Correlation between Etest®, disk diffusion, and microdilution methods for antifungal susceptibility testing of Candida species from infection and colonization

The correlation between the microdilution (MD), Etest® (ET), and disk diffusion (DD) methods was determined for amphotericin B, itraconazole and fluconazole. The minimal inhibitory concentration (MIC) of those antifungal agents was established for a total of 70 Candida spp. isolates from colonization and infection. The species distribution was: Candida albicans (n=27), C. tropicalis (n=17), C. glabrata (n=16), C. parapsilosis (n=8), and C. lusitaniae (n=2). Non-Candida albicans Candida species showed higher MICs for the three antifungal agents when compared with C. albicans isolates. The overall concordance (based on the MIC value obtainedwithin two dilutions) between the ET and the MD method was 83% for amphotericin B, 63% for itraconazole, and 64% for fluconazole. Considering the breakpoint, the agreement between the DD and MD methods was 71% for itraconazole and 67% for fluconazole. The DD zone diameters are highly reproducible and correlate well with the MD method, making agar-based methods a viable alternative to MD for susceptibility testing. However, data on agar-based tests for itraconazole and amphotericin B are yet scarce. Thus, further research must still be carried out to ensure the standardization to other antifungal agents.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - BEX 4642/06-

Susceptibility of Candida glabrata biofilms to echinocandins: alterations in the matrix composition

Candidiases are the most recurrent fungal infections, especially among immunosuppressed patients. Although Candida albicans is still the most widespread isolated species, non-Candida albicans Candida species have been increasing. The goal of this work was to determine the susceptibility of C. glabrata biofilms to echinocandins and to evaluate their effect on the biofilm matrix composition, comparing the results with other Candida species. Drug susceptibilities were assessed through the determination of minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC) and minimum biofilm eradication concentration (MBEC) of caspofungin (Csf) and micafugin (Mcf). The -1,3 glucans content of the matrices was assessed after contact with the drugs. The data suggest that, generally, after contact with echinocandins, the concentration of -1,3 glucans increased. These adjustments in the matrix composition of C. glabrata biofilms and the chemical differences between Csf and Mcf, seem responsible and may determine the effectivity of the drug responses.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 [POCI-01–0145-FEDER-006684] and BioTecNorte operation [NORTE-01–0145-FEDER-000004] funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte, Célia F. Rodrigues’ [SFRH/BD/93078/2013] PhD grant and M. Elisa Rodrigues [SFRH/BPD/95401/2013] post-doctoral grant.info:eu-repo/semantics/publishedVersio

Filamentous fungal human pathogens from food emphasising Aspergillus, Fusarium and Mucor

The following are available online at www.mdpi.com/2076-2607/5/3/44/s1,TableS1: Human pathogenic filamentous fungi isolated recorded from food. Names that are currently invalid but found in the literature are indicated by square brackets.Disease caused by filamentous fungal human pathogens (FFHP) is increasing. These organisms cause severe mycoses in immunosuppressed individuals, such as those: (a) with AIDS; (b) having undergone transplantation; and/or (c) undergoing chemotherapy. Immunocompetent people can become infected. Some FFHP are isolated from foods which may be fomites. However, the information concerning particular species on specific food is large, dispersed and difficult to obtain. Reports of filamentous fungi from food/crops and causing human disease are frequently only available in the literature of food mycology/plant pathology and medical mycology, respectively: it is seldom cross-referenced. Aspergillus contains some species with strains that are the most dangerous FFHP, with Aspergillus fumigatus causing the most serious diseases. Fusarium and Mucor also contain species of high importance and approximately 15 other genera are involved. A checklist and database of FFHP species isolated from food is presented herein with emphasis on Aspergillus, Fusarium and Mucor in summary tables to increase awareness of the connection between food and FFHP. Metadata on all FFHP is provided in a large supplementary table for updating and revision when necessary. Previous names of fungi have been revised to reflect current valid usage whenever appropriate. The information will form a foundation for future research and taxonomic revisions in the field. The paper will be highly useful for medical practitioners, food mycologists, fungal taxonomists, patients, regulators and food producers interested in reducing infectious diseases and producing high quality food.The authors thank: (a) the FCT Strategic Project UID/BIO/04469/2013 unit; (b) project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462); and (c) project “BioInd—Biotechnology and Bioengineering for improved Industrial and Agro-Food processes”, REF. NORTE-07-0124-FEDER-000028 Co-funded by the Programa Operacional Regional do Norte (ON.2—O Novo Norte), QREN, FEDER.info:eu-repo/semantics/publishedVersio

Novel, synergistic antifungal combinations that target translation fidelity

There is an unmet need for new antifungal or fungicide treatments, as resistance to existing treatments grows. Combination treatments help to combat resistance. Here we develop a novel, effective target for combination antifungal therapy. Different aminoglycoside antibiotics combined with different sulphate-transport inhibitors produced strong, synergistic growth-inhibition of several fungi. Combinations decreased the respective MICs by ≥8 fold. Synergy was suppressed in yeast mutants resistant to effects of sulphate-mimetics (like chromate or molybdate) on sulphate transport. By different mechanisms, aminoglycosides and inhibition of sulphate transport cause errors in mRNA translation. The mistranslation rate was stimulated up to 10-fold when the agents were used in combination, consistent with this being the mode of synergistic action. A range of undesirable fungi were susceptible to synergistic inhibition by the combinations, including the human pathogens Candida albicans, C. glabrata and Cryptococcus neoformans, the food spoilage organism Zygosaccharomyces bailii and the phytopathogens Rhizoctonia solani and Zymoseptoria tritici. There was some specificity as certain fungi were unaffected. There was no synergy against bacterial or mammalian cells. The results indicate that translation fidelity is a promising new target for combinatorial treatment of undesirable fungi, the combinations requiring substantially decreased doses of active components compared to each agent alone

Demethylase Inhibitor Fungicide Resistance in Pyrenophora teres f. sp. teres Associated with Target Site Modification and Inducible Overexpression of Cyp51

Pyrenophora teres f. sp. teres is the cause of net form of net blotch, an economically important foliar disease in barley (Hordeum vulgare). Net and spot forms of net blotch are widely controlled using site-specific systemic fungicides. Although resistance to succinate dehydrogenase inhibitors and quinone outside inhibitors has been addressed before in net blotches, mechanisms controlling demethylation inhibitor resistance have not yet been reported at the molecular level. Here we report the isolation of strains of net form of net blotch in Australia since 2013 resistant to a range of demethylase inhibitor fungicides. Cyp51A:KO103-A1, an allele with the mutation F489L, corresponding to the archetype F495I in Aspergillus fumigatus, was only present in resistant strains and was correlated with resistance factors to various demethylase inhibitors ranging from 1.1 for epoxiconazole to 31.7 for prochloraz. Structural in silico modelling of the sensitive and resistant CYP51A proteins docked with different demethylase inhibitor fungicides showed how the interaction of F489L within the heme cavity produced a localised constriction of the region adjacent to the docking site that is predicted to result in lower binding affinities. Resistant strains also displayed enhanced induced expression of the two Cyp51A paralogs and of Cyp51B genes. While Cyp51B was found to be constitutively expressed in the absence of fungicide, Cyp51A was only detected at extremely low levels. Under fungicide induction, expression of Cyp51B, Cyp51A2 and Cyp51A1 was shown to be 1.6-, 3- and 5.3-fold higher, respectively in the resistant isolate compared to the wild type. These increased levels of expression were not supported by changes in the promoters of any of the three genes. The implications of these findings on demethylase inhibitor activity will require current net blotch management strategies to be reconsidered in order to avoid the development of further resistance and preserve the lifespan of fungicides in use

High-Resolution SNP/CGH Microarrays Reveal the Accumulation of Loss of Heterozygosity in Commonly Used Candida albicans Strains

Phenotypic diversity can arise rapidly through loss of heterozygosity (LOH) or by the acquisition of copy number variations (CNV) spanning whole chromosomes or shorter contiguous chromosome segments. In Candida albicans, a heterozygous diploid yeast pathogen with no known meiotic cycle, homozygosis and aneuploidy alter clinical characteristics, including drug resistance. Here, we developed a high-resolution microarray that simultaneously detects ∼39,000 single nucleotide polymorphism (SNP) alleles and ∼20,000 copy number variation loci across the C. albicans genome. An important feature of the array analysis is a computational pipeline that determines SNP allele ratios based upon chromosome copy number. Using the array and analysis tools, we constructed a haplotype map (hapmap) of strain SC5314 to assign SNP alleles to specific homologs, and we used it to follow the acquisition of loss of heterozygosity (LOH) and copy number changes in a series of derived laboratory strains. This high-resolution SNP/CGH microarray and the associated hapmap facilitated the phasing of alleles in lab strains and revealed detrimental genome changes that arose frequently during molecular manipulations of laboratory strains. Furthermore, it provided a useful tool for rapid, high-resolution, and cost-effective characterization of changes in allele diversity as well as changes in chromosome copy number in new C. albicans isolates