Search

Basidiomycetes present specific problems with regard to their preservation, because most of them do not form resistant propagules in culture but exist only as mycelium. Usually these fungi can only be preserved by serial transfer on agar (labour-intensive procedures that can increase the danger of variation or loss of physiological or morphological features), or cryopreserved in liquid nitrogen (expensive). Cryopreservation at -80 degrees C and lyophilisation could be good alternatives. In this work we set up and tested six protocols of cryopreservation at -80 degrees C, and 12 protocols of lyophilisation on 15 isolates of white-rot fungi (WRF) belonging to 10 species. The tested protocols were mainly characterized by the use of different growth media, protectants, time and number of perfusion with protectants and finally by the typology and origin of the samples to be cryopreserved (mycelium/agar plug, whole colony) or to lyophilise (mycelium/agar plug, mycelium fragment, whole colony). Cryopreservation and lyophilisation outcomes were checked, at morphological (macro- and microscopic features), physiological (growth rate and laccase, Mn-independent and Mn-dependent peroxidases activities) and genetic level (Amplified Fragment Length Polymorphisms analysis - AFLP). Vitality of all fungi was successfully preserved by all cryopreservation protocols at -80 degrees C, and by two lyophilisation methods. Our results showed that cryopreservation at -80 degrees C did not produce morphological changes in any isolate, while two isolates were affected by lyophilisation. None of the physiological features were lost, even though growth rate and enzyme activities were somehow influenced by all preservation methods. AFLP analysis showed that only the two isolates that varied in their morphology after lyophilisation produced a different DNA fingerprint pattern in comparison with that obtained before lyophilisation. These findings provide evidence that cryopreservation at -80 degrees C and lyophilisation are suitable alternatives to liquid nitrogen cryopreservation for preservation of some WRF strains.

The spores and conidia of most fungi have very thick and resistant cell walls that severely impede the staining with fluorescent dyes to allow epifluorescence microscopy to be employed for their direct detection and quantification in natural habitats. In this study, oxidation by sodium hypochlorite and microwave irradiation (MWI) were used to enhance the staining of Aspergillus fumigatus and Penicillium brevicompactum conidia with six fluorescent dyes. Sodium hypochlorite resulted in high percentages of stained conidia (up to 98.8% with 4',6-diamidino-2-phenylindole [DAPI]), but had to be removed prior to staining with consequent heavy conidia losses. By contrast, MWI gave very high percentages, while its enhancement of fluorescence intensity facilitated observation by epifluorescence microscopy.

The morphological expression of human hair and nail invasion in vitro by 31 isolates of nine Scopulariopsis species was studied by light microscopy on whole material and on semi-thin sections, as well as by transmission electron microscopy. Only some isolates of Scopulariopsis brumptii, S. candida, S. carbonaria and S. koningii were keratinolytically active. They came either from nail lesions or from outdoor aerosols. The most active isolate belonged to S. koningii and was recovered from a fingernail lesion. Both hair and nail degradation followed the biochemical and morphogenetic model described by the authors for other keratinolytic fungi.

There has been a recent increase in the frequency of skin mycoses, coupled with changes in the epidemiology and distribution of the species responsible. Periodic epidemiological analyses of these disease are thus required to ensure their efficacious control. Hair fragments, skin scrapings, specimens from vesicles and blisters and nail parings were seeded on Petri dishes loaded with Sabouraud or Mycosel agar, supplemented with chloramphenicol and with chloramphenicol plus cycloheximide respectively. Parts of each specimen were also mounted in 10% potassium hydroxide for examination under the microscope. Yeasts prevailed over dermatophytes. Microsporum canis was the most frequent dermatophyte, followed by Trichophyton rubrum, T. mentagrophytes and Epidermophyton floccosum. Tinea corporis was the most common mycosis, followed by tinea unguis, tinea capitis and tinea pedis. Men were chiefly bearers of tinea cruris and tinea pedis, women of tinea corporis, and children and teenagers of tinea capitis. Some examples of the transmission of infection through interhuman contact, via animals and from the soil are also presented.

In the present study we determine the antifungal properties of two acyclic inhibitors of 2,3-oxidosqualene cyclases: 22,23-epoxy-2-aza-2,3-dihydrosqualene (EAS) and azasqualene alcohol (ASA). Fungistatic and fungicidal activity towards dermatophytes and other fungi involved in cutaneous and systemic infections was tested (48 isolates from 10 species). The tests were carried out by inoculating 10 microliters of mycelial homogenate in 1 ml of Sabouraud glucose liquid medium containing serial dilutions of 100 to 0.25 micrograms ml-1 of the substance. For each isolate, the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) of both compounds were determined. EAS was more active (MIC range 1.5-25 micrograms ml-1) than ASA (MIC range 3-50 micrograms ml-1). At the highest concentration tested, EAS also showed fungicidal action towards some isolates of Trichophyton mentagrophytes, T. terrestre, Epidermophyton floccosum, Microsporum canis and Scopulariopsis brumptii. The most sensitive species was T. mentagrophytes, the most resistant T. rubrum.

We report a preliminary study of the in vitro anti-dermatophyte activity of six squalenoid derivatives that inhibit 2,3-oxidosqualene cyclase and squalene epoxidase: 2-aza-2,3-dihydrosqualene, 22,23-epoxy-2-aza-2,3-dihydrosqualene, azasqualene alcohol, 19-aza-18,19,22,23-tetrahydrosqualene, 2,3-epoxy-19-aza-18,19,22,23-tetrahydrosqualene and hexafluorosqualene epoxide. The tests were done by inoculating 10 microliters of Trichophyton mentagrophytes (Robin) Blanchard or Microsporum canis Bodin homogenate into 1 ml of Sabouraud glucose liquid medium containing serial dilutions from 100 to 0.25 micrograms ml-1 of the substance. For each compound the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) were determined. The most effective compounds were 22,23-epoxy-2-aza-2,3-dihydrosqualene and azasqualene alcohol, with MICs respectively of 3 and 6.25 micrograms ml-1 for each of the two species of dermatophyte. The first of these compounds was the only one to show fungicidal activity over the range of concentrations tested.

The morphological expression of keratinolysis in fungi isolated from the air of Torino (98 isolates belonging to 36 species) was studied. Light microscopy on whole material and on semithin sections, as well as scanning electron microscopy was used. There were 19 keratinolytically active species, with seven in the genus Chrysosporium (C. indicum, C. keratinophilum, C. pannicola, C. tropicum, C. an. Arthroderma cuniculi, C. an. Pectinotrichum llanense, C. an. Renispora flavissima), four in the genus Malbranchea (M. arcuata, M. fulva, M. sulphurea, M. st. Uncinocarpus reesii), and three in the genus Trichophyton (T. mentagrophytes, T. rubrum, T. terrestre). In addition there were Aphanoascus fulvescens, Beauveria bassiana, Geomyces pannorum v. pannorum, Gymnoascus umbrinus and Myceliophthora vellerea. Most of these species were capable of developing structures related to surface erosion and radial penetration contemporaneously. However Gymnoascus umbrinus, Myceliophthora vellerea, an isolate of C. indicum, C. tropicum and Trichophyton mentagrophytes demonstrated only surface erosion. Different isolates of one species can vary in their production of invasive structures and in degree of keratinolytic activity. Thus such activity, like many biochemical activities of fungi, does not appear to be a constant or rigorously species-specific character.

Aerial dispersal of propagules, an important mechanism of fungal spread, has not been sufficiently studied for skin-infecting fungi. Over a period of one year, we have monitored the distribution of dermatophytes in the air of Turin. We have also examined the related aleurioconidic and arthroconidic fungi, which share physiological and morphological characters with dermatophytes and are sometimes considered to cause similar infections. A viable volumetric collector was used. Samples were taken each month; each sample collected 9.6 m3 of air. The medium was Dermasel agar supplemented with cycloheximide and chloramphenicol. Thirty-two species were isolated, within the genera Aphanoascus, Arthrographis, Chrysosporium, Geomyces, Gymnoascus, Malbranchea, Myceliophthora, Myxotrichum, Ovadendron, Trichophyton. The total mycoflora load obtained was rather low throughout the year and particularly in the hottest months. Apart from T. rubrum and T. mentagrophytes, which are dermatophytes very frequently encountered in Turin, many of the species detected have known pathogenic potential and/or keratinolytic activity.

Ecologic and hygienic interests led us to analyze the mycoflora of the sands of a sand box for children's play in Turin. Along with traditional methods, we have also employed selective techniques suitable for the isolation of cellulolytic and keratinolytic fungi. A total of 89 species were isolated. As supported by the literature references, the presence of most species is justified by the exogenous supply of vegetal residues, hairs, feathers and excrements. A good deal of species, moreover, are either well known agents of mycoses or have been isolated from various animal and human lesions; thus these sands do not represent the ideal environment for children's play.