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The aim of the study is to obtain a comprehensive characterization of Yersinia pseudotuberculosis strains from the Aksai high-mountain and Balkhash desert foci of plague in Central Asia. Materials and methods. Investigation of the cultural, morphological and biochemical properties of the examined strains was carried out using conventional methods of laboratory diagnostics. Ion S5 XL System (Thermo Fischer Scientific) was used for whole-genome sequencing. Data processing and sequence assembly of raw reads de novo were performed using Ion Torrent Suite software package 5.12 and Newbler gsAssembler 2.6 (454 Life Sciences). To search for marker SNPs, the Wombac 2.0 program based on the BioLinux 8.0 operating system was applied. When constructing the dendrogram, the Maximum Likelihood method, the PhyML 3.1 program and the HKY85 model were used. Results and discussion. The studied strains from the Aksai high-mountain and Balkhash desert foci have properties characteristic of the Y. pseudotuberculosis species, are prototrophs and do not contain the pYV plasmid. According to phylogenetic analysis based on identified 109,641 core SNPs, they are closely related to strains of the pseudotuberculosis microbe from the Sarydzhaz high-mountain focus in the Kyrgyz Republic and the Republic of Kazakhstan, as well as to the O:3 serovar strain from Turkmenistan. The cluster of these Central Asian strains on the phylogenetic tree of Y. pseudotuberculosis is localized next to strains of the pseudotuberculosis microbe of O:3 serovar from various regions of the world. The results obtained testify to the circulation of a separate phylogeographic population of Y. pseudotuberculosis in the territory of natural plague foci in Central Asia. The characterized Y. pseudotuberculosis strains and their whole genome sequences can be used as reference ones for the Aksai and Balkhash natural plague foci of Central Asia.

The aim of the study was to assemble full-length nucleotide sequences of the chromosome and plasmids for 13 Yersinia pestis strains from 11 natural plague foci located in the Russian Federation, using data from two sequencing technologies.Materials and methods. Y. pestis strains were grown on Hottinger’s agar (pH 7.2) at 37 °C. DNA was isolated using phenol-chloroform extraction. For the MinIon genetic analyzer (Oxford Nanopore), DNA fragments were prepared by ligation according to a modified protocol. For the Ion S5 genetic analyzer (IonTorrent), sample preparation was carried out according to the standard protocol for obtaining a library with a DNA fragment size of 400 nucleotide pairs (bp). The resulting single reads were filtered by average quality Q30 for IonTorrent and Q7 for Oxford Nanopore.Results and discussion. DNA fragments containing 50 000 or more nucleotide pairs were prepared for subsequent sequencing using nanopore sequencing technology (Oxford Nanopore). The Trycycler algorithm was applied for hybrid assembly of the genome of Y. pestis strains and correction of errors arising during this process, allowing the obtainment of full-length nucleotide sequences of the chromosome and plasmids for each genome of the strain. The nucleotide sequences of the chromosomes of 13 Y. pestis strains from 11 natural foci of plague located in the Russian Federation have been deposited in the international genetic database NCBI GenBank. It has been established that to assemble full-length genomes of Y. pestis strains, a significant number of reads with a size of 50 000 bp or more is required, and the use of the Trycycler algorithm allows one to generate a more accurate assembly of complete bacterial genomes.

The review summarizes the data of modern domestic and foreign studies on the mechanisms of evolutionary adaptation of the plague pathogen to transmissive spread by arthropods. The data on the molecular basis of the rapid formation of a highly pathogenic bacterium due to the acquisition of new genetic information; structural and functional changes in the genome, causing the disruption of functionality of some genes that prevent survival in the vector are presented. The stage of the complex life cycle of the pathogen associated with the peculiarities of its stay in the vector’s organism and its tactics of evasion from the action of antibacterial substances formed by the flea is considered. The importance of biofilm formation for effective transmission of the pathogen is discussed. A complex cascade of transcriptional regulators of biofilm in Yersinia pestis is considered, which includes activators and repressors of biofilm formation, as well as regulators of synthesis or modification/transport of exopolysaccharide. The hms-dependent mechanism of biofilm formation in Y. pestis is described in detail, as well as the impact on biofilm formation through the regulation of LPS-related genes and its role in the modification and transport of biofilm exopolysaccharide. The data from our own studies on the variability of genes involved in biofilm formation in the main subspecies of the plague pathogen in comparison with non-main subspecies of the plague pathogen, as well as on the ability of strains of different subspecies to form biofilm not only in the proventriculus of the flea, but also on the cuticle of soil nematodes of the Tylenchida and Rhabditida orders and the genus Panagrolaimus are presented. The latter allows us to assume the possible participation of soil and entomoparasitic nematodes in the removal of Y. pestis biofilms from the soil to the above-ground biocoenosis of the natural plague focus.

The aim of the work was to study the genetic diversity and spatial-temporal structure of Yersinia pestis in the Caspian sandy plague focus using MLVA25 and CRISPR typing methods.Materials and methods. 98 Y. pestis strains isolated in the territory of the Caspian sandy plague focus in 1925–2015 were used in the study. Whole genome sequencing was performed on Ion GeneStudio S5 System (ThermoFisher Scientific) and MGI (DNBSEQ-G50RS) platforms. Fragment sequencing was conducted using “ABI PRISM 3500XL”. The search for VNTR and CRISPR loci with subsequent alignment of nucleotide sequences was carried out in the MEGA X program. The obtained sequences were entered into the created database in the Bionumerics v7.6 (Applied Maths) software. The phylogenetic tree was constructed using the UPGMA method.Results and discussion. According to the results of MLVA25 and CRISPR analysis, 98 Y. pestis strains are divided into 60 genotypes (CS1 – CS60). Variability by 23 VNTR loci has been discovered. 7 new CRISPR spacers are described: 5 in YPa and 2 in YPb (size 31–33 bp, GC composition 34–58 %). The described spacers are designated as a108, a109, a110, a111, a112, b53, b54. The interrelation of changes in the copy number of VNTR loci depending on the place and time of isolation of strains has been revealed. The data obtained can be used to carry out molecular-genetic certification of the territory of the Caspian sandy plague focus and to study the routes and patterns of evolution and spatiotemporal circulation of Y. pestis populations in the Caspian plague foci.

The aim of the work was to study the growth dependence on amino acids in Yersinia pestis strains of different phylogenetic branches of the antiqua biovar and to determine the genetic basis of auxotrophy of these strains.Materials and methods. We used 38 strains of Y. pestis, the main subspecies of the antiqua biovar, isolated in various foci of the world in the period of 1928–2020. The nutritional requirements of the strains were determined by incubation on Difco minimal agar with different sets of amino acids. Phylogenetic analysis of the strains was carried out using the Wombac 2.0 and SeaView 5.0.5 programs. Comparative analysis of the nucleotide sequences of the genes was performed using the BLAST algorithm and the Mega 7.0 program.Results and discussion. The phylogenetic appurtenance of the investigated Y. pestis strains of antiqua biovar to the phylogenetic branches 0.ANT3, 0.ANT5, 1.ANT, 2.ANT3, 3.ANT2, 4.ANT has been determined. It is established that all studied strains have a common dependence of growth on three amino acids – phenylalanine, threonine, and methionine. In the majority of the strains of antiqua biovar of all phylogenetic branches, growth is also dependent on the presence of cysteine in the medium, except for a part of the strains of the phylogenetic branch 4.ANT. In 14 genes of sulfur and cysteine metabolism, 19 mutations have been identified. Each phylogenetic group of the antique biovar has a distinct profile of mutations in genes involved in cysteine biosynthesis. The leucine requirement of the strains belonging to phylogenetic branch 0.ANT5 has been established, possibly caused by a frame shift in the leuA gene. Strains of the 1.ANT branch isolated in the Democratic Republic of the Congo demonstrate an additional proline-dependent growth. The defined nutritional requirements in the strains and the genetic causes of auxotrophy complement the phenotypic and genetic characteristics of the phylogenetic branches of the antiqua biovar and can be used as genetic markers for the differentiation of these strains.

The paper describes a developed comprehensive system for molecular-genetic identification of Yersinia pestis strains according to their appurtenance to certain subspecies, biovars, phylo-geographic populations, using realtime PCR (RT-PCR), allele-specific RT-PCR, and multiplex PCR with hybridization fluorescent registration of results on a solid substrate. Application of this system makes it possible to establish the appurtenance of Y. pestis strains to the following phylogenetic branches: 0.ANT1, 0.ANT2, 0.ANT3, 0.ANT5, 3.ANT, 4.ANT of antique biovar of the main subspecies; 2.MED0, 2.MED1, 2.MED2, 2.MED3, 2.MED4 of medieval biovar of the main subspecies; 1.IN1, 1.IN2, 1.IN3 of intermedium biovar of the main subspecies; 1.ORI1, 1.ORI2, 1.ORI3 of oriental biovar of the main subspecies; 0.PE3 (angolica subspecies), 0.PE7 (tibetica subspecies) and 0.PE10 (qinghaica subspecies). The first stage of the studies within the frames of the developed system is indication of plague agent using registered diagnostic drugs. The second stage is the determination of belonging to individual subspecies through RT-PCR or by the method of multiplex PCR system with hybridization-fluorescent registration of results on a solid substrate, which also allows for establishing to which biovars of the main subspecies and the main phylogenetic lines of the ancient biovar the strains belong. The third stage is the identification of strain appurtenance to phylogenetic branches by the AS-RT-PCR method. The designed complex system for molecular-genetic identification of Y. pestis strains can be applied at the regional and federal levels of the laboratory network of the Russian Federation for diagnostics of infectious diseases. Its use will considerably facilitate and increase the efficiency of intraspecific differentiation of Y. pestis strains within the framework of the epidemiological investigation of outbreaks or importation of strains of plague pathogen into the territory of the Russian Federation or during the certification of strains in collection activities.

The aim of the study was to compare the nucleotide sequences of pgm‑region genes in Yersinia pestis strains isolated on the territory of the Caspian sandy and adjacent plague foci in 1925–2015. Materials and methods. 65 Y. pestis strains from the Caspian sandy and adjacent plague foci were used in the work. DNA isolation was performed using the PureLink Genomic DNA Mini Kit. Whole genome sequencing was conducted in Ion S5 XL System (Thermo Fischer Scientific). Data processing was carried out using Ion Torrent Suite software package 3.4.2 and NewblerGS Assembler 2.6. To compare the obtained sequences with the NCBI GenBank database, the Blast algorithm was used. The phylogenetic analysis was performed according to the data of whole genome SNP analysis based on 1183 identified SNPs. The search for marker SNPs was performed using the Snippy 4.6 program. The phylogenetic tree was constructed using the Maximum Likelihood algorithm, the GTR nucleotide substitution model. Results and discussion. The nucleotide sequences of pgm‑region genes of 65 Y. pestis strains from the Caspian sandy and adjacent plague foci have been assessed. Single nucleotide substitutions have been identified in Y. pestis strains from the Caspian sandy and Kobystan plain-foothill foci in the hmsR, astB, ybtS, ypo1944, ypo1943, ypo1936 genes, as well as a deletion of 5 bp in the ypo1945 gene, which is characteristic of strains of one of the phylogenetic lines of Y. pestis from the foci of Caucasus and Transcaucasia, isolated in 1968–2001. The data obtained can be used to differentiate Y. pestis strains from the Caspian sandy focus, as well as to establish the directions of microevolution of the plague pathogen in this region and adjacent foci.

The aim of the study was to test the feasibility of long-term survival and preservation of the properties of Yersinia pestis in association with soil amoeba Acanthamoeba castellanii. Materials and methods. Y. pestis strains and acanthamoeba isolated in the common area of the Gorno-Altai high-mountain plague focus were used for the study. The systematic affiliation of protozoa was determined through analyzing the 18S rRNA gene fragment sequencing data, followed by alignment with amoeba sequences from the NCBI GenBank database. A fluorescent Y. pestis strain was obtained by electroporation using the pTurboGFP-B plasmid. Co-cultivation was carried out in saline buffer in the absence of nutrients for the cells of plague pathogen. The influence of co-culturing with protozoa on Y. pestis properties was determined using microbiological, biological, and molecular-genetic methods. Results and discussion. The cell viability preservation for 22 months of the experiment in Y. pestis strain belonging to the main subspecies of the antique biovar, the 4.ANT phylogenetic line in co-culture with amoeba cells in the absence of additional nutrients has been established. Co-cultivation with amoebae did not lead to a change in the cultural, morphological, genetic and virulent properties of the plague pathogen strain. The data obtained confirm the possibility of using Acanthamoeba castellanii by the plague microbe to persist in soil biocenoses and open up the prospect of studying the mechanisms of plague pathogen surviving during extended inter-epizootic periods.

The review presents the data on domestic and foreign phenotypic classifications of Yersinia pestis strains developed in the XX century; genetic classifications of the XXI century; as well as on the genealogy of ancient strains of the plague microbe, reconstructed using paleogenomic technologies. Since the discovery of the plague agent in 1894, many classifications were created that corresponded to the level of development of microbiology at that time. The intraspecific classification schemes of the XX century were based on three principles: phenotypic differences between strains, features of the species composition of carriers, and geographical affiliation. With the development of molecular microbiology early on in the XXI century, a genetic nomenclature of the branches of the pathogen evolution was developed and a number of classifications based on the analysis of the population structure of Y. pestis were created. Through the prism of the genetic diversity of Y. pestis strains from natural plague foci in Russia, near and far abroad countries, an improved classification with a division into seven subspecies has been developed: pestis, tibetica, caucasica, qinghaica, angolica, central asiatica, ulegeica, which allocates the subspecies according to the phylogenetic principle and epidemic significance. With the advancements in paleomicrobiology, prehistoric lineages of evolution have been included in the genealogy of Y. pestis, which expand the data on the intraspecific diversity of the plague microbe.

The SNP-typing method based on the detection of stable genetic markers in the genome, i.e., single nucleotide polymorphisms, is successfully used for genotyping of pathogenic microorganisms and can be applied for SNP-profiling of Yersinia pestis strains and molecular-genetic certification of focal areas. The aim of the study was to determine the SNP profiles of Y. pestis strains of the medieval biovar isolated in the Caspian Sea region plague foci in 1912–2015 and to develop a method for identifying unique SNPs using the Sanger sequencing for molecular-genetic certification of these territories. Materials and methods. A comprehensive study of the phenotypic and genotypic properties of 190 Y. pestis strains from plague foci in the Caspian Sea region was carried out. Phylogenetic reconstruction by the Maximum Likelihood method (GTR model) in the SeaView 5.0.4 software was performed on the basis of 1621 SNPs identified among 50 Y. pestis strains according to WG-SNP analysis in the snippy 4.6 program. Primers for PCR amplification of the SNP loci selected as target were calculated using the Vector NTI program. Sanger sequencing of SNPs loci was conducted on an ABI PRISM 3500XL genetic analyzer (Applied Biosystems, USA). Results and discussion. According to phenotypic characteristics, all studied strains from the Caspian foci belonged to a highly virulent and epidemically significant medieval biovar of the main subspecies of Y. pestis. According to the results of the WG-SNP analysis, 9 SNP genotypes were identified based on the polymorphism of single nucleotides of 24 genes characteristic of the main phylopopulations, which include strains isolated during various periods of epidemic and epizootic activity in the Caspian plague foci. Determining of SNP genotypes of Y. pestis strains of the medieval biovar, obtained over a hundred years in the Caspian foci, creates the prerequisites for defining the canonical SNP profile (canSNP) and for developing an algorithm for molecular epidemiological monitoring of the foci in which this highly virulent biovar circulates.

The purpose of the work was to analyze the phylogenetic relations and origin of Yersinia pestis strains isolated in different periods of epizootic activity of the Caspian sandy natural focus (CSNF) of plague in the XX–XXI centuries.Materials and methods. We used 40 Y. pestis strains from CSNF and adjacent plague foci, isolated in 1922–2015. Carried out was whole genome sequencing of 19 Y. pestis strains from CSNF. Phylogenetic analysis was performed using whole genome SNP analysis based on 1914 identified SNPs. The search for marker SNPs was conducted using the Snippy 4.6 software. The phylogenetic tree was constructed using the Maximum Likelihood algorithm, the GTR nucleotide substitution model.Results and discussion. The whole genome SNP analysis has revealed that Y. pestis strains of the medieval biovar from CSNF belong to 2.MED1 phylogenetic lineage and fall into two major branches. One of them circulated in the focus in the first half of the XX century, and the other – in the second half of the XX – early XXI centuries. It is shown that strains of the first branch were the cause of outbreaks and individual cases of plague in the CSNF in the first half of the XX century. They are closely related to strains from the Caspian North-Western steppe and Volga-Ural sandy natural plague foci, which caused numerous outbreaks with high mortality rate in the same period. Y. pestis strains from the CSNF of the second half of the XX and early XXI centuries belong to the second phylogenetic branch of the 2.MED1 line, at the node of which the strains from the Northern Aral Sea region of 1945 lay. The latter were the predecessors of all strains isolated in the CSNF after a long inter-epizootic period that occurred in the middle of the XX century. There can also be traced a genetic relation between the strains from CSNF and the Dagestan plain-foothill focus.

The aim of the work was to conduct phylogenetic analysis of Y. pestis strains, biovar altaica ssp. central asiatica, isolated in Gorno-Altai high-mountain and Sailyugem natural plague foci on the territory of Russia and Mongolia in 1965–2020, by full-genome sequencing data.Materials and methods. To determine the population structure of the biovar altaica ssp. central asiatica, 34 whole genome sequences were used (including 20 Y. pestis strains of the biovar altaica, 18 of which were sequenced by us). To isolate DNA from the Y. pestis strains, a PureLink Genomic DNA Mini Kit (Invitrogen, USA) was applied. Sequencing of the nucleotide sequences of Y. pestis strains was carried out in Ion PGM system Lifetechnologies. The analysis and processing of the obtained data were performed with the help of Newblergs Assembler 2.6 and IonTorrent Suite software package, 3.4.2. The search for SNPs was performed using the Wombac 2.0 program. The Maximum Likelihood dendrogram was built in the PhyML 3.1. The dendrogram was visualized using the FigTree 1.4.3 software.Results and discussion. Based on the data of whole genome analysis, taking into account the 1871 revealed polymorphic nucleotides, the spatial structure of the biovar altaica ssp. central asiatica has been determined. It includes several phylogeographic branches: the Kurai-Tarkhatinskaya (cluster 0.PE4a-1) and the Ulandryk-Mongolian (0.PE4a-2), which is in agreement with the geographical regions of the isolation of strains forming these branches in the Altai Mountains. The Kurai-Tarkhatinskaya branch is further divided into the Kurai (sub-cluster 0.PE4a-1-1, formed by the strains of 2009–2018) and Tarkhatinskaya (subcluster 0.PE4a-1-2, formed by the strains of 2012–2020) sub-branches, while the Ulandryk-Mongolian branch of evolution is split into sub-branches represented by strains from the Ulandryk meso focus (sub-cluster 0.PE4a-2-2, strains 1965–2010) and the Sailyugem focus of Mongolia (sub-cluster 0.PE4a-2-1, strains 1964–1990).

The aim of the study was to develop a test system that allows for detecting plague pathogen DNA in clinical and biological samples, environmental objects with the simultaneous determination of its appurtenance to the main and non-main subspecies, differentiation of the altai biovar central asiatica subspecies separately. Materials and methods. Primer sets for specific genetic markers have been selected using the VectorNTI 10 software, optimal conditions for PCR were determined for RotorGene devices. To assess the specificity and sensitivity of the developed set of reagents, 44 strains of microorganisms were used, of which 19 were Yersinia pestis strains and 25 strains of heterologous microorganisms. The diagnostic sensitivity of “GenPest-subspecies/Altai-RGF” is 98.6 % with a confidence level of probability of 91 %. The diagnostic specificity of “GenPest-subspecies/Altai-RGF” is ≥ 99 % with a confidence level of 91 %. Results and discussion. A medical product “A set of reagents for the detection and differentiation of plague pathogen strains of the main and non-main subspecies (altai biovar, subspecies central asiatica exclusively) by the polymerase chain reaction with hybridization-fluorescent registration of results in real-time mode (GenPest-subspecies/Altai-RGF)” has been developed. The set of reagents passed the state registration in accordance with the established procedure. The use of the developed set of reagents is relevant for the Gorno-Altai high-mountain plague focus of the Russian Federation and the adjacent part of Mongolia.

The aim of the study was to characterize and carry out molecular-genetic identification of Yersinia pseudotuberculosis strains isolated in the Sarydzhaz high-mountain focus of the Tien-Shan.Materials and methods. The Y. pestis and Y. pseudotuberculosis cultures were differentiated according to their sensitivity to diagnostic bacteriophages, motility, urea fermentation, and pesticin production. Whole genome sequencing was performed using the Ion S5 XL System (Thermo Fischer Scientific). Ion Torrent Suite software package, 5.12 and Newbler gsAssembler 2.6 were used for data processing and de novo assembly of raw read sequences. The dendrogram was constructed using the Maximum Likelihood method applying the PhyML 3.1 software.Results and discussion. Strains of Y. pseudotuberculosis were isolated from museum mixed with Y. pestis cultures obtained from marmots in the Sarydzhaz high-mountain focus. According to whole genome SNP analysis, they belong to the phylogenetic group O:3 serovar, which is widespread throughout the world. Y. pseudotuberculosis is often present in mixed with Y. pestis cultures, isolated from rodents, which should be taken into account when carrying out laboratory diagnostics of plague in natural foci.

The aim of the study was a comprehensive analysis of the phenotypic and genetic properties of Yersinia pestis strains isolated in the Sarydzhaz and Upper-Naryn high-mountain foci of the Tien Shan in 2019–2020; determination of the present-day population structure and areal of these highly virulent strains of the plague pathogen.Materials and methods. Studies of biochemical properties (fermentation of carbohydrates, nutritional requirements), virulence (in vitro and in laboratory animals), molecular-genetic analysis and whole genome sequencing of Y. pestis strains isolated in the Sarydzhaz and Upper-Naryn high-mountain foci in 2019–2020 have been carried out. We used Y. pestis strains from the foci of the Tien Shan and Pamir-Alai dated 1928–2016 for the comparison. Whole genome sequencing was performed using the Ion S5 XL System. Phylogenetic analysis was performed on the basis of 1443 identified core SNPs in 36 Y. pestis strains of various phylogenetic lines included in the analysis. The construction of dendrograms was carried out using the Maximum Likelihood algorithm, PHYML program, HKY85 model.Results and discussion. It is established that all Y. pestis strains isolated in the Sarydzhaz and Upper-Naryn high-mountain foci in 2019–2020 belong to the 0.ANT5 phylogenetic branch of the ancient biovar of the main subspecies. Genome-wide sequencing revealed the presence of two 0.ANT5 clones, the first of which consists of strains from the basin of the river Kooylu in the Sarydzhaz focus, dated 2020. The second powerful clone includes the strains of 2012–2020 isolated in the Sarydzhaz and Upper-Naryn foci. The high virulence of the isolated strains has been shown. It was concluded that further study of the territories of the highmountain foci of the Tien Shan and Pamir-Alai is necessary to establish the current boundaries of the 0.ANT5 areal, as well as to identify the circulation areas of Y. pestis of other phylogenetic lineages.

Objective of the study was to analyze phylogenetic relation of the strains and dissemination routes of Yersinia pestis in Kara Kum desert focus in XX century.Materials and methods. We have carried out whole genome sequencing of 8 Yersinia pestis strains isolated in Kara Kum desert focus between 1949 and 1985. We also used nucleotide sequences of 16 Y. pestis strains from neighboring foci of East and North Caspian Sea region dated 1917–2002, sequenced at the premises of RusRAPI “Microbe” on Genestudio Ion S5XL platform (Thermo Fischer Scientific). Phylogenetic analysis was performed based on 1720 identified core SNPs. Phylogenetic dendrogram was constructed using Maximum Likelihood algorithm, PhyML v. 3.1 software package, and nucleotide substitution model GTR.Results and discussion. On the basis of whole-genome SNP analysis of Y. pestis strains from Kara Kum desert focus, West and North Caspian Sea region foci analysis of plague agent populations circulating in Turkmenistan in XX century was conducted. It has been demonstrated that three separate waves of Y. pestis dissemination swept the territory of Kara Kum desert focus. The origin of the first one, registered in 1912, plague outbreak in south-eastern part of the focus and its etiological agent remain unspecified so far. The second wave is associated with distribution of North-Aral 2.MED1 population of medieval biovar in 1949–1970, which led to the emergence of epizootic activity in 1949, as well as subsequent cases and outbreaks of plague in this territory in the middle of the XX century. In the mid-second half of the past century, one more 2. MED1 dissemination wave from the North Balkhash region hit the Kara Kum desert focus. This central-asian population has also taken root in the natural biocoenosis of the Kara Kum focus. The data obtained testify to active processes of distribution of Y. pestis of medieval biovar in East Caspian Sea region in southern sub-zone of Eurasian deserts in XX century.

Objective of the work was to identify molecular-genetic peculiarities, to conduct whole genome sequencing and phylogenetic analysis of Yersinia pestis strains isolated inVietnam between 1962 and 1989.Materials and methods. We have studied the properties of 50 Y. pestis strains, carried out whole genome sequencing of 18 and fragment sequencing of 32 strains from Vietnam. Phylogenetic analysis was performed on the basis of whole genome SNPanalysis by 1391 identified SNPs. Whole genome SNP-analysis and search for marker SNPs were conducted applying Wombac 2.0 software package. Phylogenetic diagram construction was done using Maximum Likelihood algorithm.Results and discussion. Several phylogenetic branches and Y. pestis populations coinciding with geographical and historical dissemination of the strains have been distinguished. The major part of the strains from Vietnam falls under the branch designated by us as 1.ORI2v. Two strains form a separate branch together with the strain from India belonging to 1.ORI2 line, one more strain, 55-801 Saigon, is set among the strains of 1.ORI1 line. Based on the data obtained and evidence from the literature sources it can be assumed that introduction of plague into Vietnam occurred through several waves: Nha Trang city in 1898, by sea; north provinces of the country – 1908. The second wave of Y. pestis dissemination across the territory of Vietnam began in 1960s with the emergence of the strains from the natural plague focus in Yunnan province, China.

Objective of the study was to conduct phylogenetic analysis of Yersinia pestis strains of the main subspecies, belonging to antique biovar, phylogenetic branch 0.ANT, isolated in XX – early XXI centuries in the foci of Tien-Shan and Pamir-Alay to identify the regularities of spatial-temporal circulation of plague agent in the territory of the foci. Materials and methods. We have carried out whole genome SNP-analysis of Y. pestis strains of antique biovar, isolated in natural foci of Kyrgyz Republic in 1928–2016. Phylogenetic investigation is based on 1646 identified core SNPs in 51 included in the analysis strains of different phylogenetic lines. Phylogenetic tree was constructed using Maximum Likelihood algorithm, PHYML software package, and HKY85 model. Results and discussion. All 29 studied Y. pestis strains isolated between 1928 and 2016 in the foci of Tien-Shan and Pamir-Alay fall under the phylogenetic branches 0.ANT3 and 0.ANT5 of antique biovar of the main subspecies. Strains of 0.ANT3 branch were collected predominantly in Aksay and Alay foci, while strains of 0. ANT5 – in Upper-Naryn and Sarydzhas foci of Tien-Shan. Strains of phylogenetic lines 0.ANT1 and 0.ANT2 were not found in the foci of Kyrgyz Republic. According to the results of whole genome SNP-analysis, Y. pestis strains isolated in the XXI century belong to phylogenetic branch 0.ANT5. This branch also comprises a strain obtained from a patient (lethal case) in Sarydzhas plague focus in 2013. Genetic homogeneity of the modern strains of 0.ANT5 branch and their wide dissemination testify to the extension of the areal of this population and activation of Tien-Shan foci, caused by climate warming. For the first time ever, genomes of Y. pestis 0.ANT5 branch, isolated in XXI century, including clinical strain dated 2013, have been sequenced. We have obtained the whole-genome sequence of Y. pestis strain, 0.ANT3 branch, which caused pneumonic plague outbreak in Tien-Shan in 1928.

Objective of the study was to determine the origin of Y. pestis strains that widely disseminated in natural plague foci of Northern, North-Western Caspian Sea region and Fore-Caucasus in the second half of the XX century. Materials and methods. We have carried out the investigation of properties and whole genome sequencing of 22 Y. pestis strains, isolated between 1923 and 2003 in five natural foci of the souslik type, situated in Northern and North-Western Caspian Sea region and Fore-Caucasus. Phylogenetic analysis was conducted using the data of whole genome SNP-typing, based on 1348 identified SNPs. The search of SNPs in the core genome was performed with the help of Wombac 2.0 software. Phylogenetic relations were analyzed using Maximum Likelihood dendrogram, GTR model. Results and discussion. All the studied strains from the foci of Northern, North-Western Caspian Sea region and Fore-Caucasus fall into phylogenetic branch 2. MED1of medieval biovar. On the basis of whole genome SNP-analysis, existence of two groups of closely related strains, comprising the strains dated 1923-1945 and 1962-2003, has been revealed. The predecessors of the 1962-2003 strains from the Northern, North-Western Caspian Sea region and Fore-Caucasus on the phylogenetic tree are the strains from Northern Aral Sea region that go back to 1945. It testifies to the fact that synchronized activation of a group of natural foci in Caspian Sea lowland and Fore-Caucasus in 1975-1979, after prolonged 20-37 year long breaks, could be caused by the dissemination of the strains from the Northern Aral Sea region. The data of epizootic observations indicate that activation of Volga-Ural sandy plague focus in 1960s preceded the start of registration of plague epizooties in the territory of natural foci of souslik type in Northern, North-Western Caspian sea region and Fore-Caucasus.

Objective of the study was to construct the recombinant Y. pestis strain producing fluorescent protein GFP and analysis of the prospects of its usage for investigation of interaction of the agent with the protozoa, and also macroorganism of mammals (rodents). Materials and methods. To create the strain producing fluorescent protein GFP, we used the natural Y. pestis strain isolated in 2016 in Gorno-Altai high-mountain plague focus. Fluorescent protein gen was inserted into commercial vector pTurboGFP-B via electroporation. Properties of the obtained recombinant Y. pestis strain 367 pTurboGFP-B were studied using microbiological, biological, and molecular-genetic methods. Results and conclusions. The plague agent strain, containing vector plasmid pTurboGFP-B which encodes synthesis of green fluorescent protein GFP, and being resistant to ampicillin was constructed applying electroporation. The designed Y. pestis strain, producing green fluorescent protein, does not differ from the stock strain by its cultural-morphological, biochemical properties, virulence and survivability in mixed culture with Acanthamoeba castellani. On solid nutrient media, recombinant strain and its subcultures, obtained from infected animals, formed colonies of greenish-yellow color, becoming fluorescent under UV-light. In preparations from animals and from co-cultures with amoeba, fluorescent cells of plague agent were observed. The constructed strain can be utilized as a model one for investigation of interactions between plague agent and cells of protozoa (soil amoeba, nematodes) and mammals under laboratory conditions.

Aim. The present paper provides a comparative analysis of the phylogenetic relationship between Yersinia pestis strains isolated in the Volga-Ural sandy natural focus during the periods of 1912-1945 and 1963-2003, which were characterised by different levels of epidemic activity, in order to identify the spatiotemporal patterns in the circulation of the plague pathogen in the North Caspian region. Materials and methods. We studied the properties and performed whole-genome sequencing of 18 Y. pestis strains from the Volga-Ural sandy focus, along with 12 strains from other foci in the North Caspian and North Aral regions, isolated from 1912 to 2003. The phylogenetic analysis was performed drawing on the whole-genome SNP analysis, which was conducted on the basis of 2188 SNPs identified in the core genome using the Wombac 2.0 program. Maximum Likelihood Dendrogram (GTR model) was used for the analysis of phylogenetic relationships between strains. Results and discussion. All studied strains from the foci of the North Caspian region belong to the main subspecies (biovar medievalis) of the plague pathogen. These are highly virulent and epidemiologically dangerous strains. The whole-genome sequencing and phylogenetic analysis of 30 strains from the Volga-Ural sandy focus, as well as adjacent plague foci, reveal that the strains (biovar medievalis) of two phylogenetic branches - 2.MED4 and 2.MED1 - were spread across the focus under study in the early 20th century. It is confirmed that 2.MED1 strains were the etiological agents of plague outbreaks in the Volga-Ural sandy focus during this period. The study revealed the presence of parallel evolutionary lines in 2.MED1 associated with plague outbreaks in the first half of the last century. In the second half of the 20th and early 21st centuries, the modern evolutionary line of 2.MED1 became widespread in the Volga-Ural sandy focus. The strains of this line are closely grouped, which indicates their close genetic relationship. Only sporadic cases of plague were recorded during this period. Modern strains from the Volga-Ural sandy focus (1963-2003), as well as the strains previously isolated there (1912-1945), do not originate from each other. These strains represent closely related, independent evolutionary branches, extending from the common trunk of 2.MED1. Modern strains originating from those of the North Aral desert focus (1945) form a separate cluster in the dendrogram. This suggests that, following a break in the 1950s, the Volga-Ural sandy focus was recolonised by closely related strains from the North Aral region.

Objective of the study – comparative phylogenetic analysis of Yersinia pestis strains, isolated in Precaspian North-Western steppe focus in 1924–1926, 1972, and 1986–1990 to understand the causes of focal reactivation during different time periods of the XX century.Materials and methods. The work included 30 strains of Yersinia pestis from Precaspian North-Western steppe natural focus and adjacent plague foci. Whole genome sequencing of eight Y. pestis strains from the former was carried out. Also whole-genome sequences of 16 strains from neighboring natural foci were used. Whole-genome sequencing of Y. pestis strains was conducted in Ion PGM system (Life technologies). SNPs search across the core genome was performed using software package Wombac 2.0. Tree diagram Maximum Likelihood, HKU85 model, was constructed to analyze phylogenetic relations.Results and discussion. It is established that in early XX century (1924–1926), strains of phylogenetic branches 2.MED4 and 2.MED1, belonging to medieval biovar, main subspecies, circulated on Ergenin Upland in the Precaspian North-Western steppe natural focus. Later on they became extinct in the territory. It is shown that the strains, isolated on Ergenin Upland in 1972, constituted a common subcluster on the dendrogram with the strains from low-mountain and piedmont plague foci of Caucasus and Transcaucasia, dated the same time period. It was inferred that epizootic manifestations on Ergenin upland in 1972, after a long recess since 1938, were caused by importation of Y. pestis strains from low-mountain natural plague foci of Caucasus and Transcaucasia. It was noted that expansion of Caucasian strains was of short-term character, and plague infected animals have not been found on Ergenin Upland since 1974 (including modern period). It is established that Y. pestis strains isolated in the eastern part of Precaspian North-Western steppe focus between 1986 and 1990, do not have close genetic relation to the strains that circulated on Ergenin Upland in 1924–1926 and 1972. It is determined that each epizootic period (1913–1938 and 1972–1973) in Precaspian North-Western steppe natural focus culminated in the elimination of the circulating Y. pestis strains and rehabilitation of the focal territory.

Plague agent has a complex of adhesines providing for anchoring of the pathogen to target cells in a host organism and in many ways defining the onset, character, and development of the disease. The presence of adhesines ensures translocation of effector proteins into target cells of mammalians. The review covers the literature data, both on the most studied Yersinia pestis adhesines (Ail proteins and pH6 antigen), and on recently identified auto transporting proteins of various classes, involved in adhesion processes (YadBC, Yaps, IlpP). Their significance for plague pathogenesis, genetic determinacy, structure and localization in a cell are also described in the paper. It is noted that plague agent adhesines work at different phases of infection process, have multiple functions and take part not only in anchoring to host cells, but provide for resistance to influence of immune mechanisms of a host too.

Objective of the study is to assess the effectiveness of the methods for molecular diagnostics and identification of Yersinia pestis strains in the field material obtained from Sarydzhas high-mountain focus in Kyrgyz Republic. Materials and methods. Investigated were the samples of the field material, isolated in 2016 in Sarydzhas high-mountain plague focus, using conventional methods of laboratory diagnostics and PCR with hybridization-fluorescent and electrophoretic registration of results. Results and conclusions. It is demonstrated that in a number of cases molecular-genetic method has a higher resolution as compared to conventional methods of laboratory diagnostics of plague. It proves the necessity of wider usage of molecular-diagnostic methods in epizootiological monitoring of plague in natural foci in Kyrgyz Republic.

Objective of the study is to analyze the appurtenance, numbers, and dynamics of interaction of acanthamoeba from soils of GornoAltai plague focus with Yersinia pestis 367 strain, isolated in 2016 in enzootic territory of this focus. Materials and methods. Utilized were soil amoeba from Gorno-Altai high-mountain focus and the strain Y. pestis 367 of the main subspecies of antique biovar, isolated there in 2016. Determination of systematic relation of the isolated amoeba was carried out using PCR with genus specific primers and sequencing of the obtained PCR fragments followed by identification of nucleotide sequences against GenBank database. Localization of Y. pestis cells in acanthamoeba was performed using fluorescent antibody technique by means of Axio Imager Z2 (Carl Zeiss, Germany). Results and conclusions. For the first time ever established has been the presence of Acanthamoeba castellanii in soils of burrows of Marmota altaica in the numbers of up to 300000 cells/gr in Gorno-Altai high-mountain focus. Investigated has been the dynamics of interaction of these microorganisms. Preservation of the agent in vacuoles of endoplasmatic reticulum within 14 days has been revealed. It is an indicative of the possibility of Y. pestis persistence in amoeba of Acanthamoeba subspecies in soil biocoenosis of Gorno-Altai high-mountain plague focus.

Objective of the study is to investigate phenotypic and molecular-genetic features and perform whole genome sequencing of Y. pestis strains isolated in Vietnam. Materials and methods. Studied were phenotypic and genotypic peculiarities of 20 plague agent strains isolated in different prefectures of Vietnam. Carried out was SNP-analysis of the strains, sequenced were genomes of 8 Y. pestis strains. Results and conclusions. Based on the results of studies of differential biochemical characteristics all the investigated strains were attributed to oriental biovar of the main subspecies of plague agent, which was confirmed by the presence of marker indel mutation – deletion of 93 bps in glpD gene. Investigated was also the plasmid composition of the strains. On the basis of the conducted genome sequencing and SNP-analysis appurtenance of 19 out of 20 strains under examination was determined. They belong to 1.ORI2v phylogenetic branch, relative to the strains isolated in Yunnan Province, China, which points to their common origin. Identified was a marker SNP and developed the method of SNP-typing for 1.ORI2v strains from Vietnam.

Objective of the study is to design the DNA-microarray for differentiation of Y. pestis strains of the main and non-main subspecies and biovars of the main subspecies. Materials and methods. Efficiency analysis for the devised means was conducted using 62 Y. pestis strains of various subspecies and biovars, isolated in the natural foci of Russia and neighboring countries. Results and conclusions. Selected have been the DNA-targets, probes and primers – calculated. Enhanced is the method of sub-specific and biovar differentiation of Y. pestis strains by means of DNA-microarray. DNA-chip with “Med24”, “glpD(-93)”, and “45” targets allows for prompt differentiation of the strains of the main and non-main subspecies and biovars of the main subspecies based on the presence and absence of fluorescent signal by the specific for the main subspecies and its biovars DNA-targets.

Aim. Develop a method of differentiation of Y.pestis strains of different subspecies based on PCR with hybridization-fluorescent detection in real-time. Materials and methods. DNA target search for differentiation of subspecies of plague causative agent was carried out by Mauve 2.3.1, Mega 5.0 and BLAST algorithm based on comparison offull-genome sequences of Y.pestis strains. Primers and TaqMan probes were calculated for the DNA targets found, conditions of PCR with hybridization-fluorescent detection - optimized. Results. DNA targets carrying marker mutations for the caucasus, altai, gissar, ulegei subspecies, strains from Talass alpine plague reservoir were detected. The effectiveness of the DNA targets found and the developed approach of subspecies differentiation is confirmed on 101 Y.pestis strains of different subspecies, isolated from natural foci of Russia, near and far abroad. Conclusion. The developed approach based on PCR with real-time detection allows for a rapid and effective differentiation of Y.pestis strains of various subspecies.

Yersinia pestis plasminogen activator, a surface protease (Pla) is encoded by species-specific plasmid pPla. This enzyme belongs to outer membrane protein family and has a ß-barrel structure. Pla protease interacts complexly with homeostasis system of a hostorganism and is an important factor of plague agent virulence. Represented are the literature data on the part of plasminogen activator in the development of bubonic and pneumonic plague. Described is the role of R-form lipopolysaccharide in protease Pla activity manifestation. Plasminogen activator directly contributes to fibrinolysis by activating plasminogen and inactivating plasminogen-1 activator inhibitor (PAI-1) and ά2-anti-plasmin, as well as promoted by thrombin fibrinolysis inhibitor (TAFI). Pro-coagulant Pla protein activity is caused by the destruction of tissue factor pathway inhibitor (TFPI). In addition, given is the information on non-proteolytic functions of plasminogen activator.

Training of specialists in advanced methods of plague laboratory diagnostics and in the safety of works with its agent is associated with the necessity to select strains for educational purposes, allowing for the reduction of risk of laboratory infection at tutorials. Deployment of avirulent strains or those with attenuated virulence is the guiding principle in the case. Objective of the study is to detect basic virulence genes of chromosomal and plasmid localization in plague agent strains - candidates to be used for educational purposes. Materials and methods. Utilized have been 18 strains of Yersinia pestis . Investigations have been carried out with the help of reagent panel for identification of Y. pestis strains using real-time polymerase chain reaction (RT-PCR) with hybridization-fluorescent registration of results (“Gene Yersinia pestis Identification - HFR”). Results and discussion. In the course of the study, the strains, selected as the prospective ones, have been characterized by composition of the key virulence genes of chromosomal and plasmid localization. The operation has been performed by means of the reagent panel for identification of Y. pestis strains using RT-PCR with hybridization-fluorescent registration of results. Consequently to the assessment of molecular-genetic properties of plague agent strains, distinguished have been genetic variants (the genome of which lacks one or more key virulence determinants) which are the most relevant in the reduction of biological risks of training technologies at tutorials within the frames of the educational course “Microbiology and laboratory diagnostics of plague”.

Objective of the study is to conduct phylogenetic investigation of Yersinia pestis strains (medieval biovar) from plague foci of Russia and bordering countries, using SNP-analysis of the genome-wide sequences of these strains. Materials and methods. Carried out has been sequencing of 14 Y. pestis strains, medieval biovar, from 13 natural plague foci of Russia and neighboring states, as well as their comparison to 9 strains of the same biovar, contained in the NCBI GenBank database. Using software products - Wombac 2.0 and Bionumerics 7.1, revealed is the presence of 1875 core SNPs, on the basis of which a dendrogram of phylogenetic relations between medieval strains is constructed. Results and conclusions. In consequence of genome-wide SNP-analysis, it is established that Y. pestis strains, medieval biovar, from plague foci of Russia and bordering states are assigned to 2.MED1 phylogenetic line and fall under two major evolutionary branches, the first one of which includes strains from the Caucasus and Caspian-Sea regions, and the second one - from Central Asia and China. The data obtained can be used for the development of molecular-genetic methods for differentiation of Y. pestis strains, medieval biovar.

Carried out has been genome-wide sequencing of 20 Yersinia pestis strains from all 11 natural plague foci in Russia and some foci in the neighboring states. Phylogenetic analysis on the basis of 1918 core SNPs, identified in the genomes of these strains and 16 Y. pestis strains from NCBI GenBank, has revealed 5 clusters of closely related strains. Taking these clusters into account, up-dated has been subspecific classification of plague agent. New taxonomy has combined 5 subspecies: major (ssp. pestis ), Caucasian (ssp. caucasica ), ulegeisk (ssp. ulegeica ), and two novel ones - Central Asian (ssp. central asiatica ) and Angolan (ssp. angola ). Central Asian subspecie comprises evolutionally related strains, earlier classified as altaica and hissarica, as well as the strains from Talas high-mountain focus in Kirghizia and Uzbekistan, and microtus strains from China. Central asiatica is divided into three biovars - altai, hissar, and microtus. Set forward is a serviceable designation for the strains: 0.PE4a - for altai ones, 0.PE4h - for hissar, 0.PE4t - for talas, and 0.PE4m - for microtus, and also for ulegeica subspecie - 0.PE5.

Identified are the complete nucleotide sequences of two cryptic plasmids - pCKF from the Central-Caucasian high-mountain plague focus and pTP33 - from Tuva mountain plague focus in Russia. It is established that the size of the pCKF is 5.4 kbp, and its G-C pair composition accounts for 38.4 %. The plasmid contains 8 open reading frames, encoding transport and secretion functions, in particular, secretion systems of the VI type. The size of another cryptic plasmid, pTP33, is 33.8 kbp, and G-C pair composition accounts for 50.3 %. It contains 52 open reading frames, the majority of which have been classified as phage proteins. Hence, it is inferred that pTP33 is a ring genome of phage. It also has two genes of two-component protein system, toxin-antitoxin, YoeB/YefM, that affects replication apparatus of bacteria.

Laboratory diagnostics of plague was carried out in compliance with valid operational guidelines and regulations. But its peculiarity consisted in the performance of diagnostic investigations secondary to antimicrobial therapy with application of preparations characterized by the expressed activity towards gram-negative microorganisms, including the agent of plague (ceftriaxone, ciprolet, and amikacin). The studies revealed that under antibiotic treatment during the early phase of infection the most effective method for the laboratory plague diagnostics was PCR. Based on the results of the assay it was possible to establish not only provisional, but also the final diagnosis in a patient. Obtained was genetic characteristics of the strains isolated from the patient and the marmot, withdrawn at the patient’s place, using techniques of molecular-genetic analysis, in particular PCR, multilocus VNTR, and multilocus and genome-wide sequencing. Thereupon the strains were attributed to antique biovar of the main subspecies of plague agent. In addition, close relation to Y. pestis of the main subspecies isolated in the same focus in 2012 and to the strains from Mongolian Altai and Tuvinian mountain focus was determined based on phylogenetic analysis of the isolates.

For the first time ever developed has been the method of identification of genovariants in Yersinia pestis strains belonging to the main subspecies using PCR with hybridization-fluorescent registration of results. A complex of DNA-targets, the most of which are newly detected ones, have been utilized to differentiate between genovariants of antique (0.ANT, 1.ANT, 2.ANT, 4.ANT), medievalis (2.MED, 2.MED0), and orientalis (1.ORI) biovars. Application of these targets in PCR with hybridization-fluorescent registration of results provides for a rapid, effective and reliable classifying of the strains of the genovariants, circulating in various geographical regions. Efficacy of the method has been validated via analysis of 110 Y. pestis strains, isolated in the territory of the Russian Federation, neighboring countries and beyond, including 37 strains of antique, 53 strains of medievalis, and 20 strains of orientalis biovars.

The paper contains the data on the complex phenotypic and genotypic characteristics of Yersinia pestisstrains isolated in Trans-Baikal steppe plague focus before and after the succession of the main carrier which took place in the 1960s. Based on the PCR-analysis and multilocus sequence typing of glpD, melB, napA, rhaA, and iclR genes, encoding differentially significant properties such as glycerin, melibiose, rhamnose fermentation, isocitrate lyase production, and denitrification activity, developed are the proofs of affiliation of the studied strains to the biovariant antiqua of the plague agent main subspecies. Multilocus VNTR-analysis by seven locuses of variable tandem repeats – ms01, ms04, ms06, ms07, ms46, ms62 – has been used to establish genetic relations between the strains. Thus on the basis of the results obtained, it is established that the strains of the plague agent, isolated in the Trans-Baikal steppe focus in various periods of its existence, form one common branch on the phylogenetic tree of Y. pestis evolution.

Designed is an optimized scheme for SNP analysis of the genome wide sequences of plague agent strains, based on the consecutive algorithm of clusterization of individual intraspesific Y. pestis groups. The scheme comprises 680 singular polymorphic nucleotides (SNPs), the usage of which provides not only for the clustering of closely related strains, but for the determination of phylogenetic bonds between groups of strains and order of their divergence from the common tree-trunk of the plague agent evolution. Based on the consecutive 680-SNP algorithm, carried out is the analysis of genome wide sequences of C-627 Y. pestis strain isolated in Central-Caucasian high-mountain focus (Russia), strain 1454 isolated in Altay mountain focus (Russia), strain 231 (708) – Aksay high mountain focus (Kirgizia), and vaccine EV NIIEG (Research Institute of Epidemiology and Hygiene) strain, applied for immunization in the Russian Federation and in a number of other countries. Identified are the genome variants of the strains: 1. ORI3 (EV NIIEG), 2. MED0 (C-627), 0.ANT3 (231(708)), 0.ANT4 (1454), and their place in the scheme of Y. pestis global variation. Evidenced is the absence of the chromosomal region responsible for pigmentation in the genome wide sequence of the vaccine strain EV NIIEG, which testifies to its safe use for specific plague prophylaxis.

Studied is the nutrient demand for growth factors among 185 Yersinia pestis strains of the main and non-main subspecies ( altaica, caucasica, hissarica , and ulegeica ) isolated in 38 natural plague foci of Russia and neighboring states. Revealed is the fact that all the strains of Yersinia pestis main subspecies manifest equal dependence in growth on three amino-acids such as methionine, phenylalanine, and threonine, while strains isolated from certain natural foci have an additional demand for cysteine, leucine, and arginine. Strains of non-main subspecies differ in their nutrient requirements both from the strains of the main subspecies and among themselves. Strains of subspecies caucasica stand in need of thiamine and such amino-acids as phenylalanine, tyrosine and arginine; subspecies altaica – phenylalanine, arginine and leucine; subspecies hissarica – phenylalanine, leucine and methionine; ulegeica – phenylalanine. Detected is a number of mutations that lead to auxotrophy in Yersinia pestis strains of different subspecies.

In non-pigmented and plasmid-deprived mutants – isogenic variants of highly virulent Yersinia pestis 231 strain – studied is the mechanism of biofilm formation on biotic surfaces, both in vitro (on the laboratory model of nematode Caenorhabdiitis elegans) and in vivo (inside the alimentary tract of Nosopsyllus laeviceps flea). It is determined that spontaneous loss of ability to form biofilms and generate pigmented colonies in the mutants is probably caused not only by the deletion of the whole chromosome pigmentation fragment, but also by a point(single base) mutation in structural hms operon. It is demonstrated that the absence of pCad, pFra or pPst plasmids does not have an impact on the ability of plasmid-deprived mutants to form biofilm on the cuticle of nematode C. elegans.

This paper represents a review of the current literature data concerning general principles of bacterial biofilm formation, stages of biofilm production and its structural and functional organization, as well as the data concerning involvement of different enzyme systems with the process of biofilm functioning. Carried out is the analysis of the data on the peculiarities of biofilm formation by pathogenic Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica. Displayed are the data on the role of hmsHFRS-operon genes, located in pigmentation chromosomal region of Y. pestis, in the process of biofilm structure formation, and the data on its regulation by hmsP and hmsT genes. Summarized are the results of the recent years' investigations devoted to the genetic determinancy of the plague agent biofilm formation processes, and in particular to the involvement of genes encoding synthesis of lipopolysaccharide (waaA, yrbH and gmhA) and polyamines (speA and speC), as well as rcsA and phoP-phoQ regulatory genes with biofilm formation procedures. Represented are the results of our own investigations concerning the studies of peculiarities of biofilm formation by plague agent strains of different subspecies on the abiotic surfaces and on the nematode model - Caenorhabditis elegans. Discussed is the role of the biofilm in the complex life span of the plague agent. Noted is biofilm significance not only for facilitation in plague transmission by fleas, but also for Y. pestis preservation in the environment, out of the organism of a warm-blooded host.