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Candida auris is a pathogenic yeast frequently exhibiting multidrug resistance and thus warrants special attention. The prompt detection and proper identification of this organism are needed to prevent its spread in healthcare facilities. The authors of this paper had previously developed LAMPAuris, a loop-mediated isothermal amplification assay, for the specific detection of C. auris. LAMPAuris is evaluated in this report for its ability to identify C. auris from five clades and to detect it from clinical specimens. A total of 103 skin swab samples were tested in comparison with a culture-based method and C. auris-specific SYBR green qPCR. The results show that the LAMPAuris assay had specificities ranging from 97 to 100% and sensitivities ranging from 66 to 86%. The lower sensitivity could be attributed to DNA degradation caused by the prolonged storage of the samples. In conclusion, LAMPAuris proved to be a rapid and reliable method for identifying C. auris and for detecting it in clinical specimens. Fresh specimens should ensure better yield and higher sensitivities., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Koalas are iconic mammals indigenous to Australia. These rare animals and their habitats are occasionally associated with pathogenic fungi, including species of Cryptococcus, and consequently, monitoring the mycobiota of areas inhabited by koalas is of considerable importance. In this report, we describe a novel basidiomycetous yeast isolated from a site in Kanazawa Zoo, Japan, associated with captive koalas. Swab samples were collected from koala breeding environments, from which we isolated a novel unencapsulated yeast characterized by ovoid to ellipsoidal cells (3.2-4.9 × 3.5-5 μm). These cells were observed to undergo polar budding and grow as parent bud pairs, with an optimal growth temperature of 28°C. Colonies grown on yeast extract peptone dextrose agar at 28°C have a characteristic coral pink color. On the basis of physiological, morphological, and molecular characters, the new species was placed in the genus Begerowomyces, and the name Begerowomyces aurantius JCM33898 T (LSEM1333 T =CBS16241 T ) is proposed.

Trichophyton indotineae causes dermatophytosis that is resistant to terbinafine and azole compounds. The aim of this study was to determine the mechanisms of resistance to itraconazole (ITC) and voriconazole (VRC) in strains of T. indotineae . Two azole-sensitive strains (ITC MIC < 0.125 μg/mL; VRC MIC < 0.06 μg/mL) and four azole-resistant strains (ITC MIC ≥ 0.5 μg/mL; VRC MIC ≥ 0.5 μg/mL) were used for the investigation. The expression of MDR genes encoding multidrug transporters of the ABC family for which orthologs have been identified in Trichophyton rubrum and those of CYP51A and CYP51B encoding the targets of azole antifungal compounds were compared between susceptible and resistant strains. TinMDR3 and TinCYP51B were overexpressed in T. indotineae resistant strains. Only small differences in susceptibility were observed between TinMDR3 disruptants and parental strains overexpressing TinMDR3. Whole-genome sequencing of resistant strains revealed the creation of a variable number of TinCYP51B tandem repeats at the specific position of their genomes in three resistant strains. Downregulation of TinCYP51B by RNA interference (RNAi) restored the susceptibility of azole-resistant strains. In contrast, overexpression of TinCYP51B cDNA conferred resistance to a susceptible strain of T. indotineae . In conclusion, the reduced sensitivity of T. indotineae strains to azoles is mainly due to the overexpression of TinCYP51B resulting from additional copies of this gene.

Introduction: Otomycosis is a superficial infection of the external ear caused by fungal pathogens. The genera Aspergillus and Candida are considered the main fungal causative agents, with the predominance of Aspergillus section Nigri. The present study aimed to evaluate the clinical symptoms of patients with otomycosis and predisposing factors and to identify fungal etiological agents using molecular approaches. We also present an overview of published papers on tympanic membrane perforation (TMP) secondary to otomycosis., Materials and Methods: An otorhinolaryngologist collected specimens from external ear canals of patients with suspected otomycosis based on the patient's history and clinical examinations. The specimens were collected using sterile swabs. Fungal isolates were confirmed in clinical specimens by direct microscopy and culture methods. Fungal isolates were identified based on molecular approaches., Results: In total, specimens from 211 patients with suspected otomycosis were examined. The presence of fungi was confirmed in about 51% of patients based on fungal elements in direct microscopy and culture-positive fungi. Aspergillus tubingensis was the most commonly isolated species (52.77%), followed by Aspergillus niger (25.92%). Otomycosis due to infection with Candida species was observed in 16% of cases. Of note, in 36.11% of cases, otomycosis was associated with TMP., Conclusion: A mycological examination is indispensable for a correct diagnosis in patients with otitis extern. TMP should be considered in patients with otomycosis, as it appears to be relatively common in this population., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Skin colonization by the emerging pathogen Candida auris is common in outbreaks within medical settings. Culture-based screening of patients is an effective management strategy to control the pathogen, and the newly developed CHROMagar™ Candida Plus medium is claimed to enable the presumptive identification of C. auris. Here, we evaluated the use of this medium with 63 C. auris strains comprising its four well-established clades, as well as genetically related comparators, including species from the Metschnikowia clade. The colors and halos of both confluent growth and discrete colonies of all the tested strains were compared. It was found that on CHROMagar™ Candida Plus, C. auris formed characteristic white colonies with blue-green halos that were more evident after 72 hr of incubation at 35°C than after 48 hr. However, distinguishing between closely related species such as Candida haemulonii, Candida pseudohaemulonii, and Candida duobushaemulonii required the consideration of parameters other than color, including colony size and growth ability at 35°C. In conclusion, the novel chromogenic medium CHROMagar™ Candida Plus constitutes an easy screening tool for C. auris., (© 2022 The Societies and John Wiley & Sons Australia, Ltd.)

The prevalence of fungal otitis externa, or otomycosis, has been increasing in recent decades. Fungi may act as primary pathogens in this condition, or they may occur as secondary infections after prolonged ototopical treatment with antibiotics, which alters the flora of the external auditory canal (EAC) and enables overgrowth of its fungal inhabitants. We report here a case of otomycosis by Candida parapsilosis, Malassezia obtusa, and Malassezia furfur as a secondary infection following prolonged otic ofloxacin treatment. To the best of our knowledge, although isolation of C. parapsilosis and M. furfur from the EAC is not uncommon, the recovery of M. obtusa has not yet been reported. We also conducted a literature review of the searchable data on PubMed concerning the isolation of Malassezia species from the human EAC., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The International Space Station (ISS) is a closed facility that orbits the earth carrying not only its crew but also microorganisms. We have participated in microbiota analysis projects for the Japanese Experiment Module KIBO (ISS; operations nomenclature: Microbe-I, II, III, and IV) and were in charge of fungal screening. The interior of KIBO was sampled using swabs and microbe detection sheets (MDSs) for fungal detection. The dominant genera obtained by culture were Aspergillus and Penicillium. DNA analyses of the fungal biota using a clone library showed that KIBO was dominated by Malassezia, a fungal inhabitant of human skin. Three fungal species, Aspergillus sydowii, Penicillium palitans, and Rhodotorula mucilaginosa, which grew under microgravity in KIBO were observed under a field emission-scanning electron microscope on the ground. No novel phenotypic characteristics were noted. The results of antifungal susceptibility testing of all isolates did not differ significantly from previous reports of corresponding fungi. In Microbe-I (August 2009), MDSs were culture negative, while in the next stages the CFU of MDSs were 10 for Microbe-II (February 2011), 24 for Microbe-III (October 2012), and 151 for Microbe-IV (February 2015). These results indicated that fungi inside KIBO are increasing and expanding over time, and therefore continuous surveillance is crucial., (© 2021 The Societies and John Wiley & Sons Australia, Ltd.)

Background: Candida auris is an emerging pathogen associated with outbreaks in clinical settings. Isolates of the pathogen have been geographically clustered into four clades with high intra-clade clonality. Pathogenicity varies among the clades, highlighting the importance of understanding these differences., Objectives: To examine the physiological and biochemical properties of each clade of C. auris to improve our understanding of the fungus., Methods: Optimal growth temperatures of four strains from three clades, East Asia, South Asia and South Africa, were explored. Moreover, assimilation and antifungal susceptibility properties of 22 C. auris strains from the three clades were studied., Results: The optimal growth temperatures of all strains were 35-37 °C. Assimilation testing demonstrated that the commercial API ID 32 C system can be used to reliably identify C. auris based on the biochemical properties of the yeast. Notably, C. auris can be uniquely differentiated from commonly clinical fungi by its ability to assimilate raffinose and inability to utilize D-xylose, suggesting a useful simple screening tool. The antifungal susceptibility results revealed that all strains are resistant against fluconazole (minimal inhibitory concentration (MIC) 4 to > 64 µg/mL) and miconazole (MIC 8 to > 16 µg/mL), with strains from the Japanese lineage showing relatively lower MIC values (1-4 µg/mL). Conversely, itraconazole, voriconazole, amphotericin B, micafungin and caspofungin were active against most of the tested strains. On the clade level, East Asian strains generally showed lower MICs against azoles comparing to the other clades, while they displayed MICs against flucytosine higher than those of strains from South Africa and South Asia clades., Conclusion: Our data suggest a simple identification approach of C. auris based on its physiological and biochemical properties and highlight aspects of C. auris population from various clades.

Background: Candida albicans is the most frequent pathogenic fungus in oral cavities. It adheres to dental tissues as part of dental plaques and contributes to caries formation., Objectives: To evaluate the effect of silver diamine fluoride (SDF) on reducing C. albicans adhesion on dentine surfaces., Methods: Flat dentine surfaces were prepared from bovine dental disks, and samples were divided into three groups. The first and second groups were pretreated for 3 min with 299 mM or 2.99 M SDF, respectively, and the third group (control) did not undergo any SDF pretreatment. All samples were washed, inoculated with C. albicans suspension onto their dentine surface, incubated at 30 °C for 6 h, and washed again to remove any nonadherent cells. The abundance of adherent cells was investigated using colorimetric and real-time polymerase chain reaction approaches. Subsequently, the morphological changes in C. albicans by pretreatment with SDF were observed under a scanning electron microscope (SEM)., Results: SDF inhibited candidal growth at concentrations as low as 2.99 µM. Dentine disks pretreated with 299 mM or 2.99 M SDF displayed significantly fewer adhered cells as compared with the control group. Upon pretreatment with SDF, SEM images showed severe morphological changes in the cellular walls, in a dose-dependent manner, suggesting a fungicidal effect of SDF against the yeast., Conclusion: SDF should be considered for clinical applications aimed at inhibiting dental plaque caused by C. albicans, particularly in children and elderly individuals.

Background: Dermatophytes are a group of keratinophilic fungi of medical importance. Despite a relatively long history of molecular taxonomic studies, there is still a need for information on genetic polymorphism in wider variety of genomic loci., Objectives: Our goal was to study partial DNA topoisomerase 2 gene (TOP2) polymorphism in dermatophytes., Methods: We performed DNA sequencing of TOP2 in 26 dermatophyte species along with ribosomal internal transcribed spacer (ITS) sequencing., Results: The number of polymorphic sites in TOP2 data set was similar to that one in ITS data set. Nannizzia species formed paraphyletic group in TOP2 tree. Trichophyton simii was paraphyletic in concatenated TOP2-ITS tree, one of its two clades contained solely Iranian isolates., Conclusions: Our results revealed several unresolved problems in the taxonomy of dermatophytes, including probable polyphyly of the genus Nannizzia and the species T simii., (© 2020 Blackwell Verlag GmbH.)

The mechanisms of terbinafine resistance in a set of clinical isolates of Trichophyton rubrum have been studied recently. Of these isolates, TIMM20092 also showed reduced sensitivity to azoles. The azole resistance of TIMM20092 could be inhibited by milbemycin oxime, prompting us to examine the potential of T. rubrum to develop resistance through multidrug efflux transporters. The introduction of a T. rubrum cDNA library into Saccharomyces cerevisiae allowed the isolation of one transporter of the major facilitator superfamily (MFS) conferring resistance to azoles (TruMFS1). To identify more azole efflux pumps among 39 ABC and 170 MFS transporters present within the T. rubrum genome, we performed a BLASTp analysis of Aspergillus fumigatus , Candida albicans , and Candida glabrata on transporters that were previously shown to confer azole resistance. The identified candidates were further tested by heterologous gene expression in S. cerevisiae Four ABC transporters (TruMDR1, TruMDR2, TruMDR3, and TruMDR5) and a second MFS transporter (TruMFS2) proved to be able to operate as azole efflux pumps. Milbemycin oxime inhibited only TruMDR3. Expression analysis showed that both TruMDR3 and TruMDR2 were significantly upregulated in TIMM20092. TruMDR3 transports voriconazole (VRC) and itraconazole (ITC), while TruMDR2 transports only ITC. Disruption of TruMDR3 in TIMM20092 abolished its resistance to VRC and reduced its resistance to ITC. Our study highlights TruMDR3, a newly identified transporter of the ABC family in T. rubrum , which can confer azole resistance if overexpressed. Finally, inhibition of TruMDR3 by milbemycin suggests that milbemycin analogs could be interesting compounds to treat dermatophyte infections in cases of azole resistance., (Copyright © 2019 Monod et al.)

Chronic pulmonary aspergillosis (CPA) is a common subtype of pulmonary aspergillosis and a life-threatening disease. However, its diagnosis remains difficult due to the lack of specific clinical features and radiologic findings, as well as the difficulty of isolating Aspergillus spp. We developed a novel species-specific detection method of medically important aspergilli using a loop-mediated isothermal amplification (LAMP) for CPA. Specific LAMP primer sets for Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and Aspergillus nidulans were designed. The use of the LAMP assay was validated using respiratory specimens (CPA cases, n = 21; nonaspergillosis cases, n = 23). A total of 15 cases were positive in the CPA group (A. fumigatus, n = 5; A. flavus, n = 1; A. niger, n = 1; A. terreus, n = 7; A. nidulans, n = 1), but only three in the non-CPA group (A. niger, n = 2; A. terreus n = 1). The sensitivity and specificity of the diagnosis of CPA by the LAMP system were 71.4% and 87.0%, respectively. In conclusion, we developed a species-specific detection approach for five medically important aspergilli using the LAMP method. The system showed high sensitivity and specificity for diagnosis of CPA., (© The Author(s) 2019. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Background: Filamentous basidiomycetes (f-BMs) are involved in some unexplained chronic cough (UCC) cases that can be improved by the administration of antifungal agents. The disease concept was termed fungus-associated chronic cough (FACC). The current diagnostic criteria warrant environmental fungi isolation from respiratory specimens, which is hardly conceivable for such fungi., Objectives: This study aimed to detect the f-BMs Bjerkandera adusta, the most common pathogen in FACC, from respiratory specimens of patients with UCC using real-time polymerase chain reaction (PCR). It also evaluated the applicability of the PCR system to detect antifungal agent responders among patients with unexplained cough., Methods: The PCR system specific to B. adusta was developed and its utility was evaluated using sputum samples from 23 patients with chronic cough., Results: B. adusta was detected in 10 out of 14 patients with UCC (71.4%), in contrast to only 2 out of 9 patients with non-UCC (22.2%; p < 0.05 with the Fisher's exact test). The copy number of the samples correlated with the therapeutic impact score for cough symptoms following the oral administration of itraconazole., Conclusion: Development of the real-time PCR system enabled us to demonstrate that many patients with UCC might be influenced by B. adusta, a fact evidenced by the improvement of symptoms with itraconazole administration in most PCR-positive patients. This method would help in detecting itraconazole responders among patients with UCC when the isolation of f-BMs is not achievable., (© 2018 S. Karger AG, Basel.)

Next-generation technologies have prompted efforts towards generating a large repertoire of whole-genome sequences. The dermatophyte Arthroderma vanbreuseghemii has been considered as a good model in which to conduct molecular biological studies on this fungal group. Despite the considerable repertoire of molecular tools developed for this fungus, the lack of genomic data has represented a major limitation, preventing effective implementation of those tools. Herein, the authors report the first draft whole-genome sequence of this dermatophytic species. The size of the draft genome was 23 Mb, exhibiting a GC content of 48.1%. Given the significance of secreted proteases in tissue invasion, a comparative analysis of genes encoding extracellular proteases was performed between A. vanbreuseghemii and other dermatophytes. Furthermore, genes that might be involved in DNA repair also were compared among dermatophytes. Moreover, the complete mitochondrial genome of A. vanbreuseghemii was obtained and shown to consist of 24,287 bp with a GC content of 24%. In conclusion, the availability of genomic data for A. vanbreuseghemii is expected to facilitate the implementation of the molecular tools established for this fungus, enhancing our understanding of the biology of dermatophytes.

The objective of this study was to assess the in vitro activity of the novel triazole antifungal drug, efinaconazole, and five comparators (luliconazole, lanoconazole, terbinafine, itraconazole, and fluconazole) against a large collection of Trichophyton interdigitale and Trichophyton rubrum clinical isolates. The geometric mean MICs were the lowest for luliconazole (0.0005 μg/ml), followed by lanoconazole (0.002 μg/ml), efinaconazole (0.007 μg/ml), terbinafine (0.011 μg/ml), itraconazole (0.095 μg/ml), and fluconazole (12.77 μg/ml). It appears that efinaconazole, lanoconazole, and luliconazole are promising candidates for the treatment of dermatophytosis due to T. interdigitale and T. rubrum ., (Copyright © 2018 American Society for Microbiology.)

The in vitro activities of novel azoles compared to those of five antifungal drugs against clinical ( n = 28) and environmental ( n = 102) isolates of black mold and melanized yeast were determined. Luliconazole and lanoconazole had the lowest geometric mean MICs, followed by efinaconazole, against tested isolates compared to the other drugs. Therefore, it appears that these new imidazole and triazole drugs are promising candidates for the treatment of infections due to melanized fungi and their relatives., (Copyright © 2017 American Society for Microbiology.)

Molecular evolution has dominated taxonomic studies for decades, replacing traditional methods for identification and classification. However, there is a need for better markers to resolve the problems that have limited their usefulness. In this report, we introduce the protein tag (Ptag) sequence, a highly polymorphic amino acid sequence within the C-terminal region of the largest subunit of RNA polymerase I, as a new systematic tag sequence for delineating the evolutionary history of medically important fungi. As Ptag sequences are highly polymorphic between species and low within species, 42 fungal species representing the main taxonomic groups in the phyla Ascomycota and Basidiomycota were tested. The phylogenetic tree inferred from the Ptag sequences showed high consistency with the accepted classification of the Assembling the Fungal Tree of Life (AFTOL) project. Moreover, it could resolve the interspecies phylogenetic relationships of the tested taxa. In contrast, the phylogeny inferred from the nucleotide tag (Ntag) sequence, encoding the Ptag peptide, displayed lesser discriminatory power in resolving the phylogenetic relationships among distantly related taxa. In the case of closely related fungal species, the phylogenetic trees for Ptag and Ntag sequences were consistent with the tree for ITS1 sequences of 11 dermatophytic species. Taken together, the Ptag sequences should contribute to inferring phylogeny among species whereas the Ntag sequences should be useful to analyze variations among closely related species to resolve taxonomic issues in fungi., (© The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophyte infections. The incidence of patients with tinea pedis or unguium tolerant to terbinafine treatment prompted us to screen the terbinafine resistance of all Trichophyton clinical isolates from the laboratory of the Centre Hospitalier Universitaire Vaudois collected over a 3-year period and to identify their mechanism of resistance. Among 2,056 tested isolates, 17 (≈1%) showed reduced terbinafine susceptibility, and all of these were found to harbor SQLE gene alleles with different single point mutations, leading to single amino acid substitutions at one of four positions (Leu 393 , Phe 397 , Phe 415 , and His 440 ) of the SQLE protein. Point mutations leading to the corresponding amino acid substitutions were introduced into the endogenous SQLE gene of a terbinafine-sensitive Arthroderma vanbreuseghemii (formerly Trichophyton mentagrophytes ) strain. All of the generated A. vanbreuseghemii transformants expressing mutated SQLE proteins exhibited obvious terbinafine-resistant phenotypes compared to the phenotypes of the parent strain and of transformants expressing wild-type SQLE proteins. Nearly identical phenotypes were also observed in A. vanbreuseghemii transformants expressing mutant forms of Trichophyton rubrum SQLE proteins. Considering that the genome size of dermatophytes is about 22 Mb, the frequency of terbinafine-resistant clinical isolates was strikingly high. Increased exposure to antifungal drugs could favor the generation of resistant strains., (Copyright © 2017 American Society for Microbiology.)

Purpose: We aimed to evaluate the resolving power of the translation elongation factor (TEF)-1α gene for phylogenetic analysis of Aspergillus species., Methodology: Sequences of 526 bp representing the coding region of the TEF-1α gene were used for the assessment of levels of intra- and inter-specific nucleotide polymorphism in 33 species of Aspergillus, including 57 reference, clinical and environmental strains., Results: Analysis of TEF-1α sequences indicated a mean similarity of 92.6 % between the species, with inter-species diversity ranging from 0 to 70 nucleotides. The species with the closest resemblance were A. candidus/A. carneus, and A. flavus/A. oryzae/A. ochraceus, with 100 and 99.8 % identification, respectively. These species are phylogenetically very close and the TEF-1α gene appears not to have sufficient discriminatory power to differentiate them. Meanwhile, intra-species differences were found within strains of A. clavatus, A. clavatonanicus, A. candidus, A. fumigatus, A. terreus, A. alliaceus, A. flavus, Eurotium amstelodami and E. chevalieri. The tree topology with strongly supported clades (≥70 % bootstrap values) was almost compatible with the phylogeny inferred from analysis of the DNA sequences of the beta tubulin gene (BT2). However, the backbone of the tree exhibited low bootstrap values, and inter-species correlations were not obvious in some clades; for example, tree topologies based on BT2 and TEF-1α genes were incompatible for some species, such as A. deflectus, A. janus and A. penicillioides., Conclusion: The gene was not phylogenetically more informative than other known molecular markers. It will be necessary to test other genes or larger genomic regions to better understand the taxonomy of this important group of fungi.

Dermatophytes are a group of closely related fungi that nourish on keratinized materials for their survival. They infect stratum corneum, nails, and hair of human and animals, accounting the largest portion of fungi causing superficial mycoses. Huge populations are suffering from dermatophytoses, though the biology of these fungi is largely unknown yet. Reasons are partially attributed to the poor amenability of dermatophytes to genetic manipulation. However, advancements in this field over the last decade made it possible to conduct genetic studies to satisfying extents. These included genetic transformation methods, indispensable molecular tools, i.e., dominant selectable markers, inducible promoter, and marker recycling system, along with improving homologous recombination frequency and gene silencing. Furthermore, annotated genome sequences of several dermatophytic species have recently been available, ensuring an optimal recruitment of the molecular tools to expand our knowledge on these fungi. In conclusion, the establishment of basic molecular tools and the availability of genomic data will open a new era that might change our understanding on the biology and pathogenicity of this fungal group.

As a part of a series of studies regarding the microbial biota in manned space environments, fungi were isolated from six pieces of equipment recovered from the Japanese Experimental Module "KIBO" of the International Space Station and from a space shuttle. Thirty-seven strains of fungi were isolated, identified and investigated with regard to morphological phenotypes and antifungal susceptibilities. The variety of fungi isolated in this study was similar to that of several previous reports. The dominant species belonged to the genera Penicillium, Aspergillus and Cladosporium, which are potential causative agents of allergy and opportunistic infections. The morphological phenotypes and antifungal susceptibilities of the strains isolated from space environments were not significantly different from those of reference strains on Earth., (© 2016 The Societies and John Wiley & Sons Australia, Ltd.)

Calcineurin is a serine/threonine protein phosphatase that consists of catalytic (calcineurin A) and regulatory (calcineurin B) subunits. The conserved protein plays important roles in various biological processes. Drug combination of fluconazole and the calcineurin inhibitor (FK506) showed synergistic effects against dermatophytes. In the current study, we identified the calcineurin A homologous gene (TmcanA) in the dermatophyte Arthroderma vanbreuseghemii (anamorph: Trichophyton mentagrophytes). Knockdown mutants were produced from A. vanbreuseghemii, resulting in a defection in growth properties in accordance with dose of the suppressing reagent. The TmcanA gene restored the ability of calcineurin A-deficient Cryptococcus neoformans strain to grow at elevated temperatures. Repression of TmcanA at 37°C resulted in severely stunted growth, suggesting that this protein plays a role in tolerance to elevated temperatures. In addition, TMCANA showed an interaction with high osmolarity glycerol (HOG) signalling pathway by governing the secretion of a secondary metabolite. Moreover, expression of the hydrophobin A gene (TmHF) decreased significantly under the TmcanA-repressive condition, suggesting that TMCANA is involved in its regulation. In conclusion, calcineurin A is a multifunctional gene that is involved in the regulation of several biological processes and therefore is worth being considered as a drug target for treatment of dermatophytoses., (© The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Despite the existing treatment options for onychomycosis, there remains a strong demand for potent topical medications. ME1111 is a novel antifungal agent that is active against dermatophytes, has an excellent ability to penetrate human nails, and is being developed as a topical agent for onychomycosis. In the present study, we investigated its mechanism of action. Trichophyton mentagrophytes mutants with reduced susceptibility to ME1111 were selected in our laboratory, and genome sequences were determined for 3 resistant mutants. The inhibitory effect on a candidate target was evaluated by a spectrophotometric enzyme assay using mitochondrial fractions. Point mutations were introduced into candidate genes by a reverse genetics approach. Whole-genome analysis of the 3 selected mutants revealed point mutations in the structural regions of genes encoding subunits of succinate dehydrogenase (complex II). All of the laboratory-generated resistant mutants tested harbored a mutation in one of the subunits of succinate dehydrogenase (SdhB, SdhC, or SdhD). Most of the mutants showed cross-resistance to carboxin and boscalid, which are succinate dehydrogenase inhibitors. ME1111 strongly inhibited the succinate-2,6-dichlorophenolindophenol reductase reaction in Trichophyton rubrum and T. mentagrophytes (50% inhibitory concentrations [IC50s] of 0.029 and 0.025 μg/ml, respectively) but demonstrated only moderate inhibition of the same reaction in human cell lines. Furthermore, the target protein of ME1111 was confirmed by the introduction of point mutations causing the amino acid substitutions in SdhB, SdhC, and SdhD found in the laboratory-generated resistant mutants, which resulted in reduced susceptibility to ME1111. Thus, ME1111 is a novel inhibitor of the succinate dehydrogenase of Trichophyton species, and its mechanism of action indicates its selective profile., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Acinetobacter baumannii is a Gram-negative bacterial agent involved in nosocomial infections. In this five-year retrospective study, phylogenetic relationships among carbapenem-resistant A. baumannii strains that were isolated at Teikyo University Hospital in Tokyo metropolis, Japan, were explored. A panel of 72 carbapenem-resistant A. baumannii strains that isolated from January 2006 until August 2010 was studied. Next-Generation sequencing (NGS) was employed to perform large-scale genotyping of these isolates. They were separated, according to the time of isolation, into two genetically distinct groups, one correspondent to strains of the outbreak reported to local public health department in 2010 and the other contained strains from earlier isolations, suggesting different origins of the isolates. Moreover, taxa in each group showed two main clustering patterns. Multilocus sequence typing (MLST) study on 8 isolates from the last outbreak showed that they were from one sequence type, 92, displaying less discriminatory power comparing to large-sequence typing. The clonal lineage profiles produced in this retrospective study will be used as a reference database to compare future isolations of A. baumannii. This study demonstrates the power of NGS in conducting epidemiological researches, allowing a high resolution genotyping., (Copyright © 2015 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Biological processes can be elucidated by investigating complex networks of relevant factors and genes. However, this is not possible in species for which dominant selectable markers for genetic studies are unavailable. To overcome the limitation in selectable markers for the dermatophyte Arthroderma vanbreuseghemii (anamorph: Trichophyton mentagrophytes), we adapted the flippase (FLP) recombinase-recombination target (FRT) site-specific recombination system from the yeast Saccharomyces cerevisiae as a selectable marker recycling system for this fungus. Taking into account practical applicability, we designed FLP/FRT modules carrying two FRT sequences as well as the flp gene adapted to the pathogenic yeast Candida albicans (caflp) or a synthetic codon-optimized flp (avflp) gene with neomycin resistance (nptII) cassette for one-step marker excision. Both flp genes were under control of the Trichophyton rubrum copper-repressible promoter (PCTR4). Molecular analyses of resultant transformants showed that only the avflp-harbouring module was functional in A. vanbreuseghemii. Applying this system, we successfully produced the Ku80 recessive mutant strain devoid of any selectable markers. This strain was subsequently used as the recipient for sequential multiple disruptions of secreted metalloprotease (fungalysin) (MEP) or serine protease (SUB) genes, producing mutant strains with double MEP or triple SUB gene deletions. These results confirmed the feasibility of this system for broad-scale genetic manipulation of dermatophytes, advancing our understanding of functions and networks of individual genes in these fungi., (© 2014 The Authors.)

Targeted gene deletion is now available for molecular genetic research of dermatophytes, and the physiological roles of several genes have been elucidated. However, this method cannot be applied to essential genes, which can be potential drug targets. To overcome this limitation, we have developed a conditional gene knockdown system using a copper-responsive promoter. The promoter sequence of the copper transporter gene CTR4 (P(CTR4)) and that of the copper efflux pump gene CRP1 (P(CRP1)) derived from Trichophyton rubrum were examined for their response to copper in Arthroderma vanbreuseghemii. P(CTR4) was demonstrated to repress expression of a reporter gene in the presence of copper, while the activity of P(CRP1) was induced by addition of copper. Importantly, P(CTR4) regulated the gene expression more tightly. Furthermore, when P(CTR4) was applied to regulate the expression of the endogenous genes ERG1 and TRP5, their conditional mutants exhibited decreased growth activity under the repressive conditions. These results suggest that the P(CTR4)-based gene regulation system represents a powerful tool for identification and characterization of a broad range of genes, including essential genes, in dermatophytes.

Three strains were isolated from the nostrils of a koala and the surrounding environment in a Japanese zoological park. Sequence analysis of the nuclear rDNA internal transcribed spacer (ITS) region and the large subunit rDNA D1/D2 domains in addition to physiological and morphological studies indicated that the isolates represent a single novel species belonging to the basidiomycetous genus Cryptococcus (Tremellales, Tremellomycetes, Agaricomycotina). Phylogenetic analysis based on D1/D2 and ITS regions revealed that the novel species belongs to the Fuciformis clade. The name Cryptococcus yokohamensis sp. nov. is proposed to accommodate these isolates with strain JCM 16989(T) (=TIMM 10001(T)=CBS 11776(T)=DSM 23671(T)) as the type strain.

Targeted gene disruption experiments in Trichophyton mentagrophytes are impeded by the dominant of repair of DNA double strand breaks through a nonhomologous end joining pathway (NHEJ). Inactivation of human DNA ligase IV homologs, which is involved in the final step of the NHEJ pathway, has been shown to enhance homologous recombination (HR) frequency in filamentous fungi. To improve the frequency of HR in T. mentagrophytes, the lig4 homolog (TmLIG4) was disrupted. T. mentagrophytes lacking TmLIG4 showed no discernable phenotypic differences when compared to wild-type controls. Both mutant and parent strains had almost identical growth ability, sporulation rate and sensitivity to DNA damaging agents. When four different loci were disrupted in the TMLIG4-deficient mutant, HR frequencies reached as high as 93% depending on the locus, whereas they ranged from 0%-40% in the wild-type. These results suggest that studies in strains lacking TmLIG4 would help to improve our understanding of dermatophytosis by facilitating the genetic manipulation of dermatophytes., (© 2010 The Societies and Blackwell Publishing Asia Pty Ltd.)

Dermatophytes are filamentous fungi that colonize the keratinized layer of human and animal skin. The availability of several selectable markers for dermatophyte gene manipulation would provide important tools to understand the genetic properties of this fungal group. In this study, we report the nourseothricin resistance gene nat1 that confers resistance to the aminoglycoside antibiotic nourseothricin as a dominant marker in Trichophyton mentagrophytes. The NAT cassette was introduced into T. mentagrophytes by the Agrobacterium tumefaciens-mediated transformation (ATMA) method. Transformation occurred at a frequency of 78 transformants per 1 x 10(7) cells. Molecular analysis showed integration of the NAT cassette into the genomic DNA of T. mentagrophytes. This study presents the nourseothricin resistance gene nat1 as a useful selectable marker for selection of T. mentagrophytes.

Onychomycosis is a fungal infection of fingernails or toenails caused by several species of fungi and yeasts. A Japanese monkey, Macaca fuscata, displayed severe onychomycosis in his 4 limbs. Diagnosis and etiological agent identification were performed by conventional and DNA-mediated methods. The accumulated findings of this case revealed Trichosporon montevideense, which has long been considered to be a nonpathogenic yeast. Here, we present the first report of an involvement of T. montevideense in an onychomycosis case.

Direct colony polymerase chain reaction (DCPCR) is a useful molecular biological technique for application in the field of mycology. In this study, all of the 63 fungal strains examined, including those of the genera Candida and Aspergillus, were amenable to DNA amplification using an Ampdirect(R) Plus kit, which allows direct PCR amplification with no requirement for DNA extraction, following 1 h of rapid fungal lysis. Moreover, we compared DCPCR of 35 strains, representing 20 species, using Ampdirect PCR and standard PCR with no lysis buffer. Thirty-four of these strains (97.14%) yielded positive results on Ampdirect PCR, while only 11 (including Aspergillus fumigatus TIMM1776) of the 35 strains (31.43%) showed PCR products when standard PCR reagents were used. Ampdirect DCPCR was also applicable to DNA amplification for spore and hyphal cells. This approach reduces DNA template preparation time before PCR from fungal colonies, and also reduces the cost of PCR.