Silver-Sulfamethazine-Conjugated ß-Cyclodextrin/Dextran-Coated Magnetic Nanoparticles for Pathogen Inhibition.

In the fight against antibiotic resistance, which is rising to dangerously high levels worldwide, new strategies based on antibiotic-conjugated biocompatible polymers bound to magnetic nanoparticles that allow the drug to be manipulated and delivered to a specific target are being proposed. Here, we report the direct surface engineering of nontoxic iron oxide nanoparticles (IONs) using biocompatible dextran (Dex) covalently linked to ß-cyclodextrin (ß-CD) with the ability to form non-covalent complexes with silver-sulfamethazine (SMT-Ag). To achieve a good interaction of ß-CD-modified dextran with the surface of the nanoparticles, it was functionalized with diphosphonic acid (DPA) that provides strong binding to Fe atoms. The synthesized polymers and nanoparticles were characterized by various methods, such as nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) and ultraviolet-visible (UV-Vis) spectroscopies, transmission electron microscopy (TEM), thermogravimetric analysis (TGA), atomic absorption spectroscopy (AAS), dynamic light scattering (DLS), etc. The resulting magnetic ION@DPA-Dex-ß-CD-SMT-Ag nanoparticles were colloidally stable in water and contained 24 µg of antibiotic per mg of the particles. When tested for in vitro antimicrobial activity on Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria and fungi (yeast Candida albicans and mold Aspergillus niger), the particles showed promising potential.

Aspergillus niger Environmental Isolates and Their Specific Diversity Through Metabolite Profiling.

We present a biological profile of 16 Aspergillus niger environmental isolates from different types of soils and solid substrates across a pH range, from an ultra-acidic (<3.5) to a very strongly alkaline (>9.0) environment. The soils and solid substrates also differ in varying degrees of anthropic pollution, which in most cases is caused by several centuries of mining activity at old mining sites, sludge beds, ore deposits, stream sediments, and coal dust. The values of toxic elements (As, Sb, Zn, Cu, Pb) very often exceed the limit values. The isolates possess different macro- and micromorphological features. All the identifications of Aspergillus niger isolates were confirmed by molecular PCR analysis and their similarity was expressed by RAMP analysis. The biochemical profile of isolates based on FF-MicroPlate tests from the Biolog system showed identical biochemical reactions in 50 tests, while in 46 tests the utilisation reactions differed. The highest similarity of strains isolated from substrates with the same pH, as well as the most suitable biochemical tests for analysis of the phenotypic similarity of isolated strains, were confirmed when evaluating the biochemical profile using multicriterial analysis in the Canoco program. The isolates were screened for mycotoxin production by thin-layer chromatography (TLC), as well. Two of them were able to synthesise ochratoxin A, while none produced fumonisins under experimental conditions. Presence of toxic compounds in contaminated sites may affect environmental microscopic fungi and cause the genome alteration, which may result in changes of their physiology, including the production of different (secondary) metabolites, such as mycotoxins.

Fungal infections in animals: a patchwork of different situations.

The importance of fungal infections in both human and animals has increased over the last decades. This article represents an overview of the different categories of fungal infections that can be encountered in animals originating from environmental sources without transmission to humans. In addition, the endemic infections with indirect transmission from the environment, the zoophilic fungal pathogens with near-direct transmission, the zoonotic fungi that can be directly transmitted from animals to humans, mycotoxicoses and antifungal resistance in animals will also be discussed. Opportunistic mycoses are responsible for a wide range of diseases from localized infections to fatal disseminated diseases, such as aspergillosis, mucormycosis, candidiasis, cryptococcosis and infections caused by melanized fungi. The amphibian fungal disease chytridiomycosis and the Bat White-nose syndrome are due to obligatory fungal pathogens. Zoonotic agents are naturally transmitted from vertebrate animals to humans and vice versa. The list of zoonotic fungal agents is limited but some species, like Microsporum canis and Sporothrix brasiliensis from cats, have a strong public health impact. Mycotoxins are defined as the chemicals of fungal origin being toxic for warm-blooded vertebrates. Intoxications by aflatoxins and ochratoxins represent a threat for both human and animal health. Resistance to antifungals can occur in different animal species that receive these drugs, although the true epidemiology of resistance in animals is unknown, and options to treat infections caused by resistant infections are limited.

Sterigmatocystin in foodstuffs and feed: aspects to consider.

Sterigmatocystin (STC) is a possible human carcinogen (2B) according to International Agency for Research on Cancer classification and has been associated with immunotoxic and immunomodulatory activity, together with mutagenic effects. It might be found in numerous substrates, from foods and feeds to chronically damp building materials and indoor dust. Although European Food Safety Authority concluded that the exposure to STC to be of low concern for public health, reinforces the need of data concerning exposure of European citizens. Climate change can represent an increased risk of exposure to STC since it is a crucial factor for agro-ecosystem powering fungal colonisation and mycotoxin production This aspect can represent an increased risk for European countries with temperate climates and it was already reported by the scientific community.

Discriminative potential of some PCR-based and biochemical methods at Scedosporium strains.

Three innovative PCR-based methods (fluorescent-ITS, fluorescent-CBH and ITS-PCR DGGE) were tested using a reference set of nine strains of Scedosporium from the CBS fungal collection. Cellulolytic, lipolytic and proteolytic potential and the ability to dissolve CaCO3 of the strains were evaluated in vitro by means of agar assays. f-ITS profiles almost recognized main species, although included "Pseudallescheria" ellipsoidea and the Scedosporium boydii CBS 117432 and CBS 120157 in the same cluster. All strains successfully produced DNA polymorphisms by f-CBH amplification which divided them into three different groups. The DGGE approach separated the strains studied into other five clusters which in some case were not matching with species. Strains tested were monomorphic in possessing strong proteolytic and lipolytic activities. The comparison of the three PCR-based genotyping approaches, together with biodegradation ability screening, displayed an intraspecies variability in S. boydii, interfering with unambiguous species delimitation.

Adverse health effects of indoor moulds.

Building associated illnesses - sick building syndrome (SBS) as a common example - are associated with staying in buildings with poor indoor air quality. The importance of indoor fungal growth in this phenomenon continues to be evident, even though no causative relation has been established so far. Indoor humidity is strongly associated with the symptoms of SBS. Fungal metabolites that may induce ill health in susceptible occupants comprise beta-D-glucan, mycotoxins, and volatile organic compounds as known irritants and/or immunomodulators. Indoor toxic fungal metabolites might be located in micromycetal propagules (endometabolites), in (bio-)aerosol, detritus, and house dust (exometabolites) as their particular carriers. It is highly probable that hyphal fragments, dust, and particles able to reach the alveoli have the strongest depository and toxic potential. Most fungal spores are entrapped by the upper respiratory tract and do not reach further than the bronchi because of their size, morphology, and the mode of propagation (such as slime heads and aggreggation). This is why studies of the toxic effects of fungal spores prefer directly applying metabolite mixtures over mimicking real exposure. Chronic low-level exposure to a mixture of fungal toxicants and other indoor stressors may have synergistic effects and lead to severe neuroendocrineimmune changes.

Inflammatory and haematotoxic potential of indoor Stachybotrys chartarum (Ehrenb.) Hughes metabolites.

Mould Stachybotrys chartarum (Ehrenb.) Hughes is known to pose a health risk in indoor environments. Most of its strains can produce several intra- and extracellular trichothecene mycotoxins. Complex secondary metabolites of stachybotrys isolates from mouldy dwellings/public buildings in Slovakia were intratracheally instilled in Wistar male rats (4 microg in 0.2 mL of 0.2 % dimethylsulphoxide; diacetoxyscirpenol as the positive control). After three days, haematological parameters were measured in peripheral blood and inflammatory response biomarkers in bronchoalveolar lavage fluid (BALF), and the results were statistically analysed. Exometabolites proved to suppress red blood cell (RBC), decreasing the total RBC count, haemoglobin, and haematocrit. The exposed rats showed significantly higher total BALF cell count, indicating inflammation, lower alveolar macrophage counts, and increased granulocyte count related to the BALF cells. Due to haematotoxic and inflammation-inducing properties, metabolites of S. chartarum can cause damage to the airways and haematological disorders in occupants of mouldy buildings.

An in vitro study of the toxic effects of Stachybotrys chartarum metabolites on lung cells.

During a study of indoor fungal colonisation, several isolates of Stachybotrys chartarum were recovered, and the effects of metabolites from four isolates on lung epithelial Type II cells and alveolar macrophages were studied in vitro. All the isolates showed toxic effects on both cell types, and they differed only in the extent of the changes induced. In Type II cells, the number of alkaline phosphatase positive cells was reduced, the pattern of Maclura pomifera agglutinin (MPA) binding was changed, and acid phosphatase activity in alveolar macrophages was diminished. In both cell types, the production of monocyte chemotactic protein-1 (MCP-1) and tumour necrosis factor-alpha (TNF-alpha) was changed, and parameters related to antioxidant status (superoxide dismutase, glutathione peroxidase, glutathione) were decreased.

Pulmonary cytotoxicity of secondary metabolites of Stachybotrys chartarum (Ehrenb.) Hughes.

Damp dwellings represent suitable conditions for extended indoor moulds. A cellulolytic micromycete Stachybotrys chartarum (Ehrenb.) Hughes is considered to be a tertiary colonizer of surfaces in affected buildings. Known adverse health effects of S. chartarum result from its toxins--trichothecenes or atranones, as well as spirolactams. Mechanism of their potential pathological effects on the respiratory tract has not yet been sufficiently clarified. The cytotoxic effects of complex chloroform-extractable endo- (in biomass) and exometabolites (in cultivation medium) of an indoor S. chartarum isolate of an atranone chemotype, grown on a liquid medium with yeast extract and sucrose at 25 degrees C for 14 d, on lung tissue were evaluated in the 3-day experiment. For the purpose, 4 mg of toxicants were intratracheally instilled in 200 g Wistar male rats. A trichothecene mycotoxin diacetoxyscirpenol was used as the positive control. Bronchoalveolar lavage (BAL) parameters--viability and phagocytic activity of alveolar macrophages (AM), activity of lactate dehydrogenase, acid phosphatase and cathepsin D in cell-free BAL fluid (BALF), as well as in BAL cells, were measured. Acute exposure to the metabolites caused statistically significant changes, indicating lung tissue injury in the experimental animals. Decreased AM viability and increased activity of lysosomal enzyme cathepsin D in BAL cells after fungal exometabolite exposure were the most impressive. As toxic principles were found predominantly in the growth medium, toxins were more likely responsible for lung cell damage than e.g. fungal cell wall components. S. chartarum toxic metabolites can contribute to the ill health of occupants of mouldy building after inhalation of contaminated aerosol.

Airway toxicity of house dust and its fungal composition.

House dust is an important source of different toxic metabolites as well as allergens, including those of fungal origin, in the indoor environment. A bio-assay employing 1-day-old chick tracheas was used to characterize airway effects of 2-butanone and dimethylsulphoxide (Me2SO) extracts of 23 dust samples collected from water damaged (13) and control (10) Danish schools. Direct microscopical analysis of samples, followed by cultivation on dichloran 18 % glycerol agar at 25 degrees C for 10 days to establish their mycoflora, was performed. The in vitro ciliostatic potential of the chloroform-extractable endo- and exometabolites of 41 representative fungal isolates was determined. Nine dust extracts in 2-butanone (2 from damp rooms) or 10 (6) in Me(2)SO showed some ciliostatic activity in the 3-days' experiment. Fungal composition of dust from buildings with leakage was almost identical with that from undamaged houses, as well as the fungal colony counts from the damp schools and the control samples. Aspergillus spp. were prevalent in the samples - 31 or 40 % of all fungi, followed by Penicillium spp. and Cladosporium cladosporioides. Alternaria spp., Chaetomium spp., Mucor spp., Mycelia sterilia, Paecilomyces variotii, Rhizopus sp., Ulocladium sp. and yeasts were each isolated in less than 8 % of the fungal content. No Aspergillus flavus isolate (8 in total) was aflatoxigenic,em>in vitro. Alternaria spp., Aspergillus spp., Botrytis cinerea, Penicillium spp., C. cladosporioides, Chaetomium spp. and Ulocladium sp.; in total, 88 % of all fungi tested, produced ciliostatically active metabolites. These toxigenic strains were also present in 4 dust samples from controls and 5 dust samples from water damaged buildings. Extracts of these dust samples were also toxic in bioassay. There were bio-detectable concentrations (10-20 microg of extracts/ml of the organ culture medium) of toxic compounds in house dust. Contribution of fungal metabolites to its toxic effect should be studied further.

In vitro toxicity of indoor Chaetomium Kunze ex Fr.

Microscopic fungi in the indoor environment present a serious health risk for people living in affected buildings. The potentially toxic ascomycete genus Chaetomium is supposed to be the third most frequent indoor fungal contaminant. Its brief mycological, toxicological and ecological characterization is given. The work was aimed at in vitro study of toxicity of endo- and exometabolites of 14 strains of Chaetomium spp., including 4 strains of Ch. globosum, isolated from mouldy buildings in Slovakia and Denmark, and 3 Ch. globosum strains from the Czechoslovak Collection of Microorganisms (CCM). The endometabolites of 10 isolates of Chaetomium spp. were active: 7 isolates (41% of total strain number) stopped tracheal ciliary movement of 1-d-old chickens after 24 h, 9 isolates (53%) after 48 h and 10 strains (59%) after 72 h. In the case of exometabolites, the extracts of 6 Chaetomium strains showed some ciliostatic activity: 2 isolates (12% of strains tested) after 24 h, 5 isolates (29%) after 48 h and 6 isolates (35%) after 72 h. In general, 5 isolates of Danish origin (83%) produced ciliostatically active exometabolites and 2 isolates (33%) produced such endometabolites, while only 4 strains isolated in Slovakia (50%) and 3 strains (37%) respectively did the same under experimental conditions. Most toxic metabolites were produced by Chaetomium spp. isolated from dwellings, whereas hospital isolates were not able to produce active compounds. Chaetomia as indoor contaminants can contribute to ill health of occupants of mouldy damp buildings.

Indoor fungi and their ciliostatic metabolites.

According to epidemiological studies, it is possible that some secondary metabolites of indoor airborne fungi could be responsible for health troubles which occupants suffer from. In our previous experiments, a model with tracheal rings of 1-day-old chicks in vitro was shown to be a very suitable method to study the ciliostatic chloroform-extractable endo- and/or exometabolites of filamentous fungi. In this study we isolated the filamentous fungi from walls of "mouldy" dwellings and schools (cultivation on dichloran 18% glycerol agar at 25 and 37 degrees C for 10 d) in Slovakia. We studied the ciliostatic effect of the chloroform-extractable endo- and exometabolites of 96 representative isolates (stationary cultivation on the liquid medium with 2% of yeast extract and 10% of sucrose at 25 degrees C for 10 days) on the cilia movement in tracheal organ cultures of 1-day-old chickens in vitro after 24, 48 and 72 hrs (incubation in the minimal essential medium according to Eagle with Earl s salts and 20 microg of extract of metabolites dissolved in dimethylsulfoxide per 1 mL). Strains of Penicillium Link: Fr. sp., Aspergillus versicolor (Vuill.) Tiraboschi, A. flavus Link, Cladosporium sphaerospermum Penzig and C. cladosporioides (Fres.) de Vries were isolated most frequently. Two A. flavus isolates were able to produce aflatoxins B1, B2, G1, G2 in vitro after cultivation on the liquid medium with 20% sucrose and 2% yeast extract. This is the first isolation of aflatoxigenic A. flavus strains from dwellings in Slovakia. All frequently isolated strains produced secondary metabolites with the strongest ciliostatic activity -- their exo- and endometabolites stopped tracheal ciliary movement in chicks till 24 h. There are some toxic fungal metabolites in the indoor air not only with the ability to destroy ciliary movement in the upper airways in vitro but, probably, during long-lasting exposure to cause general intoxication of macroorganism via lung tissue.

Detection of ciliostatic activity in fungal growth on building materials.

Varying ciliostatic activity was found in extracts of biomass from building materials inoculated with pure isolates of some molds previously isolated from buildings. Extracts from growth on materials composed of finely divided cellulose (plaster board and construction cardboard) were more active than those from growth on Rockwool. Extracts of biomass from all three materials demonstrated an activity greater than control samples. Based on the activity observed here, it is conceivable that the measurement of ciliostatic activity of biomass scraped from contaminated building materials could be useful for planning removal strategies.

Thermoresistant Propagules of Neosartorya fischeri ; Some Ecologic Considerations.

Neosartorya (N.) fischeri (Wehmer) Malloch and Cain is a significant agent causing molding of canned fruits. This genus made up 49.2% of all strains identified in 1989 from samples of molded canned fruit applied to the authors' laboratory by a canning plant. Ecology of this significant genus has been little studied. In the present work, 31 soil samples and 34 samples of cereal substrates from various localities were examined. Samples (10 g each) were placed into Sabouraud agar with 150 mg Rose Bengal/L and exposed to 70°C for 60 min. No colonies of N. fischeri could be isolated from one soil sample; in 30 samples the numbers of colonies ranged between 1 and 149 per sample, with an average of 27.6 colony forming units of N. fischeri per 10-g soil sample. N. fischeri as isolated from only three samples of cereal subtrates, at 1,6, and 7 colony forming units/10 g. Further studies are needed to elucidate factors determining the occurrence of thermoresistant propagule of N. fischeri in soil.