A new effective assay to detect antimicrobial activity of filamentous fungi

The search for new antimicrobial compounds and the optimization of production methods turn the use of antimicrobial susceptibility tests a routine. The most frequently used methods are based on agar diffusion assays or on dilution in agar or broth. For filamentous fungi, the most common antimicrobial activity detection methods comprise the co-culture of two filamentous fungal strains or the use of fungal extracts to test against single-cell microorganisms. Here we report a rapid, effective and reproducible assay to detect fungal antimicrobial activity against single-cell microorganisms. This method allows an easy way of performing a fast antimicrobial screening of actively growing fungi directly against yeast. Because it makes use of an actively growing mycelium, this bioassay also provides a way for studying the production dynamics of antimicrobial compounds by filamentous fungi. The proposed assay is less time consuming and introduces the innovation of allowing the direct detection of fungal antimicrobial properties against single cell microorganisms without the prior isolation of the active substance(s). This is particularly useful when performing large screenings for fungal antimicrobial activity. With this bioassay, antimicrobial activity of Hypholoma fasciculare against yeast species was observed for the first time.     

Determination of Gliotoxin in samples associated with cases of intoxication in camels

Suspected cases of mycotoxicoses, characterized by clinical symptoms such as diarrhoea, haemorrhaging and death have been observed in breeding camels in U.A.E. Hay samples, body fluids and intestinal contents were investigated for the presence of mycotoxins using a cell culture bioassay (MTT-test) and/or physicochemical methods such as high performance liquid chromatography (HPLC) and mass spectrometry (MS). Extracts of the hay samples proved to be highly cytotoxic towards the swine kidney monolayers used as target cells in the bioassay. Subsequent analyses of the extracts showed the presence of the epidithiodioxopiperazine mycotoxin gliotoxin up to 0.49 mg/kg in the hay, which is the first proven case on the natural occurrence of this mycotoxin in feed. Trace amounts of ochratoxin A were also detected, while other mycotoxins known to occur naturally (i.e. aflatoxins or trichothecenes) could not be found. Gliotoxin positive HPLC-peaks were also found in some specimens (contents of rumen and intestine, allantois fluid) from camels. Gliotoxin is characterized by a variety of biological activities including antibiotic, antifungal, antiviral, cytotoxic and immunotoxic effects. Based on the results obtained, the involvement of gliotoxin as one causative agent of the intoxications is supposed.     

Bioprospection of Cytotoxic Compounds in Fungal Strains Recovered from Sediments of the Brazilian Coast

The cytotoxic activities of extracts (50 μg/ml) from 48 fungal strains, recovered from sediments of Pecém's offshore port terminal (Northeast coast of Brazil), against HCT‐116 colon cancer cell lines were investigated. The most promising extract was obtained from strain BRF082, identified as Dichotomomyces cejpii by phylogenetic analyses of partial RPB2 gene sequence. Thus, it was selected for bioassay‐guided isolation of the cytotoxic compounds. Large‐scale fermentation of BRF082 in potato dextrose broth, followed by chromatographic purification of the bioactive fractions from the liquid medium, yielded gliotoxin ( 4 ) and its derivatives acetylgliotoxin G ( 3 ), bis(dethio)bis(methylsulfanyl)gliotoxin ( 1 ), acetylgliotoxin ( 5 ), 6‐acetylbis(dethio)bis(methylsulfanyl)gliotoxin ( 2 ), besides the quinazolinone alkaloid fiscalin B. All isolated compounds were tested for their cytotoxicities against the tumor cell lines HCT‐116, revealing 4 and 3 as the most cytotoxic ones (IC₅₀ 0.41 and 1.06 μg/ml, resp.).     

Gliotoxin contamination in and pre- and postfermented corn, sorghum and wet brewer's grains silage in Sao Paulo and Rio de Janeiro State, Brazil

Aims: The aim of this study was to determine total fungal counts and the relative density of Aspergillus fumigatus and related species in silage samples intended for bovines before and after fermentation as well as to monitor the natural occurrence of gliotoxin in silage samples (pre‐ and postfermentation). Methods and methods: The survey was performed in farms located in São Paulo and Rio de Janeiro States in Brazil. In addition, the ability of A. fumigatus strains and related species strains to produce gliotoxin was also evaluated. A total of 300 samples were taken, immediately after opening of the silo (3–5 months) and during the ensiling period. Fungal counts were done by the surface‐spread method. Gliotoxin production ability of isolates and natural contamination were determined by HPLC. Results: All postfermented samples had a total number of moulds exceeding 1 × 10⁴ CFU g^?1, with Aspergillus sp. as the most prevalent genus. Frequency of strains, among A. fumigatus and related species, was able to produce gliotoxin was similar in pre‐ and postfermented samples, except for sorghum, which showed differences between both kinds of samples. The highest toxin levels were produced by strains isolated from postfermented samples. More than 50% of the samples showed gliotoxin contamination levels that exceeded concentrations known to induce immunosuppressive and apoptotic effects in cells. Conclusions: The present data suggest that care should be taken because gliotoxin contamination in feedstuffs could affect productivity and also present a health risk for herds. Significance and Impact of the Study: Gliotoxin was found at quite important concentrations levels in pre‐ and postfermented substrates and its presence could therefore probably affect the productivity and health of herds. Current conservation and management practices do not avoid contamination with A. fumigatus on silage. Therefore, farm workers should be adequately protected during its handling.     

Cultivation-Independent Analysis of Fungal Genotypes in Soil by Using Simple Sequence Repeat Markers

Cultivation-independent analyses of fungi are used for community profiling as well as identification of specific strains in environmental samples. The objective of the present study was to adapt genotyping based on simple sequence repeat (SSR) marker detection for use in cultivation-independent monitoring of fungal species or strains in bulk soil DNA. As a model system, a fungal biocontrol agent (BCA) based on Beauveria brongniartii, for which six SSR markers have been developed, was used. Species specificity of SSR detection was verified with 15 fungal species. Real-time PCR was used to adjust for different detection sensitivities of the six SSR markers as well as for different template quantities. The limit for reliable detection per PCR assay was below 2 pg target DNA, corresponding to an estimated 45 genome copies of B. brongniartii. The cultivation-independent approach was compared to cultivation-dependent SSR analysis with soil samples from a B. brongniartii BCA-treated field plot. Results of the cultivation-independent method were consistent with cultivation-dependent genotyping and allowed for unambiguous identification and differentiation of the applied as well as indigenous strains in the samples. Due to the larger quantities of soil used for cultivation-dependent analysis, its sensitivity was higher, but cultivation-independent SSR genotyping was much faster. Therefore, cultivation-independent monitoring of B. brongniartii based on multiple SSR markers represents a rapid and strain-specific approach. This strategy may also be applicable to other fungal species or strains for which SSR markers have been developed.     

Rhodotorula rubra bioassay for T-2 toxin: Increased sensitivity and wider application

Mutant strains of Rhodotorula rubra sensitive to 0.019–2.5 μg of T-2 toxin have been selected using ethylmethane sulphonate and ultraviolet light. When used as test organism in a bioassay for mycotoxins, these strains are also sensitive to trichothecin, diacetoxyscirphenol, verrucarin A and roridin A. A modification of the bioassay suitable for the rapid screening of large numbers of fungal strains without purification of the toxin is described.     

Disruption of a nonribosomal peptide synthetase in Aspergillus fumigatus eliminates gliotoxin production

The fungal secondary metabolite gliotoxin produced by Aspergillus fumigatus has been hypothesized to be important in the development of invasive aspergillosis. In this study, we addressed this hypothesis by disrupting a nonribosomal peptide synthetase (NRPS) (encoded by gliP) predicted to be involved in gliotoxin production. Mutants with a disrupted gliP locus failed to produce gliotoxin, which confirmed the role of the NRPS encoded by gliP in gliotoxin biosynthesis. We found no morphological, developmental, or physiological defects in DeltagliP mutant strains. In addition, disruption of gliP resulted in down regulation of gene expression in the gliotoxin biosynthesis gene cluster, which was restored with addition of exogenous gliotoxin. This interesting result suggests a role for gliotoxin in regulating its own production. Culture filtrates from the DeltagliP mutant were unable to inhibit ionomycin-dependent degranulation of mast cells, suggesting a role for gliotoxin in suppressing mast cell degranulation and possibly in disease development. However, the DeltagliP mutant did not have an impact on survival or tissue burden in a murine inhalational model of invasive aspergillosis. This result suggests that gliotoxin is not required for virulence in an immunosuppressed host with an invasive pulmonary infection.     

A quantitative bioassay for extracellular metabolites that antagonize growth of filamentous fungi,and its use with biocontrol fungi

A bioassay and an empirically derived formula were developed to quantify fungitoxic effects. This bioassay can be easily performed and objectively read, and it is suitable for low-volume samples originating from aqueous or organic solvents. The formula defines the Inhibition Index (I), a single value that incorporates both the response to concentrations of the inhibitory compound and the persistence of inhibition. Antagonistic efficacy of metabolites produced by biocontrol strains of Trichoderma spp. were measured based on inhibition of growth of Rhizoctonia solani. Although the bioassay itself was not influenced by pH or light conditions, these factors affected metabolite production or activity. Aqueous extracts from light-grown germlings of Trichoderma virens inhibited R. solani more than extracts from germlings grown in the dark. Low pH increased the inhibitory activity of extracts from T. virens. Tests of fungal strains developed for biocontrol demonstrated that the bioassay reflected their activity both in the field and in other in vitro tests. The bioassay and formula are readily adapted for use with other fungi.This revised version was published online in October 2005 with corrections to the Cover Date.     

Cytotoxic Substances from Aspergillus fumigatus in Oxygenated or Poorly Oxygenated Environment

Aspergillus fumigatus often causes serious health problems. The airway of the human body, the most common initial site of damage, is always exposed to an oxygenated condition, and the oxygen concentration may play a critical role in the virulence of A. fumigatus. In this study, oxygen content, fungal growth, the production of cytotoxic substance(s) in the fungal culture, and their relationship were investigated. Two clinical strains of A. fumigatus were cultured under certain oxygen contents (10, 14 and 20%), and cytotoxicity of their culture filtrates on murine macrophages and their fungal growth were evaluated. The components of these filtrates were analyzed by gas chromatography-mass spectrometry. All culture filtrates contained gliotoxin and showed potent cytotoxicity on macrophages at very low concentration. The amount of gliotoxin in the culture filtrate prepared at 10% oxygen was markedly less, but diminutions in fungal growth and cytotoxicity of this culture filtrate were negligible. These results suggest that a well-oxygenated condition is suitable for the production of gliotoxin by A. fumigatus. A significant role of cytotoxic substances(s) other than gliotoxin is also suggested.     

Aspergillus fumigatus toxicity and gliotoxin levels in feedstuff for domestic animals and pets in Argentina

Aims:  To evaluate gliotoxin production by Aspergillus fumigatus strains isolated from feedstuff intended for domestic animals and pets, and to determine the amount of gliotoxin in these substrates.
Methods and Results:  A total of 150 feedstuff samples were collected. They were composed of 30 samples each of five different feed types (pigs, poultry, cattle, horse and pets). Aspergillus fumigatus gliotoxin production ability and gliotoxin presence in feedstuff was determined by HPLC. Aspergillus fumigatus strains were isolated from all of the tested samples. Strains from cattle, horses and pet food were able to produce gliotoxin. Corn silage samples intended for cattle did not show gliotoxin contamination. All the other tested samples had gliotoxin levels ranging from 29 to 209 μg g⁻¹. Horse and poultry feed samples had the greatest contamination frequency.
Conclusions:  Feed samples contaminated with gliotoxin are potentially toxic to animals.
Significance and Impact of the Study:  The presence of gliotoxin could affect animal productivity and health. Moreover, there are risks of contamination to farm workers handling improperly stored animal feed. Aspergillus fumigatus strains isolated from different sources should be investigated to determine prevention and control strategies.     

Detection of enterotoxigenic Clostridium perfringens in food and fecal samples with a duplex PCR and the slide latex agglutination test.

A duplex PCR procedure was evaluated for the detection of Clostridium perfringens in food and biological samples and for the identification of enterotoxigenic strains. This method uses two sets of primers which amplify in the same reaction two different DNA fragments simultaneously: the 283-bp C. perfringens phospholipase C gene fragment and the 426-bp enterotoxin gene fragment. Internal primers within the two primer sets confirmed the specificity of the method by DNA-DNA hybridization with the PCR products. No cross-reaction was observed with other Clostridium species or with other bacteria routinely found in food. The detection level was approximately 10(5) C. perfringens cells per g of stool or food sample. When overnight enrichment culture was used, 10 C. perfringens cells per g was detected in 57 artificially contaminated food samples. The duplex PCR is a rapid, sensitive, and reliable method for the detection and identification of enterotoxigenic C. perfringens strains in food samples. A slide latex agglutination test was also evaluated as a rapid, simple technique for the detection of C. perfringens enterotoxin in stool samples.     

Gliotoxin in <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>: an example that mycotoxins are potential virulence factors

Moulds produce several different mycotoxins that may improve their chance of survival in particular environments. For example, Aspergillus fumigatus, an important human pathogen, produces several mycotoxins including gliotoxin. This secondary metabolite, a small lipid soluble dipeptide, exerts toxic effects on phagocytic cells and T-lymphocytes at low concentrations in vitro. A. fumigatus also produces high levels of gliotoxin in vivo, and this suggests that host defense mechanisms might be impaired by this metabolite during host infection. In the past few years, the genes responsible for the production of gliotoxin in A. fumigatus have been identified and more recently gliotoxin-minus mutants have been used in animal experiments to ascertain the biological role of this product. Mycotoxins have also been shown to act as virulence factors in some fungal infections of insects and plants.     

Development of a new procedure based on the energy charge measurement using ATP bioluminescence assay for the detection of living mould from graphic documents

Fungal contamination is a major cause of deterioration in libraries and archives. Curators and conservators increasingly need rapid microbiological analyses. This paper presents a rapid detection method for the fungal contaminants on documents. A previous study showed that the calculation of energy charge, using bioluminescence ATP assays, provides a useful indicator to determinate the viability of fungal strains. We argue that this sensitive and time‐saving method is better than traditional culture techniques. However, the procedure needs to be modified to make it usable for lay persons. An improved and simplified protocol is proposed here for the extraction of adenylate nucleotides (AN) from fungal spores and for their measurements. Our new procedure can detect the existence of viable fungal strains on documents, presenting suspect spots within minutes. The extraction is performed by filtration with DMSO–TE solution as extractant. The different step of the measurement of AN content is carried out successively in a single test tube instead of the three tubes necessary in the initial method. The new procedure was tested on 12 strains among those most frequently found in archives and libraries and validated on swab samples from real documents. Copyright © 2008 John Wiley & Sons, Ltd.     

Ion trap MS(n) for identification of gliotoxin as the cytotoxic factor of a marine strain of Aspergillus fumigatus Fresenius.

When cultured in a marine solid medium, a strain of Aspergillus fumigatus (Fresenius) isolated from a shellfish-farming area in the Loire estuary (France) produced a highly cytotoxic exudate. To identify the origin of this activity, a cytotoxicity test on KB cells was used to monitor the purification of the exudate, together with electrospray/ion trap/mass spectrometry (ESI/IT/MS(n)) to detect and identify the toxic compound. After three purification stages, a comparison of fullscan analyses of the last six fractions showed that a monocharged compound at m/z 349 was present only in the active fraction, corresponding to the sodium adduct of gliotoxin [C(13)H(14)N(2)O(4)S(2)+Na](+). Isotopic distribution determination showed that the m/z 349 product possessed two sulphur atoms and multi-stage fragmentation confirmed the hypothesis. MS/MS analysis exhibited the characteristic gliotoxin loss of the disulphide intracyclic bridge. MS(3) analysis revealed four main ions and confirmed the identity of the m/z 349 ion. This study points out that the combined use of a KB cells bioassay and ESI/IT/MS(n) allows a fast and very specific detection and elucidation of unidentified cytotoxic products in natural samples. This method does not require total purification, and it allowed us to report the first detection of gliotoxin production in marine conditions.     

SYBR green real-time PCR method to detect Clostridium botulinum type A

Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 x 10(1) copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%).     

Measurement of nitrite and nitrate levels in biological samples by capillary electrophoresis

Nitrite is one of the products of NO-synthase in biological media. It is slowly oxidized in animals to nitrate. We developed a simple and rapid method to determine simultaneously nitrite and nitrate in biological samples. Capillary ion electrophoresis with direct UV detection at 214 nm was used employing a carrier electrolyte consisting of 10 mM sodium sulfate and an electroosmotic flow modifier. The detection limit in ultrafilates of plasma, urine and brain tissue extracts was 25 ng/ml for both compounds. Nitrate levels in human plasma and urine were in the μg/ml range. Nitrite could not be detected. Rat brain tissue extracts contained detectable amounts of nitrite and nitrate.     

Development of Dot-ELISA for the detection of human rotavirus antigen and comparison with RNA-PAGE

AIMS: Development of a simple, specific, rapid and inexpensive Dot-ELISA test for diagnosis of rotaviral antigen in stool samples. METHODS AND RESULTS: Hyperimmune rabbit antisera raised against SA-11 (Simian Agent-11) strain was used as primary antibody. The secondary antibody conjugate used was the goat anti-rabbit IgG alkaline phosphatase, and BCIP/NBT solution was used as substrate. Faecal extracts were diluted 10-fold and used for the detection of rotavirus antigen. RNA-PAGE was performed to compare the specificity and sensitivity of the diagnostic tests. Dot-ELISA positive samples were further confirmed by Western blot analysis. CONCLUSIONS: This Dot-ELISA test could be used as an alternative method for diagnosing rotaviral samples in the field. SIGNIFICANCE AND IMPACT OF THE STUDY: The Dot-ELISA test is simple, specific, rapid and cost effective. It is suitable for identifying a large number of samples obtained from epidemiological studies and hence, reducing the death rate of rotavirus-infected patients.     

Development of a F actin-based live-cell fluorimetric microplate assay for diarrhetic shellfish toxins

A new cytotoxicity assay for detection and quantitation of diarrhetic shellfish toxins (DSP) is presented. This assay is based upon fluorimetric determination of F-actin depolymerization induced by okadaic acid (OA)-class compounds in the BE(2)-M17 neuroblastoma cell line. No interferences were observed with other marine toxins such as saxitoxin, domoic acid, or yessotoxin, thus indicating a good specificity of the assay as expected by the direct relationship between protein phosphatase inhibition and cytoskeletal changes. The proposed method is rapid (<2h) and shows a linear response in the range of 50-300 nM OA. The detection limit of the assay for crude methanolic extracts of bivalves lies between 0.2 and 1.0 microg OA per gram of digestive glands, depending on the type of samples (fresh or canned), thus being similar to that of the mouse bioassay. The performance of this assay has been evaluated by comparative analysis of 32 toxic mussel samples by the F-actin assay, mouse bioassay, HPLC and PP2A inhibition assay. Results obtained by the F-actin method showed no differences with HPLC and significant correlation with PP2A inhibition assay (r(2)=0.71). No false negative results were obtained with this new cell assay, which also showed optimum reproducibility.     

Specific and dynamic detection of palytoxins by in vitro microplate assay with human neuroblastoma cells

Palytoxin is one of the most complex and biggest molecules known to show extreme acute toxicity. The dinoflagellate Ostreopsis spp., the producer organism of palytoxin, has been shown to be distributed worldwide, thus making palytoxin an emerging toxin. Rat-derived hepatocytes (Clone 9) and BE (2)-M17 human neuroblastoma cells were used to test palytoxin or palytoxin-like compounds by measuring the cell metabolic rate with Alamar Blue. The dose-dependent decrease in viability was specifically inhibited by ouabain in the case of BE (2)-M17 neuroblastoma cells. This is a functional, dynamic and simple test for palytoxins with high sensitivity (as low as 0.2 ng/ml). This method was useful for toxin detection in Ostreopsis extracts and naturally contaminated mussel samples. A comparative study testing toxic mussel extracts by LC (liquid chromatography)-MS/MS (tandem MS), MBA (mouse bioassay), haemolysis neutralization assay and a cytotoxicity test indicated that our method is suitable for the routine determination and monitoring of palytoxins and palytoxin-like compounds.