transport ATPase.