No abstract

The genus Stachybotrys produces a broad diversity of secondary metabolites, including macrocyclic trichothecenes, atranones, and phenylspirodrimanes. Although the class of the phenylspirodrimanes is the major one and consists of a multitude of metabolites bearing various structural modifications, few investigations have been carried out. Thus, the presented study deals with the quantitative determination of several secondary metabolites produced by distinct Stachybotrys species for comparison of their metabolite profiles. For that purpose, 15 of the primarily produced secondary metabolites were isolated from fungal cultures and structurally characterized in order to be used as analytical standards for the development of an LC-MS/MS multimethod. The developed method was applied to the analysis of micro-scale extracts from 5 different Stachybotrys strains, which were cultured on different media. In that process, spontaneous dialdehyde/lactone isomerization was observed for some of the isolated secondary metabolites, and novel stachybotrychromenes were quantitatively investigated for the first time. The metabolite profiles of Stachybotrys species are considerably influenced by time of growth and substrate availability, as well as the individual biosynthetic potential of the respective species. Regarding the reported adverse effects associated with Stachybotrys growth in building environments, combinatory effects of the investigated secondary metabolites should be addressed and the role of the phenylspirodrimanes re-evaluated in future research.

Stachybotrys (S.) chartarum had been linked to severe health problems in humans and animals, which occur after exposure to the toxic secondary metabolites of this mold. S. chartarum had been isolated from different environmental sources, ranging from culinary herbs and improperly stored fodder to damp building materials. To access the pathogenic potential of isolates, it is essential to analyze them under defined conditions that allow for the production of their toxic metabolites. All Stachybotrys species are assumed to produce the immunosuppressive phenylspirodrimanes, but the highly cytotoxic macrocyclic trichothecenes are exclusively generated by the genotype S of S. chartarum. In this study, we have analyzed four genotype S strains initially isolated from three different habitats. We grew them on five commonly used media (malt-extract-agar, glucose-yeast-peptone-agar, potato-dextrose-agar, cellulose-agar, Sabouraud-dextrose-agar) to identify conditions that promote mycotoxin production. Using LC-MS/MS, we have quantified stachybotrylactam and all S-type specific macrocyclic trichothecenes (satratoxin G, H, F, roridin E, L-2, verrucarin J). All five media supported a comparable fungal growth and sporulation at 25 °C in the dark. The highest concentrations of macrocyclic trichothecenes were detected on potato-dextrose-agar or cellulose-agar. Malt-extract-agar let to an intermediate and glucose-yeast-peptone-agar and Sabouraud-dextrose-agar to a poor mycotoxin production. These data demonstrate that the mycotoxin production clearly depends on the composition of the respective medium. Our findings provide a starting point for further studies in order to identify individual components that either support or repress the production of mycotoxins in S. chartarum.

The Bacillus (B.) cereus group is genetically highly homogenous and consists of nine recognized species which are present worldwide. B. cereus sensu stricto play an important role in food-borne diseases by producing different toxins. Yet, only a small percentage of B. cereus strains are able to produce the heat stable depsipeptide cereulide, the causative agent of emetic food poisonings. To minimize the entry of emetic B. cereus into the food chain, food business operators are dependent on efficient and reliable methods enabling the differentiation between emetic and non-emetic strains.Currently, only time-consuming cell bioassays, molecular methods and tandem mass spectrometry are available for this purpose. Thus, the aim of the present study was to establish a fast and reliable method for the differentiation between emetic and non-emetic strains by MALDI-TOF MS. Selected isolates/strains of the B. cereus group (total n=110, i.e. emetic n=45, non-emetic n=65) were cultured on sheep blood agar for 48h.Subsequently, the cultures were directly analyzed by MALDI-TOF MS without prior extraction steps (direct smear method). The samples were measured in linear positive ionization mode in the mass range of m/z 800 -1,800 Da. Using ClinProTools 3.0 statistical software and flex analyst, a differentiation between emetic and non-emetic isolates was possible with a rate of correct identification of 99.1 % by means of the evaluation of two specific biomarkers (m/z 1171 and 1187 Da). ImportanceBacillus (B.) cereus plays an important role in food-borne diseases due to the production of different toxins, e.g. the heat stable depsipeptide cereulide. Only a small number of B. cereus strains are able to produce this toxin, the causative agent of emetic food poisonings. To minimize the entry of emetic B. cereus into the food chain, food business operators require efficient and reliable methods enabling the differentiation between emetic and non-emetic strains. The aim of the present study was to develop a fast and reliable method for the differentiation between emetic and non-emetic strains by MALDI-TOF MS. A differentiation between emetic and non-emetic isolates was possible with a rate of correct

The fungus Stachybotrys (S.) chartarum was isolated from culinary herbs, damp building materials, and improperly stored animal forage. Two distinct chemotypes of the fungus were described that produced either high-cytotoxic macrocyclic trichothecenes (S type) or low-cytotoxic atranones (A type). Recently, two distinct gene clusters were described that were found to be necessary for the biosynthesis of either macrocyclic trichothecenes (21 SAT (Satratoxin) genes) or atranones (14 ATR (Atranone) genes). In the current study, PCR primers were designed to detect SAT and ATR genes in 19 S. chartarum chemotype S and eight S. chartarum chemotype A strains. Our analysis revealed the existence of three different genotypes: satratoxin-producing strains that harbored all SAT genes but lacked the ATR gene cluster (genotype S), non-satratoxin-producing strains that possessed the ATR genes but lacked SAT genes (genotype A), and a hitherto undescribed hybrid genotype among non-satratoxin-producing strains that harbored all ATR genes and an incomplete set of SAT genes (genotype H). In order to improve the discrimination of genotypes, a triplex PCR assay was developed and applied for the analysis of S. chartarum and S. chlorohalonata cultures. The results show that genes for macrocyclic trichothecenes and atranones are not mutually exclusive in S. chartarum. Correlation of the new genotype-based concept with mycotoxin production data shows also that macrocyclic trichothecenes are exclusively produced by S. chartarum genotype S strains.

No abstract

Antibodies represent an important element in the adaptive immune response and a major tool to eliminate microbial pathogens. For many bacterial and viral infections, efficient vaccines exist, but not for fungal pathogens. For a long time, antibodies have been assumed to be of minor importance for a successful clearance of fungal infections; however this perception has been challenged by a large number of studies over the last three decades. In this review, we focus on the potential therapeutic and prophylactic use of monoclonal antibodies. Since systemic mycoses normally occur in severely immunocompromised patients, a passive immunization using monoclonal antibodies is a promising approach to directly attack the fungal pathogen and/or to activate and strengthen the residual antifungal immune response in these patients.

Starch gelatinization is a major determinant of carbohydrate digestibility and varies with diet processing. Laboratory rodent diets are often marketed as identical, but are sold in different forms, regardless of the markedly higher starch gelatinization in extruded than in pelleted diets. Our hypothesis was that this would impact energy and nutrient digestibility in mice fed pellets or extrudate, respectively. Trial 1 showed that feeding C57BL/6 mice a standard maintenance diet in extruded form results in a significantly higher digestibility of organic matter, energy, and carbohydrates than the identical diet in pelleted form. The replication of the experiment, however, revealed a variation between batches of the same pelleted diet regarding starch and total dietary fiber contents. Given the significant differences in diet digestibility and the potential impacts of digestibility on nutrient utilization, the intestinal microbiome, and intermediary metabolism, trials performed with differently processed diets are not comparable. This might partly explain failures to reproduce results, especially in gastrointestinal or microbiome research. Considering this impact on experimental animals, the degree of starch gelatinization should be declared in the diet information for laboratory animal diets. The differences between batches of laboratory animal diets as observed in the pellets are not acceptable.