MALDI-TOF MS application for identification of filamentous fungi

Introduction. MALDI-TOF identification method is widely used in microbiology due to its accuracy and rapid results achievement. However, applying this method to mold fungi faces some difficulties and is not always effective.

Purpose of the study was to evaluate the profits of using the long cultivation and protein extraction protocol in routine identification of mold fungi isolates from environment.

Materials and methods. The analysis of molds museum collection from Centre for Strategic Planning of FMBA of Russia was performed by MALDI-TOF mass-spectrometry Biotyper (Bruker Daltonics) with cultivation in liquid media and long optimized protein extraction protocol with acetonitrile and formic acid.

Results. One hundred thirty seven isolates were analyzed. Quality spectra were achieved for 71.5% of samples. Identification with MBT Filamentous Fungi Library database with the high confidence score (> 1.7) was achieved for 55% of isolates (26% with score > 2). Samples analyzed included members of nineteen families and 27 genera. 16% of samples were not identified despite producing high-quality spectra.

Limitations. When studying the possibility of using the time-of-flight mass spectrometry method to identify mold fungi, a sample of 137 isolates of mold fungi from the environment was analyzed, which is a sufficient reference sample. The analyzed samples included representatives of 19 families and 27 genera, which makes it possible to apply the findings to at least these representatives of micellar fungi. In this study 22 samples with good quality spectra, were not identified with MBT Filamentous Fungi Library database. In the future studies, these samples, along with other samples like that, will be identified by genetic molecular methods and added to the new home-made database for filamentous fungi MALDI-TOF identification.

Conclusion. Effective identification of filamentous fungi by mass-spectrometry methods requires pure culture achieved from liquid media, long optimized protocol of protein extraction and building an in-house database of spectra not presented in Bruker database.

Contribution:
Kurbatova I.V. — research concept and design, material collection and microscopic identification data processing, statistical processing, text writing, editing;
Rakitina D.V. — research concept and design, material collection and data processing, identification by MALDI-TOF statistical processing, text writing, editing;
Kravchenko E.S. — cultivation of samples;
Maniya T.R. — writing text, editing;
Aslanova M.M. — concept and design of the study, editing;
Yudin S.M. — editing.
All authors are responsible for the integrity of all parts of the manuscript and approval of the manuscript final version.

Conflict of interest. The authors declare no conflict of interest.

Acknowledgments. The research was carried out with the support of the State Assignment Reg. No. АААА-А21-121011190012-3, theme “Development of unified methods, including sampling, for the determination of microbiological and parasitological contamination of wastewater” (code “Wastewater”).

Received: March 5, 2022 / Accepted: April 12, 2022 / Published: May 31, 2022