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Structome analysis, the quantitative three-dimensional structural analysis of whole cells at the electron microscopic level, of Exophiala dermatitidis (black yeast), Saccharomyces cerevisiae, Mycobacterium tuberculosis (MTB) and Myojin spiral bacteria (MSB) have already been reported. Here, the results of the structome analysis of Escherichia coli cells based on transmission electron microscope observation of serial ultrathin sections was reported, and compared with the data obtained from phase contrast microscopy and scanning electron microscopy. On average, the cells had 0.89 μm in diameter, 2.47 μm in length and 1.16 fl (μm3) in cell volume in the structome analysis. Furthermore, E. coli cells had 26 100 ribosomes per whole cell with density of 2840 per 0.1 fl cytoplasm. The total ribosome number per cell was 15 times larger than that of MTB and about one-eighth of those of the yeast cells above. On the other hand, the ribosome density of E. coli cells are more than 13 times, 4 times, 2.5-times and 1.5-times higher than MSB, MTB, E. dermatitidis and S. cerevisiae, respectively. Finally, our ribosome enumeration data were compared between the structome-analyzed species and the relationship between the ribosome density and the growth rate among these species was discussed., (© The Author 2017. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Azole resistance of Aspergillus fumigatus isolates has been reported worldwide and it would appear to be mainly due to a point mutation in the 14α-sterol demethylase (CYP51A) gene, which is the target enzyme for azoles. The mutation has been confirmed in isolates from patients who received long-term itraconazole (ITZ) therapy and from agricultural fields where high levels of azole fungicides were employed. However, the relationship between farm environments and azole-resistant A. fumigatus has not been fully studied. In this investigation, 50 isolates of A. fumigatus were obtained from a farm where tetraconazole has been sprayed twice a year for more than 15 years. The mean minimum inhibitory concentration (MIC) of isolates was 0.74 (0.19-1.5) mg/L against ITZ, which was below the medical resistance level of ITZ. The sequence of CYP51A from isolates indicated no gene mutations in isolates from the farm. Antifungal susceptibility of isolates to tetraconazole showed that spraying with tetraconazole did not induce resistance to tetraconazole or ITZ in A. fumigatus., (© The Author 2014. 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.)

Malassezia yeasts are part of the cutaneous microflora commonly found on animals and human and may sometimes cause various opportunistic skin diseases. As most of Malassezia species show lipid-dependency, lipolytic enzymes such as lipase and phospholipase are necessary for them to obtain useful lipids from the environment. Consequently, these enzymes are thought to play an important role in the growth and pathogenicity of Malassezia. Here we analyze and compare extracellular lipase and phospholipase activities of several Malassezia species cultivated under common growth conditions. M. globosa showed the highest lipase activity of all of the Malassezia species included in our studies. The lipid-independent M. pachydermatis also showed high lipase and phospholipase activity. These results indicate that this Malassezia species are capable of utilizing lipids well in contrast to the other lipid-dependent species of the genus. Our data suggest that lipase may be a pathogenic factor in the skin disease associated with Malassezia and provide an explanation as to why M. globosa is an important pathogenic species in several human skin diseases despite its slow rate of growth.

A variety of methods are utilized for DNA strain subtyping of Candida spp. because no 'gold standard' exists. Random amplified polymorphic DNA (RAPD) or restriction enzyme analysis (REA) are useful to determine the source of an outbreak, but more reproducible and discriminatory methods such as Southern hybridization and pulsed field gel electrophoresis (PFGE) may be required. When applied to some nosocomial Candida infections, multiple strains and species have been identified. Microevolution of yeast species occurs and epidemiologically related isolates may show minor pattern differences, creating uncertainty as to whether they are distinct strains. Approximately 1000 isolates of Aspergillus fumigatus from environmental and clinical sources were typed by REA probed with an A. fumigatus-specific retrotransposon-like sequence. Patients with no symptom of aspergillosis may carry several strains, whereas patients with pulmonary aspergillosis may carry one or two strains; nocosomial transmission of aspergillosis was proven in 39% of the patients studied; any given environmental strain can be infectious; the environmental population of A. fumigatus is extremely diverse and no specific niche was found in the hospital. A PCR assay was designed to target conserved 18S-ribosomal DNA (rDNA) sequences shared by most fungi and a 687 bp product was amplified from 25 medically important fungal species. Studies with blood, cerebrospinal fluid and sputum specimens from patients with mycoses indicated that the PCR assay is more sensitive in diagnosing invasive fungal infections than blood culture methods. More specific identification is obtainable with genus/species-specif c probes designed from within the PCR-amplified sequences for C. albicans, C. krusei, C. lusitaniae, Pneumocystis carinii, Cryptococcus neoformans, Aspergillus/Penicillium spp. and C. glabrata/Saccharomyces cerevisiae. A. fumigatus and A. niger were differentiated by denaturing gradient gel electrophoresis. In situ hybridization (ISH) detected a 648 bp fragment of the 18S rDNA of C. neoformans and a 568 bp fragment of the alkaline proteinase gene of A. fumigatus in tissues from experimentally infected animals. In ISH, the entire process can be automated, making this procedure rapid and easy. The difficulty in establishing a diagnosis of invasive candidiasis has prompted the quest for a clinically useful PCR test for candidaemia. The universal fungal oligonucleotide primer pair, ITS3 and ITS4, amplifies portions of the 5.8S ad 28S rDNA subunits, and the ITS2 region. Although rRNA genes are highly conserved, the ITS regions are distinctive. DNA probes were designed from ITS2 that were specific for 16 different Candida species. Simple, rapid sample preparation was suitable for PCR analysis of BacT/Alert blood culture bottles. Sample preparation, PCR, and EIA detection of the amplicon from five different Candida species was accomplished in 7 h, 2.5 days sooner than by conventional culture methods. As well as saving time, minor yeast species among a major species, or among bacteria, were simultaneously detected. PCR-EIA using a microtitration plate format had sensitivity 10-times greater than that obtained with ethidium bromide-stained agarose gels. Taqman combines in one step PCR, probe hybridization, and fluorescent signal generation. Taqman PCR had sensitivity equivalent to PCR-EIA and required only 5 h, including sample preparation.