Alexander N. Mokrievich

The aim of the study was to create and study the immunogenic properties of Francisella tularensis strain 15 NIIEG without the sodC gene encoding CuZn-dependent superoxide dismutase. Materials and methods. Deletion of the sodC gene from the chromosome of the tularemia microbe was performed applying the allelic exchange method using the suicide plasmid vector pGM5. The modified chromosome fragment without the structural part of the sodC gene was constructed by combining amplicons synthesized via PCR using two pairs of primers. Electroporation was used to transfer plasmids into Escherichia coli and F. tularensis bacteria. BALB/c mice were used for immunization and infection. To assess the reactogenicity of the strains for mice, the average weight of animals in groups was determined. Results and discussion. The allelic exchange method was used to create the F. tularensis 15ΔsodC strain, in the chromosome of which a 545 bp fragment with the structural part of the sodC gene was missing. This strain did not differ in virulence and reactogenicity from the original strain 15 NIIEG, but mice immunized with the deleted version of the vaccine strain more easily tolerated the experimental infection caused by the natural strain F. tularensis subsp. mediasiatica A-678. It has been suggested that deletion of the sodC gene may improve the immunogenic properties of the tularemia microbe strain in the targeted design of a promising candidate for the creation of a modern tularemia vaccine with reduced reactogenicity.

The goal of the study is phylogenetic typing of Francisella tularensis subsp. holarctica strains isolated in the Russian Federation in 1966–2023. In

The bacterium Francisella tularensis is the etiological agent of tularemia, a natural focal, especially dangerous infection, which, “thanks to” its low infectious dose and ability to be transmitted to humans via all possible routes, is a potential bioterrorism agent. This pathogen has been known to mankind for over a hundred years, but it is still impossible to prevent massive human disease outbreaks and sporadic incidence cases, whereas tularemia diagnosis may be verified within several days-to-weeks. The basis for tularemia causative agent virulence is based on its ability to disrupt phagocyte function. In animals and humans, the various Francisella tularensis systems work together to bypass host immune system, attach to and enter eukaryotic cells, block phagosome-lysosome fusion, multiply in various host cells without being detected, inhibit their destruction and cause host cell death to release bacteria and infect neighboring tissue cells, thus developing an infectious disease in different organs. This is achieved through a unique complement-dependent penetration process into host cell, called loop phagocytosis, and an unusual inert endotoxin as well as variation in diverse forms of “free” lipid A modifications and lipid A in the LPS composition, its dynamic acyl chain length regulation, and specifically combined regulatory factors to induce the “pathogenicity island” protein synthesis. Accumulated point mutations, intragenomic rearrangements, deletions, insertions, duplications, transpositions, gene degradation, variation in the number of copies in repeated DNA sequences, as well as homologous and non-homologous recombinations underlie a markedly expanded potential for existence of the tularemia causative agent: they contribute to the holarctic subspecies strain survival in varying conditions, including osmotic shock, to form multiple resistance to various toxic substances and alter F. tularensis subspecies virulence. Analyzing a whole body of publications on the abovementioned aspects for tularemia causative agent life activity attempts to combine the differences, structural features and “tricks” of the F. tularensis species cells allowing them to be a powerful pathogen, with a high potential to adapt upon low pathogen variability and a limited genome length compared with other specially dangerous bacteria.

PURPOSE OF THE STUDY Phylogenetic typing of F. tularensis subsp. holarctica strains. isolated on the Russian Federation territory in the period from 1966 to 2023. MATERIALS AND METHODS The work used the nucleotide sequences of the genomes of 36 strains isolated on the Russian Federation territory. The phylogenetic analysis also included the genomes of 27 strains obtained from the NCBI database. Based on 6626 single nucleotide polymorphisms (SNPs) identified in the F. tularensis genome, a dendrogram of the relationships of the studied strains was constructed. RESULTS By analyzing whole-genome sequencing data of F. tularensis subsp. holarctica isolated on the Russian Federation territory from 1966 to 2023, it was shown that erythromycin-sensitive strains isolated in the territories of Altai in 2011 and Khabarovsk Territory in 1975 belong to the main phylogenetic group B.4. Erythromycin-sensitive strains of F. tularensis subsp. holarctica, isolated in the territories of the Rostov region in 1996 and the Khabarovsk Territory in 1966, belong to the main phylogenetic group B.6, subgroup B.7. The studied erythromycin-resistant strains of F. tularensis subsp. holarctica belong to group B.12, subgroups B.26-B.20, B.26-B.23 and B.27. On the territory of the Rostov region, in addition to erythromycin-sensitive ones, there are circulating strains of the tularemia pathogen belonging to the subgroup B.26-B.20, as well as those belonging to the subgroup B.27, which are also common in the territory of the Donetsk People’s Republic. Erythromycin-resistant strains of F. tularensis subsp. holarctica, isolated on the territory of the Khanty-Mansiysk Autonomous Okrug in 2013 and containing different numbers of tandem repeats of the M-3 locus, belong to different lines of the B.26 subgroup. The strains obtained on the territory of Kalmykia in 1987 belong to the subgroup B.26 (B.23). CONCLUSION The identity of the F. tularensis subsp. holarctica strains, isolated on the territory of Russia, to three main phylogenetic groups B.4, B.6 and B.12 was determined. The genetic diversity of tularemia causative agent strains is shown, as well as the circulation of several genotypes in one territory for a long time.

In 2023, 305 cases of human infection with tularemia agent were registered in the Russian Federation. Epizootic manifestations of the infection, varying in the degree of intensity, were identified in 65 constituent entities of the country, and sporadic cases of human tularemia were reported in 15 regions of Russia. The unfavorable epidemic situation persists in the Republic of Karelia (189 patients with tularemia), Stavropol Territory (35 people), and a surge in the incidence is also recorded in the Donetsk People’s Republic (53 patients), which accounts for 91 % of all tularemia cases in the country. A total of 82 Francisella tularensis cultures were isolated in 9 entities of the Russian Federation. 923,729 people were vaccinated and revaccinated against tularemia in 2023. Based on the data analysis, obtained in 2023, epidemic complications in the form of sporadic cases of the disease among the unvaccinated population in the following territories are most likely to occur in 2024: the Central Federal District – in the Vladimir, Oryol, Ryazan and Tver Regions, and Moscow city; Northwestern Federal District – in the Republic of Karelia, Arkhangelsk, Vologda Regions and in St. Petersburg; Southern Federal District – in the Republic of Crimea, Volgograd and Rostov Regions; North Caucasus Federal District – in the Stavropol Territory; Volga Federal District – in the Republic of Mordovia, as well as in the Kirov and Saratov Regions; Ural Federal District – in the Yamal-Nenets Autonomous District, KhantyMansi Autonomous District and Tyumen Region; Siberian Federal District – in the territories of certain districts of the Krasnoyarsk Territory, Omsk, Kemerovo, Tomsk, Novosibirsk and Irkutsk Regions; Far Eastern Federal District – in the Republic of Sakha (Yakutia).

The review provides concise information on the innate ability of cells of the tularemia pathogen, Francisella tularensis subsp. Holarctica, to resist antimicrobials through a variety of mechanisms, leading to its multi-resistance. In total, taking into account new territories, 120 cases of human infection were registered in the Russian Federation in 2022. Epizootic manifestations of the infection of varying degrees of intensity were detected in 58 constituent entities. Against this background, sporadic cases of tularemia in humans were reported in 18 regions of the country. An outbreak of tularemia occurred in the Stavropol Territory; the disease of mild and moderate severity was found in 76 people. The increased incidence of tularemia persists in the Republic of Karelia with severe cases of the disease in the absence of immunoprophylaxis of this infection in the region. A total of 61 cultures of the tularemia pathogen F. tularensis subsp. holarctica, out of which 20 erythromycin-resistant strains were isolated in the Stavropol Territory. In addition, 8 cultures of F. tularensis subsp. mediasiatica from a silt sample and mites Dermacentor silvarum and Haemaphysalis concinna caught in the Republic of Altai were isolated. On the territory of the Russian Federation in 2022, 930 999 people were vaccinated and revaccinated against tularemia. Based on the analysis of the data obtained in 2022, epidemic complications in 2023 in the form of sporadic cases of the disease among the unvaccinated population are most likely to occur in the territories of the Central Federal District – in the Vladimir, Ryazan and Smolensk Regions; Northwestern Federal District – in the Arkhangelsk Region and the Republic of Karelia; Southern Federal District – in the Volgograd and Rostov Regions. The situation in the North Caucasian Federal District will remain tense in the Stavropol Territory; in the Volga Federal District – in the territories of the Saratov Region, as well as in the Kirov Region and the Republic of Mordovia; Ural Federal District – in Khanty-Mansi and Yamalo-Nenets Autonomous Districts; Siberian Federal District – in certain territories of Omsk, Kemerovo, Tomsk, Novosibirsk, Irkutsk Regions, Altai, Krasnoyarsk Territories; in the Far Eastern Federal District, the most intense epizootic activity of natural tularemia foci is in Primorsky Krai.

Tularemia is a natural focal zoonotic infection that can cause epidemic manifestations of an emergency nature. The purpose of the study is to develop a met

Tularemia is a severe infectious disease caused by the Gram-negative bacteria Francisella tularensis. F. tularensis is currently divided into three subspecies, holarctica, tularensis, and mediasiatica, which differ in their virulence and geographic distribution. Subspecies mediasiatica is the least studied because of its very low documented virulence for humans and limited geographic distribution. It was discovered in sparsely populated regions of Central Asia. Since 2011, a new subsp. mediasiatica lineage was identified in Altai (Russia). In 2021, we isolated one subsp. mediasiatica strain in Krasnoyarsk Territory. In spite of its geographic origin, 500 km east from Altai, this strain belongs to the Altai lineage and contributes surprisingly little genetic diversity to previous knowledge.

Tularemia is a severe infectious disease caused by the Gram-negative bacteria Francisella tularensis. F. tularensis is currently divided into three subspecies, holarctica, tularensis and mediasiatica which differ in their virulence and geographic distribution. Subspecies mediasiatica is the least studied because of its very low documented virulence for humans and limited geographic distribution. It was discovered in sparsely populated regions of Central Asia. Since 2011, a new subsp. mediasiatica lineage was identified in Altai (Russia). In 2021, we isolated one subsp. mediasiatica strain in Krasnoyarsk Territory. In spite of its geographic origin, 500 km east from Altai, this strain belongs to the Altai lineage and contributes surprisingly little genetic diversity to previous knowledge. This improved knowledge of the phylogeography of subsp. mediasiatica led us to propose a scenario in which the two zoonotic lineages, holarctica and tularensis, independently emerged from the mediasiatica lineage in Siberia or Central Asia, and make predictions which will allow to challenge this hypothesis