A single strain of a novel ascomycetous yeast species belonging to the genus Candida was isolated from the external ear canal of an inpatient in a Japanese hospital. Analyses of the 26S rDNA D1/D2 domain, nuclear ribosomal DNA ITS region sequences, and chemotaxonomic studies indicated that this strain represents a new species with a close phylogenetic relationship to Candida ruelliae and Candida haemulonii in the Metschnikowiaceae clade. This strain grew well at 40 °C, but showed slow and weak growth at 42 °C. The taxonomic description of Candida auris sp. nov. is proposed (type strain JCM15448T= CBS10913T= DSM21092T).

Agrobacterium tumefaciens-mediated transformation (ATMT) was used to facilitate gene transfer into the clinically important dermatophyte, Trichophyton mentagrophytes (teleomorph: Arthroderma vanbreuseghemii). A binary vector containing a hygromycin B resistance cassette was introduced into A. tumefaciens, and the resultant strain was co-cultivated with fungal small conidia. Transformation yielded a large number of hygromycin B-resistant transformants. Hybridization analysis showed that most of the transformants harboured a single copy of T-DNA randomly integrated into the genome. Transformation frequency was increased to more than 200 per 10(7) conidia by optimizing the co-cultivation time and temperature. ATMT was then used for targeted gene disruption mediated by homologous recombination. Using a PCR-based strategy, we isolated the areA/nit-2-like nitrogen regulatory gene (tnr:Trichophytonnitrogen regulator) from T. mentagrophytes. A binary vector containing two regions of the tnr locus flanking the hygromycin B resistance cassette was constructed and introduced into T. mentagrophytesvia ATMT. Transformants with disruption of the areA/nit-2-like gene (tnr) were obtained in three of four independent disruption experiments, most of which showed homologous recombination via double crossover without additional ectopic integration of the disruption construct.

Allergic bronchopulmonary mycosis, characterized by excessive mucus secretion, airflow limitation, bronchiectasis, and peripheral blood eosinophilia, is predominantly caused by a fungal pathogen, Aspergillus fumigatus. Using DNA microarray analysis of NCI-H292 cells, a human bronchial epithelial cell line, stimulated with fungal extracts from A. fumigatus, Alternaria alternata, or Penicillium notatum, we identified a mucin-related MUC5AC as one of the genes, the expression of which was selectively induced by A. fumigatus. Quantitative RT-PCR, ELISA, and histochemical analyses confirmed an induction of mucin and MUC5AC expression by A. fumigatus extracts or the culture supernatant of live microorganisms in NCI-H292 cells and primary cultures of airway epithelial cells. The expression of MUC5AC induced by A. fumigatus extracts diminished in the presence of neutralizing Abs or of inhibitors of the epidermal growth factor receptor or its ligand, TGF-α. We also found that A. fumigatus extracts activated the TNF-α–converting enzyme (TACE), critical for the cleavage of membrane-bound pro–TGF-α, and its inhibition with low-molecular weight inhibitors or small interfering RNA suppressed the expression of MUC5AC. The protease activity of A. fumigatus extracts was greater than that of other fungal extracts, and treatment with a serine protease inhibitor, but not with a cysteine protease inhibitor, eliminated its ability to activate TACE or induce the expression of MUC5AC mRNA in NCI-H292. In conclusion, the prominent serine protease activity of A. fumigatus, which caused the overproduction of mucus by the bronchial epithelium via the activation of the TACE/TGF-α/epidermal growth factor receptor pathway, may be a pathogenetic mechanism of allergic bronchopulmonary mycosis.

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Prototheca cutis sp. nov., a newly discovered pathogen of protothecosis isolated from inflamed human skin Protothecosis is a sporotrichosis-like dermatitis in humans and animals caused by infection with achlorophyllic algae of the genus Prototheca. The genus Prototheca consists of achlorophyllous algae closely related to the green algae of the genus Chlorella (Pore, 1998). Species of the genus Prototheca exist in the environment as ubiquitous detritus inhabitants and contaminants of various substrates. General protothecosis is caused in humans by Prototheca wickerhamii and in domestic animals by Prototheca zopfii. The general symptom is dermatitis or bovine mastitis, and mortal cases are extremely rare. On the other hand, P. wickerhamii is a serious pathogen which rarely causes meningitis (Kaminski et al., 1992). Their roles as pathogens of human diseases are largely unknown. However, the incidence of infections caused by unusual organisms is likely to increase with the increasing numbers of immunocompromised individuals throughout the world (LassFlörl & Mayr, 2007).In this study, we characterized a pathogenic strain isolated from inflamed skin caused by protothecosis in a patient in a Japanese hospital. Phenotypic, chemotaxonomic, and phylogenetic analyses indicated the strain to be affiliated with the genus Prototheca. The data obtained also suggested that the isolate represents a novel species as a pathogen of dermatitis.Most of the morphological, biochemical, and physiological characteristics were examined using the methods described by Yarrow (1998). Skin samples obtained by biopsy were subjected to both histopathological and microbiological examinations. For histopathological examination, the sample was digested in diastase and paraffin-embedded pathological specimens were subjected to periodic acidSchiff staining. For microbiological examination, samples were inoculated onto Sabouraud dextrose agar (SDA), and incubated at 35 u C. Assimilation pattern-based identification of the isolate was performed with API 20C AUX V4.0 strips (bioMérieux). Fatty acid methyl esters were determined by TechnoSuruga Laboratory (Shizuoka, Japan). Yeast nitrogen base with amino acids and ammonium sulfate, vitamin free (Y2035; United States Biological), was used as vitamin-free medium.Genomic DNA from the isolate was extracted and purified according to the protocol recommended for the FTA Classic Card (Whatman). DNA fragments covering the nuclear 18S rDNA gene and the 26S rDNA gene D1/D2 domain were amplified by using primers for the 18S rDNA gene (SSU-F1 and SSU-R1) and the D1/D2 domain (28SF1 and 635), respectively, as described previously (Sugita The GenBank/EMBL/DDBJ accession number for the 18S rDNA gene and 26S rDNA gene D1/D2 domain sequences of strain JCM 15793 T are AB470468 and AB470469, respectively.Larger versions of Figures 1 and 2, and a phylogenetic tree based on 26S rRNA gene D1/D2 domain sequences are available with the online version of this paper.

The objective of this study was to assess the activity of the novel triazole antifungal drug, efinaconazole, and five comparators (luliconazole, lanoconazole, terbinafine, itraconazole, and fluconazole) against a large collection of and 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 and .

C andida auris is an emerging, multidrug-resistant pathogen associated with a high mortality rate. Since this yeast's first identification and classification by our research group in 2009 (1), there have been several outbreaks linked to this pathogen in health care facilities around the world (2-10). It has been reported that most clinical isolates are resistant to azoles, and about half of the isolates also are resistant to more than one class of antifungal agent, limiting the therapeutic options (2-8, 10). Moreover, the pathogen can persist on environmental surfaces for weeks, resulting in the yeast's spread among patients in health care facilities (11). Therefore, accurate identification of C. auris is critical for controlling this pathogen's prevalence around the globe and preventing further outbreaks. Traditional methods have proven to be unsuitable for accurate identification of C. auris. Automated identification systems popularly used in clinical laboratories, like the Vitek 2 YST card (bioMérieux, Marcy I'Etoile, France) or API20C AUX (bioMérieux), commonly misidentify C. auris as Candida haemulonii or Rhodotorula glutinis, respectively (2, 4-7, 12), and MicroScan misidentifies C. auris as any of several different Candida species (12). On the other hand, specialized methods can provide accurate identification. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is useful for identifying C. auris, if a proper reference database is available (13-15). Moreover, sequencing of the genes for the D1/D2 region of large subunit ribosomal DNA (rDNA) or of the internal transcribed spacer (ITS) region of rDNA is a reliable option. Real-time PCR assays also are useful for detection of C. auris (16, 17). However, these methods may not be suitable for local or small clinical settings due to financial and technical issues. As shown in the present study, we have successfully devised and assessed the reliability of a loop-mediated isothermal amplification (LAMP)-based identification approach specific to C. auris, enabling distinction of the pathogen from closely related species and other fungi. To design the LAMP primers, the genome sequences of four Candida species, C. auris (PRJNA342691), C. tropicalis (GCF_000006335.2), C. albicans (GCA_000182965.3), and C. lusitaniae (LYUB00000000.2), were aligned and compared using Mauve (version 20150226) (18). An 869-bp DNA fragment of the C. auris genome (accession no. XM_018317007) that encodes a pyruvate:ferredoxin oxidoreductase domain (19) was identified as sharing low similarity with other Candida species. This DNA fragment was amplified using EmeraldAmp PCR master mix (TaKaRa Bio, Inc.

Indoor bioaerosols have recently received considerable interest because of their impact on health. In this study, concentrations of bioaerosols in relation to airborne particulate matter in various indoor environments were investigated. The comparative performance of two common biosamplers, including the single-stage Andersen impactor and the all-glass impinger (AGI) for bioaerosol sampling, was also evaluated. The average levels of airborne bacteria and fungi sampled by Andersen were 516 and 176 colony forming units (CFU) m -3 and by AGI were 163 and 151 CFU m -3 , respectively. The highest bacterial levels were measured in residence apartments. The most predominant bacteria were belonged to Staphylococcus sp. and Arthrobacter sp. The Andersen impactor appeared to yield fungal concentrations that were comparable to the results obtained using the AGI biosampler. Meanwhile, Andersen impactor counts for bacteria were significantly higher than those obtained by AGI. Particle count data generated by the optical particle counter indicated that 95% of airborne particles were < 1 µm in diameter. Statistical analysis revealed a significant correlation between particle counts of PM 1 and concentrations of culturable airborne bacteria measured with the both bioaerosol samplers.Based on these results, the Andersen impactor performed much better than the AGI for sampling airborne bioaerosols in low-contaminated indoor environments. Accurate measurement of microbial concentrations in indoor environments should be performed by bioaerosol monitoring; however, combining particle counting with bioaerosol sampling could provide prompt information about rapid variations of air quality.