Takamasa Kaneko

Revised Culture-Based System for Identification of Malassezia Species

ABSTRACT 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%) lipid-dependent 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.

Tween 40-based precipitate production observed on modified chromogenic agar and development of biological identification kit for Malassezia species

We developed a simple identification kit for nine species of Malassezia (M. furfur, M. slooffiae, M. sympodialis, M. restricta, M. obtusa, M. globosa, M. pachydermatis, M. dermatis, and M. japonica) based on their biological features. This method utilizes Tween 40-based precipitate production on modified chromogenic agar (CHROMagar) Malassezia medium, growth on specific agars (Sabourauds dextrose agar, Cremophor EL agar, Tween 60-esculin agar), and catalase reactions. This identification kit was verified with 11 type and reference strains of nine Malassezia species. An additional 26 clinical isolates were also successfully identified using the kit and the results were confirmed by molecular biological analysis.

Vital growth factors of Malassezia species on modified CHROMagar Candida

A comparison of several media, i.e., potato dextrose agar with olive oil (Oil-PDA), modified Dixon agar (mDIX) and variations of Leeming and Notman agar (LNA) for the isolation and growth of Malassezia and Candida species was examined. Since LNA supported the highest growth of Malassezia species its key components, i.e., ox bile, glycerol monostearate, glycerol and Tween 60, were added to CHROMagar Candida. All 7 species of Malassezia grew well on this modified medium (LN-CHROM) after incubation for 4 days at 30 degrees C and development was equal to that observed on LNA. Colonies on LN-CHROM were smooth and from pink to dark purple in color. Furthermore, the use of LN-CHROM did not alter the colony characteristics of Candida species as compared to that found on CHROMagar Candida. The results of the present investigation indicate that the use of LN-CHROM would make possible the simultaneous isolation and identification of Malassezia and Candida species.

Genetic and biological features of catheter-associated Malassezia furfur from hospitalized adults.

Malassezia furfur, an etiological agent of catheter-associated fungemia, requires long-chain fatty acids for in vitro growth. We examined the applicability of rDNA sequence analysis, autoaggregation testing in liquid culture, utilization of parenteral lipid emulsions, and phospholipase activity for discrimination of catheter-associated M. furfur strains. The rDNA sequence types of catheter-associated M. furfur strains were distinct from those of other isolates. All M. furfur isolates recovered from blood culture bottles and the tips of catheters from patients receiving fat emulsion therapy were type I-3. Only M. furfur isolate GIFU 01 from a blood culture bottle showed no autoaggregation in liquid culture. All strains of M. furfur examined grew well on Sabourauds dextrose agar supplemented with Intralipid lipid emulsion as compared to individual Tweens (20, 40, 60, 80) and Cremophor EL. A high percentage of type I-3 M. furfur strains (80.0%) showed very high phospholipase activity compared to type I-1 and I-4 strains obtained from healthy skin of the same subjects or healthy control subjects (20.0% and 0.0%, respectively). The blood culture bottle isolate GIFU 01 showed very high lipolytic enzymes activity for Intralipid but no phospholipase activity. These results suggest that particular factors, such as non-autoaggregation and very high lipolytic enzyme activity for parenteral lipid emulsions, play important roles in the growth and pathogenicity of Malassezia-related sepsis.

Human external ear canal as the specific reservoir of Malassezia slooffiae.

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.

Etiological bacterial level in enterohemorrhagic Escherichia coli infection feces and chromogenic culture medium CHROMagar STEC usefulness

We report a case of enterohemorrhagic Escherichia coli (EHEC) infection in which EHEC was not detected by culture on DHL agar medium. The proportion of EHEC bacterial count to enterobacterial count in feces was 1.7%, and the detection probability by 5-colony angling was low (8.1%). The probability of angling detection using CHROMagar STEC, a chromogenic medium for detecting EHEC, was high (100%). An additional and collection test was done using E. coli bacterial solutions to which two main sera groups--O157 and O26 were added. The maximum detectable level in the bacterial solution with O157 was 10(3)-10(4) CFU/mL in DHL and 10(2) CFU/mL in CHROMagar STEC. Bacterial solution levels with O26 were 10(3) CFU/mL in DHL and 10(2) CFU/mL in CHROMagar STEC. Assuming that the EHEC bacterial amount in feces of those with EHEC infection is low, we speculated that CHROMagar STEC may be useful as on EHEC screening medium.