Biological Properties and Genetic Characteristics of Francisella tularensis Strains Isolated in the Territory of the Rostov Region in 2020

Objective of the study was to investigate biological properties and genetic characteristics of tularemia agent strains isolated from natural foci of the Rostov Region in 2020.

Materials and methods. Field material from natural foci of the Rostov Region was examined by serological, bacteriological, biological, and molecular-genetic methods. Cultural-morphological, biochemical, antigenic and pathogenic properties of isolated cultures were studied. Protein profles were obtained through MALDI-TOF MS using mass spectrometer Autoflex speed III Bruker Daltonics and Flex Control of Biotyper software. The genetic characteristics of the strains were determined by VNTR and INDEL typing and SNP analysis.

Results and discussion. Six strains of tularemia pathogen were isolated from mouse-like rodents using biological method. The investigation of their biological features and data of PCR analysis and INDEL typing with canonical markers showed that all strains are typical representatives of the Francisella tularensis subsp. holarctica biovar EryR. VNTR typing by six genetic loci revealed that all strains belong to four individual genotypes. The strain isolated in 2020 in the Salsky district was identical to the strain which was isolated in the same area in 1989. Based on the whole genome sequencing of two strains, we established that they are closest to the cultures isolated in Turkey (2009, 2012) and Khanty-Mansiysk (2013) by the studied set of SNP markers. Thus, we found that both identical (or closely related) clones of the tularemia agent and new strains with unique genotypes which previously were not described for the Rostov Region can circulate in natural foci of this region for a long period of time.

1. Mesheryakova I.S., Dobrovolsky A.A., Demidova T.N., Kormilitsyna M.I., Mikhailova T.V. Vector-borne epidemic outbreak of tularemia in the town of Khanty-Mansiysk in 2013. Epidemiologiya i Vaktsinoproflaktika. [Epidemiology and Vaccinal Prevention]. 2014; 5(78):14–20.

2. Mokrievich A.N., Kravchenko T.B., Firstova V.V., Titareva G.M., Dyatlov I.A., Timofeev V.S. Tularemia: State of the Problem and Research Methods. Obolensk; M .: “Dynasty”; 2019. 263 p.

3. Kudryavtseva T.Yu., Popov V.P., Mokrievich A.N., Pakskina N.D., Kholin A.V., Mazepa A.V., Kulikalova E.S., Kosilko S.A., Birkovskaya Yu. A., Trankvilevsky D.V., Khramov M.V., Dyatlov I.A. Epidemic activity of natural tularemia foci in the territory of the Russian Federation in 2018 and forecast of the situation for 2019. Problemy Osobo Opasnykh Infektsii. [Problems of Particularly Dangerous Infections]. 2019; 1:32–41. DOI: 10.21055/0370-1069-2019-1-32-41.

4. Kovalev E.V., Karpushchenko G.V., Schwager M.M., Polonsky A.V., Sidelnikov V.V., Goncharov A.Yu., Polovinka N.V. Features of distribution of tularemia infection in the Rostov Region. Epidemiologiya i Vaktsinoproflaktika. [Epidemiology and Vaccinal Prevention]. 2017; 16(6):37–40. DOI: 10.31631/2073-3046-2017-16-6-37-40.

5. Rodionova I.V. Differentiation of the geographical races of Francisella tularensis based on the activity of citrulline ureidase. Laboratornoe delo [Laboratory Practice]. 1970; 1: 42–3.

6. Tsimbalistova M.V., Pavlovich N.V. The phosphatase activity in representatives of the genus Francisella. Zhurnal Mikrobiologii, Epidemiologii i Immunobiologii. [Journal of Microbiology, Epidemiology and Immunobiology]. 1998; 1:10–3.

7. Tsimbalistova M.V., Pavlovich N.V. Features of resistance formation to β-lactam antibiotics in Francisella tularensis subsp. mediasiatica. Zhurnal Mikrobiologii, Epidemiologii i Immunobiologii. [Journal of Microbiology, Epidemiology and Immunobiology]. 2014; 1:3–8.

8. Directive of the European Parliament and of the Council of the European Union on the protection of animals used for scientifc purposes 2010/63 / EU. St. Petersburg; 2010.48 p.

9. Vogler A.J., Birdsell D., Wagner D.M., Keim P. An optimized, multiplexed multi‐locus variable‐number tandem repeat analysis system for genotyping Francisella tularensis. Lett. Appl. Microbiol. 2009; 48(1):140–4. DOI: 10.1111/j.1472-765X.2008.02484.x.

10. Grissa I., Bouchon P., Pourcel C., Vergnaud G. On-line resources for bacterial micro-evolution studies using MLVA or CRISPR typing. Biochimie. 2008; 90(4):660–8. DOI: 10.1016/j.biochi.2007.07.014.

11. Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 2011; 28:2731–9. DOI: 10.1093/molbev/msr121.

12. Vodop’yanov A.S., Pisanov R.V., Vodop’yanov S.O., Mishankin B.N., Oleynikov I.P., Kruglikov V.D., Titova S.V. Molecular epidemiology of Vibrio cholerae – development of the algorithm for data analysis of whole genome sequencing. Epidemiologiya i Infektsionnye Bolezni. [Epidemiology and Infectious Diseases]. 2016; 21(3):146–52. DOI: 10.18821/1560-9529-2016-21-3-146-152.

13. Trankvilevsky D.V., Tsarenko V.A., Zhukov V.I. The current state of epizootic monitoring of natural foci of infections in the Russian Federation. Meditsinskaya Parazitologiya I Parazitarnye Bolezni. [Medical Parasitology and Parasitic Diseases]. 2016; 2: 19-24.

14. Pavlovich N.V., Tsimbalistova M.V., Aronova N.V., Gorbatov A.A. Method for in vitro virulence assessment of tularemia microbe strains of subspecies Francisella tularensis subsp. tularensis, subsp. mediasiatica, subsp. holarctica. RF patent No. 2018118456, publ. 07/25/2019. Bul. No. 21.

15. 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-modifed strains of tularemia agent. Problemy Osobo Opasnykh Infektsii. [Problems of Particularly Dangerous Infections]. 2017; 4:92–6. DOI: 10.21055/0370-1069-2017-4-92-96.

16. Gyuranecz M., Birdsell D.N., Splettstoesser W., Seibold E., Beckstrom-Sternberg S.M., Makrai L., Fodor L., Fabbi M., Vicari N., Johansson A., Busch J.D., Vogler A.J., Keim P., Wagner D.M. Phylogeography of Francisella tularensis subsp. holarctica, Europe. Emerg. Infect. Dis. 2012; 18(2):290–3. DOI: 10.3201/eid1802.111305.

17. Sissonen S., Rossow H., Karlsson E., Hemmilä H., Henttonen H., Isomursu M., Kinnunen P.M., Pelkola K., Pelkonen S., Tarkka E., Myrtennäs K., Nikkari S., Forsman M. Phylogeography of Francisella tularensis subspecies holarctica in Finland, 1993–2011. Infect. Dis. (Lond). 2015; 47:701–6. DOI: 10.3109/23744235.2015.1049657.

18. Johansson A., Farlow J., Larsson P., Dukerich M., Chambers E., Byström M., Fox J., Chu M.C., Forsman M., Sjöstedt A., Keim P. Worldwide genetic relationships among Francisella tularensis isolates determined by multiple-locus variable-number tandem repeat analysis. J. Bacteriol. 2004; 186:5808–18. DOI: 10.1128/JB.186.17.5808-5818.2004.

19. Schulze C., Heuner K., Myrtennäs K., Karlsson E., Jacob D., Kutzer P., Große K., Forsman M., Grunow R. High and novel genetic diversity of Francisella tularensis in Germany and indication of environmental persistence. Epidemiol. Infect. 2016; 144(14):3025–36. DOI: 10.1017/S0950268816001175.

20. Gnusareva O.A., Kotenev E.S., Volynkina A.S., Chishenyuk T.I., Kulichenko A.N. Molecular-epidemiological analysis of the tularemia outbreak in the Stavropol Territory in 2017. Infektsionnye Bolezni: Novosti, Mneniya, Obuchenie. [Infectious Diseases: News, Opinions, Training]. 2018; 7(3):57–61. DOI: 10.24411/2305-3496-2018-13008.