Valeria Filipello Marchisio

Fungi responsible for skin mycoses in Turin (Italy)

Summary. 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 diseases 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.

Development and Use of Flow Cytometry for Detection of Airborne Fungi

ABSTRACT Traditional methods for the enumeration of airborne fungi are slow, tedious, and rather imprecise. In this study, the possibility of using flow cytometry (FCM) for the assessment of exposure to the fungus aerosol was evaluated. Epifluorescence microscopy direct counting was adopted as the standard for comparison. Setting up of the method was achieved with pure suspensions of Aspergillus fumigatus and Penicillium brevicompactum conidia at different concentrations, and then analyses were extended to field samples collected by an impinger device. Detection and quantification of airborne fungi by FCM was obtained combining light scatter and propidium iodide red fluorescence parameters. Since inorganic debris are unstainable with propidium iodide, the biotic component could be recognized, whereas the preanalysis of pure conidia suspensions of some species allowed us to select the area corresponding to the expected fungal population. A close agreement between FCM and epifluorescence microscopy counts was found. Moreover, data processing showed that FCM can be considered more precise and reliable at any of the tested concentrations.

Vitality and genetic fidelity of white-rot fungi mycelia following different methods of preservation.

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.

Bioremediation potential of basidiomycetes isolated from compost

The potential of a consortium of three basidiomycete mycelia isolated from compost to degrade polycyclic aromatic hydrocarbons (PAH) was first evaluated using a test based on decolorization of Poly R-478 dye. When pre-grown on straw, the consortium decolorized the dye by 83% in 7 days and generated a laccase activity of 663 IU l(-1). Its ability to degrade naphthalene was investigated in soil microcosms specially suited for this volatile PAH. The kinetic study was conducted at a maximal naphthalene concentration of 500 mg kg(-1) of soil. Naphthalene concentration, CO(2) evolution and phytotoxicity (germination index, GI%) on Lepidium sativum seeds were monitored. The naphthalene concentration decreased by about 70% in three weeks in the presence of metabolic activity, while the GI% increased indicating reduced phytotoxicity.

Characterization of Fungal Biodiversity In Compost and Vermicompost

The ability of fungi to break down complex carbon sources makes them of vital importance in both the generation and application of compost. This paper illustrates the diversity and functions of the mycoflora of both a green compost (made solely thermophilically from plant debris) and a vermicompost (made mesophilically by the action of earthworms on plant and animal wastes). The soil dilution plate technique was applied on 3 media (PDA, CMC, PDA plus cycloheximide) and at 3 incubation temperatures (24°C, 37°C and 45°C) to isolate and identify fungal entities. Enzymatic activities (amylase, cellulase, chitinase, esterase, ligninase, pectinase, phosphatase, plastic degradation and xylanase) of most species from both composts were evaluated with a semiquantitative method on 15 substrates. There were substantial qualiquantitative differences in the species composition of the two composts. A total of 193 entities were isolated: 54 from green compost only, 77 from vermicompost only, 62 from both. This taxonomic diversity was reflected in the metabolic potential. Amilase, cellulase, protease and esterase activities were significantly higher in the green compost. Lignin and plastic polymer degradation were significantly higher in the vermicompost. Structural and functional characterization of this kind is of assistance in determining both the most appropriate application of composts and their hygienic quality.

Preliminary survey of airborne fungal propagules in Turin, Italy

Four samplings of the Turin atmosphere were carried out on the roof of a hospital, 14 m above ground level. Two types of volumetric sampler placed close together were used to collect a set amount of air over the surface of solid culture media. Minimum and maximum CFU peaks varied from 123 to 386 m−3 air. Seventy-seven species were isolated, mostly belonging to the Deuteromycetes and to the genera Penicillium, Aspergillus, Cladosporium, Alternaria, Rhinocladiella, Hormonema, Botrytis and Paecilomyces. Most isolates are common in soil and/or on decaying plant material. A number of species are newly reported as aerial fungi (Chrysosporium carmichaelii, Engyodontium album, Rhinocladiella mansonii, Scopulariopsis carbonaria, Tilletiopsis washingtonensis and some Penicillium and Phialophora species). About a third of the species are known allergens. No actual human pathogens were present, though many species (about a third) are to be classed as opportunistic pathogens. However, CFU m−3 values of both allergens and opportunists indicate that no one species reaches a worrying concentration.

Outdoor airborne fungi: sampling strategies

SummaryA six-stage Andersenss sampler was compared with a single stage type (SAS) for the collection of airborne fungi particles.The efficiency of two non selective culture media, and namely malt agar (MA) and potato dextrose agar (PDA), which were both modified in order to inhibit bacterial growth, was compared as well.An overall assessment of quantitative results suggests that the two samplers have a comparable degree of efficiency. However, the SAS appears to collect a greater number of CFU/m3 in absence of wind and viceversa the Andersen one appears to be more efficient in the presence of wind.The number of species detected with the SAS device is smaller, thus requiring an increased number of suctions.Andersen sampler sieving, according to the aerodynamic characteristics of the particles, appears to be not too accurate: there is an overlap from stage to stage of the sampler and some larger particles settle on smaller particle-collecting stages and viceversa.The PDA, with streptomycin and chloramphenicol, is better and closer to the natural distribution pattern: the number of CFU/m3 is higher although the number of the collected species is about the same.

Basidiomycetes from Compost and their Dye Degradation and Enzyme Activities

The degree of decolorization of Poly R-478, a highly recalcitrant anthraquinone dye, by three basidiomycetes belonging to Polyporales isolated from compost was investigated together with the enzymes involved. Decolorization tests in two liquid cultures, one with a simple mycelium inoculum, the other with an inoculum of mycelium grown on straw, resulted in 70% and 87% decolorization respectively in 7 days. However, the efficiency did not increase significantly in the presence of the lignocellulose substrate. The three strains produced laccase and/or manganese peroxidase activity during the decolorization, whereas lignin peroxidase activity was not observed. Previous growth on straw enhanced the synthesis of ligninolytic enzymes, though there was no correlation between enzyme activity and decolorization. The three fungi can be proposed as promising candidates for the treatment of colored industrial effluents and probably for soils contaminated by complex polymers, such as polycyclic aromatic hydrocarbons.

Antifungal activity of bis-azasqualenes, inhibitors of oxidosqualene cyclase.

The antifungal activity and in vitro toxicity toward animal cells of two inhibitors of oxidosqualene cyclase, squalene bis‐diethylamine (SBD) and squalene bis‐diethylmethylammonium iodide (SBDI) were studied. Minimum inhibitory concentration (MIC) against dermatophytes and other fungi involved in cutaneous and systemic infections (12 isolates from seven species) were determined by the broth microdilution method based on the reference documents M38‐A and M27‐A2 of Clinical and Laboratory Standards Institute (CLSI). Both compounds exerted fungistatic activities, although with different action. SBDI was the more active compound and displayed low MIC values (in the 3.12–12.5 μg ml−1 range) against Microsporum canis, Trichophyton mentagrophytes and one isolate of Scopulariopsis brevicaulis, while SBD showed MIC values against these species in the 3.12–25 μg ml−1 range. Toxicity was tested on Madin‐Darby canine kidney (MDCK) epithelial cells and human microvascular endothelial cells (HMEC). SBDI proved the less toxic compound: it inhibited M. canis, T. mentagrophytes and S. brevicaulis at concentrations below those found toxic for MDCK cells. HMEC were the more sensitive cells.