Trichoderma spp. Antagonism to the Dermatophyte Trichophyton rubrum: Implications in Treatment of Onychomycosis

Onychomycosis--the dermatophytic invasion of the nail--is difficult to eradicate with drug treatment. The hyphae of the main invading pathogen, Trichophyton rubrum, are often interwoven with the nail plate, preventing effective anti-mycotic agents from reaching its growing tips. An alternative approach to treat onychomycosis may possibly be the application of a biological control agent against the pathogen. In analogy with the success of biocontrol of phytopathogenic fungi, we screened a series of commercially available Trichoderma strains for potential antagonism between Trichoderma and Trichophyton spp. A wide spectrum of antagonism capacity, ranging from effective overgrowth to no interaction was found, with Trichoderma virens NRRL 26672 being the most effective against the Trichophyton strains tested e.g. T. rubrum NCPF118. Furthermore, T. virens NRRL 26672 grown with T. rubrum NCPF118 hyphae as a carbon source, exhibited enhanced induced secretion of active extracellular chitinases and beta-glucosidases, affecting lysis and sporulation on T. rubrum NCPF118 hyphae. Growth of Trichod. virens NRRL 26672 in poor medium also resulted in secretion of antibiotics active in arresting the growth of T. rubrum NCPF118 inoculum. Our findings may open new directions for the treatment of onychomycosis, either in combination with known medications or as a new "natural" route.     

Biotransformation of pentacyclic terpene isolated from Alstonia scholaris (R.BR.)

Abstract

Betulin (1) a pentacyclic triterpene was isolated from medicinal plant Alstonia scholaris (R.BR.) and its structural modification by five filamentous fungi was investigated using flask shake and stirred bioreactor methods. Screening-scale and preparative-scale biotransformation with a standard two-stage protocol yielded betulinic acid (2). Out of five fungal strains (Microsporum canis, Trichophyton tonsurans, Aspergillus niger, A. niger NIAB-280 and Penicillium spp.) only two strains, M. canis and T. tonsurans, showed significant yield of (2). Samples withdrawn from fermentation medium were extracted with ethyl acetate and purified using column chromatography. Compound 2 was extracted from fermentation medium after 5–10 days. Flasks and the bioreactor were stirred at 250 rpm and 28°C. The yield of (2) gradually increased with incubation time. A stirred bioreactor was found to be convenient and simple for compound 1 oxidation. A validated analytical HPLC method was employed to confirm the biotransformation of compound 1 to 2.     

Studies on antidermatophytic activity of waste leaves of Curcuma longa L.

During antidermatophytic screening of some essential oils, Curcuma longa L. exhibited the strongest antifungal activity, completely inhibiting the mycelial growth of ringworm, caused by the fungi- Microsporum gypseum and Trichophyton mentagrophytes. The essential oil from leaves of Curcuma longa was fungicidal at 2.5 μl/ml at which it tolerated heavy doses of inoculum. The fungicidal activity of the oil was thermostable up to 80 °C and self life up to 24 months in storage. The oil also showed a broad fungitoxic spectrum, inhibiting the mycelial growth of other fungi, viz., Epidermophyton floccosum, M. nanum, T. rubrum, T. violaceum. Moreover, up to 5 % concentration it did not exhibit any adverse effect on mammalian skins. The oil has been formulated in the form of an ointment, 1 % w/v and subjected to topical testing on patients of the Out Patient Department (OPD) at Moti Lal Nehru Medical College, Allahabad. Patients were selected on the basis of KOHpositive results and diagnosed tenia corporis. After the second week of treatment, all patients were KOH- negative. At the end of medication, 75 % of patients recovered completely while 15 % showed significant improvement from the disease. The ointment thus, can be exploited commercially after ongoing successful clinical trials. Relationship of the dermatophytes to the toxicity of the oil vis-a-vis phylogeny using molecular data of the pathogens have also been discussed.     

The First Isolation of Arthroderma benhamiae in Japan

A clinical isolate of Trichophyton mentagrophytes from rabbit was examined by polymerase chain reaction (PCR) analysis and a mating experiment. The species-specific primers designed from the nucleotide sequences of the chitin synthase 1 (CHS1) gene in the teleomorph of Arthroderma benhamiae amplified a fragment from genomic DNA samples of A. benhamiae and the clinical isolate but not from those of A. simii and A. vanbreuseghemii. On the other hand, the species-specific primers of A. simii and A. vanbreuseghemii did not amplify any fragment from the genomic DNA of the clinical isolates. When the isolate was respectively crossed with (+) or (-) tester strains of A. benhamiae, A. simii and A. vanbreuseghemii, ascospores were produced in the crossing with the A. benhamiae (+) strain. Therefore, the isolate was identified to be A. benhamiae (-), confirming the result of molecular analysis. This is the first report on the isolation of A. benhamiae in Japan.     

Characterization of the cyclophilin of Trichophyton mentagrophytes

A genetic approach to cyclophilins in a dermatophyte, Trichophyton mentagrophytes, was carried out. The nucleotide and deduced amino acid sequences of the cyclophilin of T. mentagrophytes shared about 70% sequence similarity with those of Schizosaccharomyces pombe, Saccharomyces cerevisiae and Candida albicans. However, the first 21 amino acid and the C-terminal amino acid regions of 188 to 226 of the T. mentagrophytes cyclophilin were distinct from those of the other fungal cyclophilins. The recombinant glutathione S-transferase (GST)-T. mentagrophytes cyclophilin fusion protein produced by Escherichia coli was purified. The protease digest of the fusion protein had a molecular weight of about 13 kDa and peptidyl-prolyl cis-trans isomerase (PPI) activity. This digest protein from T. mentagrophytes was confirmed to be cyclophilin by proving PPI activity.     

Genotype analysis of the variable internal repeat region in the rRNA gene of Trichophyton tonsurans isolated from Japanese Judo practitioners

Tinea capitis due to Trichophyton tonsurans is currently epidemic among Japanese Judo practitioners. T. tonsurans has seven genotypes in a variable internal repeat (VIR) region of the rRNA gene. All 101 isolates obtained from Japanese Judo practitioners had the identical genotype. This suggests that a specific genotype strain occurs throughout Japan.