whole-genome-sequencing-candida-glabrata-for-detection-markers

Whole Genome Sequencing of <em>Candida glabrata</em> for Detection of Markers of Antifungal Drug Resistance

Candida glabrata can rapidly acquire mutations that result in drug resistance, especially to azoles and echinocandins. Identification of genetic mutations ...

Westmead hospital

Ten bis(alkylpyridinium)alkane compounds were tested for antifungal activity against 19 species (26 isolates) of yeasts and molds. We then determined the MICs and minimum fungicidal concentrations (MFCs) of four of the most active compounds (compounds 1, 4, 5, and 8) against 80 Candida and 20 cryptococcal isolates, in comparison with the MICs of amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole, and caspofungin, using Clinical Laboratory and Standards Institutes broth microdulition M27-A3 (yeasts) or M38-A2 (filamentous fungi) susceptibility protocols. The compounds were more potent against Candida and Cryptococcus spp. (MIC range, 0.74 to 27.9 microg/ml) than molds (0.74 to 59.7 microg/ml). MICs against Exophiala were 0.37 to 5.9 microg/ml and as low as 1.48 microg/ml for Scedosporium but >or=25 microg/ml for zygomycetes, Aspergillus, and Fusarium spp. Compounds 1, 4, 5, and 8 exhibited good fungicidal activity against Candida and Cryptococcus, except for Candida parapsilosis (MICs of >44 mug/ml). Geometric mean (GM) MICs were similar to those of amphotericin B and lower than or comparable to fluconazole GM MICs but 10- to 100-fold greater than those for the other azoles. GM MICs against Candida glabrata were <1 microg/ml, significantly lower than fluconazole GM MICs (P<0.001) and similar to those of itraconazole, posaconazole, and voriconazole (GM MIC range of 0.4 to 1.23 microg/ml). The GM MIC of compound 4 against Candida guilliermondii was lower than that of fluconazole (1.69 microg/ml versus 7.48 microg/ml; P=0.012). MICs against Cryptococcus neoformans and Cryptococcus gattii were similar to those of fluconazole. The GM MIC of compound 4 was significantly higher for C. neoformans (3.83 mug/ml versus 1.81 microg/ml for C. gattii; P=0.015). This study has identified clinically relevant in vitro antifungal activities of novel bisalkypyridinium alkane compounds.

In vitro antifungal activities of bis(alkylpyridinium)alkane compounds against pathogenic yeasts and molds.

Miltefosine (MI) is a novel, potential antifungal agent with activity against some yeast and filamentous fungal pathogens. We previously demonstrated in the model yeast, Saccharomyces cerevisiae, that MI causes disruption of mitochondrial membrane potential and apoptosis-like cell death via interaction with the Cox9p sub-unit of cytochrome c oxidase (COX). To identify additional mechanisms of antifungal action, MI resistance was induced in S. cerevisiae by exposure to the mutagen, ethyl methanesulfonate, and gene mutation(s) responsible for resistance were investigated. An MI-resistant haploid strain (H-C101) was created. Resistance was retained in the diploid strain (D-C101) following mating, confirming dominant inheritance. Phenotypic assessment of individual D-C101 tetrads revealed that only one mutant gene contributed to the MI-resistance phenotype. To identify this gene, the genome of H-C101 was sequenced and 17 mutated genes, including metacaspase-encoding MCA1, were identified. The MCA1 mutation resulted in substitution of asparagine (N) with aspartic acid (D) at position 164 (MCA1(N164D)). MI resistance was found to be primarily due to MCA1(N164D), as single-copy episomal expression of MCA1(N164D), but not two other mutated genes (FAS1(T1417I) and BCK2(T104A)), resulted in MI resistance in the wild-type strain. Furthermore, an MCA1 deletion mutant (mca1Δ) was MI-resistant. MI treatment led to accumulation of reactive oxygen species (ROS) in MI-resistant (MCA1(N164D)-expressing and mca1Δ) strains and MI-susceptible (MCA1-expressing) strains, but failed to activate Mca1 in the MI-resistant strains, demonstrating that ROS accumulation does not contribute to the fungicidal effect of MI. In conclusion, functional disruption of Mca1, leads to MI resistance and inability to mediate MI-induced apoptotic effects. Mca1-mediated apoptosis is therefore a major mechanism of MI-induced antifungal action.

Functional disruption of yeast metacaspase, Mca1, leads to miltefosine resistance and inability to mediate miltefosine-induced apoptotic effects.

Protothecosis is a rare algal infection, affecting primarily immunocompromised hosts. Optimal management is unclear: in-vitro antimicrobial breakpoints are not established and therapeutic decisions are primarily based on case reports. We present a case of cutaneous Prototheca wickerhamii infection in an immunosuppressed 63 year old male, successfully treated with liposomal amphotericin and prolonged itraconazole. Inoculation may have been through frequent hot-tub use, highlighting hot-tub exposure as an infection risk for the immunocompromised host.

Successful treatment of cutaneous protothecosis with liposomal amphotericin and oral itraconazole.

Multi-antifungal drug resistance in Candida glabrata is increasing. We examined the feasibility of next-generation sequencing (NGS) to investigate the presence of antifungal drug resistance markers in C. glabrata.

Identification of genetic markers of resistance to echinocandins, azoles and 5-fluorocytosine in Candida glabrata by next-generation sequencing: a feasibility study.

Article	Year
In vitro antifungal activities of bis(alkylpyridinium)alkane compounds against pathogenic yeasts and molds. Antimicrobial agents and chemotherapy\| PubMed ID: 20530227	2010
Functional disruption of yeast metacaspase, Mca1, leads to miltefosine resistance and inability to mediate miltefosine-induced apoptotic effects. Fungal genetics and biology : FG & B\| PubMed ID: 24731805	2014
Successful treatment of cutaneous protothecosis with liposomal amphotericin and oral itraconazole. Medical mycology case reports\| PubMed ID: 27642561	2016
Identification of genetic markers of resistance to echinocandins, azoles and 5-fluorocytosine in Candida glabrata by next-generation sequencing: a feasibility study. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases\| PubMed ID: 28344162	2017

Chayanika Biswas

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