A conventional and easy method to establish a murine oral candidiasis model, which has not only a stable yeast population in the oral cavity but also symptoms characteristic of oral thrush, was developed by using a sedative agent. Mice were immunosuppressed with prednisolone and were given tetracycline hydrochloride. They were orally infected with 106 viable cells of Candida albicans by means of a cotton swab and enough chlorpromazine chloride had been injected to keep them in a sedative state about for 3 hr after inoculation. From day 3 to day 7 post inoculation, 105–106 colony forming units of Candida were recovered from the oral cavity of each mouse and whitish, curd‐like patches were observed on most parts of tongue. Microscopically, germ tubes had appeared on the tongue surface. This model would be a useful experimental oral candidiasis for investigating the pathogenesis of C. albicans oral infection and the efficacy of various antifungal agents microbiologically and symptomatically.

ABSTRACTOral candidiasis is often accompanied by severe inflammation, resulting in a decline in the quality of life of immunosuppressed individuals and elderly people. To develop a new oral therapeutic option for candidiasis, a nonpathogenic commensal oral probiotic microorganism,Streptococcus salivariusK12, was evaluated for its ability to modulateCandida albicansgrowthin vitro, and its therapeutic activity in an experimental oral candidiasis model was tested.In vitroinhibition of mycelial growth ofC. albicanswas determined by plate assay and fluorescence microscopy. Addition ofS. salivariusK12 to modified RPMI 1640 culture medium inhibited the adherence ofC. albicansto the plastic petri dish in a dose-dependent manner. Preculture ofS. salivariusK12 potentiated its inhibitory activity for adherence ofC. albicans. Interestingly,S. salivariusK12 was not directly fungicidal but appeared to inhibitCandidaadhesion to the substratum by preferentially binding to hyphae rather than yeast. To determine the potentially anti-infective attributes ofS. salivariusK12 in oral candidiasis, the probiotic was administered to mice with orally induced candidiasis. Oral treatment withS. salivariusK12 significantly protected the mice from severe candidiasis. These findings suggest thatS. salivariusK12 may inhibit the process of invasion ofC. albicansinto mucous surfaces or its adhesion to denture acrylic resins by mechanisms not associated with the antimicrobial activity of the bacteriocin.S. salivariusK12 may be useful as a probiotic as a protective tool for oral care, especially with regard to candidiasis.

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...

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This study indicates that most of the affected nail plates of patients with onychomycosis were positive for specific fungal DNAs, and suggests that nondermatophytes detected at high rates may be involved in the pathogenesis of onychomycosis.

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Thus, some essential oils used as antiinflammatory remedies suppress neutrophil activation by TNF-alpha at a low concentration (0.0125-0.025 %) in vitro.

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.