Forty-six strains of Malassezia spp. with atypical biochemical features were isolated from 366 fresh clinical isolates from human subjects and dogs. Isolates obtained in this study included 2 (4.7%) lipiddependent M. pachydermatis isolates; 1 (2.4%) precipitate-producing and 6 (14.6%) non-polyethoxylated castor oil (Cremophor EL)-assimilating M. furfur isolates; and 37 (34.3%) M. slooffiae isolates that were esculin hydrolyzing, 17 (15.7%) that were non-tolerant of growth at 40°C, and 2 (1.9%) that assimilated polyethoxylated castor oil. Although their colony morphologies and sizes were characteristic on CHROMagar Malassezia medium (CHROM), all strains of M. furfur developed large pale pink and wrinkled colonies, and all strains of M. slooffiae developed small (<1 mm) pale pink colonies on CHROM. These atypical strains were distinguishable by the appearance of their colonies grown on CHROM. Three clinically important Malassezia species, M. globosa, M. restricta, and M. furfur, were correctly identified by their biochemical characteristics and colony morphologies. The results presented here indicate that our proposed identification system will be useful as a routine tool for the identification of clinically important Malassezia species in clinical laboratories.Members of the genus Malassezia are among the microbiological flora of the skin of homoiothermic animals. Most species of this genus are lipid-dependent yeasts which colonize the seborrheic part of the skin, and they have been reported to be associated with pityriasis versicolor, seborrheic dermatitis, Malassezia folliculitis, and atopic dermatitis (1,6,19,20,24,29). Although M. furfur was previously thought to be the causative agent or trigger factor in all of these skin disorders, Guého et al. (9) reclassified this genus into five species in 1996. Malassezia has since been reclassified into seven species based on molecular biological analysis of nuclear ribosomal DNA/ RNA (9, 10), and the results agreed with those of mitochondrial ribosomal DNA analyses (30). As members of the genus Malassezia share similar morphological and biochemical characteristics, it was thought that differentiating between them based on phenotypic features would be difficult. While molecular biological techniques are the most reliable for the identification of Malassezia, they are not available in most clinical laboratories. Therefore, culture methods for the identification of Malassezia species are required. Some of these identification or differentiation methods have been reported previously. Guillot et al. reported a method of identification based on lipid usage pattern, catalase reaction, growth temperature, and cell shape (11). Hammer and Riley reported the production of a precipitate by some Malassezia strains on Dixon's agar (12); for example, M. furfur, M. obtusa, and M. slooffiae were precipitate-negative strains, while M. sympodialis and M. globosa were precipitate-positive strains. Mayser et al. reported that some Malassezia species hydrolyzed esculin and assimilated pol...

To determine the infection source of a sporadic Legionella pneumonia case associated with a hot spring bath, we used five molecular methods, including repetitive element polymerase chain reaction (rep-PCR), arbitrarily primed PCR (AP-PCR), ribotyping, restriction endonuclease analysis (REA), and macrorestriction endonuclease analysis (MREA) by pulsed-field gel electrophoresis. L. pneumophila serogroup (SG) 3 strain EY 3702, isolated from an intratracheal specimen of a 71-year-old Japanese female who developed pneumonia after nearly drowning in a hot spring spa bath, produced rep-PCR and AP-PCR fingerprints identical to those of L. pneumophila SG 3 strains EY 3768 and EY 3769 isolated from the bath water. Four epidemiologically unrelated L. pneumophila SG 3 strains showed different rep-PCR or AP-PCR fingerprints from those of the three EY strains (EY 3702, 3768, and 3769). The three EY strains were also genotypically indistinguishable by ribotyping with EcoRI and PstI, by REA with EcoRI or Hindill, and by MREA with Nod. Based on these results, we identified the bath water of the hot spring spa as the source of infection of this patient, even though the viable number of the organisms in the bath water was low (3 CFU/100 ml) when determined 27 days after her nearly drowning.

A 71-year-old Japanese female, was found unconscious by drawing, in a hot spring spa, at around noon of 20 October 1994. She recovered by emergency cardiopulmonary resuscitation, and admitted to the Takinomiya General Hospital, with adult respiratory distress syndrome (ARDS). Although she recovered from ARDS within 4 days after her admission, she developed severe pneumonia accompanied with the second attack of ARDS. Ordinary bacteriological culture of her respiratory specimens failed to yield any significant pathogen for her pneumonia, and neither cefazolin nor imipenem/cilastatin was effective. Thus minocyclin was given on the 7th hospital-day and this was effective for blood gas and C-reactive protein (CRP) levels. Intratracheal exsudate inoculated on BCYE alpha agar plate yielded grayish white colonies. Cells of the colonies were clearly agglutinated by anti-Legionella pneumophila serogroup (SG) 3 serum. Antibody titers of patient's paired sera against the strain L. pneumophila SG3 Bloomington-2 and the patient's strain (Y-1) were determined by microplate agglutination test, and a significant rise from 1:20 to 1:320 was demonstrated. Patient recovered by erythromycin treatment and was discharged on the 59th hospital day. L. pneumophila SG3 organisms were again isolated from the spa water where the patient drawn. From these findings described above, we diagnosed the patient as pneumonia due to L. pneumophila SG3, and the spa water was the most probable source of infection.

The incidence of Malassezia species recovered from the external ear canal was characterized using culture medium optimized for Malassezia spp., CHROMagar Malassezia. The results of this study indicated that in healthy individuals M. slooffiae was the dominant Malassezia species followed by M. restricta.

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