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We announce the whole genome hybrid sequences of 11 highly virulent Yersinia pestis strains of the ancient phylogenetic branch 0.ANT5, one of the closest to the strains of the First Plague Pandemic. Nine strains were isolated in 2013-2023 and two in 1953 and 1971 in the Tien Shan plague focus in Kyrgyzstan., Competing Interests: The authors declare no conflict of interest.

The 2.MED1 phylogenetic branch of Yersinia pestis of the medieval biovar became widespread in the Caspian Sea region, the Caucasus, and the Northern Aral Sea region in the 20th century, causing outbreaks and epizootics of plague there. Some of the formed natural foci of 2.MED1 still show epizootic activity and retain their epidemic potential. In this work, we carried out a phylogenetic analysis of 46 Y. pestis strains of the medieval biovar isolated in the Caucasus, the Caspian Sea, and the Northern Aral Sea regions during epidemic outbreaks and epizootics from 1922-2014. The obtained phylogenetic data, together with epidemiological and epizootological data accumulated over a period of about a hundred years, indicate the presence of two waves of penetration of the 2.MED1 branch into the Caucasus. The first occurred, apparently, in the first half of the 20th century as a result of the penetration of 2.MED1 from the foci of the Northern and North-Western Caspian Sea. The second wave was caused by the spread of 2.MED1 from the Northern Aral to the foci of the North-Western, Northern and Eastern Caspian Sea regions at the beginning of the second half of the 20th century, followed by introduction into the Pre-Caucasus and Transcaucasia. The rapid spread of 2.MED1 could be associated with the transfer of the pathogen by land and sea transport in the process of economic activity of the population., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Eroshenko et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

We announce the genome sequences of five historical highly virulent Yersinia pestis strains of the phylogroups 2.MED4 and 2.MED1 of the medieval biovar. They were the etiological agents of plague outbreaks with high mortality rates in the Northern Caspian Sea region at the end of the 19th century and beginning of the 20th.

According to the whole genome SNP analysis of 38 Yersinia pestis strains isolated in the foci of the Northern Caspian and Northern Aral Sea regions in the 20th-early 21st centuries, between 1912 and 2015, the spatial and temporal structure of the 2.MED population of a medieval biovar in this region was determined. A phylogenetic branch 2.MED4 was identified which preceded the 2.MED1 branch that diverged later. 2.MED1 strains became the etiological agent of high-mortality plague outbreaks that occurred in the Northern Caspian region at the beginning of the 20th century. Later in the 20th century, the 2.MED1 branch became widespread in the Caspian Sea region, Caucasus, and vast areas of Central Asia. Based on the data of phylogenetic analysis, as well as epidemiological and epizootiological data, we reconstructed the paths of spread of the 2.MED1 branch in the Northern Caspian Sea region and in the Northern subzone of the Central Asian deserts. It is shown, that the reason for the activation of plague foci in the Northern Caspian region in the second half of the 20th century after a long inter-epizootic period caused by cyclical climate warming was the return of 2.MED1 from the foci of the Northern Aral Sea region. This led to the formation of stable plague foci in the Northern Caspian Sea region and Pre-Caucasus, which manifested epizootic activity in the second half of the 20th and early 21st centuries., Competing Interests: The authors have declared that no competing interests exist.

The established phylogeny of the etiological agent of plague, Yersinia pestis , is not perfect, as it does not take into account the strains from numerous natural foci of Commonwealth of Independent States (CIS). We have carried out PCR and SNP typing of 359 strains and whole genome sequencing of 51 strains from these plague foci and determined the phylogenetic diversity of the strains circulating here. They belong to 0.ANT3, 0.ANT5, 2.ANT3, 4.ANT branches of antique biovar, 2.MED0, 2.MED1 branches of medieval biovar and to 0.PE2, 0.PE4a. 0.PE4h, 0.PE4t branches. Based on the studies of 178 strains from 23 plague foci of CIS countries, it was determined that the population structure of 2.MED strains is subdivided into Caucasian-Caspian and Central Asian-Chinese branches. In Central-Caucasian high-mountain plague foci in the Russian Federation (RF) the most deeply diverged branch of medieval biovar, 2.MED0, has been found. With the data obtained, the current population structure of Y. pestis species has been refined. New subspecies classification is developed, comprising seven subspecies: pestis, caucasica (0.PE2), angolica (0.PE3), central asiatica (0.PE4), tibetica (0.PE7), ulegeica (0.PE5), and qinghaica (0.PE10).

Fifty six Yersinia pestis strains, isolated over the period of more than 50 years in three high-mountain foci of Kyrgyzstan (Tien Shan, Alai, and Talas), have been characterized by means of PCR and single nucleotide polymorphism (SNP) typing methods. Seven of these strains were also characterized by means of whole genome sequencing and genome-wide SNP phylogenetic analysis. It was found that forty two strains belong to 0.ANT2, 0.ANT3 and 0.ANT5 phylogenetic branches. From these, strains of 0.ANT2 and 0.ANT3 branches were earlier detected in China only, whereas 0.ANT5 phylogenetic branch was identified for Y. pestis phylogeny for the first time. According to the results of genome-wide SNP analysis, 0.ANT5 strains are ones of the most closely related to Y. pestis strain responsible for the Justinianic Plague. We have also found out that four of the studied strains belong to the phylogenetic branch 2.MED1, and ten strains from Talas high-mountain focus belong to the phylogenetic branch 0.PE4 (sub-branch 0.PE4t). Established diversity of Y. pestis strains and extensive dissemination of the strains pertaining to the 0.ANT branch confirm the antiquity of the mentioned above plague foci and suggest that strains of the 0.ANT branch, which serve as precursors for all highly virulent Y. pestis strains, had their origin in the Tien Shan mountains.

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 of full-genome sequenc- es 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 Ypestis 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 Ypestis strains of various subspecies.

There is evidence that in 1923-2014 the sharp aggravations of the epizootic situation of plague in the area of its Caspian sandy natural focus after long interepizootic periods are in time with the ups of the Caspian Sea in the extrema of 11-year solar cycles. There were cases of multiple manifestations of plague in the same areas in the epizootic cycles of 1946-1954, 1979-1996, 2001, and 2013-2014. The paper considers the possible role of amebae of the genus Acanthamoeba and nematodes, the representatives of the orders Rhabditida and Tylenchida in the microfocal pattern of plague manifestations.

An analysis of a 5.4-kbp cryptic plasmid detected in the course of whole-genome sequencing of the Yersinia pestis medieval biovar isolated in the Russian Central Caucasian high-mountain plague focus was performed. The identification of the nucleotide sequence of this cryptic plasmid and its structural and functional analysis revealed that it contained eight open reading frames, among which the following genes were identified: the rep gene of a replication protein, the virB6 gene of a type-IV secretion system inner membrane protein, the virB5gene of the type-IV secretion system minor pilin, and a number of genes probably associated with secretion and transport. A general analysis of the pCKF plasmid DNA showed that the adenine content was 28.34%, the cytosine content was 20.5%, the guanine content was 17.87%, and that of thymine was 33.28%, while the total G+C content appeared to be 38.38%. The G+C content of the chromosome of the Y pestis strain C-627 is 47.6%, which indicates that the pCKF plasmid was obtained from a microorganism equally-phylogenetically distant from the Yersinia bacteria andany other bacteria from the Enterobacteriaceae family. A comparison of the amino acid sequences.of hypothetical proteins encoded by pCKF plasmid with analogous proteins encoded by other bacteria was carried out. The possible contribution of the pCKF plasmid to the maintenance of the most ancient known phylogenetic line of Y. pestis medieval biovar, 2.MEDO, was discussed.

The genetic diversity of Yersinia pestis strains from the Mongolian natural plague foci has been investigated. A total of 32 strains isolated from western, eastern, and central aimaks, as well as from the territory of the Gobi region, have been studied. Twenty-four strains belong to the main Y. pestis subspecies, while eight belong to other subspecies. There is only one strain of biovar medievalis (genovariant 2.MED1) among the strains of the main subspecies, while the rest of the subspecies belong to the biovar antiqua. Biovar antiqua strains are split into three groups. Strains from the eastern part of the country were classified as genovariant 2.ANT3, and those from the western and central regions were classified as genovariant 3.ANT2, which was endemic for Mongolia. One strain from the Bayan-Ulegeiskii aimak had the rare genovariant 4.ANT. None of the strains of the biovar antiqua belonged to its ancient 0.ANT branch, which is inconsistent with the commonly accepted idea that ancient marmot's plague agent race originates from Mongolia. Six out of eight strains of the minor subspecies belonged to the ulegeica subspecies, which are endemic to Mongolia, one strain belonged to the microtus group, and the last belonged to a previously uncharacterized variant of the minor subspecies.

The results of a study on the taxonomy and quantitative abundance of free-living amoebas in soil samples from the Russian plague foci of the northwestern Caspian steppe, the Caspian sand, and the Volga-Ural steppe are presented. Amoebas of the Willaertia and Hartmanella genera, as well as representatives of myxomycetes, were isolated from samples. From these, amoebas of the Acanthamoeba genus predominated and could be as abundantas 300000 cells per 1 g of soil. Sequencing of the 18S rRNA gene region showed that Acanthamoeba from the Volga-Ural steppe focus belonged to the A. castellanii species. Phylogenetic analysis confirmed that amoebas from two other Caspian foci belonged to the species of Acanthamoeba spp.

The genetic basis of the varying ability to reduce nitrate in strains belonging to different biovars and subspecies of plague-causing microbe has been investigated and the inability to reduce nitrate observed in different intraspecies groups of Yersinia pestis has been shown to stem from mutations in different genes involved in the expression of this trait. The absence of denitrifying activity in strains of altaica and hissarica subspecies was not due to a mutation at position 613 of the periplasmic reductase napA observed in the strains of the biovar medievalis of the main subspecies, but rather was due to a mutation in the sequence encoding the nitrate-binding domain of the ABC transporter protein SsuA; a thymine insertion (+T) was detected at position 302 from the start of the ssuA gene. Five strains of biovar antiqua isolated at different times in Mongolia, China, and Africa were shown to lack the ability to reduce nitrate. A PCR test targeting two chromosomal regions containing deletions of 19 and 24 bp in size has been developed for the identification of strains of the biovar medievalis. This test can be combined with the test for the marker mutation in the napA gene for a more reliable detection of Y. pestis strains belonging to this biovar.

Entomoparasitic nematodes are natural control agents for many insect pests, including fleas that transmit Yersinia pestis, a causative agent of plague, in the natural foci of this extremely dangerous zoonosis. We examined the flea samples from the Volga-Ural natural focus of plague for their infestation with nematodes. Among the six flea species feeding on different rodent hosts (Citellus pygmaeus, Microtus socialis, and Allactaga major), the rate of infestation varied from 0 to 21%. The propagation rate of parasitic nematodes in the haemocoel of infected fleas was very high; in some cases, we observed up to 1,000 juveniles per flea specimen. Our study of morphology, life cycle, and rDNA sequences of these parasites revealed that they belong to three distinct species differing in the host specificity. On SSU and LSU rRNA phylogenies, these species representing three genera (Rubzovinema, Psyllotylenchus, and Spilotylenchus), constitute a monophyletic group close to Allantonema and Parasitylenchus, the type genera of the families Allantonematidae and Parasitylenchidae (Nematoda: Tylenchida). We discuss the SSU-ITS1-5.8S-LSU rDNA phylogeny of the Tylenchida with a special emphasis on the suborder Hexatylina.

Here, we report the characterization of 22 clinical toxigenic V. cholerae non-O1/non-O139 strains isolated in the Middle Asia (Uzbekistan) in 1971-1990. PCR analysis has revealed that these strains contain the main virulence genes such as ctxA, zot, ace (CTXφ); rstC (RS1φ); tcpA, toxT, aldA (pathogenicity island VPI), but they lack both pandemic islands VSP-I and VSP-II specific to epidemic strains of O1 serogroup of El Tor biotype and O139 serogroup. Only two of the twenty two toxigenic strains have tcpA gene of El Tor type, one strain has tcpA gene of classical type, while nineteen other strains carry a new variant of this gene, designated as tcpA(uzb).. Nucleotide sequences analysis of virulence genes in toxigenic V. cholerae non-O1/non-O139 strains from Uzbekistan showed that they differ significantly from the sequences of these genes in epidemic O1 and O139 strain indicating that they belong to a separate line of evolution of virulent V. cholerae strains. For the first time it is shown that V. cholerae non-O1/non-O139 toxigenic strains of different serogroups may belong to the same clone.

Contemporary features of distribution of various subspecies and biovars of plague causative agent by landscape-geographical zones and mountain belts on the territory of Russia and other CIS countries are examined. The most widely spread in plain and mountain natural foci were noted to be Yersinia pestis main subspecies medieval biovar strains. Strains of Y. pestis non-main subspecies are spread in mountain landscapes of Altai, Caucasus, Tian Shan. Change of dominating species of rodents considered as the main carriers of plague was noted not to result in change of genetic and biochemical characteristics of Y. pestis strains. Perspectives of study of "micro-focality" of plague are emphasized for deciphering the mechanism of the enzootic.

Literature data and results of our experimental studies on genetic base of biochemical differentiation of Yersinia pestis strains of various subspecies and biovars are summarized in the review. Data on variability of genes coding biochemical features (sugar and alcohol fermentation, nitrate reduction), the differential development of which are the base of existing phenotypic schemes of Y. pestis strains classification, are presented. Variability of these genes was shown to have possible use for the development of genetic classification of Y. pestis strains of various subspecies and biovars.

Aim: Development of the standard algorithm of molecular typing of Yersinia pestis that ensures establishing of subspecies, biovar and focus membership of the studied isolate. Determination of the characteristic strain genotypes of plague infectious agent of main and nonmain subspecies from various natural foci of plague of the Russian Federation and the near abroad., Materials and Methods: Genotyping of 192 natural Y. pestis strains of main and nonmain subspecies was performed by using PCR methods, multilocus sequencing and multilocus analysis of variable tandem repeat number., Results: A standard algorithm of molecular typing of plague infectious agent including several stages of Yersinia pestis differentiation by membership: in main and nonmain subspecies, various biovars of the main subspecies, specific subspecies; natural foci and geographic territories was developed. The algorithm is based on 3 typing methods--PCR, multilocus sequence typing and multilocus analysis of variable tandem repeat number using standard DNA targets--life support genes (terC, ilvN, inv, glpD, napA, rhaS and araC) and 7 loci of variable tandem repeats (ms01, ms04, ms06, ms07, ms46, ms62, ms70). The effectiveness of the developed algorithm is shown on the large number of natural Y. pestis strains. Characteristic sequence types of Y. pestis strains of various subspecies and biovars as well as MLVA7 genotypes of strains from natural foci of plague of the Russian Federation and the near abroad were established., Conclusion: The application of the developed algorithm will increase the effectiveness of epidemiologic monitoring of plague infectious agent, and analysis of epidemics and outbreaks of plague with establishing the source of origin of the strain and routes of introduction of the infection.

Based on the results of computer analysis of nucleotide sequences in strains Yersinia pestis and Y. pseudotuberculosis recorded in the files of NCBI GenBank database, differences between genes argA, aroG, aroF, thiH, and thiG of strain Pestoides F (subspecies caucasica) were found, compared to other strains of plaque agent and pseudotuberculosis microbe. Using PCR with calculated primers and the method of sequence analysis, the structure of variable regions of these genes was studied in 96 natural Y. pestis and Y. pseudotuberculosis strains. It was shown that all examined strains of subspecies caucasica, unlike strains of plague-causing agent of other subspecies and pseudotubercolosis microbe, had identical mutations in genes argA (integration of the insertion sequence IS100), aroG (insertion of ten nucleotides), aroF (inserion of IS100), thiH (insertion of nucleotide T), and thiG (deletion of 13 nucleotides). These mutations are the reason for the absence in strains belonging to this subspecies of the ability to synthesize arginine, phenylalanine, tyrosine, and vitamin B1 (thiamine), and cause their auxotrophy for these growth factors.

Comparative analysis of nucleotide sequences of genes participating in melibiose fermentation and isocitrate lyase production was conducted in 90 natural Yersinia pestis strains of main and non main subspecies. It was ascertained that the lack of the ability to utilize disaccharide melibiose in strains of the main subspecies is caused by integration of the insertion sequence IS285 at 73 bp from the beginning of the structural gene melB that encodes the transport protein galactoside permease. In contrast, strains of non main subspecies (caucasica, altaica, and ulegeica) contain the intact gene melB and are capable of fermenting melibiose. Differences in the manifestation of the other differential trait, production of isocitrate lyase, are connected with the presence of mutation (insertion of two nucleotides +CC) in the regulatory gene iclR encoding repressor protein of the acetate operon, which is the reason for constitutive synthesis of this enzyme. Strains of non main subspecies do not contain mutations in gene iclR, and this correlates in these strains with their capacity for inducible synthesis of isocitrate lyase.

Structural and functional organization of genes responsible for biosynthesis of amino acid methionine, which plays a leading role in cellular metabolism of bacteria, was studied in 24 natural Yersinia pestis strains of the major and minor subspecies from various natural plague foci located in the territory of Russian Federation and neighbouring foreign countries, and also in Y. pestis and Y. pseudotuberculosis strains recorded in the files of NCBI GenBank database. Conservatism of genes metA, metB, metC, metE, and metH as well as regulatory genes metR and metJ involved in biosynthesis of this amino acid was established. Sequencing of the variable locus of gene metB in natural Y. pestis strains of major and minor subspecies revealed that the reason for the methionine dependence of strains belonging to the major subspecies is a deletion of a single nucleotide (-G) in the 988 position from the beginning of the gene, whereas this dependence in strains belonging to subspecies hissarica results from the appearance of a single nucleotide (+G) insertion in the 989 position of gene metB. These mutations are absent in strains of the caucasica, altaica, and ulegeica subspecies of the plague agent and in strains of pseudotuberculosis microbe, which correlates with their capacity for methionine biosynthesis.

The nucleotide sequences of the Tc's insect toxin complex genes have been analyzed in 18 natural strains of the main and non-main subspecies of Yersinia pestis isolated in different natural foci in the Russian Federation, as well as neighboring and more remote countries, as compared to the data on Y. pestis and Y. pseudotuberculosis strains stored in the NCBI GenBank database. The nucleotide sequences of these genes in plague agent strains have been found to be highly conserved, in contrast to those of the pseudotuberculosis agent. The sequences of two genes, tcaC and tccC2, have been found to be almost identical in Y. pestis strains, whereas other three genes (tcaA, tcaB, and tccC1) contain a few mutations, which, however, are not common for all strains of the plague agent. Exceptions are only strains of the Y. pestis biovar orientalis, whose tcaB gene is in a nonfunctional state due to a nucleotide deletion. The results suggest that the formation of the species Y. pestis as an agent of a natural focal infection with a transmissive mechanism has not resulted in degradation of the Tc's complex genes. Instead, these genes are likely to have been altered as the plague agent have been adapting to the new environment.

The biofilm-forming phenotype of 14 isolates from four 'nonmain' subspecies of Yersinia pestis was compared with eight isolates from the more commonly studied 'main' or epidemic subspecies of Y. pestis in this study. The four nonmain subspecies are more geographically limited, and are associated with certain mammalian hosts and regions of the Caucasus and Central Asia, whereas the main subspecies spread worldwide during the historic plague pandemics. With the main subspecies pestis, pigmentation on Congo red medium (CR(+)) correlated with biofilm formation on both abiotic and biotic surfaces. Main subspecies pestis strains that do not produce pigmentation on Congo red medium (CR(-)) have a deletion that includes the hmsF and hmsS genes known to be required for biofilm formation. CR(-) strains of the nonmain subspecies, altaica and ulegeica, differed however from pestis and, while defective for biofilms on the two surfaces, both had intact hmsF and hmsS genes. The presence of rcsA was also investigated and results showed that it occurred with a 30-bp insertion in all forms of the subspecies. These findings suggest that biofilms are regulated differently in altaica and ulegeica than they are in pestis and also indicate that the rcsA pseudogene arose early in Y. pestis evolution, increasing the ability of the strain to form biofilm and thereby increasing its effective transmission.

The nucleotide sequences of the inv, yadA, and ail adhesin-invasin genes were analyzed in 24 strains of the main and nonmain Yersinia pestis subspecies, which were isolated from natural plague foci in Russia and neighbor countries, and ten Y. pseudotuberculosis strains. All of the five plague agent subspecies (main, caucasica, altaica, ulegeica, and hissarica) had the inv and yadA genes altered by insertion of the IS element and a single nucleotide deletion, respectively, as was earlier observed for the Y. pestis strains KIM and CO92. Consequently, the strains lacked functional activity of the Inv and YadA proteins. The ail gene of the main and ulegeica subspecies had a missense mutation, which replaced Val138 with Phe in the Ail protein. The strains of the caucasica subspecies had an AGT insertion in the ail gene, resulting in Ser148 insertion in the polypeptide chain. The changes in the ail sequence probably exerted no effect on ail expression, since the strains of all subspecies were resistant to blood serum complement.

Aim: To study biofilm formation in strains of Yersinia pestis isolated in 2009 in Astrakhan region., Materials and Methods: Study of biofilm formation was performed on abiotic surfaces as well as on cuticle of nematode Caenorhabditis elegans. Detection of genes was performed by PCR with specific primers., Results: Study of phenotypic (fermentation of sugars and alcohols) as well as genetic (presence of plasmids, genes of chromosome region of pigmentation) characteristics of Y. pestis strains showed that they are typical for strains isolated in Astrakhan region. All isolated in 2009 strains formed well developed biofilm on abiotic surfaces and cuticle of C. elegans nematode. They contained genes of hms operon and regulatory genes hmsT and hmsP, which are necessary for formation of pigmented colonies on Congo red medium as well as biofilm on abiotic and biotic surfaces., Conclusion: Strains of Y. pestis isolated in 2009 in Astrakhan region formed well developed biofilm on different types of surfaces, which could facilitate their survival in complex parasitic biocenosis of plague natural focus.

Aim: To compare biofilm formation in main and non-main subspecies of Yersinia pestis strains as well as in Yersinia pseudotuberculosis strains and to study influence of different genes on expression of this characteristic in different subspecies of Y. pestis., Materials and Methods: Study of biofilm formation was performed bygrowing cultures on LB broth in polystyrene Petri dishes with subsequent staining of biofilms formed on the dishes' bottom with crystal violet as well as by electron microscopy. Pigment-sorption sign was detected on differential medium with Congo red., Results: It was shown that the majority of Y. pestis strains and all strains of Y. pseudotuberculosis form well-expressed biofilms on abiotic surface. Formation of biofilms by Y. pestis strains is clearly correlateswith their ability to form pigmented colonies on solid medium with dyestuff. Genes which according to literature data are necessary for biofilm formation by Y. pestis and Y. pseudotuberculosis were found in genome of non-main species., Conclusion: Ability of Y. pestis strains belonging to main and non-main subspecies to form biofilm on abiotic surface was revealed.

Aim: To perform a comparison of genetic characteristics of vaccine strain EV and its putative "virulent derivatives", obtained after passages through highly susceptible animals, in order to identify the strains-"revertants" and establish their possible origin., Materials and Methods: Yersinia pestis EV strains and its putative "virulent derivatives" were used in the study. Polymerase chain reaction and DNA-DNA hybridization were used for genetic analysis., Results: Comparison of several genetic characteristics of vaccine strain EV and its putative "virulent derivatives" allowed to establish that virulent "revertants" are not derivatives of vaccine strain EV because they do not belong to East biovar, do not have ribotype characteristic for EV strain and contain pigmentation area, which is absent in vaccine strain., Conclusion: Obtained results evidence against possibility of reversion of vaccine EV strain to virulent forms in organisms of highly susceptible animals and confirm its safety for vaccination.

Microbe Russian Anti-Plague Research Institute, Saratov, Russia The literature data and experimental results of the authors on the molecular basis of plague agent interaction with invertebrates are discussed. The details of the plague agent life cycle, its genome organization, and molecular genetic mechanisms of its survival in flea vector and on the nematode cuticule are discussed. The experimental data about the ability to form biofilms at abiotic and biotic surfaces in the Yersinia pestis strains of the main and non-main subspecies are presented. Mechanisms of horizontal and vertical transmission of plague agent are considered. The suggestion about participation of the new member in the complex parasitic biocenosis (nematode, vector parasite) is put forward.

Comparative analysis of distribution of pseudogenes (YPO1582, YPO1728, YPO1967, and YPO4008) of strains of basic and supplementary species of the plague infection agent and pseudotuberculosis infection agent was performed. The genome of basic subspecies of plague infection agent species strain contains 3 different variants: intact genes, genes with IS-element inserts, or individual fragments. The pseudogene profile can be used as genetic marker of the Y. Pestis strains of basic subspecies from natural foci of plague. Strains of supplementary subspecies of Y. Pestis and Y. pseudotuberculosis contain these genes as the wild-type gene alleles. In addition to other factors, this fact can be regarded as evidence of ancient origin of plague infection agent supplementary subspecies and their similarity to pseudotuberculosis infection agent.

Structural and functional analysis of the araN gene involved in regulation of expression of diagnostically significant symptom (arabinose fermentation) was performed in the Yersinia pestis microorganism. Lack of arabinose fermentation in the Altai substrain, Hissar substrain, and Talas strains was shown to be due to solitary nucleotide insert into the araN gene. The insert is in the position 763 bp. The strains of the main, Caucasian, and Ulege substrains do not contain this mutation of the araN gene. The absence of the mutation correlates with ability to ferment arabinose.

Prevalence of vcs genes coding the type III secretion system (T3SS) in cholera vibrios of different serogroups isolated in Russia and neighboring countries was studied for the first time. Virulent strains of O1 and O139 serogroups as well as toxigenic Vibrio cholerae strains of other serogroups contained no T3SS genes. Unlike mentioned strains, 29.2% of atoxigenic non O1/non O139 cholera vibrios isolated from patients in Russia and neighboring countries contained the T3SS genes cluster, which might contribute to the pathogenic properties of these strains.

A study of the structural and regulatory genes, determining rhamnose fermentation, that are located in the rha locus of the chromosome of Yersinia pestis main and non-main subspecies and of Yersinia pseudotuberculosis of serogroups I-III was performed. The nucleotide sequence of Y. pestis main subspecies differs substantially from those of non-main subspecies and Y. pseudotuberculosis by the presence of a nucleotide substitution in 671 bp location of rhaS gene resulting presumably in the Y. pestis non-main subsp inability to utilize rhamnose. This results in incapability of Y. pestis non-main subspecies to utilize rhamnose. Other nucleotide substitutions found in Y. pestis non-main subspecies strains influence only upon expression efficiency of this diagnostic criterion.

Study of molecular-epidemiological characteristics of Vibrio cholerae non O1/non O139 serogroup with complete and limited set of virulence genes was performed. Differences of their genes composition as compared to these of O1 serogroup (classic and El Tor biovars) were revealed, which points to their origin from avirulent environmental cholera vibrios.

Analysis of restriction fragment length polymorphism of rRNA genes of Yersinia pestis and Y. pseudotuberculosis strains, circulating in Russian Federation and abroad revealed the effectiveness of ribotyping for differentiation between these microorganisms, as well as for differentiation between different Y. pestis biovars and main and nonmain subspecies of this agent. Use of this method was shown to be promising as a component for the complex molecular typing system of Y. pestis. Variant ribotypes of main and non-main subspecies of Y. pestis strains are presented.

Genetic organization of 52 Vibrio cholerae El Tor biotype preseventh and seventh pandemic strains isolated in various periods was studied by PCR assay and DNA-DNA hybridization. It was established that the genome of most ancient of analyzed strains isolated from a diarrhea patient in 1910 was devoid of CTX and RS1 prophages, vibrio pathogenicity islands (VPI and VPI-2), and pandemic islands (VSP-1 and VSP-2) that contain key virulence genes. The appearance of pathogenic properties in cholera vibrios for the first time causing a local outbreak of cholera in 1937 is connected with the acquisition of VPI and CTX that carried genes tcpA and ctx-AB, respectively, which are responsible for the colonization of small intestine and encode the production of cholera toxin. The appearance of seventh pandemic agent for cholera was shown to correlate with the acquisition by its precursor of two additional blocks of genes VSP-1 and VSP-2. This finding strongly supports the involvement of these genes in formation of the pandemic potential in strains. Molecular typing methods allowed elucidation of differences in the genetic organization between prepandemic and pandemic strains. The detected variability of the genome of contemporary virulent strains may be a reason for the occurrence of etiological agent for cholera with new properties.

New data were obtained concerning cell sensitivity of pathogenic strains of cholera vibrions, which belong to the serogroup O1 of classical biovar, to the temperate bacteriophage K139, the native host of which is Vibrio cholerae O139. Molecular-genetic and biochemical studies showed that phage 139 integrated into the chromosome of strains V. cholerae O1 can change their toxigenic properties. A change in the production of cholera toxin (CT) in lysogens is associated both with an increase in the activity of the toxR regulatory gene and with a distortion of the structure of a chromosomal DNA region that contains a copy of the operon ctxAB encoding CT biosynthesis.

A comparative analysis of the genome of V. cholerae O139 strains isolated in Russia's territory from patients with cholera and from the environment showed essential differences in their structures. The genome of clinical strains possessed all tested genes associated with virulence (ctxAB, zot, ace, rstC, rtxA, hap, toxR and toxT) and the at-tRS site for the CTXp phage DNA integration. As for the O139 V. cholerae chromosome strains isolated from water, 70% of the studied genes (ctxAB, zot, ace, rstC, tcpA, and toxT) and the attRS sequence were not detected in them. A lack of the key virulence genes in O139-serogroup "water" vibrios, including genes of toxin-coregulated adhesion pili. (that are receptors for the CTXp phage), and of the attachment site of the above phage are indicative of that the O139 V. cholerae strains isolated from open water sources located in different Russia's regions are epidemically negligible.

Infection of V. cholerae 01 (classical and eltor biovars) cells with the temperate cholera phage 139 derived from V. cholerae serogroup 0139 followed by integration of the phage genome into the bacterial chromosome significantly increased the production of cholera toxin, the main virulence factor. The level of toxin biosynthesis in the lysogenic V. cholerae classical strain increased 3-fold and that in V. eltor thirty times in comparison with the parental strains. Increased production of cholera toxin was not associated with an increase in the number of copies of genes involved in its biosynthesis but seemed to be due to changes in toxinogenesis regulation.

To find out stable and effective producers of major protective antigens intended for use as components of cholera chemical vaccine against V. cholerae strains of serogroups O and O139, the comparative analysis of the production of cholera toxin, toxin-coregulated pili (TCP), antigens O1 and O139, polysaccharide capsule and outer membrane protein OmpU in different V. cholerae strains groups O1 and O139 has been made. V. cholerae strain KM68, serogroup O1, has been found capable of the production of antigen O1, serovar Ogawa, protein OmpU at a sufficiently high level and the hyperproduction of cholera toxin and TCP, and thus suitable for use in the manufacture of cholera bivalent vaccine as the source of these antigens. Specially selected alysogenic noncapsular strain KM137 of serogroup O139, characterized by a high and stable level of the biosynthesis of this somatic antigen when grown in both laboratory and production conditions, may serve as the produces of antigen O139.

Restriction analysis of temperate cholera phage 139 isolated from Vibrio cholerae P16064, serogroup 0139, showed its DNA to be double-stranded linear with cohesive terminals. DNA-DNA hybridization on nylon membranes revealed that many V. cholerae strains of serogroup 0139 isolated in different regions contained a temperate cholera phage 139 in their genomes. Southern blot hybridization of chromosomal DNA PST-fragments with phage probe showed that the temperate phage 139 was identical to the temperate phage of serogroup II V. eltor. The phage integrated in the chromosome near genes encoding motility (mot) and production of the capsule (cap) and purine (pur). Phage genome is apparently responsible for instability of cap, pur, and mot genes whose products are important for the development of an infectious process in cholera.

A conjugational gene transfer system consisting of donor and recipient strains has been developed for genetic analysis of Vibrio cholerae 0139 serogroup, a new cholera agent. Donor strains constructed using the Tn5-Mob carrying the origin of transfer (ori T) of plasmid RP4 and helper plasmid pRP4-4 were able to perform a directed transfer of chromosomal markers. Recipient strains carried mutations in auxotrophic genes as well as in virulence genes. Based on this gene transfer system, a genetic map of V. cholerae chromosome has been created showing the order of 17 gene markers. Relationship between the genetic structure of V. cholerae 0139 and 01 is discussed.

Conjugational-mating experiments showed mrh genes coding for the synthesis of mannose and fucose resistant hemagglutinin/adhesin and vibrio motility gene mot to be localized in the chromosome of V. cholerae Dacca strain 35 close to tox-2 mutation providing a high level of toxin production and rfb locus which controls 01 antigen synthesis. The results indicate the presence of a block of virulence genes within ura-pur chromosomal region of V. cholerae.

A clinical isolate Vibrio cholerae P16064 serogroup 0139 was shown to produce two different kinds of colonies: opaque encapsulated and translucent devoid of capsule. Low virulence of translucent colonies seems to be due to not only loss of capsule which determines serum resistance, but also to decreased expression of genes controlling the motility, and production of protease and mannose-sensitive adhesion pili. Analysis of the lysogenic properties of the two types of colonies permitted us to propose that simultaneous spontaneous alteration of capsule production and the above-mentioned virulence factors in translucent colonies may be caused by a temperate phage in turbid clones of strain P16064.

The study of 27 V. cholerae strains, isolated from cholera patients and found to be hemolytically inactive, with a view to establish their capacity for the production of cholera toxin has revealed that 4 strains (V. cholerae cholerae Dacca 35, V. cholerae cholerae Dacca 3, V. cholerae cholerae B1307, V. cholerae cholerae J89) produce this protein. The quantitative determination of enterotoxin has been made with the use of GM1 ELISA technique. Strain Dacca 35 has been found to be highly toxigenic and, as regards the amount of exotoxin it produces, no different from V. cholerae cholerae strain 569B, a well-known producer of cholera toxin. In strain Dacca 35 correlation between the capacity of the cells for toxin production and the morphology of colonies has been established. The study has revealed that the chromosome of strain Dacca 35 contains two copies of gene vctAB responsible for the synthesis of cholera toxin.

Two replicons, pOX38 (in F-factor derivative lacking all IS elements) and pCT105 (containing cholera toxin operon cloned in pBR322) have been fused to produce recombinant plasmid, pCO109, which was then introduced into Vibrio cholerae eltor by conjugation. Restriction analysis showed pCO109 to dissociate in V. cholerae cells at a higher frequency than in Escherichia coli strains, its pOX38 component being lost, while the pCT105 component demonstrated relative stability. V. cholerae eltor RV79 (pCT105) produced 4-5 micrograms/ml of cholera toxin. Occasional insertion of cloned vctA, B operon into RV79 chromosome was also observed.

The results of cholera vibrio chromosomal mapping using Vibrio cholerae classica and V. cholerae eltor donor strains obtained with the help of various R. plasmids, are summarized in the paper. A genetic map of V. cholerae chromosome was established showing the order of 35 gene markers. The relationship between the genetic structures of cholera eltor and classical vibrio biotypes is discussed.

Melanin-producing V. eltor mutants obtained by means of the transposon which determines resistance to tetracycline (Tn 10) are described. Gene mel is believed to be localized on the chromosome of V. eltor in the region of markers his trp met ura rif arg ilv.

V. cholerae multiple-labeled mutants 569B with altered toxin production have been obtained by the method of induced mutagenesis with the use of nitrosoguonidine. These mutants can be used for the genetic mapping of tox genes on the chromosome of V. cholerae.