Efficacy of the combination of amphotericin B and echinocandins against Candida auris in vitro and in the Caenorhabditis elegans host model.

IMPORTANCE: Multidrug resistance is a rising problem among non-Candida albicans species, such as Candida auris. This therapeutic problem has been very important during the COVID-19 pandemic. The World Health Organization has included C. auris in its global priority list of health-threatening fungi, to study this emerging multidrug-resistant species and to develop effective alternative therapies. In the present study, the synergistic effect of the combination of amphotericin B and echinocandins has been demonstrated against blood isolates of C. auris. Different susceptibility responses were also observed between aggregative and non-aggregative phenotypes. The antifungal activity of these drug combinations against C. auris was also demonstrated in the Caenorhabditis elegans host model of candidiasis, confirming the suitability and usefulness of this model in the search for solutions to antimicrobial resistance.

Integrated post-genomic cell wall analysis reveals floating biofilm formation associated with high expression of flocculins in the pathogen Pichia kudriavzevii.

The pathogenic yeast Pichia kudriavzevii, previously known as Candida krusei, is more distantly related to Candida albicans than clinically relevant CTG-clade Candida species. Its cell wall, a dynamic organelle that is the first point of interaction between pathogen and host, is relatively understudied, and its wall proteome remains unidentified to date. Here, we present an integrated study of the cell wall in P. kudriavzevii. Our comparative genomic studies and experimental data indicate that the general structure of the cell wall in P. kudriavzevii is similar to Saccharomyces cerevisiae and C. albicans and is comprised of ß-1,3-glucan, ß-1,6-glucan, chitin, and mannoproteins. However, some pronounced differences with C. albicans walls were observed, for instance, higher mannan and protein levels and altered protein mannosylation patterns. Further, despite absence of proteins with high sequence similarity to Candida adhesins, protein structure modeling identified eleven proteins related to flocculins/adhesins in S. cerevisiae or C. albicans. To obtain a proteomic comparison of biofilm and planktonic cells, P. kudriavzevii cells were grown to exponential phase and in static 24-h cultures. Interestingly, the 24-h static cultures of P. kudriavzevii yielded formation of floating biofilm (flor) rather than adherence to polystyrene at the bottom. The proteomic analysis of both conditions identified a total of 33 cell wall proteins. In line with a possible role in flor formation, increased abundance of flocculins, in particular Flo110, was observed in the floating biofilm compared to exponential cells. This study is the first to provide a detailed description of the cell wall in P. kudriavzevii including its cell wall proteome, and paves the way for further investigations on the importance of flor formation and flocculins in the pathogenesis of P. kudriavzevii.

Usefulness of Chromogenic Media with Fluconazole Supplementation for Presumptive Identification of Candida auris.

Introduction:Candida auris is a major threat to public health. Rapid detection is essential for early treatment and transmission control. The use of chromogenic media allows the presumptive identification of this new species. The aim of this study is to describe the morphological characteristics of C. auris colonies on three commercial chromogenic media. Methods: Nineteen C. auris isolates from different countries/clades and 18 isolates of other species were cultivated in CHROMagarTM Candida Plus, HiCromeTM Candida, CHROMagar-Candida, and fluconazole-supplemented (32 mg/L) CHROMagar-Candida media. Results: On CHROMagarTM Candida Plus and HiCromeTM Candida, C. auris isolates from Colombia, Venezuela, India, Korea, and Japan displayed blue-shaded colonies, while isolates from Spain and Germany exhibited light pink shades with a bluish halo. All isolates showed white to pink colonies on CHROMagar-Candida. On CHROMagar Candida supplemented with fluconazole, whilst C. auris, C. glabrata, or C. krusei showed a similar pink color at 48 h incubation, phenotypic differentiation was possible by the rough, paraffin-like texture or the intense purple color acquired by C. krusei and C. glabrata, respectively. Moreover, in this medium, the presence of C. auris in combination with other species of similar color was not limiting for its early identification, due to this medium selecting only strains resistant to this antifungal. Conclusions: The use of chromogenic media such as CHROMagarTM Candida Plus facilitates a presumptive identification of C. auris. However, this identification can be difficult in the presence of mixed cultures. In these cases, the use of CHROMagarTM Candida medium with 32 mg/L fluconazole offers better performance for the identification of C. auris by inhibiting fluconazole-susceptible strains and selecting rare or high fluconazole MIC (>32 mg/L) isolates.

Chromosome-level assemblies from diverse clades reveal limited structural and gene content variation in the genome of Candida glabrata.

BACKGROUND: Candida glabrata is an opportunistic yeast pathogen thought to have a large genetic and phenotypic diversity and a highly plastic genome. However, the lack of chromosome-level genome assemblies representing this diversity limits our ability to accurately establish how chromosomal structure and gene content vary across strains. RESULTS: Here, we expanded publicly available assemblies by using long-read sequencing technologies in twelve diverse strains, obtaining a final set of twenty-one chromosome-level genomes spanning the known C. glabrata diversity. Using comparative approaches, we inferred variation in chromosome structure and determined the pan-genome, including an analysis of the adhesin gene repertoire. Our analysis uncovered four new adhesin orthogroups and inferred a rich ancestral adhesion repertoire, which was subsequently shaped through a still ongoing process of gene loss, gene duplication, and gene conversion. CONCLUSIONS: C. glabrata has a largely stable pan-genome except for a highly variable subset of genes encoding cell wall-associated functions. Adhesin repertoire was established for each strain and showed variability among clades.

Characterization of Awp14, A Novel Cluster III Adhesin Identified in a High Biofilm-Forming Candida glabrata Isolate.

Candida glabrata is among the most prevalent causes of candidiasis. Unlike Candida albicans, it is not capable of changing morphology between yeast and hyphal forms but instead has developed other virulence factors. An important feature is its unprecedented large repertoire of predicted cell wall adhesins, which are thought to enable adherence to a variety of surfaces under different conditions. Here, we analyzed the wall proteome of PEU1221, a high biofilm-forming clinical strain isolated from an infected central venous catheter, under biofilm-forming conditions. This isolate shows increased incorporation of putative adhesins, including eight proteins that were not detected in walls of reference strain ATCC 2001, and of which Epa22, Awp14, and Awp2e were identified for the first time. The proteomics data suggest that cluster III adhesin Awp14 is relatively abundant in PEU1221. Phenotypic studies with awp14Δ deletion mutants showed that Awp14 is not responsible for the high biofilm formation of PEU1221 onto polystyrene. However, awp14Δ mutant cells in PEU1221 background showed a slightly diminished binding to chitin and seemed to sediment slightly slower than the parental strain suggesting implication in fungal cell-cell interactions. By structural modeling, we further demonstrate similarity between the ligand-binding domains of cluster III adhesin Awp14 and those of cluster V and VI adhesins. In conclusion, our work confirms the increased incorporation of putative adhesins, such as Awp14, in high biofilm-forming isolates, and contributes to decipher the precise role of these proteins in the establishment of C. glabrata infections.

Virulence of Candida auris from different clinical origins in Caenorhabditis elegans and Galleria mellonella host models.

Candida auris is an emerging multidrug-resistant fungal pathogen responsible for nosocomial outbreaks of invasive candidiasis. Although several studies on the pathogenicity of this species have been reported, the knowledge on C. auris virulence is still limited. This study aims to analyze the pathogenicity of C. auris, using one aggregating isolate and eleven non-aggregating isolates from different clinical origins (blood, urine and oropharyngeal specimens) in two alternative host models of candidiasis: Caenorhabditis elegans and Galleria mellonella. Furthermore, possible associations between virulence, aggregation, biofilm-forming capacity, and clinical origin were assessed. The aggregating phenotype isolate was less virulent in both in vivo invertebrate infection models than non-aggregating isolates but showed higher capacity to form biofilms. Blood isolates were significantly more virulent than those isolated from urine and respiratory specimens in the G. mellonella model of candidiasis. We conclude that both models of candidiasis present pros and cons but prove useful to evaluate the virulence of C. auris in vivo. Both models also evidence the heterogeneity in virulence that this species can develop, which may be influenced by the aggregative phenotype and clinical origin.

High Biofilm Formation of Non-Smooth Candida parapsilosis Correlates with Increased Incorporation of GPI-Modified Wall Adhesins.

Candida parapsilosis is among the most frequent causes of candidiasis. Clinical isolates of this species show large variations in colony morphotype, ranging from round and smooth to a variety of non-smooth irregular colony shapes. A non-smooth appearance is related to increased formation of pseudohyphae, higher capacity to form biofilms on abiotic surfaces, and invading agar. Here, we present a comprehensive study of the cell wall proteome of C. parapsilosis reference strain CDC317 and seven clinical isolates under planktonic and sessile conditions. This analysis resulted in the identification of 40 wall proteins, most of them homologs of known Candida albicans cell wall proteins, such as Gas, Crh, Bgl2, Cht2, Ecm33, Sap, Sod, Plb, Pir, Pga30, Pga59, and adhesin family members. Comparative analysis of exponentially growing and stationary phase planktonic cultures of CDC317 at 30 °C and 37 °C revealed only minor variations. However, comparison of smooth isolates to non-smooth isolates with high biofilm formation capacity showed an increase in abundance and diversity of putative wall adhesins from Als, Iff/Hyr, and Hwp families in the latter. This difference depended more strongly on strain phenotype than on the growth conditions, as it was observed in planktonic as well as biofilm cells. Thus, in the set of isolates analyzed, the high biofilm formation capacity of non-smooth C. parapsilosis isolates with elongated cellular phenotypes correlates with the increased surface expression of putative wall adhesins in accordance with their proposed cellular function.

Candida parapsilosis Colony Morphotype Forecasts Biofilm Formation of Clinical Isolates.

Candida parapsilosis is a frequent cause of fungal bloodstream infections, especially in critically ill neonates or immunocompromised patients. Due to the formation of biofilms, the use of indwelling catheters and other medical devices increases the risk of infection and complicates treatment, as cells embedded in biofilms display reduced drug susceptibility. Therefore, biofilm formation may be a significant clinical parameter, guiding downstream therapeutic choices. Here, we phenotypically characterized 120 selected isolates out of a prospective collection of 215 clinical C. parapsilosis isolates, determining biofilm formation, major emerging colony morphotype, and antifungal drug susceptibility of the isolates and their biofilms. In our isolate set, increased biofilm formation capacity was independent of body site of isolation and not predictable using standard or modified European Committee on Antimicrobial Susceptibility Testing (EUCAST) drug susceptibility testing protocols. In contrast, biofilm formation was strongly correlated with the appearance of non-smooth colony morphotypes and invasiveness into agar plates. Our data suggest that the observation of non-smooth colony morphotypes in cultures of C. parapsilosis may help as an indicator to consider the initiation of anti-biofilm-active therapy, such as the switch from azole- to echinocandin- or polyene-based strategies, especially in case of infections by potent biofilm-forming strains.

Identification of Candida auris and related species by multiplex PCR based on unique GPI protein-encoding genes.

BACKGROUND: The pathogen Candida auris is rapidly gaining clinical importance because of its resistance to antifungal treatments and its persistence in hospital environments. Early and accurate diagnosis of C. auris infections is crucial, and however, the fungus has often been misidentified by commercial systems. OBJECTIVES: To develop conventional and real-time PCR methods for accurate and rapid identification of C. auris and its discrimination from closely related species by exploiting the uniqueness of certain glycosylphosphatidylinositol (GPI)-modified protein-encoding genes. METHODS: Species-specific primers for two unique putative GPI protein-encoding genes per species were designed for C. auris, C. haemulonii, C. pseudohaemulonii, C. duobushaemulonii, C. lusitaniae and C. albicans. Primers were blind tested for their specificity and efficiency in conventional and real-time multiplex PCR set-up. RESULTS: All primers combinations showed excellent species specificity. In multiplex mode, correct identification was aided by different-sized amplicons for each species. Efficiency of the C. auris primers was validated using a panel of 155 C. auris isolates, including all known genetically diverse clades. In real-time multiplex PCR, different melting points of the amplicons allowed the distinction of C. auris from four related species. C. auris limit of detection was 5 CFU/reaction with a threshold value of 32. The method was also able to detect C. auris in spiked blood and serum. CONCLUSIONS: PCR identification based on unique GPI protein-encoding genes allows for accurate and rapid species identification of C. auris and related species without need for expensive equipment when applied in conventional PCR set-up.

Candida duobushaemulonii: An Old But Unreported Pathogen.

Candidiasis caused by species of the Candida haemulonii complex (Candida haemulonii and Candida duobushaemulonii) and closely related species, Candida auris and Candida pseudohaemulonii are increasing. These species often show reduced susceptibility to antifungal drugs, such as azoles and amphotericin B or, less frequently, echinocandins. However, conventional phenotypic identification methods are unable to accurately differentiate these species and, therefore, their prevalence may have been underestimated. In this study, 150 isolates that were probably misidentified were reanalyzed using two novel PCR approaches. We found that one isolate previously identified in 1996 as Candida intermedia was C. duobushaemulonii, being one of the oldest isolates of this species described to date. We also found that this isolate had reduced susceptibility to fluconazole, itraconazole, and amphotericin B.

MicroMundo Upside Down: Targeted Searching for Antibiotics-Producing Bacteria From Soil With Reverse Antibiosis Approaches.

Tiny Earth (TE) is a popular international citizen science program aimed at improving public awareness on the growing antimicrobial resistance problem of which MicroMundo Albacete is a Spanish node. With a protocol that is focused on the isolation of antibiotics-producing actinomycetes from soil, 70% of the high school students in MicroMundo Albacete 2020 isolated colonies with antagonistic activity against Gram-positive tester bacteria. However, no activity was found against Gram-negative bacteria. Here, we further adapted the protocol toward a more targeted screening that also enables isolation of antagonistic bacteria against Gram negatives using two different reverse-antibiosis approaches involving a spraying technique or flipping soil sample disks upside down. Exploiting the soil samples from MicroMundo Albacete 2020, the new approaches yielded isolation of actinomycete bacteria with antagonistic activity against Gram-negative as well as Gram-positive tester bacteria. We propose that (educational) science programs which aim to search for antibiotic-producing bacteria may implement these approaches in their protocol to promote a successful and stimulating outcome of the experiment for the participating students.

Optimizing Small World Initiative service learning by focusing on antibiotics-producing actinomycetes from soil.

Small World Initiative and Tiny Earth are popular citizen science programs that are implemented worldwide in response to the global antibiotic resistance crisis. When starting up the program in Albacete (Spain), we noted that rates of isolated antibiotic-producing bacteria are generally low. To make the activity more stimulating for participating students, we modified the protocol to obtain more positive results by focusing on isolation of actinomycetes, the main producers of most clinically used antibiotics. Adaptations involved redesigning culture media, incubation times and temperatures, and modification of the ESKAPE antibiosis experiment by employing an agar-transplantation step. Of 390 bacterial isolates tested, almost 6% tested positive in antibiosis experiments and DNA sequence analysis confirmed that all positives are actinomycetes, demonstrating that our protocol is efficient toward isolating antibiotic-producing actinomycetes from soil. Evaluation forms filled by participating students indicated that the program was received very positively and that our modifications contribute to make this educational program more stimulating and efficient.

Deletion of GLX3 in Candida albicans affects temperature tolerance, biofilm formation and virulence.

Candida albicans is a predominant cause of fungal infections in mucosal tissues as well as life-threatening bloodstream infections in immunocompromised patients. Within the human body, C. albicans is mostly embedded in biofilms, which provides increased resistance to antifungal drugs. The glyoxalase Glx3 is an abundant proteomic component of the biofilm extracellular matrix. Here, we document phenotypic studies of a glx3Δ null mutant concerning its role in biofilm formation, filamentation, antifungal drug resistance, cell wall integrity and virulence. First, consistent with its function as glyoxalase, the glx3 null mutant showed impaired growth on media containing glycerol as the carbon source and in the presence of low concentrations of hydrogen peroxide. Importantly, the glx3Δ mutant showed decreased fitness at 37°C and formed less biofilm as compared to wild type and a reintegrant strain. At the permissive temperature of 28°C, the glx3Δ mutant showed impaired filamentation as well as increased sensitivity to Calcofluor white, Congo red, sodium dodecyl sulfate and zymolyase, indicating subtle alterations in wall architecture even though gross quantitative compositional changes were not detected. Interestingly, and consistent with its impaired filamentation, biofilm formation and growth at 37°C, the glx3Δ mutant is avirulent. Our results underline the role of Glx3 in fungal pathogenesis and the involvement of the fungal wall in this process.

Molecular identification of Candida auris by PCR amplification of species-specific GPI protein-encoding genes.

The emerging multidrug-resistant pathogenic yeast Candida auris causes life-threatening invasive infections and shows a capacity for hospital transmission that is uncommon in other Candida species. Rapid and accurate diagnosis of C. auris infections is crucial; however, the fungus is frequently misidentified. Here, we present a rapid and easily applicable PCR assay for reliable identification of C. auris by designing primers from unique GPI protein-encoding genes. Specificity of the used primers for C. auris was verified with a panel of 19 different Candida species including the clinically most relevant and phylogenetically closely related species. Efficacy of the PCR approach was validated by correctly identifying 112 C. auris isolates from an outbreak in a Spanish hospital, 20% of which were not reliably identified by MALDI-TOF MS, and 27 genotypically diverse C. auris isolates originating from hospitals in various countries, in a test that included (blind) negative controls. By employing two GPI protein primer pairs in a single PCR, a double screening can be performed, which enhances the robustness of the PCR assay and avoids potential false negatives due to recent evolutionary events, as was observed for two isolates. Our PCR method, which is based on the uniqueness of selected GPI protein-encoding genes, is useful for easy, low-cost, and accurate identification of C. auris infections in a clinical setting.

Identification of Secreted Candida Proteins Using Mass Spectrometry.

Analysis of fungal secretomes using mass spectrometry is a useful technique in cell biology. Knowledge of the secretome of a human fungal pathogen may yield important information of host-pathogen interactions and may be useful for identifying vaccines candidates or diagnostic markers for antifungal strategies. In this chapter, with a main focus on sample preparation aspects, we describe the methodology that we apply for gel-independent batch identification and quantification of proteins that are secreted during growth in liquid cultures. Using these techniques with Candida and other yeast species, the majority of the identified proteins are classical secretory proteins and cell wall proteins containing N-terminal signal peptides for secretion, although dependent on sample preparation quality and the mass spectrometric analysis also usually, a number of nonsecretory proteins are identified.

Proteomic analysis of hyperadhesive Candida glabrata clinical isolates reveals a core wall proteome and differential incorporation of adhesins.

Attachment to human host tissues or abiotic medical devices is a key step in the development of infections by Candida glabrata. The genome of this pathogenic yeast codes for a large number of adhesins, but proteomic work using reference strains has shown incorporation of only few adhesins in the cell wall. By making inventories of the wall proteomes of hyperadhesive clinical isolates and reference strain CBS138 using mass spectrometry, we describe the cell wall proteome of C. glabrata and tested the hypothesis that hyperadhesive isolates display differential incorporation of adhesins. Two clinical strains (PEU382 and PEU427) were selected, which both were hyperadhesive to polystyrene and showed high surface hydrophobicity. Cell wall proteome analysis under biofilm-forming conditions identified a core proteome of about 20 proteins present in all C. glabrata strains. In addition, 12 adhesin-like wall proteins were identified in the hyperadherent strains, including six novel adhesins (Awp8-13) of which only Awp12 was also present in CBS138. We conclude that the hyperadhesive capacity of these two clinical C. glabrata isolates is correlated with increased and differential incorporation of cell wall adhesins. Future studies should elucidate the role of the identified proteins in the establishment of C. glabrata infections.

Rasagiline meta-analysis: a spotlight on clinical safety and adverse events when treating Parkinson's disease.

INTRODUCTION: Rasagiline (Azilect, AGN 1135) is a selective irreversible inhibitor of monoamine oxidase B (MAO-B). MAO-B regulates the brain concentrations of important neurotransmitters that are related to movement, emotion, and cognition. Oral rasagiline, as monotherapy or as adjunctive therapy to levodopa, was effective in the symptomatic treatment of adult patients with Parkinson's disease participating in double-blind, placebo-controlled, international studies. AREAS COVERED: This article reviews the reported adverse effects of rasagiline. A MEDLINE search was performed for all articles from 1990 to present, which reported any adverse effects from rasagiline or related references. We conducted an analysis of the adverse effects of rasagiline based on the reported clinical studies. Furthermore, we compared the incidence of adverse events in clinical trials for rasagiline and placebo. EXPERT OPINION: Among the most frequently reported adverse effects for rasagiline as monotherapy are headache, dizziness, and insomnia. Depression, dizziness, somnolence, and other sleep disorders are reported when used in combination therapy. Our analysis demonstrates that the most frequently reported adverse effects in trials did not occur more often with rasagiline than placebo. In conclusion, rasagiline is a well-tolerated MAO-B inhibitor that may help to achieve the desired level of clinical benefit in Parkinson's disease.

Adhesins in human fungal pathogens: glue with plenty of stick.

Understanding the pathogenesis of an infectious disease is critical for developing new methods to prevent infection and diagnose or cure disease. Adherence of microorganisms to host tissue is a prerequisite for tissue invasion and infection. Fungal cell wall adhesins involved in adherence to host tissue or abiotic medical devices are critical for colonization leading to invasion and damage of host tissue. Here, with a main focus on pathogenic Candida species, we summarize recent progress made in the field of adhesins in human fungal pathogens and underscore the importance of these proteins in establishment of fungal diseases.

Genomic insights into the atopic eczema-associated skin commensal yeast Malassezia sympodialis.

UNLABELLED: Malassezia commensal yeasts are associated with a number of skin disorders, such as atopic eczema/dermatitis and dandruff, and they also can cause systemic infections. Here we describe the 7.67-Mbp genome of Malassezia sympodialis, a species associated with atopic eczema, and contrast its genome repertoire with that of Malassezia globosa, associated with dandruff, as well as those of other closely related fungi. Ninety percent of the predicted M. sympodialis protein coding genes were experimentally verified by mass spectrometry at the protein level. We identified a relatively limited number of genes related to lipid biosynthesis, and both species lack the fatty acid synthase gene, in line with the known requirement of these yeasts to assimilate lipids from the host. Malassezia species do not appear to have many cell wall-localized glycosylphosphatidylinositol (GPI) proteins and lack other cell wall proteins previously identified in other fungi. This is surprising given that in other fungi these proteins have been shown to mediate interactions (e.g., adhesion and biofilm formation) with the host. The genome revealed a complex evolutionary history for an allergen of unknown function, Mala s 7, shown to be encoded by a member of an amplified gene family of secreted proteins. Based on genetic and biochemical studies with the basidiomycete human fungal pathogen Cryptococcus neoformans, we characterized the allergen Mala s 6 as the cytoplasmic cyclophilin A. We further present evidence that M. sympodialis may have the capacity to undergo sexual reproduction and present a model for a pseudobipolar mating system that allows limited recombination between two linked MAT loci. IMPORTANCE: Malassezia commensal yeasts are associated with a number of skin disorders. The previously published genome of M. globosa provided some of the first insights into Malassezia biology and its involvement in dandruff. Here, we present the genome of M. sympodialis, frequently isolated from patients with atopic eczema and healthy individuals. We combined comparative genomics with sequencing and functional characterization of specific genes in a population of clinical isolates and in closely related model systems. Our analyses provide insights into the evolution of allergens related to atopic eczema and the evolutionary trajectory of the machinery for sexual reproduction and meiosis. We hypothesize that M. sympodialis may undergo sexual reproduction, which has important implications for the understanding of the life cycle and virulence potential of this medically important yeast. Our findings provide a foundation for the development of genetic and genomic tools to elucidate host-microbe interactions that occur on the skin and to identify potential therapeutic targets.

Minocycline mediated mitochondrial cytoprotection: premises for therapy of cerebrovascular and neurodegenerative diseases.

In the last decades, emerging molecular targets for ischemic neuroprotection and regeneration have been postulated. This fact allowed that classical drugs with well established therapeutic applications might be used in cerebrovascular diseases as well as neurodegenerative diseases. Minocycline is a commonly used antibiotic of the tetracycline family (7-dimethylamino-6-dimethyl-6-deoxytetracycline) which reveals cytoprotective capability and potential use in treatment of different diseases. Here, we discuss the literature concerning minocycline. The available data indicate that the antibiotic has multi-faceted effects on cell functions and, consequently, a number of clinical properties that are useful and/or could be useful for treatment of different diseases including bacterial infections, cancer, autoimmune disorders, ischemia as well as neurodegenerative and psychiatric diseases. Thus, application of minocycline as a therapeutic agent is the subject of clinical trials for various diseases. It is also evident that minocycline-mediated cytoprotection, including neuroprotection, is an important aspect of its clinical application. Here, we have reviewed the basis of the minocycline activity as well as different studies indicating that minocycline can be used as potential therapeutic agent in both cerebrovascular and neurodegenerative diseases in human.