Amino acid- or protein-dependent growth of Trichophyton mentagrophytes and Trichophyton rubrum

Culture conditions were examined for Trichophyton mentagrophytes and Trichophyton rubrum, which are major pathogens involved in dermatophytosis. They grew well in Sabouraud's dextrose broth or RPMI 1640. Growth in phosphate-buffered yeast nitrogen base supplemented with glucose was very slow, although growth improved significantly with the addition of amino acids or proteins to the medium. The fungi could also grow using human nail fragments as the only source of nutrition. Examination of proteases by substrate gel electrophoresis indicated that distinct sets of proteases are secreted from the dermatophytes in two different media, Sabouraud's dextrose broth and nail fragments. A protease inhibitor, phenylmethanesulfonyl fluoride, inhibited the growth of the fungi on nail fragments, but it did not inhibit their growth in Sabouraud's dextrose broth.     

Antigenic relationship between downy and granular forms of Trichophyton mentagrophytes and Trichophyton rubrum

One strain each of granular and downy varieties of Trichophyton mentagrophytes and two strains each of granular and floccose colonial types Trichophyton rubrum were used for preparation of antigens. The antigens were analyzed by double diffusion against homologous and heterologous antisera raised in rabbits. A total of 17 distinct antigens were recognized, seven of which were common to two dermatophytes. Granular and downy varieties of the Trichophyton species produced on additional line of nonidentity. Cross reactions failed to establish any relationship between similar morphologic forms of different dermatophytes. One isolate of T. rubrum, which was originally the floccose culture type but changed into the granular form after artificial infection, showed an antigenic relationship to the floccose culture.     

Enzymes of Trichophyton rubrum

Jensen, Altschuler &Bard (1957) have surveyed the respiratory enzymes ofTrichophyton mentagrophytes. Enzymes of the hexosediphosphate, hexosemonophosphate, and tricarboxylic acid pathways were found plus a group of miscellaneous enzymes. In view of the close morphological, immunological, and nutritional similarities ofT. mentagrophytes toTrichophyton rubrum, we felt that a study of the enzymology of the latter organism had potential value in forming a basis for the comparison of the two dermatophytes. Our results showed that cell-free extracts ofT. rubrum contain glucokinase, TPN-dependent glucose and fructose dehydrogenases and separate TPN- and DPN-linked glycerol dehydrogenases. These results are in accord with findings of other investigators. ATPase activity was minimal, and shikimic acid kinase, and shikimic and quinic acid dehydrohydrogenases were not detected.     

Use of arbitrarily primed polymerase chain reaction to differentiate Trichophyton dermatophytes

Abstract Dermatophytes such as Trichophyton species are common human pathogens, the infection of which results in dermatophytosis (also known as ringworm). Several laboratory tests are used routinely for the diagnosis of dermatophytosis, but they are either slow or lacking specificity. Through examination of genomic DNA from Trichophyton dermatophytes and other fungi in arbitrarily primed PCR, it was shown that a random primer 5'-ACCCGACCTG-3' produced bands of 4.3 kb, 1.9 kb, 1.7 kb and 0.7 kb in T. rubrum DNA, bands of 2.5 kb, 1.9 kb and 0.8 kb in T. mentagrophytes var. interdigitale and T. mentagrophytes var. mentagrophytes DNA, and bands of 2.5 kb, 1.9 kb, 1.5 kb and 0.9 kb in T. tonsurans DNA. This primer amplified bands of different sizes in other fungal DNA. Therefore, based on the distinct band patterns observed in arbitrarily primed PCR using this primer, T. rubrum , T. mentagrophytes and T. tonsurans dermatophytes could be rapidly differentiated.     

Analytical isoelectric focusing of secreted dermatophyte proteins applied to taxonomic differentiation of Microsporum and Trichophyton species (preliminary studies)

Culture filtrates were prepared from dermatophytes under standard conditions and adapted for analytical isoelectric focusing in thin layer polyacrylamide gels over the pH range 3.5-9.5. Dermatophytes grown in trypticase soy broth secreted a large number of proteins displaying a wide range of isoelectric points (pIs). Trichophyton megninii extracts contained a triplet of proteins focusing in the pH 8.0-8.5 range that were absent in taxonomically related T. kuryangei isolates. Single ascospore isolates and standard tester strains of Nannizzia otae (+) mating type were differentiated from the (-) mating type by proteins focusing at pH 6.5 and 8.4. These were markedly reduced in the (+) type. The isofocused pattern of Microsporum canis conformed closely to the (-) mating type of N. otae. The protein patterns of T. megninii and T. kuryangei were distinct from those obtained with M. canis and M. equinum because of an intense-staining broad protein band, pI 7.2, and three periodic acid-Schiff-positive glycoproteins focusing in the acidic range which were absent in the Microsporum species. A characteristic protein or doublet (pI 8.7) was present in the Microsporum species and absent in the Trichophyton species. Analytical isoelectric focusing is a potentially useful method to distinguish inter- and intra-species differences in the pattern of secreted dermatophyte proteins present in culture filtrates and in trichophytins. The information derived may be useful in the classification of species.     

Flurbiprofen,a unique non-steroidal anti-inflammatory drug with antimicrobial activity against Trichophyton,Microsporum and Epidermophyton species

AIMS: The objective of this study was to determine the antifungal activity of flurbiprofen against dermatophytes like Trichophyton, Microsporum and Epidermophyton species. METHODS AND RESULTS: Susceptibility tests were performed against dermatophytes like Trichophyton, Microsporum and Epidermophyton species by the microbroth dilution method. Among the dermatophytes tested, Trichophyton, Microsporum and Epidermophyton species are remarkably susceptible to this compound (MIC(50): 8-16 microg ml(-1)). A yeast pathogen, Candida albicans, and a bacterium, Staphylococcus aureus, are also susceptible to flurbiprofen. CONCLUSIONS: Flurbiprofen is a non-steroidal anti-inflammatory compound with strong antifungal activity, which is not found in two well known and medically used antifungal organic acids like benzoic and salicylic acids. SIGNIFICANCE AND IMPACT OF THE STUDY: The present action of flurbiprofen on microbes indicates its future prospects as an antimicrobial agent against dermatophytes and yeast pathogens. However, in view of the anti-inflammatory property of flurbiprofen, its antifungal action may provide an additional advantage for use as a skin ointment.     

Molecular differentiation of Keratinomyces (Trichophyton) species

The taxonomy of the form-genus Keratinomyces (Trichophyton) within the group of the dermatophytes is based on morphological features which remain insufficient for the distinction of these anamorphic species. The three species included in the genus Keratinomyces, namely K. ajelloi, K. ceretanicus and K. longifusus were examined by means of their mitochondrial-like DNA diversity and compared to few other dermatophytes. The analysis of the mtDNA restriction fragments confirmed that the three species are different and well separate from the other dermatophytes.     

Enhanced gene replacements in Ku80 disruption mutants of the dermatophyte, Trichophyton mentagrophytes

The frequency of targeted gene disruption via homologous recombination is low in the clinically important dermatophyte, Trichophyton mentagrophytes . The Ku genes, Ku70 and Ku80 , encode key components of the nonhomologous end-joining pathway involved in DNA double-strand break repair. Their deletion increases the homologous recombination frequency, facilitating targeted gene disruption. To improve the homologous recombination frequency in T. mentagrophytes , the Ku80 ortholog was inactivated. The nucleotide sequence of the Ku80 locus containing a 2788-bp ORF encoding a predicted product of 728 amino acids was identified, and designated as TmKu80 . The predicted TmKu80 product showed a high degree of amino acid sequence similarity to known fungal Ku80 proteins. Ku80 disruption mutant strains of T. mentagrophytes were constructed by Agrobacterium tumefaciens -mediated genetic transformation. The average homologous recombination frequency was 73.3 ± 25.2% for the areA/nit-2 -like nitrogen regulatory gene ( tnr ) in Ku80⁻ mutants, about 33-fold higher than that in wild-type controls. A high frequency ( c . 67%) was also obtained for the Tri m4 gene encoding a putative serine protease. Ku80 ⁻ mutant strains will be useful for large-scale reverse genetics studies of dermatophytes, including T. mentagrophytes , providing valuable information on the basic mechanisms of host invasion.     

In Vitro Fungicidal Photodynamic Effect of Hypericin on Trichophyton spp

Hypericin is a natural photosensitizer used in photodynamic therapy (PDT), which has shown in vitro antifungal effect against Candida spp. The aim of this study was to evaluate the in vitro fungicidal effect of hypericin-PDT on dermatophytes. Trichophyton rubrum and Trichophyton mentagrophytes strains were incubated with different concentrations of hypericin for different times and exposed to light-emitting diode lamp (602 ± 10 nm, 10.3 mW cm^?2, and fluence 37 J cm^?2). Using the optimal incubation time, 60 min, a 3-log fungicidal effect was achieved with hypericin concentration ranges of 10–20 μM for T. rubrum and 20–50 μM for T. mentagrophytes (p = 0.95). Confocal fluorescence microscopy showed the localization of hypericin inside the dermatophytes diffusely distributed in the cytoplasm of conidia and hyphae and outside the nucleus. In conclusion, hypericin-PDT has a fungicidal effect in vitro on dermatophytes. Hypericin seems to be a promising photosensitizer to treat localized dermatophytic infections such as tinea pedis and onychomycosis.     

Immunological Studies on Dermatophytes III. Further Analyses of the Reactivities of Neutral Polysaccharides with Rabbit Antisera to Microsporum quinckeanum, Trichophyton schoenleinii, Trichophyton rubrum, Trichophyton interdigitale, and Trichophyton granulosum

The serological reactivities of polysaccharides isolated from five species of dermatophytes, Microsporum quinckeanum, Trichophyton granulosum, T. interdigitale, T. rubrum, and T. schoenleinii, with rabbit antisera to these species were studied qualitatively by precipitation in gel and quantitatively by complement-fixation analyses. Significant differences in the serological reactivities of the galactomannans I were detected with antisera to T. schoenleinii and T. interdigitale. The differences appeared to be related to the specificity of these antisera for the galactofuranose residues in the polysaccharides. Antisera to M. quinckeanum, T. granulosum, or T. rubrum did not detect differences between the galactomannans I. The serological reactivities of the galactomannans II were different with each of the five antisera. The reactivities of the glucans could be correlated with the amount of alpha 1 --> 6 linked glucopyranose residues when antisera to T. schoenleinii and M. quinckeanum were used.     

Trichophytin Extraction: Biological Comparison of Trichophytin Extracted from Trichophyton mentagrophytes Grown in a Complex Medium and a Defined Medium

The extraction of antigens (trichophytin) from Trichophyton mentagrophytes var. asteroides grown in both a complex medium and a defined medium proved to be reproducible and feasible. These extracts were first evaluated by skin testing in both infected and noninfected guinea pigs. Further evaluation and standardization was accomplished in selected humans by comparing these antigens to a trichophytin known to elicit allergic reactions to the dermatophytes. The trichophytins produced by this extraction procedure gave no false positive (or negative) skin test reactions. These antigens did, however, reliably elicit allergic sensitivity of both the immediate and delayed type. The potency of both complex and defined media antigen was shown to be equal to that of the standard trichophytin.     

Antifungal activity of 5 new synthetic compounds vs. Trichophyton rubrum and Epidermophyton floccosum

The antifungal activity of five new synthetic compounds was evaluated on two dermatophytes: Epidermophyton floccosum and Trichophyton rubrum. The data showed that the imidazo-pyrazole and pyrazolo-thiazoles were not particularly effective, while the two pyrazole-thiocyanates proved highly active on both fungi. The most active 5-amino-3-methyl-1-phenylpyrazolo-4-thiocyanate was chosen to perform SEM and TEM morphological studies on both fungi. Both SEM and TEM observations revealed interesting alterations on the two dermatophytes, particularly involving the endomembrane system. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro susceptibility to antimycotic drug undecanoic acid,a medium-chain fatty acid,is nutrient-dependent in the dermatophyte Trichophyton rubrum

The antimycotic activity of fatty acids has long been known, and their presence in human skin and sweat appears to protect the host against superficial mycoses. Undecanoic acid is a medium-chain fatty acid that has been used in the treatment of dermatophytoses in humans. In this study, we selected one Trichophyton rubrum undecanoic acid-resistant strain that showed a marked reduction in its capacity to grow on human nail fragments, which correlated with the reduced activity of secreted keratinolytic proteases. Moreover, the susceptibility of T. rubrum to undecanoic acid is also dependent on the carbon source utilized by both control and resistant strains. The growth of the control strain was strongly inhibited by undecanoic acid in Sabouraud medium or in cultures supplemented with low-fat milk, whereas it was ineffective when the cultures were supplemented with Tween 20 or keratin as the carbon source, suggesting that nutrient conditions are crucial in establishing a susceptibility to antifungal drugs, which is helpful for the isolation and characterization of resistant strains, and in the screening for new antifungal drugs.