Genotyping of Francisella tularensis subsp. mediasiatica Strains Using Marker SNPs

The aim of the study was the genotyping of Francisella tularensis subsp. mediasiatica strains using marker SNPs based on whole genome sequencing data. Materials and methods. The work used 50 complete genomes (WGS) of F. tularensis subsp. mediasiatica strains from the NCBI database and 25 genomes, which were sequenced directly by specialists from the Rostov-on-Don Anti-Plague Institute of the Rospotrebnadzor. The allocation of individual clusters on the dendrogram was carried out with a bootstrap support value of at least >90 % (when using 1000 replications). Results and discussion. A total of 5,251 SNPs identified in a representative sample of 75 F. tularensis subsp. mediasiatica genomes were selected, with each SNP detected in at least two strains. During the bioinformatics analysis, genomes with more than 500 SNPs in regions not covered by whole-genome sequencing (WGS) were excluded. Phylogenetic analysis resulted in a dendrogram that identified 11 major clusters named after the geographic locations of early or dominant strains. These clusters were supported by bootstrap values >90 % (1,000 replicates). For strain typing within the clusters, “marker” SNPs specific to each cluster and its subgroups – but absent in others – were identified. A software tool, “SNP Genotyper”, was developed to automate genotype determination based on those marker SNPs. Genetic variability of F. tularensis subsp. mediasiatica isolates of diverse origins was studied using whole-genome SNP (wgSNP) analysis. Genetic diversity was observed among subsp. mediasiatica strains isolated in Altai Territory (Russian Federation) and the Republic of Kazakhstan. Based on marker SNPs, an algorithm has been developed for the operational analysis of WGS data from F. tularensis subsp. mediasiatica genomes. The developed method for determining genetic lineages can be a useful tool both for operational analysis when isolating fresh strains, determining the phylogenetic relationships of strains, studying the genetic diversity of a population, and conducting retrospective studies.

1. Gürcan Ş. Epidemiology of tularemia. Balkan Med. J. 2014; 31(3):3–10. DOI: 10.5152/balkanmedj.2014.13117.

2. Kilic S., Birdsell D.N., Karagöz A., Çelebi B., Bakkaloglu Z., Arikan M., Sahl J.W., Mitchell C., Rivera A., Maltinsky S., Keim P., Üstek D., Durmaz R., Wagner D.M. Water as source of Francisella tularensis infection in humans, Turkey. Emerg. Infect. Dis. 2015; 21(12):2213–6. DOI: 10.3201/eid2112.150634.

3. Dennis D.T., Inglesby T.V., Henderson D.A., Bartlett J.G., Ascher M.S., Eitzen E., Fine A.D., Friedlander A.M., Hauer J., Layton M., Lillibridge S.R., McDade J.E., Osterholm M.T., O’Toole T., Parker G., Perl T.M., Russell P.K., Tonat K.; Working Group on Civilian Biodefense. Tularemia as a biological weapon: medical and public health management. JAMA. 2001; 285(21):2763–73. DOI: 10.1001/jama.285.21.2763.

4. Degabriel M., Valeva S., Boisset S., Henry T. Pathogenicity and virulence of Francisella tularensis. Virulence. 2023; 14(1):2274638. DOI: 10.1080/21505594.2023.2274638.

5. Keim P., Johansson A., Wagner D.M. Molecular epidemiology, evolution, and ecology of Francisella. Ann. N.Y. Acad. Sci. 2007; 1105(1):30–66. DOI: 10.1196/annals.1409.011.

6. Timofeev V., Bakhteeva I., Titareva G., Kopylov P., Christiany D., Mokrievich A., Vergnaud G. Russian isolates enlarge the known geographic diversity of Francisella tularensis subsp. mediasiatica. PloS One. 2017; 12(9):e0183714. DOI: 10.1371/journal.pone.0183714.

7. Kudryavtseva T.Yu., Mokrievich A.N. Molecular-genetic bases of differences between tularaemia pathogen subspecies and Francisella tularensis subsp. holarctica strain typing. Mol. Genet. Microbiol. Virol. 2022; 37(1):10–8. DOI: 10.3103/s0891416822010049.

8. Aikimbaev M.A. Taxonomy of genus Francisella. Rep. Acad. Sci. Kaz. SSR. Ser. Biol. 1966; 5:42–4.

9. Olsufjev N.G., Meshcheryakova I.S. Subspecific taxonomy of Francisella tularensis McCoy and Chapin 1912. Int. J. Syst. Evol. Microbiol. 1983; 33(4):872–4. DOI: 10.1099/00207713-33-4-872.

10. Timofeev V., Bakhteeva I., Mokrievich A., Vakhrameeva G., Gritskova E., Anisimov Yu., Rozhdestvensky E., Bazarova G., Zhumakaev R., Dyatlov I., Vergnaud G. The first finding of Francisella tularensis subsp. mediasiatica in Krasnoyarsk Territory, Siberia, and an update of the subspecies genetic diversity. Bacteria. 2022; 1(4):242–9. DOI: 10.3390/bacteria1040018.

11. Mokrievich A.N., Timofeev V.S., Kudryavtseva T.Yu., Ulanova G.I., Karbysheva S.B., Mironova R.I., Vakhrameeva G.M., Gubareva T.I., Pavlov V.M., Dyatlov I.A. [Isolation of Central Asian subspecies of tularemia agent in the Altai Territory]. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2013; (1):66–9. DOI: 10.21055/0370-1069-2013-1-66-69.

12. Shevtsov A., Izbanova U., Amirgazin A., Kairzhanova A., Dauletov A., Kiyan V., Vergnaud G. Genetic homogeneity of Francisella tularensis subsp. mediasiatica strains in Kazakhstan. Pathogens. 2024; 13(7):581. DOI: 10.3390/pathogens13070581.

13. Borzenko M.A., Zarva I.D., Holin A.V., Kulikalova E.S., Mazepa A.V., Syngeeva A.K., Naumova K.V., Rozhdestvensky E.N., Bazarova G.Kh., Sanarov P.P., Polkovnikov E.S., Ivanitskaya Yu.N., Sbitneva S.V., Krasil’nikova N.Yu., Pashhenko I.G. [Current epidemiological situation on tularemia in Altai]. In: [Sanitary and Epidemiological Welfare of the Population and Consumer Rights Protection: Regional Aspects. Proceedings of the All-Russian Scientific and Practical Conference]. Irkutsk: LLC Publishing House “IRKUT”; 2022. P. 223–6.

14. Timofeev V., Titareva G., Bahtejeva I., Kombarova T., Kravchenko T., Mokrievich A., Dyatlov I. The comparative virulence of Francisella tularensis subsp. mediasiatica for vaccinated laboratory animals. Microorganisms. 2020; 8(9):1403. DOI: 10.3390/microorganisms8091403.

15. Larsson P., Elfsmark D., Svensson K., Wikström P., Forsman M., Brettin T., Johansson A. Molecular evolutionary consequences of niche restriction in Francisella tularensis, a facultative intracellular pathogen. PLoS Pathog. 2009; 5(6:)e1000472. DOI: 10.1371/journal.ppat.1000472.

16. Bankevich A., Nurk S., Antipov D., Gurevich A.A., Dvorkin M., Kulikov A.S., Lesin V.M., Nikolenko S.I., Pham S., Prjibelski A.D., Pyshkin A.V., Sirotkin A.V., Vyahhi N., Tesler G., Alekseyev M.A., Pevzner P.A. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 2012; 19(5):455–77. DOI: 10.1089/cmb.2012.0021.

17. Seemann T. Snippy: Fast Bacterial Variant Calling from NGS Reads. GitHub; 2022. [Internet]. Available from: https://github.com/tseemann/snippy.

18. Tamura K., Stecher G., Kumar S. MEGA11: Molecular Evolutionary Genetics Analysis version 11. Mol. Boil. Evol. 2021; 38(7):3022–7. DOI: 10.1093/molbev/msab120.

19. Huerta-Cepas J., Serra F., Bork P. ETE 3: reconstruction, analysis, and visualization of phylogenomic data. Mol. Boil. Evol. 2016; 33(6):1635–8. DOI: 10.1093/molbev/msw046.

20. Lärkeryd A., Myrtennäs K., Karlsson E., Dwibedi C.K., Forsman M., Larsson P., Sjödin A. CanSNPer: a hierarchical genotype classifier of clonal pathogens. Bioinformatics. 2014; 30(12):1762–4. DOI: 10.1093/bioinformatics/btu113.

21. Di Giallonardo F., Puglia I., Curini V., Cammà C., Mangone I., Calistri P., Cobbin J.C.A., Holmes E.C., Lorusso A. Emergence and spread of SARS-CoV-2 lineages B.1.1.7 and P.1 in Italy. Viruses. 2021; 13(5):794. DOI: 10.3390/v13050794.

22. Alieva B., Hodzhaeva G.A. [Main demographic indicators of the Republic of Karakalpakstan]. Ekonomika i Sotsium [Economics and Society]. 2024; 6-2(121):843–6.