Mycotoxin Res (2015) 31:23–32 DOI 10.1007/s12550-014-0213-3
Occurrence of Stachybotrys chartarum chemotype S in dried culinary herbs Barbara Biermaier & Christoph Gottschalk & Karin Schwaiger & Manfred Gareis
Received: 30 July 2014 / Revised: 6 October 2014 / Accepted: 7 October 2014 / Published online: 28 October 2014 # Society for Mycotoxin Research and Springer-Verlag Berlin Heidelberg 2014
Abstract Stachybotrys (S.) chartarum is an omnipresent cellulolytic mould which produces secondary metabolites, such as the highly toxic macrocyclic trichothecenes. While it is known to occur in animal feed like hay and straw as well as in water-damaged indoor environments, there is little knowledge about the occurrence of S. chartarum and its secondary metabolites in food. The objective of the present study was to examine selected dried culinary herbs for the presence of S. chartarum chemotype S, to assess the potential risk of a contamination of foods with macrocyclic trichothecenes. In total, 50 Stachybotrys isolates from different types of culinary herbs (n=100) such as marjoram (Origanum majorana Linné (L.)), oregano (Origanum vulgare L.), thyme (Thymus vulgaris L.), and savory (Satureja hortensis L.) were examined by MTT-cell culture test (effect-based bioassay), ELISA, and by liquid chromatography tandem mass spectrometry (LC-MS/MS). Selected toxic and non-toxic isolates (n=15) were genetically characterized by PCR and sequencing. Five isolates (10 %) were highly toxic in the MTT-cell culture test, and the production of macrocyclic trichothecenes was proven by ELISA and LC-MS/MS. These five isolates were genetically confirmed as S. chartarum chemotype S. To the best of our knowledge, this is the first report about a contamination of dried culinary herbs with toxigenic S. chartarum.
Keywords Stachybotrys chartarum . Stachybotrys chlorohalonata . Culinary herbs . Mycotoxin . Macrocyclic trichothecenes . Satratoxin
B. Biermaier (*) : C. Gottschalk : K. Schwaiger : M. Gareis Chair of Food Safety, Faculty of Veterinary Medicine, Department of Veterinary Science, Ludwig-Maximilians-University Munich (LMU), Schoenleutnerstr. 8, D-85764 Oberschleissheim, Germany e-mail: [email protected]
Introduction Herbs and spices are known to be a source of food contamination with microorganisms and their metabolites, because they grow close to the soil, or are sun-dried in open environments directly after harvesting (Little et al. 2003; Weber 2003). The quality of the raw materials strongly depends on factors such as cultivation technique, time and mode of harvest, and climatic conditions (Schweiggert et al. 2007). Available studies regarding the fungal contamination of herbs and spices focused on herbal drugs, medicinal plants (Halt 1998; Rizzo et al. 1998; Stevic et al. 2012), and spices such as pepper (Elshafie et al. 2002; Hashem and Alamri 2010; Mandeel 2005). However, studies on the fungal contamination of dried culinary herbs are rare (de Boer et al. 1985; WojcikStopczyńska et al. 2009). Although dried culinary herbs are a good substrate for cellulolytic moulds such as Stachybotrys chartarum, its occurrence has been reported for spices and herbal medicines, without further differentiation (Abdel-Hafez and El-Said 1997; Ramos et al. 2013), but has not been described in culinary herbs. S. chartarum chemotype S is able to produce macrocyclic trichothecenes (Fig. 1), which are highly toxic (Jarvis et al. 1983; Vesper et al. 1999), and therefore is considered to be responsible (or a cofactor) for various illnesses in animals and humans exposed to mouldy materials. Compared to the simple trichothecenes, macrocyclic trichothecenes possess a 100-fold higher cytotoxicity, because of their irreversible inhibition of protein biosynthesis (Gareis 2006; Jarvis et al. 1998). Horses in particular, but also other animals such as swine, ruminants, poultry, and dogs, can develop stachybotryotoxicosis after exposure to mouldy feed and straw. Stachybotryotoxicosis is caused by the toxic effects of macrocyclic trichothecenes
Mycotoxin Res (2015) 31:23–32
Fig. 1 Chemical structures of the macrocyclic trichothecenes formed by S. chartarum chemotype S (according to Cole et al. 2003). Isomers not shown
and is characterized by necroses and ulcers in nose and mouth, hemorrhages of mucous membranes, and even deaths (Forgacs and Carll 1962; Harrach et al. 1983; Schneider et al. 1979; Tantaoui-Elaraki et al. 1994). Since 1990, Stachybotrys was also reported to play a critical role for human health because of its ability to grow on cellulose-rich building materials at high humidity. It is suspected to cause various symptoms related to the sick building syndrome (SBS) such as headache, nausea, fatigue, joint pain, arthritis, depression, and also severe pulmonary hemorrhages, which even lead to the death of infants (Dearborn et al. 2002; Etzel et al. 1998). However, most cases lack an unambiguous etiology, because there are other environmental pollutants such as microbial volatile organic compounds (MVOCs), bacteria, endotoxins, and volatile organic compounds (VOCs), which also play a role in triggering these illnesses (Kuhn and Ghannoum 2003; Lorenz et al. 2013; Terr 2001). To study the possibility that humans may be exposed to S. chartarum toxins via the alimentary route, we examined marjoram (Origanum majorana Linné (L.)), oregano (Origanum vulgare L.), thyme (Thymus vulgaris L.), and savory (Satureja hortensis L.) for their mycological quality, with special emphasis on the occurrence of toxigenic S. chartarum chemotype S.
Materials and methods Culinary herb samples A total of 80 samples (each 20 of marjoram, oregano, thyme, and savory) were purchased between January and December 2011 from retail shops and from online shops. All samples were available in dried and rubbed form; package sizes were between 7.0 and 100 g. Fifty percent originated from conventional production and 50 % from organic production. Stachybotrys isolates and reference strains In addition to the Stachybotrys spp. isolated in this study, we included 24 Stachybotrys isolates, all obtained from marjoram (n=20), from a previous study (unpublished data). Reference isolates of S. chartarum chemotype A and S and Stachybotrys chlorohalonata were kindly provided by the BioCentrumDTU, Lyngby, Denmark. Determination of the mycobiota and isolation of Stachybotrys spp. Five grams of the culinary herb samples were mixed with 45 ml peptone water (0.1 %) on a laboratory bag mixer and
Mycotoxin Res (2015) 31:23–32
1:10 dilutions were prepared (10−1 to 10−3). From each dilution, 1 ml was spread out on malt extract agar containing 125 mg novobiocin/l agar (MEA+), and on dichlorane glycerol agar (DG-18). The plates were incubated at 25 °C; moulds were analyzed both by stereo microscopy and standard light microscopy beginning at day 4 of cultivation, according to Samson (2010). Additionally, 15–20 leaflets of each sample were put on two round filter papers (Ø 70 mm, Whatman, Freiburg, Germany) soaked with nutrient solutions according to van Iterson (Forgacs and Carll 1962; Hintikka 1977) and to Ohff and Weissbach (1984), respectively, to prevent fastidious, cellulolytic moulds like Stachybotrys spp. from being overgrown (Bata et al. 1985). These plates were also incubated at 25 °C during 4 weeks and weekly analyzed as described above. In case of a positive identification of Stachybotrys spp., the fungal material was transferred onto MEA+ and incubated at 25 °C to obtain pure cultures. For further examination, threepoint cultures were prepared on MEA and incubated at 25 °C for 21 days. Preparation of crude extracts For further examination of the Stachybotrys isolates by MTTcell culture test, ELISA, and liquid chromatography tandem mass spectrometry (LC-MS/MS), the three-point cultures were extracted with chloroform (Gareis et al. 1999). For that purpose, the content of three complete agar plates was mixed two times with 75 ml chloroform for 5 min, respectively, and filtered through a cellulose filter containing 10 g water-free sodium sulphate into a round-bottom flask, and the solvent was evaporated by a rotary evaporator at 40 °C. The dried extract was transferred into glass tubes after resolving in 3× 2 ml chloroform, filtered through a nylon syringe filter (Carl Roth, Karlsruhe, Germany), and aliquoted to portions corresponding to each 15 g agar. Then, the aliquots were evaporated again in a vacuum centrifuge, and the dry material was used for MTT test, ELISA, and LC-MS/MS. Effect-based bioassay (MTT test) The cytotoxicity of the Stachybotrys isolates was examined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-cell culture test with swine kidney target cells, which are particularly sensitive to cytotoxic mycotoxins (Gareis 2006; Hanelt et al. 1994). The cells were cultivated in minimum essential medium Eagle (MEM; Sigma-Aldrich, Taufkirchen, Germany) and prepared as described by Hanelt et al. (1994). The crude extracts were dissolved in 1 ml special MEM (MEM + 1.7 % ethanol + 0.3 % DMSO+double concentrated fetal calf serum), and serial 1:2 dilutions were prepared. An aliquot of 100 μl of each dilution (starting with the dilution 1:2
corresponding to a concentration of 7.5 g agar/ml medium) and solvent controls were added to the cells in the microtiter plate and incubated at 37 °C (5 % CO2) for 48 h. MTT (20 μl) was subsequently added to each well and the plates were incubated for 4 h under the same conditions as mentioned before. After carefully removing the fluid from each well, 100 μl of DMSO was added to dissolve the formazans. The optical density was measured at 510 nm (Hanelt et al. 1994; Reubel et al. 1989). The results of the cytotoxicity measurements are presented as inhibitory concentration 50 values (IC50). These values correspond to the amount of crude extract which leads to a 50 % reduction of the metabolism of MTT to formazans compared to the control cells (Gareis 2006). Extracts with IC50 values of ≥7.50 g/ml, corresponding to IC50 values obtained for pure agar samples and solvent controls, were classified as non-toxic. Extracts with values between