The Effectiveness of MALDI ToF Mass Spectrometry in Identification of <i>Francisella tularensis</i> 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

RETROSPECTIVE ANALYSIS OF BIOLOGICAL PROPERTIES OF FRANCISELLA TULARENSIS COLLECTION STRAINS ISOLATED IN SOUTH SIBERIA (1950 -2015)

Aim. Study taxonomic belonging of collection strains of tularemia causative agent based on proteomic and molecular-genetic methods of identification. Materials and methods. 23 strains of tularemia were used in the study, isolated from Krasnoyarsk region and Altai Republic from 1950 to 2015. FT-agar was used for the cultivation. Spectra for time-of-flight mass-spectrometry were collected using Microflex LT (Bruker Daltonics, Germany) and analyzed compared with previously collected enhanced database of MALDI Biotyper 3.0. PC R with specific primers was carried out with electrophoretic visualization of results in real time. Results. F. tularensis strains isolated from south of western Siberia from 1950 to 2010 were established to belong to subspecies holarctica, and 56.3% of those - erythromycin sensitive (1 biovar Erys), the rest - erythromycin-resistant (II biovar EryR). 7 strains isolated after 2011 by citrulline ureidase activity, cleavage of glycerin and presence of pdpA and pdpD fragments of pathogenicity island (FP1) were determined as Central Asian subspecies. Conclusion. Results of a retrospective study of biological properties of tularensis strains isolated from south Siberia have shown the lack of Central Asian subspecies tularemia causative agent in the collection of Irkutsk Institute for Plague Control before 2011. Detection of this subspecies in Russian Federation gives evidence on the necessity to study and analyze problems of epidemiology, ecology and epizootology of Central Asian subspecies tularemia causative agent as well as determination of borders of its spread

Present-Day Epidemiological-Epizootological Peculiarities of Natural Tularemia Foci in Altai

Relevance Natural tularemia foci are widespread in Altai Krai and Republic Altai and characterized by high functional stability. Aim. To study modern epidemiological and epizootological features of natural foci of tularemia in the Altai region Russia. Materials and Methods The current state of small mammal number in the region is examined. Results of complex studying of 14 Francisella tularensis strains isolated in Republic Altai from 1950 to 2015 (from the collection of Museum of live cultures in Irkutsk Antiplague Research Institute of Rospotrebnadzor) and 21 strains isolated in 2016–2018 in the Altai region are presented. For the first time the tularemia causative agent was isolated at this territory in 1940s. Water vole (Arvicola amphibius) is the most epidemiologically significant as the infection source in these foci. Ixodes ticks: Dermacentor silvarum, D. reticulatus, D. marginatus, Haemaphysalis concinna, Ixodes persulcatus are the main vectors. Now Francisella tularensis subspecies holarctica circulates at Altai territory, and since 2011 additionally F. tularensis mediasiatica. Conclusions The functioning of the natural center of tularemia in the territory of the Altai region in the modern period is characterized by coupled circulation of the causative agent of tularemia of two subspecies, sporadic cases of tularemia diseases and infection by the causative agent of environmental objects (bloodsucking arthropods, open water, small mammals). All this indicates a high activity of natural foci with respect to this especially dangerous natural focal infection