Development of molecular methods for identification of Schizophyllum commune from clinical samples

Abstract

In the last 50 years, to our knowledge, only 16 cases of diseases caused by Schizophyllum commune in humans have been reported. Within only 6 months, we found four isolates of this basidiomycetous fungus, obtained from patients suffering from chronic sinusitis. The cultures of the isolated fungi showed neither clamp connections nor fruiting bodies (basidiocarps), which are distinctive features for S. commune, but fast-growing cottony white mycelium only. This was harvested, and DNA was extracted. The internal transcribed spacer region of the ribosomal DNA (rDNA) was amplified with fungus-specific primers, and the PCR products were sequenced. Two strains of S. commune, collected from branches of a European hornbeam (Carpinus betulus) and a tree of heaven (Ailanthus altissima), respectively; four specimens from the herbarium of the Institute of Botany, Karl-Franzens-University Graz; and two strains from internationally known culture collections (CBS 340.81 [ATCC 44201] and CBS 405.96) were investigated in the same way. The sequence data of all strains were compared and showed homology of over 99% in this 660-bp-long fragment of rDNA. With these results, a map of restriction enzyme cutting sites and a primer set specific for S. commune were created for reliable identification of this human pathogenic fungus.

FIG. 1

(a) Three-week-old culture of S. commune on SGA. (b) Clamp connections (indicated by arrows) on dikaryotic hyphae, derived through mating two monokaryotic isolates (HNO 34 and HNO 62).

FIG. 2

Sequences of S. commune ITS region. ITS1 and ITS2 are in uppercase; small-subunit, 5.8S, and large-subunit rDNA are in lowercase. Priming sites for scom1 (positions 126 to 145) and scom2r (positions 412 to 431) are underlined. Dots represent similarity with the first sequence, dashes represent unknown characters, and underlined spaces represent gaps introduced for alignment. Sequences: 1, GZU 42-2000/339; 2, GZU 42-2000/342; 3, GZU 29-88; 4, GZU 196-80; 5, GZU 79-96; 6, GZU 22-91; 7, HNO 34; 8, HNO 62; 9, HNO 323; 10, HNO 104; 11, CBS 340.81; 12, CBS 405.96.

FIG. 3

(a) Restriction map of the ITS region for the restriction enzymes EcoRI and AvaI. (b) RFLP pattern of the clinical isolates of S. commune. Lane 0, 100-bp DNA ladder; lanes 1 and 5, HNO 62; lanes 2 and 6, HNO 34; lanes 3 and 7, HNO 323; lanes 4 and 8, HNO 104.

FIG. 4

Agarose gel showing the amplicons of 12 isolates of S. commune, amplified with the species-specific primers scom1 and scom2r. Lane 0, 100-bp DNA ladder; lane 13, open tube control. The numbering of samples 1 to 12 is the same as in Table 1.