scholarly journals Itraconazole, Voriconazole, and Posaconazole CLSI MIC Distributions for Wild-Type and Azole-Resistant Aspergillus fumigatus Isolates

2018 ◽  
Vol 4 (3) ◽  
pp. 103 ◽  
Author(s):  
Jochem Buil ◽  
Ferry Hagen ◽  
Anuradha Chowdhary ◽  
Paul Verweij ◽  
Jacques Meis
Keyword(s):  
Aspergillus Fumigatus ◽  
Minimal Inhibitory Concentration ◽  
Susceptibility Testing ◽  
Inhibitory Concentration ◽  
Azole Resistance ◽  
Wild Type ◽  
Gene Encoding ◽  
Environmental Resistance ◽  
Resistance Selection ◽  
Susceptibility Profiles

Azole resistance in Aspergillus fumigatus is most frequently conferred by mutations in the cyp51A gene encoding 14α-sterol demethylases. TR34/L98H and TR46/Y121F/T289A are the two most common mutations associated with environmental resistance selection. We studied the minimal inhibitory concentration (MIC) distribution of clinical A. fumigatus isolates to characterize the Clinical and Laboratory Standards Institute (CLSI) susceptibility profiles of isolates with the wild-type (WT) cyp51A genotype, and isolates with the TR34/L98H and TR46/Y121F/T289A cyp51A mutations. Susceptibility testing was performed according to CLSI M38-A2. The MICs of 363 A. fumigatus isolates were used in this study. Based on the CLSI epidemiological cut-off values (ECVs), 141 isolates were phenotypically non-WT and 222 isolates had a phenotypically WT susceptibility. All isolates with the TR34/L98H mutation had an itraconazole MIC > 1 mg/L which is above the CLSI ECV. Eighty-six of 89 (97%) isolates with the TR34/L98H mutation had voriconazole and posaconazole MICs above the CLSI ECV, i.e., MICs of 1 and 0.25 mg/L, respectively. The isolates with a TR46/Y121F/T289A mutation showed a different phenotype. All 37 isolates with a TR46/Y121F/T289A mutation had a voriconazole MIC above the CLSI ECV, while 28/37 (76%) isolates had an itraconazole MIC > 1 mg/L. Interestingly, only 13 of 37 (35%) isolates had a posaconazole MIC > 0.25 mg/L.

scholarly journals In VitroBiochemical Study of CYP51-Mediated Azole Resistance in Aspergillus fumigatus

2015 ◽  
Vol 59 (12) ◽  
pp. 7771-7778 ◽  
Author(s):  
Andrew G. S. Warrilow ◽  
Josie E. Parker ◽  
Claire L. Price ◽  
W. David Nes ◽  
Steven L. Kelly ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Inhibitory Concentration ◽  
Point Mutations ◽  
Residual Activity ◽  
Azole Resistance ◽  
Wild Type ◽  
Biochemical Basis ◽  
Content Type ◽  
Resistant Phenotype

ABSTRACTThe incidence of triazole-resistantAspergillusinfections is increasing worldwide, often mediated through mutations in the CYP51A amino acid sequence. New classes of azole-based drugs are required to combat the increasing resistance to existing triazole therapeutics. In this study, a CYP51 reconstitution assay is described consisting of eburicol, purified recombinantAspergillus fumigatusCPR1 (AfCPR1), andEscherichia colimembrane suspensions containing recombinantA. fumigatusCYP51 proteins, allowingin vitroscreening of azole antifungals. Azole-CYP51 studies determining the 50% inhibitory concentration (IC50) showed thatA. fumigatusCYP51B (Af51B IC50, 0.50 μM) was 34-fold more susceptible to inhibition by fluconazole thanA. fumigatusCYP51A (Af51A IC50, 17 μM) and that Af51A and Af51B were equally susceptible to inhibition by voriconazole, itraconazole, and posaconazole (IC50s of 0.16 to 0.38 μM). Af51A-G54W and Af51A-M220K enzymes were 11- and 15-fold less susceptible to inhibition by itraconazole and 30- and 8-fold less susceptible to inhibition by posaconazole than wild-type Af51A, confirming the azole-resistant phenotype of these two Af51A mutations. Susceptibility to voriconazole of Af51A-G54W and Af51A-M220K was only marginally lower than that of wild-type Af51A. Susceptibility of Af51A-L98H to inhibition by voriconazole, itraconazole, and posaconazole was only marginally lower (less than 2-fold) than that of wild-type Af51A. However, Af51A-L98H retained 5 to 8% residual activity in the presence of 32 μM triazole, which could confer azole resistance inA. fumigatusstrains that harbor the Af51A-L98H mutation. The AfCPR1/Af51 assay system demonstrated the biochemical basis for the increased azole resistance ofA. fumigatusstrains harboring G54W, L98H, and M220K Af51A point mutations.

scholarly journals A Novel Environmental Azole Resistance Mutation inAspergillus fumigatusand a Possible Role of Sexual Reproduction in Its Emergence

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Jianhua Zhang ◽  
Eveline Snelders ◽  
Bas J. Zwaan ◽  
Sijmen E. Schoustra ◽  
Jacques F. Meis ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Sexual Reproduction ◽  
Crop Protection ◽  
Resistance Mutation ◽  
Azole Resistance ◽  
Wild Type ◽  
Resistance Mutations ◽  
Resistance Selection ◽  
Genotype 2

ABSTRACTThis study investigated the dynamics ofAspergillus fumigatusazole-resistant phenotypes in two compost heaps with contrasting azole exposures: azole free and azole exposed. After heat shock, to which sexual but not asexual spores are highly resistant, the azole-free compost yielded 98% (49/50) wild-type and 2% (1/50) azole-resistant isolates, whereas the azole-containing compost yielded 9% (4/45) wild-type and 91% (41/45) resistant isolates. From the latter compost, 80% (36/45) of the isolates contained the TR46/Y121F/T289A genotype, 2% (1/45) harbored the TR46/Y121F/M172I/T289A/G448S genotype, and 9% (4/45) had a novel pan-triazole-resistant mutation (TR463/Y121F/M172I/T289A/G448S) with a triple 46-bp promoter repeat. Subsequent screening of a representative set of clinicalA. fumigatusisolates showed that the novel TR463mutant was already present in samples from three Dutch medical centers collected since 2012. Furthermore, a second new resistance mutation was found in this set that harbored four TR46repeats. Importantly, in the laboratory, we recovered the TR463mutation from a sexual cross between two TR46isolates from the same azole-containing compost, possibly through unequal crossing over between the double tandem repeats (TRs) during meiosis. This possible role of sexual reproduction in the emergence of the mutation was further implicated by the high level of genetic diversity of STR genotypes in the azole-containing compost. Our study confirms that azole resistance mutations continue to emerge in the environment and indicates compost containing azole residues as a possible hot spot. Better insight into the biology of environmental resistance selection is needed to retain the azole class for use in food production and treatment ofAspergillusdiseases.IMPORTANCEComposting of organic matter containing azole residues might be important for resistance development and subsequent spread of resistance mutations inAspergillus fumigatus. In this article, we show the dominance of azole-resistantA. fumigatusin azole-exposed compost and the discovery of a new resistance mutation with clinical relevance. Furthermore, our study indicates that current fungicide application is not sustainable as new resistance mutations continue to emerge, thereby threatening the use of triazoles in medicine. We provide evidence that the sexual part of the fungal life cycle may play a role in the emergence of resistance mutations because under laboratory conditions, we reconstructed the resistance mutation through sexual crossing of two azole-resistantA. fumigatusisolates derived from the same compost heap. Understanding the mechanisms of resistance selection in the environment is needed to design strategies against the accumulation of resistance mutations in order to retain the azole class for crop protection and treatment ofAspergillusdiseases.

scholarly journals Contributions of both ATP-Binding Cassette Transporter and Cyp51A Proteins Are Essential for Azole Resistance in Aspergillus fumigatus

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Sanjoy Paul ◽  
Daniel Diekema ◽  
W. Scott Moye-Rowley
Keyword(s):  
Aspergillus Fumigatus ◽  
Azole Resistance ◽  
Atp Binding ◽  
Wild Type ◽  
Gene Encoding ◽  
Pathogenic Yeast ◽  
Genetic Changes ◽  
Content Type ◽  
Transporter Protein ◽  
Atp Binding Cassette

ABSTRACT While azole drugs targeting the biosynthesis of ergosterol are effective antifungal agents, their extensive use has led to the development of resistant organisms. Infections involving azole-resistant forms of the filamentous fungus Aspergillus fumigatus are often associated with genetic changes in the cyp51A gene encoding the lanosterol α14 demethylase target enzyme. Both a sequence duplication in the cyp51A promoter (TR34) and a substitution mutation in the coding sequence (L98H) are required for the full expression of azole resistance. A mechanism commonly observed in pathogenic yeast such as Candida albicans involves gain-of-function mutations in transcriptional regulatory proteins that induce expression of genes encoding ATP-binding cassette (ABC) transporters. We and others have found that an ABC transporter protein called Cdr1B (here referred to as AbcG1) is required for wild-type azole resistance in A. fumigatus. Here, we test the genetic relationship between the TR34 L98H allele of cyp51A and an abcG1 null mutation. Loss of AbcG1 from a TR34 L98H cyp51A-containing strain caused a large decrease in the azole resistance of the resulting double-mutant strain. We also generated antibodies that enabled the detection of both the wild-type and L98H forms of the Cyp51A protein. The introduction of the L98H lesion into the cyp51A gene led to a decreased production of immunoreactive enzyme, suggesting that this mutant protein is unstable. Our data confirm the importance of AbcG1 function during azole resistance even in a strongly drug-resistant background.

scholarly journals First Detection of TR46/Y121F/T289A and TR34/L98H Alterations in Aspergillus fumigatus Isolates from Azole-Naive Patients in Denmark despite Negative Findings in the Environment

2014 ◽  
Vol 58 (9) ◽  
pp. 5096-5101 ◽  
Author(s):  
K. M. T. Astvad ◽  
R. H. Jensen ◽  
T. M. Hassan ◽  
E. G. Mathiasen ◽  
G. M. Thomsen ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Transplant Patient ◽  
Susceptibility Testing ◽  
Reference Method ◽  
Ecological Niches ◽  
Azole Resistance ◽  
Wild Type ◽  
Content Type ◽  
Negative Findings ◽  
Microbroth Dilution

ABSTRACTAzole-resistantAspergillus fumigatusharboring the TR34/L98H or TR46/Y121F/T289A alterations is increasingly found in Europe and Asia. Here, we present the first clinical cases of TR46/Y121/T289A and three cases of TR34/L98H outside the cystic fibrosis (CF) population in Denmark and the results of environmental surveys. Four patients (2012 to 2014) with 11A. fumigatusand 4Rhizomucor pusillusisolates and 239 soil samples (spring 2010 and autumn 2013, respectively) with a total of 113A. fumigatusisolates were examined.Aspergillusisolates were screened for azole resistance using azole-containing agar. Confirmatory susceptibility testing was done using the EUCAST microbroth dilution EDEF 9.1 reference method. For relevantA. fumigatusisolates,CYP51Asequencing and microsatellite genotyping were performed. Three patients harbored TR34/L98H isolates. Two were azole naive at the time of acquisition and two were coinfected with wild-typeA. fumigatusorR. pusillusisolates, complicating and delaying diagnosis. The TR46/Y121F/T289A strain was isolated in 2014 from a lung transplant patient. Genotyping indicated that susceptible and resistantAspergillusisolates were unrelated and that no transmission between patients occurred. Azole resistance was not detected in any of the 113 soil isolates. TR34/L98H and TR46/Y121F/T289A alterations appear to be emerging in the clinical setting in Denmark and now involve azole-naive patients. Two recent soil-sampling surveys in Denmark were unable to indicate any increased prevalence of azole-resistantA. fumigatusin the environment. These findings further support the demand for real-time susceptibility testing of all clinically relevant isolates and for studies investigating the seasonal variation and ecological niches for azole-resistant environmentalA. fumigatus.

scholarly journals Structural Insights into the Azole Resistance of the Candida albicans Darlington Strain Using Saccharomyces cerevisiae Lanosterol 14α-Demethylase as a Surrogate

2021 ◽  
Vol 7 (11) ◽  
pp. 897
Author(s):  
Danyon O. Graham ◽  
Rajni K. Wilson ◽  
Yasmeen N. Ruma ◽  
Mikhail V. Keniya ◽  
Joel D. A. Tyndall ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Crystal Structures ◽  
Differential Susceptibility ◽  
Azole Resistance ◽  
Wild Type ◽  
Gene Encoding ◽  
High Level ◽  
Structural Insights ◽  
Level Resistance

Target-based azole resistance in Candida albicans involves overexpression of the ERG11 gene encoding lanosterol 14α-demethylase (LDM), and/or the presence of single or multiple mutations in this enzyme. Overexpression of Candida albicans LDM (CaLDM) Y132H I471T by the Darlington strain strongly increased resistance to the short-tailed azoles fluconazole and voriconazole, and weakly increased resistance to the longer-tailed azoles VT-1161, itraconazole and posaconazole. We have used, as surrogates, structurally aligned mutations in recombinant hexahistidine-tagged full-length Saccharomyces cerevisiae LDM6×His (ScLDM6×His) to elucidate how differential susceptibility to azole drugs is conferred by LDM of the C. albicans Darlington strain. The mutations Y140H and I471T were introduced, either alone or in combination, into ScLDM6×His via overexpression of the recombinant enzyme from the PDR5 locus of an azole hypersensitive strain of S. cerevisiae. Phenotypes and high-resolution X-ray crystal structures were determined for the surrogate enzymes in complex with representative short-tailed (voriconazole) and long-tailed (itraconazole) triazoles. The preferential high-level resistance to short-tailed azoles conferred by the ScLDM Y140H I471T mutant required both mutations, despite the I471T mutation conferring only a slight increase in resistance. Crystal structures did not detect changes in the position/tilt of the heme co-factor of wild-type ScLDM, I471T and Y140H single mutants, or the Y140H I471T double-mutant. The mutant threonine sidechain in the Darlington strain CaLDM perturbs the environment of the neighboring C-helix, affects the electronic environment of the heme, and may, via differences in closure of the neck of the substrate entry channel, increase preferential competition between lanosterol and short-tailed azole drugs.

scholarly journals Evaluation of MIC Strip Isavuconazole Test for Susceptibility Testing of Wild-Type and Non-Wild-Type Aspergillus fumigatus Isolates

2016 ◽  
Vol 61 (1) ◽  
Author(s):  
Maiken Cavling Arendrup ◽  
Paul Verweij ◽  
Henrik Vedel Nielsen
Keyword(s):  
Growth Inhibition ◽  
Aspergillus Fumigatus ◽  
Susceptibility Testing ◽  
Wild Type ◽  
Content Type

ABSTRACT We evaluated the MIC Strip Isavuconazole test against EUCAST E.Def 9.3 by using 40 wild-type and 39 CYP51A mutant Aspergillus fumigatus strains. The strip full inhibition endpoint (FIE) and 80% growth inhibition endpoint were determined by two independent readers, reader 1 (R1) and R2. The essential (within ±0, ±1, and ±2 twofold dilutions) and categorical agreements were best with the FIE (for R1/R2, 42%/41%, 75%/73%, and 90%/89% for essential agreement, and 91.1%/92.4% categorical agreement, with 6.3/8.9% very major errors and 0/1.3% major errors, respectively). The MIC Strip Isavuconazole test with the FIE appears to be useful.

scholarly journals Epidemiology and Molecular Characterizations of Azole Resistance in Clinical and Environmental Aspergillus fumigatus Isolates from China

2016 ◽  
Vol 60 (10) ◽  
pp. 5878-5884 ◽  
Author(s):  
Yong Chen ◽  
Zhongyi Lu ◽  
Jingjun Zhao ◽  
Ziying Zou ◽  
Yanwen Gong ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Antifungal Susceptibility ◽  
Public Health Problem ◽  
Wide Distribution ◽  
Resistance Mechanisms ◽  
Azole Resistance ◽  
Common Mutation ◽  
Content Type ◽  
Environmental Resistance ◽  
Resistant Strains

ABSTRACTAzole resistance inAspergillus fumigatushas emerged as a worldwide public health problem. We sought here to demonstrate the occurrence and characteristics of azole resistance inA. fumigatusfrom different parts of China. A total of 317 clinical and 144 environmentalA. fumigatusisolates from 12 provinces were collected and subjected to screening for azole resistance. Antifungal susceptibility,cyp51Agene sequencing, and genotyping were carried out for all suspected azole-resistant isolates and a subset of azole-susceptible isolates. As a result, 8 (2.5%) clinical and 2 (1.4%) environmentalA. fumigatusisolates were identified as azole resistant. Five azole-resistant strains exhibit the TR34/L98H mutation, whereas four carry the TR34/L98H/S297T/F495I mutation in thecyp51Agene. Genetic typing and phylogenetic analysis showed that there was a worldwide clonal expansion of the TR34/L98H isolates, while the TR34/L98H/S297T/F495I isolates from China harbored a distinct genetic background with resistant isolates from other countries. High polymorphisms existed in thecyp51Agene that produced amino acid changes among azole-susceptibleA. fumigatusisolates, with N248K being the most common mutation. These data suggest that the wide distribution of azole-resistantA. fumigatusmight be attributed to the environmental resistance mechanisms in China.

scholarly journals Unexpected Link between Metal Ion Deficiency and Autophagy in Aspergillus fumigatus

2007 ◽  
Vol 6 (12) ◽  
pp. 2437-2447 ◽  
Author(s):  
Daryl L. Richie ◽  
Kevin K. Fuller ◽  
Jarrod Fortwendel ◽  
Michael D. Miley ◽  
Jason W. McCarthy ◽  
...  
Keyword(s):  
Metal Ions ◽  
Aspergillus Fumigatus ◽  
Stress Responses ◽  
Metal Ion ◽  
Nitrogen Sources ◽  
Hyphal Growth ◽  
Wild Type ◽  
Serine Threonine Kinase ◽  
Gene Encoding ◽  
Carbon And Nitrogen

ABSTRACT Autophagy is the major cellular pathway for bulk degradation of cytosolic material and is required to maintain viability under starvation conditions. To determine the contribution of autophagy to starvation stress responses in the filamentous fungus Aspergillus fumigatus, we disrupted the A. fumigatus atg1 gene, encoding a serine/threonine kinase required for autophagy. The ΔAfatg1 mutant showed abnormal conidiophore development and reduced conidiation, but the defect could be bypassed by increasing the nitrogen content of the medium. When transferred to starvation medium, wild-type hyphae were able to undergo a limited amount of growth, resulting in radial expansion of the colony. In contrast, the ΔAfatg1 mutant was unable to grow under these conditions. However, supplementation of the medium with metal ions rescued the ability of the ΔAfatg1 mutant to grow in the absence of a carbon or nitrogen source. Depleting the medium of cations by using EDTA was sufficient to induce autophagy in wild-type A. fumigatus, even in the presence of abundant carbon and nitrogen, and the ΔAfatg1 mutant was severely growth impaired under these conditions. These findings establish a role for autophagy in the recycling of internal nitrogen sources to support conidiophore development and suggest that autophagy also contributes to the recycling of essential metal ions to sustain hyphal growth when exogenous nutrients are scarce.

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