Emerging Threats in Antifungal-Resistant Fungal Pathogens.

The use of antifungal drugs in the therapy of fungal diseases can lead to the development of antifungal resistance. Resistance has been described for virtually all antifungal agents in diverse pathogens, including Candida and Aspergillus species. The majority of resistance mechanisms have also been elucidated at the molecular level in these pathogens. Drug resistance genes and genome mutations have been identified. Therapeutic choices are limited for the control of fungal diseases, and it is tempting to combine several drugs to achieve better therapeutic efficacy. In the recent years, several novel resistance patterns have been observed, including antifungal resistance originating from environmental sources in Aspergillus fumigatus and the emergence of simultaneous resistance to different antifungal classes (multidrug resistance) in different Candida species. This review will summarize these current trends

Novel Approaches for Fungal Transcriptomics from Host Samples.

Candida albicans adaptation to the host requires a profound reprogramming of the fungal transcriptome as compared to in vitro laboratory conditions. A detailed knowledge of the C. albicans transcriptome during the infection process is necessary in order to understand which of the fungal genes are important for host adaptation. Such genes could be thought of as potential targets for antifungal therapy. The acquisition of the C. albicans transcriptome is, however, technically challenging due to the low proportion of fungal RNA in host tissues. Two emerging technologies were used recently to circumvent this problem. One consists of the detection of low abundance fungal RNA using capture and reporter gene probes which is followed by emission and quantification of resulting fluorescent signals (nanoString). The other is based first on the capture of fungal RNA by short biotinylated oligonucleotide baits covering the C. albicans ORFome permitting fungal RNA purification. Next, the enriched fungal RNA is amplified and subjected to RNA sequencing (RNA-seq). Here we detail these two transcriptome approaches and discuss their advantages and limitations and future perspectives in microbial transcriptomics from host material

Azole and fungicide resistance in clinical and environmental Aspergillus fumigatus isolates

Aspergillus fumigatus is a human pathogen but it is also a widespread filamentous fungus in the environment. A. fumigatus can therefore be exposed to antifungals used in medical and agricultural environments. Only the class of azoles is used in both of these environments (i.e., voriconazole and itraconazole in medicine; prochloraz, propiconazole or imazalil in agriculture). Exposure to azoles provides the potential for the development of resistance. Several clinical itraconazole-resistant isolates have been reported in A. fumigatus and their resistance mechanisms have been partially resolved. Since limited data exist on the susceptibility of A. fumigatus to both medical and agricultural antifungals, we undertook a drug susceptibility study including clinical (400) and agricultural (150) A. fumigatus isolates (Swiss origin). We tested azoles and also compounds of major antifungal classes used in agriculture (i.e., azoxystrobin, iprodione, benalaxyl or cyprodinil). The results showed that all A. fumigatus isolates were intrinsically resistant to iprodione, benalaxyl or cyprodinil (MIC90>32 µg.ml−1) and that azoxystrobin minimal inhibitory concentrations (MICs) showed a wide range (0.06 to 32 µg.ml−1). MIC ranges of azoles were compound-dependent. MIC90 for voriconazole, itraconazole, imazalil and prochloraz were within a range of 0.125 to 1 µg.ml−1 and similar between clinical and environmental isolates, whereas propiconazole was the least active compound (MIC90: 4-8 µg.ml−1). Ten clinical and 36 environmental isolates with high itraconazole MIC (≥2 µg.ml−1) were detected. In clinical isolates, no cross-resistance was observed between itraconazole and all others azoles tested. Several patterns of azole MICs for were, however, observed in the environmental isolates. Unexpectedly, a single environmental isolate was voriconazole-resistant (MIC of 16 µg.ml−1) but still susceptible to itraconazole (MIC of 2 µg.ml−1). Taken together, our results demonstrate the absence of susceptibility of A. fumigatus isolates to non-azole agricultural agents and that there is little impact of azole resistance in both clinical and environmental isolates. When detected, azole resistance was compound-specifi

Comparative Genomics of Two Sequential <i>Candida glabrata</i> Clinical Isolates.

&lt;i&gt;Candida glabrata&lt;/i&gt; is an important fungal pathogen which develops rapid antifungal resistance in treated patients. It is known that azole treatments lead to antifungal resistance in this fungal species and that multidrug efflux transporters are involved in this process. Specific mutations in the transcriptional regulator &lt;i&gt;PDR1&lt;/i&gt; result in upregulation of the transporters. In addition, we showed that the &lt;i&gt;PDR1&lt;/i&gt; mutations can contribute to enhance virulence in animal models. In this study, we were interested to compare genomes of two specific &lt;i&gt;C. glabrata&lt;/i&gt; -related isolates, one of which was azole susceptible (DSY562) while the other was azole resistant (DSY565). DSY565 contained a &lt;i&gt;PDR1&lt;/i&gt; mutation (L280F) and was isolated after a time-lapse of 50 d of azole therapy. We expected that genome comparisons between both isolates could reveal additional mutations reflecting host adaptation or even additional resistance mechanisms. The PacBio technology used here yielded 14 major contigs (sizes 0.18-1.6 Mb) and mitochondrial genomes from both DSY562 and DSY565 isolates that were highly similar to each other. Comparisons of the clinical genomes with the published CBS138 genome indicated important genome rearrangements, but not between the clinical strains. Among the unique features, several retrotransposons were identified in the genomes of the investigated clinical isolates. DSY562 and DSY565 each contained a large set of adhesin-like genes (101 and 107, respectively), which exceed by far the number of reported adhesins (63) in the CBS138 genome. Comparison between DSY562 and DSY565 yielded 17 nonsynonymous SNPs (among which the was the expected &lt;i&gt;PDR1&lt;/i&gt; mutation) as well as small size indels in coding regions (11) but mainly in adhesin-like genes. The genomes contained a DNA mismatch repair allele of &lt;i&gt;MSH2&lt;/i&gt; known to be involved in the so-called hyper-mutator phenotype of this yeast species and the number of accumulated mutations between both clinical isolates is consistent with the presence of a &lt;i&gt;MSH2&lt;/i&gt; defect. In conclusion, this study is the first to compare genomes of &lt;i&gt;C. glabrata&lt;/i&gt; sequential clinical isolates using the PacBio technology as an approach. The genomes of these isolates taken in the same patient at two different time points exhibited limited variations, even if submitted to the host pressure

Probing the role of point mutations in the cyp51A gene from Aspergillus fumigatus in the model yeast Saccharomyces cerevisiae

Azole-resistant strains of Aspergillus fumigatus have been detected and the underlying molecular mechanisms of resistance characterized. Point mutations in the cyp51A gene have been proved to be related to azole resistance in A. fumigatus clinical strains and with different resistance profiles depending on the amino acid change (G54E, G54V, G54R, G54W, M220V, M220K, M220T, M220I). The aim of this work was to express A. fumigatus cyp51A genes in the yeast Saccharomyces cerevisiae in order to better assess the contribution of each independent amino acid substitution to resistance. A tetracycline regulatable system allowing repression of the endogenous essential ERG11 gene was used. The expression of Aspergillus cyp51A alleles could efficiently restore the absence of ERG11 in S. cerevisiae. In general, S. cerevisiae clones expressing. A. fumigatus cyp51A alleles from azole-resistant isolates showed higher MICs to all azoles tested than those expressing alleles from susceptible isolates. The azole susceptibility profiles obtained in S. cerevisiae upon expression of specific cyp51A alleles recapitulated susceptibility profiles observed from their A. fumigatus origins. In conclusion this work supports the concept that characteristics of specific A. fumigatus cyp51A alleles could be investigated in the heterologous host S. cerevisia

DAMA detection claim is still compatible with all other DM searches

We show that the annual modulation signal observed by DAMA can be reconciled with all other negative results from dark matter searches with a conventional halo model for particle masses around 5 to 9 GeV. We also show which particular dark matter stream could produce the DAMA signal.Comment: Talk given at TAUP2005, Sept. 10-14 2005, Zaragoza (Spain). 3 pages, 4 figure

Red-Shifted Firefly Luciferase Optimized for Candida albicans In vivo Bioluminescence Imaging.

Candida albicans is a major fungal pathogen causing life-threatening diseases in immuno-compromised patients. The efficacy of current drugs to combat C. albicans infections is limited, as these infections have a 40-60% mortality rate. There is a real need for novel therapeutic approaches, but such advances require a detailed knowledge of C. albicans and its in vivo pathogenesis. Additionally, any novel antifungal drugs against C. albicans infections will need to be tested for their in vivo efficacy over time. Fungal pathogenesis and drug-mediated resolution studies can both be evaluated using non-invasive in vivo imaging technologies. In the work presented here, we used a codon-optimized firefly luciferase reporter system for detecting C. albicans in mice. We adapted the firefly luciferase in order to improve its maximum emission intensity in the red light range (600-700 nm) as well as to improve its thermostability in mice. All non-invasive in vivo imaging of experimental animals was performed with a multimodal imaging system able to detect luminescent reporters and capture both reflectance and X-ray images. The modified firefly luciferase expressed in C. albicans (Mut2) was found to significantly increase the sensitivity of bioluminescence imaging (BLI) in systemic infections as compared to unmodified luciferase (Mut0). The same modified bioluminescence reporter system was used in an oropharyngeal candidiasis model. In both animal models, fungal loads could be correlated to the intensity of emitted light. Antifungal treatment efficacies were also evaluated on the basis of BLI signal intensity. In conclusion, BLI with a red-shifted firefly luciferase was found to be a powerful tool for testing the fate of C. albicans in various mice infection models

Examining the virulence of Candida albicans transcription factor mutants using Galleria mellonella and mouse infection models.

The aim of the present study was to identify Candida albicans transcription factors (TFs) involved in virulence. Although mice are considered the gold-standard model to study fungal virulence, mini-host infection models have been increasingly used. Here, barcoded TF mutants were first screened in mice by pools of strains and fungal burdens (FBs) quantified in kidneys. Mutants of unannotated genes which generated a kidney FB significantly different from that of wild-type were selected and individually examined in Galleria mellonella. In addition, mutants that could not be detected in mice were also tested in G. mellonella. Only 25% of these mutants displayed matching phenotypes in both hosts, highlighting a significant discrepancy between the two models. To address the basis of this difference (pool or host effects), a set of 19 mutants tested in G. mellonella were also injected individually into mice. Matching FB phenotypes were observed in 50% of the cases, highlighting the bias due to host effects. In contrast, 33.4% concordance was observed between pool and single strain infections in mice, thereby highlighting the bias introduced by the "pool effect." After filtering the results obtained from the two infection models, mutants for MBF1 and ZCF6 were selected. Independent marker-free mutants were subsequently tested in both hosts to validate previous results. The MBF1 mutant showed impaired infection in both models, while the ZCF6 mutant was only significant in mice infections. The two mutants showed no obvious in vitro phenotypes compared with the wild-type, indicating that these genes might be specifically involved in in vivo adapt

Potential Use of MALDI-ToF Mass Spectrometry for Rapid Detection of Antifungal Resistance in the Human Pathogen Candida glabrata.

The echinocandins are relatively new antifungal drugs that represent, together with the older azoles, the recommended and/or preferred agents to treat candidaemia and other forms of invasive candidiasis in human patients. If "time is of the essence" to reduce the mortality for these infections, the administration of appropriate antifungal therapy could be accelerated by the timely reporting of laboratory test results. In this study, we attempted to validate a MALDI-ToF mass spectrometry-based assay for the antifungal susceptibility testing (AFST) of the potentially multidrug-resistant pathogen Candida glabrata against anidulafungin and fluconazole. The practical applicability of the assay, reported here as MS-AFST, was assessed with a panel of clinical isolates that were selected to represent phenotypically and genotypically/molecularly susceptible or resistant strains. The data show the potential of our assay for rapid detection of antifungal resistance, although the MS-AFST assay performed at 3 h of the in vitro antifungal exposure failed to detect C. glabrata isolates with echinocandin resistance-associated FKS2 mutations. However, cell growth kinetics in the presence of anidulafungin revealed important cues about the in vitro fitness of C. glabrata isolates, which may lead to genotypic or phenotypic antifungal testing in clinical practice

An analysis method for time ordered data processing of Dark Matter experiments

The analysis of the time ordered data of Dark Matter experiments is becoming more and more challenging with the increase of sensitivity in the ongoing and forthcoming projects. Combined with the well-known level of background events, this leads to a rather high level of pile-up in the data. Ionization, scintillation as well as bolometric signals present common features in their acquisition timeline: low frequency baselines, random gaussian noise, parasitic noise and signal characterized by well-defined peaks. In particular, in the case of long-lasting signals such as bolometric ones, the pile-up of events may lead to an inaccurate reconstruction of the physical signal (misidentification as well as fake events). We present a general method to detect and extract signals in noisy data with a high pile-up rate and qe show that events from few keV to hundreds of keV can be reconstructed in time ordered data presenting a high pile-up rate. This method is based on an iterative detection and fitting procedure combined with prior wavelet-based denoising of the data and baseline subtraction. {We have tested this method on simulated data of the MACHe3 prototype experiment and shown that the iterative fitting procedure allows us to recover the lowest energy events, of the order of a few keV, in the presence of background signals from a few to hundreds of keV. Finally we applied this method to the recent MACHe3 data to successfully measure the spectrum of conversion electrons from Co57 source and also the spectrum of the background cosmic muons