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Identification of cheese-associated fungi using selected ion monitoring of volatile terpenes.

The cheese-associated fungi Penicillium commune, P. roqueforti, P.solitum, P. discolor and Aspergillus versicolor have been investigated for production of volatile terpenes for chemical identification, when grown on yeast extract agar. Volatiles were collected by headspace solid-phase microextraction. Selected ion monitoring of four to seven of the most characteristic ions of mainly sesquiterpenes made it possible to identify the fungi to species level within 2 d. In a mixed culture of P. roqueforti and P. commune, inoculated in a ratio of 1000:1, volatiles from both fungi could be detected within 3 d, making identification possible.

Aspergillus↗

Use of an electronic nose for the early detection and differentiation between spoilage fungi.

Six spoilage fungi (four Eurotium species, a Penicillium sp. and Wallemia sebi) were grown as spore lawn surface cultures at 0.95 water activity and 25 degrees C. Prior to and during visible growth (24 and 48, and 72 h), single cultures were enclosed in polyethylene bags, the head space was sampled with an electronic nose unit, consisting of 14 polymer sensors, and the data analysed. There was good replication between volatile patterns of the same species and using principal component, discriminant function and cluster analyses it was possible to differentiate between the agar blanks, three Eurotium spp., the Penicillium sp. and W. sebi during microscopic growth for the first time. This suggests that there is potential for the early detection of the activity of spoilage fungi in general, as well as possible differentiation between related xerophilic spoilage fungi, by detection of the patterns of volatile odours produced using an electronic nose system.

Bread↗

Measurement of fungi by an indirect conductimetric assay.

AIMS: To investigate the growth of fungi using an indirect conductimetric assay and derive, experimentally and theoretically, the relationship between microbial concentration and electrical conductivity change. METHODS AND RESULTS: The indirect assay, in which change in electrical conductivity of an alkaline solution (NaOH) is produced by absorption of CO2 from microbial metabolism, was conducted with the Bactometer (bioMerieux, Marcy-l'Etoile, France) for the enumeration of fungi. A linear relationship was obtained between detection time and logarithmic initial microbial concentration. This indirect assay used growth media, which could not be used in the direct conductimetric assay, to monitor fungal growth. CONCLUSIONS: The indirect assay does not depend on the growth media and the turbidity of sample and could offer a simple and rapid assay for the measurement of fungal growth under various conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The indirect assay is applicable for rapid detection of fungi, estimation of the growth rate and evaluation of antifungal activity.

Colony Count, Microbial↗

Effect of Eubacterium limosum, a ruminal hydrogenotrophic bacterium, on the degradation and fermentation of cellulose by 3 species of rumen anaerobic fungi.

The degradation and fermentation of cellulose filter paper were studied in axenic cultures of 3 species of rumen anaerobic fungi, Neocallimastix frontalis, Piromyces communis and Caecomyces communis, and in cocultures containing 1 of these fungal strains and Eubacterium limosum, a hydrogenotrophic rumen bacterial species. When E limosum was introduced into fungal cultures a slight decrease in fungal cellulolytic activity was observed. The end products of the fermentation of cellulose found in the cocultures were different from those found in the fungal monocultures. E limosum used formate and part of the hydrogen produced by the fungi and probably created a shift in the metabolism of the fungi resulting in a reduction of lactate and ethanol production.

Anaerobiosis↗

Environmental surveillance of filamentous fungi in three tertiary care hospitals in Greece.

The environmental fungal load (FL) of three hospitals was studied in representative regions in Greece (Thessalonika, Northern Greece, Athens, Central Greece and Heraklion, Southern Greece). Air, surfaces and tap water from high-risk departments were sampled monthly during one year. Air FL was [median (range)] 10.6 (1.2-37), 5.5 (3-28.8) and 7.7 (3.1-12.1) cfu/m(3) at Thessalonika, Athens and Heraklion, respectively. Air FL was lower in winter and higher in summer and autumn but seldom above acceptable levels. Aspergillus spp. constituted 70.5% of the filamentous fungi isolated. Aspergillus niger was the most prevalent species in the air of all the hospitals followed by Aspergillus flavus and Aspergillus fumigatus. The least contaminated departments were the intensive care units, whilst most contaminated were the solid organ transplantation in Athens and haematology departments in Thessalonika. No correlation between fungal species, season, hospital or departments was observed. Sixty per cent of all surfaces examined yielded filamentous fungi and/or blastomycetes. While no fungi were recovered from water in Thessalonika and Athens, one-third of the samples in Heraklion (apart from those of ICU) yielded multiple fungal species. The higher air FL in Thessalonika and Athens was recorded in departments located close to renovation works. These findings suggest that the air and surface FL fluctuates over the year, is due to varying fungal species, but does not differ greatly among hospitals. The variation among hospitals, as well as the role of hospital water fungal contamination and appropriate measures to eliminate it, need further study.

Air Microbiology↗

Exposure to airborne fungi, MVOC and mycotoxins in biowaste-handling facilities.

Health impacts due to fungi in indoor air can only be estimated reliably, if both fungal propagules and fungal secondary metabolites are qualified and quantified. In the present study, the fungal species composition in a compost facility is compared to the spectrum of microbial metabolites in the air with regard to the physiological properties of different fungal species. A number of relevant fungi was tested for the production of both volatile and non-volatile metabolites on different substrata. The profiles of mycotoxins and microbial volatile organic compounds (MVOC) turned out to be specific for certain species in pure culture. Consequently, the fungi may have different toxicological health impacts, though information on the relevance of microbial volatiles is still limited.

Air Microbiology↗

The predatory capability of three nematophagous fungi in the control of Haemonchus contortus infective larvae in ovine faeces.

The effect of oral administration of three different nematode-trapping fungi, in aqueous suspension containing either Dactylaria sp. or Arthrobotrys oligospora conidia or Duddingtonia flagrans chlamydospores, on the number of Haemonchus contortus infective larvae in sheep faeces, was evaluated. The three selected species of fungi produce three-dimensional adhesive nets in the presence of nematodes. Sixteen Creole sheep were divided into four groups of four animals each. Groups 1 and 2 were orally drenched with a suspension containing 2x10(7) conidia of either A. oligospora or Dactylaria sp. Group 3, received a similar treatment, with D. flagrans chlamydospores, instead of conidia, being administered, at the same dose. Group 4 acted as control, without any fungi. Faecal samples were collected directly from the rectum of each sheep and faecal cultures were prepared and incubated at 15 and 21 days. Larvae were recovered from faecal cultures and counted. The highest reduction of the nematode population occurred in the D. flagrans group, reaching reductions of 96.3% and 91.4% in individual samplings in plates incubated for 15 and 21 days, respectively. Arthrobotrys oligospora showed moderate reductions in the faecal larval population, ranging between 25-64% at 15 days incubation. In general, Dactylaria sp., was less efficient in its trapping ability. Despite the inconsistent results with Dactylaria sp., reduction percentages of 73.4% and 80.7% were recorded in individual samplings during the first and second days, in plates incubated for 15 days. Duddingtonia flagrans, was shown to be a potential biological control agent of H. contortus infective larvae.

Animals↗

Keratinophilic fungi from Orissa, India, I: Isolation from soils.

Results of a preliminary survey of keratinophilic fungi in soils collected from five districts of Orissa, India, are reported. A total of 80 soil samples were examined, of which 51 samples (63.7%) were positive for keratinophilic fungi. Twelve species of fungi classified in seven genera were isolated: Aphanoascus reticulisporus (2.4%), Aph. terreus (1.2%), Chrysosporium state of Arthroderma tuberculatum (4.8%), Chrysosporium indicum (31.3%), Chrys. tropicum (18.0%), Chrys. evolceanui (1.2%), Gymnoascoideus petalosporus (9.6%), Microsporum gypseum (20.4%), Nannizzia spp. (3.6%), Pseudoarachniotus flavoluteus (3.6%), Pseudoarachniotus sp. (1.2%) and a new genus of gymnoascaceae (2.4%). Our isolation of Aph. reticulisporus is the first recorded in India.

Fungi↗

Susceptibility of dermatiaceous fungi to amphotericin B, miconazole, ketoconazole, flucytosine and rifampin alone and in combination.

The dematiaceous fungi comprise a group of organisms that are deeply pigmented and found in soil or on decaying organic material, such as wood. The majority of infections with these fungi presumably results from traumatic inoculation. Although various forms of infection have been appreciated for some time, none of the presently available antifungal drugs have been shown to have predictable activity against these organisms. We report on the activity in vitro of various antifungal agents alone and in combination against various dematiaceous fungi.

Amphotericin B↗

Mode of action of 5-fluorocytosine and 5-fluorouracil in dematiaceous fungi.

The mode of action of 5-fluorocytosine (5FC) and 5-fluorouracil (5FU) in dematiaceous fungi was studied and compared with results of experiments in yeasts and Aspergillus species. In dematiaceous fungi 5FU is more potent than 5FC. The high activity of 5FU is related to a good and rapid uptake of this compound into the fungus cell. Both compounds exert fungistatic and fungicidal activity. A correlation exists between the amount of 5FU incorporated into RNA and its antifungal activity. The resistance frequency to 5FC varies from 2 x 10(-3) to 1 x 10(-7); resistance frequency to 5FU is generally lower. Addition of 5FC and 5FU to logarithmically multiplying cells inhibits increases of cell numbers and cell constituents after a delay period. The effects on the increase of protein and carbohydrate are more delayed than on the increase of DNA and RNA, indicating unbalanced growth. The concept of a dual biochemical mechanism, i.e. incorporation of 5FU into RNA and formation of 5-fluorodeoxy UMP leading to inhibition of DNA synthesis, previously proposed for the antifungal action of 5FC is also applicable to the action of 5FC and 5FU on the dematiaceous fungi.

Cytosine↗

Incidence of toxigenic fungi and ochratoxin A in dried fruits sold in Brazil.

A total of 117 dried fruit samples (black sultanas, white sultanas, dates, dried plums, dried figs and apricots) from different origins were analysed both for toxigenic fungi and for the presence of ochratoxin A. Amongst the fungi found, Aspergillus niger was predominant, with 406 isolates, of which 15% were ochratoxin A producers. They were followed by A. ochraceus, with 15 isolates and 87% ochratoxigenics, and A. carbonarius, with only five isolates of which 60% were ochratoxin A producers. The average infection rates for A. niger in black sultanas, plums, figs, dates and white sultanas were 22.0, 8.0, 4.0, 1.5 and 0.5%, respectively. The apricot samples were not contaminated by any fungi or ochratoxin A. Black sultana and dried figs contained the highest contamination with ochratoxin A, with 33 and 26.3% of the samples containing more than 5 microg kg(-1) respectively, while all the white sultanas, dates and plums had no sample that exceeded this limit.

Aspergillus↗

Multi-element survey of wild edible fungi and blackberries in the UK.

A survey of 12 metals including lead (Pb), copper (Cu), cadmium (Cd), mercury (Hg), arsenic (As) and platinum (Pt) was carried out using ICP-MS in 34 samples of wild fungi and 48 samples of wild blackberries collected from sites across the UK. On a fresh weight basis (mg/kg) levels of Pb were in the range 0.003-5.990, Cu 0.596-34.800, Cd<0.001-19.6, Hg<0.001-4.150, As 0.001-0.972 and Pt (microg/kg) 0.006-0.200, with higher concentrations found in fungi than in blackberries. The results of the survey showed that the concentrations of the metals were consistent with previous studies, where available. Concentrations in wild fungi of Pt, tin (Sn), and titanium (Ti) were significantly higher at urban sites than at rural sites. Urban blackberries had significantly higher levels of Pb, Ti, and Cd than rural ones, but lower levels of manganese (Mn). Pb, Ti and Sn concentrations were significantly higher in blackberries sampled near main roads rather than in rural areas.

Environmental Monitoring↗

The incidence of fungi and mycotoxins in South Africa wheat and wheat-based products.

This investigation was undertaken to survey the fungal and mycotoxin contamination of South African wheat ranging from that growing in the field to processed wheat products. Samples of wheat were taken from various growing areas in South Africa and screened for fungi and mycotoxins, using a range of methodologies, including chromatography, immunoaffinity/fluorimetry, and cytotoxicity testing. Similar samples were taken from supermarkets and retail outlets in South Africa and analyzed in a similar manner. The result showed that a range of fungi and mycotoxins could be detected in wheat in all these sample types. The major fungal contaminants were Fusarium spp. and their attendant mycotoxins, in particular deoxynivalenol, which is in keeping with the observations made in the rest of the world. An interesting observation was that samples of wheat taken from the field with heavy Fusarium contamination were contaminated with fumonisin B1, which is not normally associated with this crop. Of more concern were the low but persistent levels of mycotoxins and fungi in wheat-based products sold directly to the public.

Consumer Product Safety↗

Oxidative stress in submerged cultures of fungi.

It has been known for many years that oxygen (O2) may have toxic effects on aerobically growing microorganisms, mainly due to the threat arising from reactive oxygen species (ROS). In submerged culture industrial fermentation processes, maintenance of adequate levels of O2 (usually measured as dissolved oxygen tension (DOT)) can often be critical to the success of the manufacturing process. In viscous cultures of filamentous cultures, actively respiring, supplying adequate levels of O2 to the cultures by conventional air sparging is difficult and various strategies have been adopted to improve or enhance O2 transfer. However, adoption of those strategies to maintain adequate levels of DOT, that is, to avoid O2 limitation, may expose the fungi to potential oxidative damage caused by enhanced flux through the respiratory system. In the past, there have been numerous studies investigating the effects of DOT on fungal bioprocesses. Generally, in these studies moderately enhanced levels of O2 supply resulted in improvement in growth, product formation and acceptable morphological changes, while the negative impact of higher levels of DOT on morphology and product synthesis were generally assumed to be a consequence of "oxidative stress." However, very little research has actually been focused on investigation of this implicit link, and the mechanisms by which such effects might be mediated within industrial fungal processes. To elucidate this neglected topic, this review first surveys the basic knowledge of the chemistry of ROS, defensive systems in fungi and the effects of DOT on fungal growth, metabolism and morphology. The physiological responses of fungal cells to oxidative stress imposed by artificial and endogenous stressors are then critically reviewed. It is clear that fungi have a range of methods available to minimize the negative impacts of elevated ROS, but also that development of the various defensive systems or responses, can itself have profound consequences upon many process-related parameters. It is also clear that many of the practically convenient and widely used experimental methods of simulating oxidative stress, for example, addition of exogenous menadione or hydrogen peroxide, have effects on fungal cultures quite distinct from the effects of elevated levels of O2, and care must thus be exercised in the interpretation of results from such studies. The review critically evaluates our current understanding of the responses of fungal cultures to elevated O2 levels, and highlights key areas requiring further research to remedy gaps in knowledge.

Adaptation, Physiological↗

Selection and identification of fungi isolated from sugarcane bagasse and their application for phenanthrene removal from soil.

This work investigated the identification and selection of fungi isolated from sugarcane bagasse and their application for phenanthrene (Phe) removal from soil. Fungi were identified by PCR amplification of ITS regions as Aspergillus terrus, Aspergillus fumigatus and Aspergillus niger, Penicillium glabrum and Cladosporium cladosporioides. A primary selection of fungi was accomplished in plate, considering Phe tolerance of every strain in two different media: potato dextrose agar (PDA) and mineral medium (MM). The radial extension rate (r(r)) in PDA exhibited significant differences (p<0.05) at 200 and 400 ppm of Phe. A secondary selection of A. niger, C. cladosporoides, and P. glabrum sp. was achieved based on their tolerance to 200, 400, 600 and 800 ppm of Phe, in solid culture at a sugarcane bagasse/contaminated soil ratio of 95:5, in Toyamas, Czapeck and Wunder media. Under these conditions, a maximum (70%) Phe removal by A. niger was obtained. In addition C. cladosporioides and A. niger were able to remove high (800 ppm) Phe concentrations.

Agar↗

Rapid identification of fungi by sequencing the ITS1 and ITS2 regions using an automated capillary electrophoresis system.

We developed a standardized DNA sequence-based approach for the accurate and timely identification of medically important fungi by sequencing polymerase chain reaction (PCR) products with a rapid automated capillary electrophoresis system. A simple DNA extraction method and PCR amplification using universal fungal primers was used to amplify ribosomal DNA from a range of clinical isolates and reference strains. The entire internal transcribed spacer (ITS) 1-5.8S-ITS2 ribosomal DNA region was sequenced using automated dye termination sequencing for 89 clinical isolates. These had previously been identified by traditional methods and included 12 ascomycetous yeast species, three basidiomycetous yeast species, eight dermatophyte species and two thermally dimorphic fungi, Scedosporium prolificans and S. apiospermum. Furthermore, 21 reference strains representing 19 different Candida species, Geotrichum candidum and Malassezia furfur were also sequenced as part of this study and were used either as standards for sequence-based comparisons, or as assay controls. Sequence-based identification was compared to traditional identification in a blinded manner. Of the clinical isolates tested, 88/89 had DNA sequences that were highly homologous to those of reference strains accessioned in GenBank, and 87/89 gave a sequence-based identification result that correlated with the traditional identification. In contrast to relatively slow conventional methods of identification, a sequence-based identification from a pure culture can be obtained within 24 h of a DNA extraction carried out after a minimal period of culture growth. We conclude that this approach is rapid, and may be a more accurate cost-effective alternative than most phenotypic methods for identification of many medically important fungi frequently encountered in a routine diagnostic microbiology laboratory.

Ascomycota↗

In vitro effects of fungi isolated from equine hooves on primary human keratinocytes.

The effects of two dermatophytes (Microsporum gypseum and Trichophyton mentagrophytes) and four moulds (Scopulariopsis brevicaulis, Alternaria alternata, Geotrichum candidum and Penicillium spp.) on living keratinocyte cultures were examined in vitro using primary human keratinocytes. Rates of apoptosis of infected cells were determined using a colorimetric TUNEL system which detects the characteristic nuclear DNA fragmentation of apoptotic cells. The cytotoxicity of the individual fungi was tested by quantitatively measuring cytosolic enzyme lactate dehydrogenase, released upon cell lysis, in culture supernatants. Additionally, the cell structures within the infected keratinocytes in cultures were examined by scanning electron microscopy. All of the fungi exhibited high cytotoxicity, whereas the development of only the two dermatophytes and the mould Scopulariopsis brevicaulis resulted in distinctly increased apoptosis. Electron microscopy showed that all fungi studied caused similar alterations in the cell structure, with Microsporum gypseum being the most harmful. Increasing loss of cell adhesion as a consequence of a decreasing number of reticulating cell appendices and a reduced cell plasticity were the most evident alterations.

Animals↗

Indoor air quality in two urban elementary schools--measurements of airborne fungi, carpet allergens, CO2, temperature, and relative humidity.

This article presents measurements of biological contaminants in two elementary schools that serve inner city minority populations. One of the schools is an older building; the other is newer and was designed to minimize indoor air quality problems. Measurements were obtained for airborne fungi, carpet loadings of dust mite allergens, cockroach allergens, cat allergens, and carpet fungi. Carbon dioxide concentrations, temperature, and relative humidity were also measured. Each of these measurements was made in five classrooms in each school over three seasons--fall, winter, and spring. We compared the indoor environments at the two schools and examined the variability in measured parameters between and within schools and across seasons. A fixed-effects, nested analysis was performed to determine the effect of school, season, and room-within-school, as well as CO2, temperature and relative humidity. The levels of all measured parameters were comparable for the two schools. Carpet culturable fungal concentrations and cat allergen levels in the newer school started and remained higher than in the older school over the study period. Cockroach allergen levels in some areas were very high in the newer school and declined over the study period to levels lower than the older school. Dust mite allergen and culturable fungal concentrations in both schools were relatively low compared with benchmark values. The daily averages for temperature and relative humidity frequently did not meet ASHRAE guidelines in either school, which suggests that proper HVAC and general building operation and maintenance procedures are at least as important as proper design and construction for adequate indoor air quality. The results show that for fungi and cat allergens, the school environment can be an important exposure source for children.

Air Microbiology↗