Evaluation of species diversity among Indian clinical Schizophyllum commune using matrix-assisted laser desorption ionization–time of flight mass spectrometry and MLST
Authors: Pradeep Kumar singh1, Jacques F. Meis2, Anuradha Chowdhary1
Affiliation: 1Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India; 2Radboudumc/CWZ, Nijmegen, Netherlands.

Objectives:
Schizophyllum is an important plant and animal pathogen in the genus basidiomycetes.  Schizophyllum commune is the best-known species causing allergic and invasive mycoses since 1950. Key morphological features necessary for the recognition of these fungi are the presence of clamp connections on hyphae and the development of fruiting bodies. However, the final identification needs confirmation by molecular methods. The aim of this study was to determine the species diversity among Indian clinical isolates of Schizophyllum using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and multilocus phylogenetic analysis.
Methods:
Thirty-seven molecularly identified clinical isolates of S. commune were selected from the culture collection of Medical Mycology, V. P. Chest Institute, Delhi to determine the species diversity and the reference strain of Schizophyllum radiatum (CBS 301.32) was included. The markers used for sequencing were the internal transcribed spacer (ITS), a portion of the nuclear large subunit (LSU) of ribosomal DNA, the RNA polymerase II second-largest subunit (RPB2), and the translation elongation factor 1α (EF-1α) gene. The neighbor joining (NJ) phylogenetic tree was constructed using MEGA (v.6) software. Support of the internal branches was assessed by the bootstrap method with 1,000 replications, where values of ≥70 were considered significant. The isolates were also processed for identification and to upgrade the in-house database by creating the main spectrum (MSP).
Results:
The LSU, EF-1α, and RPB2 markers showed consistency and concatenated sequences were analyzed. The analyses revealed two main clades in the phylogenetic tree. One clade included all the Indian strains of S. commune (n=37), and the other clade included only reference strain of S. radiatum (CBS 301.32) used in the study. Both clades showed high bootstrap values. The reference strains of S. umbrinum and S. fasciatum acted as outgroups since the genetic distance to the clades of S. radiatum and S. commune were elevated. MALDI-TOF MS showed all the isolates as S. commune including S. radiatum reference strain as the Bruker database does not contain main spectrum (MSPs) for S. radiatum. MSP was created for S. radiatum isolate and again identification was done. MALDI-TOF MS correctly identified all the isolates after the upgradation of database. MSP dendogram also showed two different clusters for S. commune and S. radiatum, which was in concordance with the NJ phylogenetic tree based on concatenated sequences. 
Conclusion:
The importance of Schizophyllum in the clinical setting has been demonstrated by the number of reported cases and strains isolated. In Indian scenario, S. commune is the prevalent species in clinical settings instead of S. radiatum which is most frequently recovered species from USA. It was also observed that EF1 and RPB2 alone can differentiate between these two species of Schizophyllum and thus the two markers can be used for the identification of Schizophyllum species. MALDI-TOF MS plays an important role in rapid identification of Schizophyllum species as it takes only half an hour for the identification but it has limited database. Creation of in-house database can improve the identification rate drastically.

Evaluation of species diversity among Indian clinical Schizophyllum commune using matrix-assisted laser desorption ionization–time of flight mass spectrometry and MLST