Identification of Stachybotrys spp. by MALDI-TOF mass spectrometry.

Stachybotrys (S.) spp. are omnipresent cellulolytic molds. Some species are highly toxic owing to their ability to synthesize various secondary metabolites such as macrocyclic trichothecenes or hemolysins. The reliable identification of Stachybotrys at species level is currently limited to genome-based identification. This study aimed to establish a fast and reliable MALDI-TOF MS identification method by optimizing the pre-analytical steps for protein extraction for subsequent generation of high-quality fingerprint mass spectra. Eight reference strains of the American Type Culture Collection and the Technical University of Denmark were cultivated in triplicate (biological repetitions) for 2 days in malt extract broth. The mycelia (1.5 ml) were first washed with 75 % ethanol and an additional washing step with dimethyl sulfoxide (10 %) was added to remove unspecific low weight masses. Furthermore, mycelia were broken with roughened glass beads in formic acid (70 %) and acetonitrile. The method was successfully applied to a total of 45 isolates of Stachybotrys originating from three different habitats (indoor, feed, and food samples; n = 15 each): Twenty-seven isolates of S. chartarum and 18 isolates of S. chlorohalonata could be identified by MALDI-TOF MS. The data obtained exactly matched those obtained by genome-based identification. The mean score values for S. chartarum ranged from 2.509 to 2.739 and from 2.148 to 2.622 for S. chlorohalonata with a very good reproducibility: the relative standard deviations were between 0.3 % and 6.8 %. Thus, MALDI-TOF MS proved to be a fast and reliable alternative to identification of Stachybotrys spp. by nucleotide amplification and sequencing.