MALDI-TOF mass spectrometry fingerprinting: A diagnostic tool to differentiate dematiaceous fungi Stachybotrys chartarum and Stachybotrys chlorohalonata

Journal of Microbiological Methods 115 (2015) 83–88

Contents lists available at ScienceDirect

Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth

MALDI-TOF mass spectrometry fingerprinting: A diagnostic tool to differentiate dematiaceous fungi Stachybotrys chartarum and Stachybotrys chlorohalonata Maike Gruenwald ⁎, Andreas Rabenstein, Markko Remesch, Jan Kuever Bremen Institute for Materials Testing, Microbiology Department, Paul-Feller-Straße 1, 28199 Bremen, Germany

a r t i c l e

i n f o

Article history: Received 17 February 2015 Received in revised form 27 May 2015 Accepted 29 May 2015 Available online 31 May 2015 Keywords: Darkly pigmented Fingerprint Fungi MALDI-TOF mass spectrometry Stachybotrys

a b s t r a c t Stachybotrys chartarum and Stachybotrys chlorohalonata are two closely related species. Unambiguous identification of these two species is a challenging task if relying solely on morphological criteria and therefore smarter and less labor-intensive approaches are needed. Here we show that even such closely related species of fungi as S. chartarum and S. chlorohalonata are unequivocally discriminated by their highly reproducible MALDI-TOFMS fingerprints (matrix assisted laser desorption/ionization time-of-flight mass spectrometry fingerprints). We examined 19 Stachybotrys and one Aspergillus isolate by MALDI-TOF-MS. All but one isolate produced melanin containing conidia on malt extract agar. Mass spectra were obtained in good quality from the analysis of hyaline and darkly pigmented conidia by circumventing the property of melanin which causes signal suppression. MALDI-TOF fingerprint analysis clearly discriminated not only the two morphologically similar species S. chartarum and S. chlorohalonata from each other but separated them precisely from Stachybotrys bisbyi and Aspergillus versicolor isolates. Furthermore, even S. chartarum chemotypes A and S could be differentiated into two distinct groups by their MALDI-TOF fingerprints. The chemotypes of S. chartarum isolates were identified by trichodiene synthase 5 (tri5) sequences prior to mass spectra analysis. Additionally, species identities of all isolates were verified by their 18S rRNA and tri5 gene sequences. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Stachybotrys and its mycotoxins are involved in plant (De Silva et al., 1995), animal, and human diseases e.g. idiopathic pulmonary hemorrhage (Dearborn et al., 1999; Vesper et al., 1999, 2000) and sick building syndrome (Cooley et al., 1998). For that reason, much effort was directed towards better understanding of the biochemistry and the characterization of Stachybotrys (Andersen et al., 2002, 2003; Black et al., 2008; Cruse et al., 2002, 2003; Koster et al., 2003, 2009; Nielsen and Smedsgaard, 2003; Vesper et al., 1999, 2000). In this study, 19 Stachybotrys isolates covering 3 different species were identified using Abbreviations: MALDI-TOF MS, matrix-assisted laser desorption/ionization time-offlight mass spectrometry; ATCC, American Type Culture Collection; BSA, bovine serum albumin; BTS, Bruker bacterial test sample; CHCA, α-cyano-4-hydroxycinnamic acid; dpi, days past inoculation; DSMZ, Leibniz-Institute DSMZ — German Collection of Microorganisms and Cell Culture; IBT, IBT Culture Collection of Fungi, Technical University of Denmark; MEA, malt extract agar; ML, maximum likelihood analysis; PMK, Qiagen DNeasy Plant Mini Kit; tri5, trichodiene synthase 5 gene sequence. ⁎ Corresponding author at: Julius Kühn-Institut, Federal Research Centre for Cultivated Plants, Institute for Ecological Chemistry, Plant Analysis and Stored Product Protection, Königin-Luise-Straße 19, 14195 Berlin, Germany. E-mail addresses: [email protected] (M. Gruenwald), [email protected] (A. Rabenstein), [email protected] (M. Remesch), [email protected] (J. Kuever).

http://dx.doi.org/10.1016/j.mimet.2015.05.025 0167-7012/© 2015 Elsevier B.V. All rights reserved.

molecular biological methods and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) fingerprints. Stachybotrys chartarum and Stachybotrys chlorohalonata are dematiaceous molds which can grow on substrates containing cellulose such as hay, straw, or moist wallpapers and fabric in water damaged houses (Andersen et al., 2003; Dearborn et al., 1999; Murtoniemi et al., 2003). Stachybotrys bisbyi is a fungus with hyaline mycelia and conidia. It can be found on Oryza sativa (De Silva et al., 1995). S. chartarum and S. chlorohalonata produce various mycotoxins. S. chartarum chemotype S generates i.a. macrocyclic trichothecenes, satratoxins and roridins and S. chartarum chemotype A and S. chlorohalonata synthesize atranones and dolabellanes (Andersen et al., 2003). S. chartarum chemotypes cannot be distinguished morphologically. Furthermore, the two species S. chartarum and S. chlorohalonata can be easily mistaken for each other on some culture media such as malt extract agar (MEA), though, they are distinguishable by colony diameter, pigmentation, and conidia on media described elsewhere (Andersen et al., 2002, 2003). Hence, a fast, simple, and reliable identification method is needed. Aspergillus versicolor is also commonly detected in water damaged buildings and exudes mycotoxins like sterigmatocystin (Bloom et al., 2007; Engelhart et al., 2002). It is therefore included in this study and used as a phylogenetic outgroup. On molecular biological base, the S. chlorohalonata and S. chartarum and its chemotypes can be separated

84

M. Gruenwald et al. / Journal of Microbiological Methods 115 (2015) 83–88

by trichodiene synthase 5 (tri5) sequence analysis. S. bisbyi lacks tri5 and does not produce macrocyclic trichothecene (Andersen et al., 2002; Koster et al., 2009). This can also be considered for A. versicolor. For that reason, species identification for all isolates was additionally done by 18S rRNA sequences. MALDI-TOF fingerprint analysis is a simple method to identify fungal species (Cassagne et al., 2011) and is considerably less time consuming and labor-intensive than molecular biological identifications (Panda et al., 2015). MALDI-TOF-MS of fungal conidia produces highly reproducible spectra, when culture and analysis conditions are kept constant. Therefore, MALDI-TOF fingerprinting identification method has already been applied to a variety of fungi on species and strain level, e.g. Aspergillus, Penicillium, and Neoscytalidium (Alshawa et al., 2012; Chen and Chen, 2005; Hettick et al., 2008a, 2008b, 2011; Valentine et al., 2002; Welham et al., 2000). However, only low intensity spectra of darkly pigmented fungi have thus far been obtained (Alshawa et al., 2012; Hettick et al., 2008b, 2011; Valentine et al., 2002). An approach to circumvent the inhibiting effect of melanin aims at suppressing the melanin production during cultivation (Alshawa et al., 2012; Buskirk et al., 2011; Hettick et al., 2011; Panda et al., 2015). During the analysis of darkly pigmented spores or cell material the melanin diminishes the ionization capability of the MALDI matrix by interfering with the energy transfer from the matrix to the sample molecules. The melanin compounds act as an energy sink (Buskirk et al., 2011). Thus, sample molecules are ionized poorly due to an insufficient energy transfer. Here we introduce a simple method for generating MALDI-TOF fingerprints with good intensity from melanized and hyaline fungal spores using them for unambiguous identification of morphological closely related species of Stachybotrys. 2. Materials and methods 2.1. Reagents α-Cyano-4-hydroxycinnamic acid (CHCA) and Bruker bacterial test standard (BTS) were purchased from Bruker Daltonik GmbH (Bremen, Germany). Acetonitrile (liquid chromatography mass spectrometry [LC–MS] grade Chromasolv®), ethanol (gradient grade, 99,9%), formic acid (98%), trifluoroacetic acid (ReagentPlus®, 99%), water (LC–MS Chromasolv®), albumin bovine serum, REDTaq™ Genomic DNA Polymerase and the appropriate PCR buffer were purchased from Sigma Aldrich (Steinheim, Germany). Chloroform, malt extract, glass beads with 3 mm diameter and magnesium chloride (MgCl2) were purchased from Merck KGaA (Darmstadt, Germany). Glass beads with 0.5 mm diameter were purchased from Scientific Industries Inc. (New York, USA). Agar-Agar Kobe I was purchased from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). Qiagen Taq PCR Master Mix, DNeasy® Plant Mini Kit (containing AE elution buffer, AP1 lysis buffer, AP2 precipitation buffer, AP3/E binding buffer, AW wash buffer, RNase A), and MinElute® PCR Purification Kit (containing EB elution buffer, PBI binding buffer, PE wash buffer) were purchased from Qiagen GmbH (Hilden, Germany). dNTP Mix (10 mM each) was purchased from Fermentas GmbH (St. Leon-Rot, Germany). The PCR enhancer Yellow Sub™ was purchased from Geneo BioTechProducts (Hamburg, Germany). 2.2. Fungal culture 19 Stachybotrys isolates and one Aspergillus strain (Table 3) were cultured in the dark for 10 days at 25 °C on malt extract agar (20 g malt extract with 15 g Agar-Agar Kobe I in 1 l distilled water). Two Stachybotrys isolates were purchased from Leibniz-Institute DSMZ — German Collection of Microorganisms and Cell Culture (DSMZ): S. chartarum DSM 2144 and S. bisbyi DSM 63042. All other strains were subcultured isolates from contaminated indoor environments. 18S rRNA and tri5 gene sequence analysis verified their morphologic identification as S. chartarum, S. chlorohalonata and A. versicolor. S. bisbyi DSM 63042 and A. versicolor were both used as outgroup.

2.3. Phylogenetic analysis DNA extraction of all Stachybotrys cultivars and of the Aspergillus strain was obtained by using Qiagen DNeasy Plant Mini Kit (PMK) according to the manufactures' protocol with the following modifications: 80–100 mg spores (10 dpi) were disrupted with two or three glass pearls (3 mm diameter), and a spatula tip of small glass pearls (0.5 mm diameter) by a MM301 bead mill (Retsch GmbH, Haan, Germany) for 2.5 min at 30 Hz. DNA purity and concentration were analyzed according to the PMK instructions by mixing 2 μl eluate with 4 μl LC–MS water and checking the extinction at 260 nm and 280 nm with a ScanDrop® spectrophotometer (Analytik Jena AG, Jena, Germany). In all cases DNA eluate was directly used for PCR reactions. 18S rRNA gene fragments identified the isolate species (Black et al., 2008; Haugland and Heckman, 1998; Wu et al., 2003). Trichodiene synthase 5 fragment (tri5) was used to distinguish S. chlorohalonata from S. chartarum and to identify the chemotypes of the later (Andersen et al., 2003; Cruse et al., 2002, 2003; Koster et al., 2003). PCR amplification of tri5 was done by using primers described by Cruse et al. (2002). The forward primer sequence (NS1f) for 18S rRNA is described by White et al. (1990). The reverse primer sequence (F1Ra) for 18S rRNA is described by De Souza et al. (2004). All primers used in this study are listed in Table 1. 18S rRNA gene sequences of Stachybotrys were amplified in a PCR reaction mixture containing 12 μl DNA eluate (30 ng genomic DNA), 1.5 mM MgCl2, 200 μM per dNTP, 0.08 U/μl REDTaq™, 0.08 μg/μl BSA, and Sigma PCR buffer. Each primer was concentrated to 0.8 μM. To amplify A. versicolor 20 μl PCR Mix was used which contained 1.5 ng DNA/μl, Qiagen Taq PCR Master Mix comprising 200 μM of each of the four dNTPs, 1.5 mM MgCl2, PCR buffer, Qiagen TaqDNA Polymerase, 0.75 fold Yellow Sub™, and 0.1 μg/μl BSA. Each primer was concentrated to 0.75 μM. Tri5 sequences were amplified using PCR Mix based on Cruse et al. (2002) with some alterations as follows: 20 μl reaction solution contained 1.5 ng DNA/μl, Qiagen Taq PCR Master Mix comprised 200 μM of each of the four dNTPs, 1.5 mM MgCl2, PCR buffer, Qiagen TaqDNA Polymerase, supplementary 1 mM MgCl2, 0.75 fold Yellow Sub™, 0.1 μg/μl BSA, and 0.75 μM of each primer. PCR was performed with a Mastercycler® gradient (Eppendorf AG, Hamburg, Germany) with touchdown temperature programs (Don et al., 1991) for all three PCR programs. Temperature program for the amplification of the 18S sequences was: 4 min at 94.0 °C, 14 cycles 94.0 °C/20 s, 60 °C/20 s reducing temperature by 0.5 °C per cycle, 72 °C/60 s, following 25 cycles 94.0 °C/20 s, 53.0 °C/20 s, 72 °C/50 s, and a final extension step with 72 °C/5 min. Temperature program for tri5 sequences: 94.0 °C/3 min, 20 touchdown cycles 94.0 °C/45 s, 55 °C/40 s reducing temperature by 0.5 °C per cycle, 72 °C/60 s, following 15 cycles of 94.0 °C/45 s, 45 °C/30 s, 72 °C/60 s, and a final extension step with 72 °C/3 min. PCR products were prepared for sequencing using Qiagen MinElute PCR Purification Kit. Qiagen instructions were followed. Solutions with 28.57 ng DNA/μl and 1.43 mM primer for 18S rRNA and 14.29 ng DNA/μl with 1.43 mM primer for tri5 gene sequences were prepared. Sequencing was done by LGC Genomics (Berlin, Germany). Reference sequences were downloaded from NCBI GenBank. 18S rRNA gene reference sequences had the accession numbers GenBank: AF548096.1 (S. chartarum strain IBT 7711), GenBank: AF548097.1 (S. chlorohalonata IBT 9299), GenBank: GU227343.1 (A. versicolor HDJZ-ZWM-16) and tri5 DNA reference sequences were GenBank: EU288831 (S. chartarum chemotype S), GenBank: EU288836

Table 1 18S rRNA and tri5 primers and their sequences used in this study. Primer

Sequence

Reference

tri5 forward tri5 reverse Ns1f forward F1Ra reverse

5′-CATCAATCCAACAGTTTCAC-3′ 5′-GCAACCTTCAAAGACTATTG-3′ 5′-GTAGTCATATGCTTGTCTC-3′ 5′-CTTTTACTTCCTCTAAATGACC-3′

Cruse et al. (2002) Cruse et al. (2002) White et al. (1990) De Souza et al. (2004)

M. Gruenwald et al. / Journal of Microbiological Methods 115 (2015) 83–88

85

(S. chartarum chemotype S), GenBank: EU288807 (S. chartarum chemotype A), GenBank: EU288811 (S. chartarum chemotype A), GenBank: EU288847 (S. chlorohalonata), and GenBank: EU288848 (S. chlorohalonata). Sequences were aligned with ClustalW Multiple alignment in BioEdit 7.0.5 (Thomson et al., 1994). Maximum Likelihood analysis was performed with MEGA6.06 (Tamura et al., 2013). 2.4. Mass spectrometry Spores of all isolates were sampled 10 days past inoculation and prepared following in general the Bruker Daltonik Instruction for ethanol/ formic acid extraction as described in McTaggart et al. (2011). Though, alternative conditions were used for centrifugation (12,000 × g for 4 min and 2 min), pellets were dried for 30 min with a Savant SpeedVac SC110 concentrator (Savant Instruments, Inc. New York, USA), and 2 μl CHCA/μl sample was used instead of 1 μl CHCA/μl sample. Samples were analyzed with a Microflex LT-mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany). Calibration of mass spectra and conditions for acquiring mass spectra and identifying fungal samples are described in Blaettel et al. (2013). Used software version was MALDI Biotyper 2.0. Laser frequency for ionization was 60 Hz with a 150 ns pulse, target potential was 20.11 kV, potential at the second electrode was 16.80 kV, and potential at the subsequent slit was 7.03 kV. Final mass spectra composed of 240 single spectra, each of them showed a relative intensity above 1500 for the two most intense peaks. The mass spectra generated from the conidia were compared with reference spectra in the Biotyper database using the program's default settings. Classification results are created including the first 10 ranks listing the species represented in the reference spectra and the log(score) value. The log(score) values rank from 0 to 3. According to the manufactures' protocol identification at the species level is achieved at a log(score) N 2.0 and at the genus level at a log(score) N 1.7 (Blaettel et al., 2013; Panda et al., 2015; Pavlovic et al., 2014).

Fig. 1. Maximum likelihood tree for 18S rRNA. Fungal strains are identified as S. chlorohalonata, S. chartarum, and A. versicolor by reference sequences from GenBank (GenBank: AF548096.1, GenBank: AF548097.1, GenBank: GU227343.1). Reference strains 2144 S. chartarum and 63,042 S. bisbyi are included. All species are clearly distinguished from each other.

3. Results and discussion 3.1. Molecular biological analysis Nearly the complete 18S rRNA gene sequences (1632–1638 bp) were obtained from sequencing for all isolates, except isolate 815 which had only 815 bp. The reverse primer produced a sequence with only 193 bp for isolate 815. For that reaction an insufficient amount of template was accidently used and an alignment of forward and reverse sequence could not be accomplished. ML analyses included GenBank reference sequences and confirmed that the fungi sampled from contaminated indoor environments were 7 S. chlorohalonata and 10 S. chartarum cultivars. Isolate 1023 was similar to reference sequence GenBank: GU227343.1 and was thereby identified as A. versicolor. Results of ML analysis are shown in Fig. 1. In 18S rRNA sequences S. chlorohalonata and S. chartarum have 4 nucleotide variations (Isolate 813 has 5 variations), S. bisbyi and S. chartarum differ in 38 positions from each other and A. versicolor has 160 variations from S. chartarum. The length of partial tri5 DNA sequences was 579–580 bp for S. chlorohalonata and 582–583 bp for S. chartarum isolates. No tri5 PCR product could be obtained from A. versicolor and S. bisbyi, which is in accordance with results that neither macrocyclic trichothecene nor tri5 have been detected in S. bisbyi isolates (Andersen et al., 2002; Koster et al., 2009) and A. versicolor samples (Black et al., 2008). ML analysis of tri5 sequences distinguished the S. chartarum from the S. chlorohalonata cultivars by 25–27 nucleotides. A dendrogram derived from ML analysis is shown in Fig. 2. Isolates of S. chartarum separate into two chemotype clusters. Each chemotype is divided again into two subgroups. Subgroup A1 consists of isolates 475, 476, 521, and 522. The smaller subgroup A2 contains only isolate 261. The subgroups A1 and A2 differ in 2 nucleotide positions. At position 5 isolate 261 has a guanine instead of a cytosine base and at position 521 the isolate shows a

Fig. 2. Maximum likelihood tree for tri5. Fungal strains are identified as S. chlorohalonata and S. chartarum chemotype A or S by reference sequences from GenBank (GenBank: EU288807, GenBank: EU288811, GenBank: EU288836, GenBank: EU288831, GenBank: EU288847, and GenBank: EU288848). Reference strain 2144 S. chartarum is included and identified as chemotype S. Species and chemotypes are unambiguously separated.

86

M. Gruenwald et al. / Journal of Microbiological Methods 115 (2015) 83–88

Table 2 Chemotype subgroups of S. chartarum and the 5 nucleotide positions where their tri5 sequences vary from each other. Chemotype group

S1 S2 A1 A2

Isolate #

178, 232, 235, 523 231, 2144 475, 476, 521, 522 261

Nucleotide position 5

35

319

331

521

C C T T

T C C C

C C G C

G C C G

A C C C

cytosine instead of a guanine base. Subgroup S1 contains isolates 178, 232, 235, and 523, whereas the subgroup S2 is composed of the isolates 231 and 2144. The two subgroups S1 and S2 differentiate in 3 nucleotide positions. S2 has a cytosine base at positions 5, 35 and 331, whereas the bigger subgroup S1 has a guanine, an adenosine and a thymine base, respectively. The two main chemotype groups S1 and A1 differ at 5 positions from each other (Table 2). S1 and A2 have 3 mismatches and S2 varies in 2 nucleotide positions from both subgroups A1 and A2. 3.2. MALDI-TOF fingerprints of S. chartarum and S. chlorohalonata From all darkly pigmented species MALDI-TOF fingerprints with only poor quality were generated by following the Bruker instruction strictly. Hettick et al. (2008b) reported that the melanized conidia of Aspergillus chevalieri and Aspergillus niger gave spectra with very low intensity and therefore, could not be distinguished unambiguously from

Fig. 3. MALDI-TOF fingerprint spectra of darkly pigmented and hyaline fungi. (A) S. chartarum isolate 231; spectrum obtain with 1 μl CHCA, (B) S. chartarum isolate 231; spectrum obtained with 2 μl CHCA, (C) A. versicolor; spectrum obtained with 2 μl CHCA (D) S. bisbyi; spectrum obtained with 2 μl CHCA. Note the different magnitudes of abundance.

each other. In darkly pigmented conidia the melanin diminishes the ionization capability of the MALDI matrix by acting as an energy sink and sample molecules are ionized poorly. Therefore, the amount of CHCA matrix was doubled to increase the energy, which could possibly be transferred from CHCA to the surrounding molecules. Fingerprints of all three darkly pigmented species (S. chartarum, S. chlorohalonata, A. versicolor) and the hyaline species (S. bisbyi) were then obtained with good intensity by this simple modification of the Bruker instructions. Chromatograms of S. chartarum 231 with 1 μl and 2 μl CHCA respectively as well as fingerprints of S. bisbyi and A. versicolor with 2 μl CHCA are given in Fig. 3. MALDI-TOF fingerprints of all isolates were highly reproducible. The dendrogram in Fig. 4 gives the relative distance levels between the fingerprint reference spectra of the four species. All species are clearly separated from each other. The branch of S. chartarum has 3 subgroups (i) 523, (ii) 261, 475, 476, 521, and 522, (iii) 178, 231, 232, 235, and 2144. Subgroups (i) and (iii) consist of all S. chartarum, which are identified as chemotype S by tri5 sequences. All MALDI-TOF fingerprints of S. chartarum isolates identified as chemotype A by tri5 sequence analysis are clustered in subgroup (ii). The spectra of chemotype A have the most dominant peak at 7316 m/z. Spectra of S. chartarum chemotype S do not show this peak, but a dominant peak with a lower mass of 7284 m/z. These two peaks can be used as the main biomarkers for the two chemotypes of S. chartarum. The fingerprints of fresh conidia were matched against the Biotyper database. Identification of the spectra derived from conidia was based on the best match of the calculation result table. Although the log(score) values for several isolates were b 2.0 in the first rank, the identification of all isolates always matched its corresponding species as identified by 18S rRNA and tri5 gene sequences (Table 3). Accurate species identification with lowered log(score) values is reported for some microorganisms, e.g. clinical yeast isolates (Rosenvinge et al., 2013). MALDI-TOF identifications of Stachybotrys and Aspergillus isolates were repeated twice. The species of each strain were always correctly

Fig. 4. Dendrogram of the relative distance level between MALDI-TOF fingerprint spectra of tested S. chlorohalonata, S. chartarum, S. bisbyi, and A. versicolor. Isolate numbers are noted together with species (and chemotype) identification. Species as well as the different chemotypes of S. chartarum clustered at separated branches.

M. Gruenwald et al. / Journal of Microbiological Methods 115 (2015) 83–88 Table 3 Isolate numbers and species of examined Stachybotrys and Aspergillus isolates as well as the chemotype of S. chartarum strains verified by RNA/DNA sequencing. The first rank [highest log(score)] of match against the Biotyper reference spectrum was used for identification. MALDI-TOF-MS fingerprinting

RNA/DNA sequencing c,d

Isolate #

Species

177 178 231 232 235 261 284 475 476 521 522 523 524 749 813 814 815 1023 2144a,b 63042a

S. chlorohalonata S. chartarum S. chartarum S. chartarum S. chartarum S. chartarum S. chlorohalonata S. chartarum S. chartarum S. chartarum S. chartarum S. chartarum S. chlorohalonata S. chlorohalonata S. chlorohalonata S. chlorohalonata S. chlorohalonata A. versicolor S. chartarum S. bisbyi Total n = 20

a b c d

Chemotype

d

First rank species (log(score))

First rank chemotype

S. chlorohalonata (1.955) S. chartarum (1.691) A S. chartarum (2.441) S S. chartarum (1.875) S S. chartarum (2.026) S S. chartarum (1.967) A S. chlorohalonata (2.348) A S. chartarum (1.859) A A S. chartarum (1.859) A A S. chartarum (1.979) A A S. chartarum (1.776) A S S. chartarum (1.813) S S. chlorohalonata (2.196) S. chlorohalonata (2.166) S. chlorohalonata (2.251) S. chlorohalonata (2.023) S. chlorohalonata (1.991) A. versicolor (1.886) S S. chartarum (1.713) S S. bisbyi (1.594) Total n = 11 20/20 [100%] 10/11 [91%] S S S S A

Isolate numbers of DSMZ. Other designation ATCC 16026. Identification by 18S rRNA. Identification by tri5 gene sequence.

determined. Chemotypes were designated accurately in 10 of 11 isolates. The best match for chemotype of S. chartarum 178 was chemotype A. Tri5 analysis defined this strain as chemotype S. The log(score) value for this sample was 1.691, which is the second smallest value of all samples. Only S. bisbyi has a smaller value of 1.594. It seems that log(score) values b 1.7 are insufficient to determine correctly the chemotype of S. chartarum.

4. Conclusion In contrast to earlier reports (Alshawa et al., 2012; Hettick et al., 2008b, 2011) it is readily possible to retrieve MALDI-TOF spectra of darkly pigmented conidia of fungi. Fingerprints of the melanized fungi S. chartarum and S. chlorohalonata as well as A. versicolor were obtained in accurate intensity by mixing 1 μl of conidia extract with 2 μl of CHCA. This simple method can also be applied to generate spectra from conidia of hyaline fungi such as S. bisbyi. The two species S. chartarum and S. chlorohalonata have easily confusable morphologies on MEA culture. MALDI-TOF fingerprints do not only discriminate accurately S. chartarum or S. chlorohalonata from S. bisbyi and A. versicolor but discern precisely the morphological resembling species S. chartarum and S. chlorohalonata from each other. Furthermore, the S. chartarum chemotypes A and S which are morphologically not separable can be identified adequately by their MALDI-TOF MS fingerprints. Reliable species identification by MALDI-TOF-MS fingerprints succeeded in 100%, chemotype identification was accurate for 91% of the tested isolates.

References Alshawa, K., Beretti, J.-L., Lacroix, C., Feuilhade, M., Dauphin, B., Quesne, G., Hassouni, N., Nassif, X., Bougnoux, M.-E., 2012. Successful identification of clinical dermatophyte and Neoscytalidium species by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J. Clin. Microbiol. 50, 2277–2281. http://dx.doi.org/10. 1128/jcm.06634-11.

87

Andersen, B., Nielsen, K.F., Jarvis, B.B., 2002. Characterization of Stachybotrys from waterdamaged buildings based on morphology, growth, and metabolite production. Mycologia 94, 392–403. http://dx.doi.org/10.2307/3761773. Andersen, B., Nielsen, K.F., Thrane, U., Szaro, T., Taylor, J.W., Jarvis, B.B., 2003. Molecular and phenotypic descriptions of Stachybotrys chlorohalonata sp nov and two chemotypes of Stachybotrys chartarum found in water-damaged buildings. Mycologia 95, 1227–1238. http://dx.doi.org/10.2307/3761923. Black, J.A., Dean, T.R., Foarde, K., Menetrez, M., 2008. Detection of Stachybotrys chartarum using rRNA, tri5, and β-tubulin primers and determining their relative copy number by real-time PCR. Mycol. Res. 112, 845–851. http://dx.doi.org/10.1016/j.mycres.2008.01.006. Blaettel, V., Petri, A., Rabenstein, A., Kuever, J., Koenig, H., 2013. Differentiation of species of the genus Saccharomyces using biomolecular fingerprinting methods. Appl. Microbiol. Biotechnol. 97, 4597–4606. http://dx.doi.org/10.1007/s00253-013-4823-z. Bloom, E., Bal, K., Nyman, E., Must, A., Larsson, L., 2007. Mass spectrometry-based strategy for direct detection and quantification of some mycotoxins produced by Stachybotrys and Aspergillus spp. in indoor environments. Appl. Environ. Microbiol. 73, 4211–4217. http://dx.doi.org/10.1128/aem.00343-07. Buskirk, A.D., Hettick, J.M., Chipinda, I., Law, B.F., Siegel, P.D., Slaven, J.E., Green, B.J., Beezhold, D.H., 2011. Fungal pigments inhibit the matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry analysis of darkly pigmented fungi. Anal. Biochem. 411, 122–128. http://dx.doi.org/10.1016/j.ab.2010.11.025. Cassagne, C., Ranque, S., Normand, A.-C., Fourquet, P., Thiebault, S., Planard, C., Hendrickx, M., Piarroux, R., 2011. Mould routine identification in the clinical laboratory by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. PLoS One 6. http://dx.doi.org/10.1371/journal.pone.0028425. Chen, H.Y., Chen, Y.C., 2005. Characterization of intact Penicillium spores by matrixassisted laser desorption/ionization mass spectrometry. Rapid Commun. Mass Spectrom. 19, 3564–3568. http://dx.doi.org/10.1002/rcm.2229. Cooley, J.D., Wong, W.C., Jumper, C.A., Straus, D.C., 1998. Correlation between the prevalence of certain fungi and sick building syndrome. Occup. Environ. Med. 55, 579–584. Cruse, M., Telerant, R., Gallagher, T., Lee, T., Taylor, J.W., 2002. Cryptic species in Stachybotrys chartarum. Mycologia 94, 814–822. http://dx.doi.org/10.2307/3761696. Cruse, M., Telerant, R., Gallagher, T., Lee, T., Taylor, J.W., 2003. Errata: cryptic species in Stachybotrys chartarum (vol 94, pg 814, 2002). Mycologia 95, 559. De Silva, L.B., Herath, W.H.M.W., Gunawardena, D.S.S., Wijesundera, R.L.C., Medis, S.A., Choudhary, M.I., Clardy, J., 1995. Bisbynin, a novel secondary metabolite from the fungus Stachybotrys bisbyi (Srinivasan) Barron. Tetrahedron Lett. 36, 1997–2000. http:// dx.doi.org/10.1016/0040-4039(95)00211-T. De Souza, F.A., Kowalchuk, G.A., Leeflang, P., Van Veen, J.A., Smit, E., 2004. PCR-denaturing gradient gel electrophoresis profiling of inter- and intraspecies 18S rRNA gene sequence heterogeneity is an accurate and sensitive method to assess species diversity of arbuscular mycorrhizal fungi of the genus Gigaspora. Appl. Environ. Microbiol. 70, 1413–1424. http://dx.doi.org/10.1128/aem.70.3.1413-1424.2004. Dearborn, D.G., Yike, I., Sorenson, W.G., Miller, M.J., Etzel, R.A., 1999. Overview of investigations into pulmonary hemorrhage among infants in Cleveland, Ohio. Environ. Health Perspect. 107, 495–499. Don, R.H., Cox, P.T., Wainwright, B.J., Baker, K., Mattick, J.S., 1991. ’Touchdown’ PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 19, 4008. Engelhart, S., Loock, A., Skutlarek, D., Sagunski, H., Lommel, A., Farber, H., Exner, M., 2002. Occurrence of toxigenic Aspergillus versicolor isolates and sterigmatocystin in carpet dust from damp indoor environments. Appl. Environ. Microbiol. 68, 3886–3890. http://dx.doi.org/10.1128/aem.68.8.3886-3890.2002. Haugland, R.A., Heckman, J.L., 1998. Identification of putative sequence specific PCR primers for detection of the toxigenic fungal species Stachybotrys chartarum. Mol. Cell. Probes 12, 387–396. http://dx.doi.org/10.1006/mcpr.1998.0197. Hettick, J.M., Green, B.J., Buskirk, A.D., Kashon, M.L., Slaven, J.E., Janotka, E., Blachere, F.M., Schmechel, D., Beezhold, D.H., 2008a. Discrimination of Penicillium isolates by matrixassisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting. Rapid Commun. Mass Spectrom. 22, 2555–2560. http://dx.doi.org/10.1002/rcm.3649. Hettick, J.M., Green, B.J., Buskirk, A.D., Kashon, M.L., Slaven, J.E., Janotka, E., Blachere, F.M., Schmechel, D., Beezhold, D.H., 2008b. Discrimination of Aspergillus isolates at the species and strain level by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting. Anal. Biochem. 380, 276–281. http://dx.doi.org/ 10.1016/j.ab.2008.05.051. Hettick, J.M., Green, B.J., Buskirk, A.D., Slaven, J.E., Kashon, M.L., Beezhold, D.H., 2011. Discrimination of fungi by MALDI-TOF mass spectrometry. In: Fenselau, C., Demirev, P.A. (Eds.), Rapid Characterization of Microorganisms by Mass Spectrometry vol. 1065. American Chemical Society, pp. 35–50. Koster, B., Scott, J., Wong, B., Malloch, D., Straus, N., 2003. A geographically diverse set of isolates indicates two phylogenetic lineages within Stachybotrys chartarum. Can. J. Bot. 81, 633–643. http://dx.doi.org/10.1139/b03-056. Koster, B., Wong, B., Straus, N., Malloch, D., 2009. A multi-gene phylogeny for Stachybotrys evidences lack of trichodiene synthase (tri5) gene for isolates of one of three intrageneric lineages. Mycol. Res. 113, 877–886. http://dx.doi.org/10.1016/j.mycres. 2009.04.003. McTaggart, L.R., Lei, E., Richardson, S.E., Hoang, L., Fothergill, A., Zhang, S.X., 2011. Rapid identification of Cryptococcus neoformans and Cryptococcus gattii by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J. Clin. Microbiol. 49, 3050–3053. http://dx.doi.org/10.1128/jcm.00651-11. Murtoniemi, T., Nevalainen, A., Hirvonen, M.R., 2003. Effect of plasterboard composition on Stachybotrys chartarum growth and biological activity of spores. Appl. Environ. Microbiol. 69, 3751–3757. http://dx.doi.org/10.1128/aem.69.7.3751-3757.2003. Nielsen, K.F., Smedsgaard, J., 2003. Fungal metabolite screening: database of 474 mycotoxins and fungal metabolites for dereplication by standardised liquid chromatography-UV-mass spectrometry methodology. J. Chromatogr. 1002, 111–136. http://dx.doi.org/10.1016/s0021-9673(03)00490-4.

88

M. Gruenwald et al. / Journal of Microbiological Methods 115 (2015) 83–88

Panda, A., Ghosh, A.K., Mirdha, B.R., Xess, I., Paul, S., Samantaray, J.C., Srinivasan, A., Khalil, S., Rastogi, N., Dabas, Y., 2015. MALDI-TOF mass spectrometry for rapid identification of clinical fungal isolates based on ribosomal protein biomarkers. J. Microbiol. Methods 109, 93–105. http://dx.doi.org/10.1016/j.mimet.2014.12.014. Pavlovic, M., Mewes, A., Maggipinto, M., Schmidt, W., Messelhäußer, U., Balsliemke, J., Hörmansdorfer, S., Busch, U., Huber, I., 2014. MALDI-TOF MS based identification of food-borne yeast isolates. J. Microbiol. Methods 106, 123–128. http://dx.doi.org/10. 1016/j.mimet.2014.08.021. Rosenvinge, F.S., Dzajic, E., Knudsen, E., Malig, S., Andersen, L.B., Løvig, A., Arendrup, M.C., Jensen, T.G., Gahrn-Hansen, B., Kemp, M., 2013. Performance of matrix-assisted laser desorption-time of flight mass spectrometry for identification of clinical yeast isolates. Mycoses 56, 229–235. http://dx.doi.org/10.1111/myc.12000. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. Thomson, J.D., Higgins, D.G., Gibson, T.J., 1994. Clustal W: improving the sensitivity or progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Valentine, N.B., Wahl, J.H., Kingsley, M.T., Wahl, K.L., 2002. Direct surface analysis of fungal species by matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun. Mass Spectrom. 16, 1352–1357. http://dx.doi.org/10.1002/rcm.721.

Vesper, S.J., Dearborn, D.G., Yike, I., Sorenson, W.G., Haugland, R.A., 1999. Hemolysis, toxicity, and randomly amplified polymorphic DNA analysis of Stachybotrys chartarum strains. Appl. Environ. Microbiol. 65, 3175–3181. Vesper, S.J., Dearborn, D.G., Elidemir, O., Haugland, R.A., 2000. Quantification of siderophore and hemolysin from Stachybotrys chartarum strains, including a strain isolated from the lung of a child with pulmonary hemorrhage and hemosiderosis. Appl. Environ. Microbiol. 66, 2678–2681. http://dx.doi.org/10.1128/aem.66.6.26782681.2000. Welham, K.J., Domin, M.A., Johnson, K., Jones, L., Ashton, D.S., 2000. Characterization of fungal spores by laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 14, 307–310. http://dx.doi.org/10.1002/(sici)10970231(20000315)14:5b307::aid-rcm823N3.3.co;2-v. White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (Eds.), PCR Protocols: A Guide to Methods and Applications. Academic Press, New York, N.Y., pp. 315–322. Wu, Z.H., Tsumura, Y., Blomquist, G., Wang, X.R., 2003. 18S rRNA gene variation among common airborne fungi, and development of specific oligonucleotide probes for the detection of fungal isolates. Appl. Environ. Microbiol. 69, 5389–5397. http://dx.doi. org/10.1128/aem.69.9.5389-5397.2003.