The Effectiveness of MALDI ToF Mass Spectrometry in Identification of Francisella tularensis Strains

The aim of the study was to evaluate the effectiveness of MALDI‑ToF mass spectrometry in the identification of collection and newly isolated strains of tularemia pathogen using the database “Protein profiles of mass spectra of microorganisms belonging to I–II pathogenicity groups for the MALDI Biotyper software”.

Materials and methods. We investigated 142 strains of Francisella tularensis, including 59 collection strains and 83 newly isolated ones. Bacteriological, molecular-genetic and proteomic research methods were used to identify them. The acquisition of mass spectra, analysis, generation and expansion of reference libraries were performed on a mass analyzer “Microflex LT” using FlexControl v. 3.3, FlexAnalysis v. 3.3, and MALDI Biotyper 3.0 software packages. The cluster analysis was performed using the BioNumerics 7.6 software.

Results and discussion. The possibility of identifying tularemia pathogen has been assessed using the extended database for MALDI Biotyper 3.0 “Protein profiles of mass spectra of microorganisms belonging to I–II pathogenicity groups for the MALDI Biotyper software”. During identification to the species level, the significance of mass spectrometry results for collection strains and newly isolated ones was 91.5 % and 97.6 %, respectively. In determining the genus appurtenance, the reliability of identification was 100 %. Thus, the MALDI‑ToF mass spectrometry method allows for accurate species and genus identification of F. tularensis strains. Based on the cluster analysis of 66 F. tularensis strains in BioNumerics 7.6 software using «Pearson correlation» and the UPGMA algorithm, the possibility of subspecies differentiation has been evaluated. Due to the similarity of protein profiles of F. tularensis strains, a clear differentiation into subspecies could not be achieved. It is necessary to use other options for sample preparation, new generation devices with higher resolution, as well as apply additional approaches and analysis tools for successful subspecific differentiation.

1. Onishchenko G.G., Kutyrev V.V., editors. [Laboratory Diagnostics of Dangerous Infectious Diseases. Practice Guideliness]. Moscow: Shiko; 2013. 560 p.

2. Balakhonov S.V., Mironova L.V., Afanas’ev M.V., Kulikalova E.S., Ostyak A.S. [MALDI ToF mass spectrometric identification of pathogen species in improving the epidemiological surveillance of dangerous infectious diseases]. Bakteriologia [Bacteriology]. 2016; 1(1):88–94. DOI: 10.20953/2500-1027-2016-1-88-94.

3. Nano F.E., Zhang N., Cowley S.C., Klose K.E., Cheung K.K., Roberts M.J., Ludu J.S., Letendre G.W., Meierovics A.I., Stephens G., Elkins K.L. A Francisella tularensis pathogenicity island required for intramacrophage growth. J. Bacteriol. 2004; 186(19):6430–6. DOI: 10.1128/JB.186.19.6430-6436.2004.

4. Seibold E., Maier T., Kostrzewa M., Zeman E., Splettstoesser W. Identification of Francisella tularensis by wholecell matrix-assisted laser desorption ionization-time of flight mass spectrometry: fast, reliable, robust, and cost-effective differentiation on species and subspecies levels. J. Clin. Microbiol. 2010; 48(4):1061–9. DOI: 10.1128/JCM.01953-09.

5. Karatuna O., Celebi B., Can S., Akyar I., Kilic S. The use of matrix-assisted laser desorption ionization-time of flight mass spectrometry in the identification of Francisella tularensis. Bosn. J. Basic Med. Sci. 2016; 16(2):132–8. DOI: 10.17305/bjbms.2016.894.

6. Gekenidis M.-T., Studer P., Wüthrich S., Brunisholz R., Drissner D. Beyond the matrix-assisted laser desorption ionization (MALDI) biotyping workflow: in search of microorganism-specific tryptic peptides enabling discrimination of subspecies. Appl. Environ. Microbiol. 2014; 80(14):4234–41. DOI: 10.1128/AEM.00740-14.

7. Gao W., Li B., Ling L., Zhang L., Yu S. MALDI TOF MS method for differentiation of methicillin-sensitive and methicillin-resistant Staphylococcus aureus using (E)-Propyl α-cyano4-Hydroxyl cinnamylate. Talanta. 2022; 244:123405. DOI: 10.1016/j.talanta.2022.123405.

8. Tsimbalistova M.V., Pavlovich N.V., Aronova N.V., Chaika I.A., Chaika S.O., Vodop’yanov A.S. [Mass-spectrometric analysis of natural and antigen-altered strains of the tularemia microbe]. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2017; (4):92–6. DOI: 10.21055/0370-10692017-4-92-96.

9. Seibold E., Bogumil R., Vorderwülbecke S., Al Dahouk S., Buckendahl A., Tomaso H., Splettstoesser W. Optimized application of surface-enhanced laser desorption/ionization time-of-flight MS to differentiate Francisella tularensis at the level of subspecies and individual strains. FEMS Immunol. Med. Microbiol. 2017; 49(3):364– 73. DOI: 10.1111/j.1574-695X.2007.00216.x.