Pathogenicity of Ochroconis gallopava isolated from hot springs in Japan and a review of published reports.

Four strains of Ochroconis gallopava from 3 out of 15 Japanese hot springs were isolated. Colonies of the hot spring isolates were uniformly floccose and dark olive green on the surface and dark brown on their reverse side on potato dextrose agar (PDA) plates, however, they became felty, flat, and brownish-black, and produced a reddish-brown pigment after several times of subculture at room temperature. Shapes and sizes of conidia of the four strains were individual, while the D1/D2 domain of the large subunit ribosomal RNA gene sequences showed 99.7% identity in the GenBank database. The DNA pattern of the hot spring isolates amplified by species specific loop mediated isothermal amplification method were as the same pattern as that of a clinical isolate. The minimum inhibitory concentrations of antifungal agents to O. gallopava isolated from the hot springs were ranged from 0.5 to 1 microg/ml in amphotericin B, 1 to 16 microg/ml in flucytosine, 0.125 to 0.25 microg/ml in itraconazole, 1 to 4 microg/ml in miconazole, 16 to 64 microg/ml in flconazole and 0.03 to 0.5 microg/ml in micafungin. The isolates had fatal outcome in experimentally infected mice intravenously with severe invasiveness to brains and kidneys. These findings suggested that O. gallopava habitats in hot springs could be one of sources for infection.

Rapid identification of Ochroconis gallopava by a loop-mediated isothermal amplification (LAMP) method.

Ochroconis gallopava is a species of dematiaceous fungi recognized as a causative agent of zoonotic and emerging fungal infections. It affects the central nervous system and respiratory tracts of humans, birds and cats. We designed O. gallopava species-specific primer sets to aid in its identification by a loop-mediated isothermal amplification (LAMP) method based on the D1/D2 domain of the LSU rDNA sequence. The LAMP method successfully detected the gene from both fungal DNA and experimentally infected brains and spleens of mice and will be helpful in the diagnosis of O. gallopava infection.

Detection of gp43 of Paracoccidioides brasiliensis by the loop-mediated isothermal amplification (LAMP) method.

Paracoccidioidomycosis is a deep mycosis caused by the thermo-dependent dimorphic fungus Paracoccidioides brasiliensis and is prevalent in Latin American countries. We detected the species specific gp43 gene of P. brasiliensis by loop-mediated isothermal amplification (LAMP) in 22 clinical and seven armadillo-derived isolates. The amplified DNA appeared as a ladder with a specific banding pattern. The advantage of the LAMP method is speed; only 3 h were necessary for identification of the organism and diagnosis of the disease. We were also able to obtain positive results from DNA extracted from a paraffin-embedded tissue sample of paracoccidioidomycosis, suggesting that this method may achieve clinical application in the near future.

Histoplasma capsulatum variety duboisii isolated in Japan from an HIV-infected Ugandan patient.

A strain of Histoplasma capsulatum var. duboisii (deposited as IFM 50954 in Chiba University) was isolated from the cerebrospinal fluid of a female Ugandan patient infected with HIV. The isolate had in vitro urease activity on Christensen's urea agar slants, although the common belief is that H. capsulatum var. duboisii is urease negative, and is, considered one of the characteristic markers that distinguishes the three varieties of H. capsulatum. Forty H. capsulatum var. capsulatum, five H. capsulatum var. duboisii, and five H. capsulatum var. farciminosum isolates were evaluated for urease activity on Christensen's urea agar slants and for other qualitative and quantitative urease assays of activity. All 50 isolates of H. capsulatum used in this study were positive for urease activity, suggesting that urease activity may be universal characteristic of H. capsulatum. We also compared the urease activity and pathogenicity of seven H. capsulatum isolates that convert into yeast-form cells. Although isolate IFM 50954 showed moderate virulence in mice and moderate urease activity among tested H. capsulatum isolates, there was no correlation between level of urease activity and pathogenicity. In addition, scanning electron microscopy revealed that some microconidia of isolate IFM 50954 formed "double-cell" configurations that were attached to each other by narrow bases.

Distribution and organization of auxotrophic genes on the multichromosomal genome of Burkholderia multivorans ATCC 17616.

The Burkholderia multivorans strain ATCC 17616 carries three circular chromosomes with sizes of 3.4, 2.5, and 0.9 Mb. To determine the distribution and organization of the amino acid biosynthetic genes on the genome of this beta-proteobacterium, various auxotrophic mutations were isolated using a Tn5 derivative that was convenient not only for the determination of its insertion site on the genome map but also for the structural analysis of the flanking regions. Analysis by pulsed-field gel electrophoresis revealed that 20 out of 23 insertion mutations were distributed on the 3.4-Mb chromosome. More detailed analysis of the his, trp, arg, and lys mutations and their flanking regions revealed the following properties of these auxotrophic genes: (i) all nine his genes were clustered on the 3.4-Mb chromosome; (ii) seven trp genes were organized within two distinct regions, i.e., a trpEGDC cluster on the 3.4-Mb chromosome and a trpFBA cluster on the 2.5-Mb chromosome; (iii) the leu gene cluster, leuCDB, was also located close to the trpFBA cluster; and (iv) lysA and argG genes were located on the 2.5-Mb chromosome, in contrast to the argH gene, which was located on the 3.4-Mb chromosome. Southern hybridization analysis, allelic exchange mutagenesis of ATCC 17616, and complementation tests demonstrated that all of the genes examined were functional and existed as a single copy within the genome. The present findings also indicated that the 2.5-Mb chromosome carried various auxotrophic genes with no structural or functional counterparts on the remaining two chromosomes.