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Background. The study of the phylogeography of the tularemia microbe is not only practical, but also of scientific interest, as it allows us to evaluate the evolutionary processes of Francisella tularensis in modern conditions.Aim. Phylogenetic analysis of strains of the causative agent of tularemia in the Rostov region and identification of erythromycin-sensitive genovariants.Results. A representative collection of 122 cultures, isolated from various sources from 1945 to 2022, was studied. As it was established, all the studied strains of the causative agent of tularemia had typical cultural, morphological, and biological characteristics. The results of the study of bacteria antibiograms showed that all strains were resistant to β-lactams, polymyxin, clindamycin, and sensitive to aminoglycosides, rifampicin, and fluoroquinolones. In relation to erythromycin, only 2 erythromycin-sensitive strains were found. Phylogenetic analysis of 34 European and Russian strains of F. tularensis made it possible to determine whether these strains belong to subgroup B.7 of the main genetic group B.6.Conclusion. For the first time, in the Tselinsky district of the Rostov region, the presence of «atypical» genovariants of the tularemia microbe belonging to subgroup B.7 of the main group B.6 of the subspecies holarctica was registered, according to the modern scheme of genetic typing of F. tularensis subsp. holarctica.

The aim of the work was to identify genotypic features of clonal complexes formed by CTX–VPI+ strains of Vibrio cholerae isolated from water bodies of the Rostov Region in different years, based on bioinformatics analysis of their whole genome sequences (WGSs).Materials and methods. Whole genome sequencing was performed on the MiSeq (Illumina) platform; identification of genetic determinants in WGSs and bioinformatics analysis – by means of BioEdit, BLASTN, BLASTP, CARD, Vector NTI software packages.Results and discussion. The strains were almost identical in each group of 2002-, 2005-, 2007-, 2020-, 2021-, and 2022-isolates and were found at different points of water sampling over a longer or shorter periods of time (from 3 weeks to 2 months). Bioinformatics analysis of WGSs of representative strains divided them into 4 clusters, which can be considered as separate clonal complexes sharing a number of characteristic genetic markers: 1) 2002-isolates containing one SNP in regulatory rpoS gene; 2) 2007-strains carrying tandemly duplicated preCTX prophage; 3) strains isolated in 2020 in Rostov-on-Don and in 2005, during epidemiological complications in the Kamensk district of the Rostov Region, which had a “hybrid” pathogenicity island including the cluster of type 3 secretion system and the nan-nag region of the VPI-2 island, and 9 SNPs in rpoS gene; 4) 2021- and 2022-strains and a single 2018-isolate containing a 9 bp deletion in vchC collagenase gene. Despite the revealed differences, representatives of all clonal complexes possessed sufficient sets of intact determinants of pathogenicity/persistence factors to realize virulent properties and ability to survive in water bodies. Detection of clonal complexes of potentially pathogenic strains in surface water bodies indicates occasional emergence of favorable conditions for their long-term circulation, which emphasizes the importance of constant monitoring of V. cholerae in the territory of Russia.

The aim of the work was to study the structure of the alkyl sulfatase (asu) gene in Vibrio cholerae strains of various serogroups, as well as to compare nucleotide and amino acid sequences of alkyl sulfatases using various methods of bioinformatic analysis.Materials and methods. 483 strains of V. cholerae O1, O139 and nonO1/nonO139 serogroups were employed in the work. The search for the gene, its recurrence, and localization was carried out applying the Blast software. The nucleotide and corresponding amino acid sequences of the gene, as well as its structure, were studied using bioinformatic analysis. Sequencing was performed on the MiSeq (Illumina) platform. The enzymatic activity was detected using a medium, confirming the presence/absence of the gene by PCR in vitro and in silico.Results and discussion. Bioinformatic analysis of the nucleotide and corresponding amino acid sequences of the asu gene has been carried out and its structure investigated. Four functional domains have been identified. In the beta-lactamase domain, a conservative amino acid sequence -HAHADH- has been found in all strains of cholera vibrios, which is part of the Zn2+ binding motif. It has been established that the alkyl sulfatase of cholera vibrios belongs to the family of Zn2+-dependent β-lactamases. Blast analysis has revealed the similarity of nucleotide and amino acid sequences of alkyl sulfatases in representatives of V. cholerae O1 and O139 serogroups (ctxAB+tcpA+) and representatives of the genera Aeromonas and Pseudomonas, which is in the line with the data of 3D modeling of the amino acid sequence structures of the alkyl sulfatase enzyme in these microorganisms. The bioinformatic analysis of nucleotide and amino acid sequences of alkyl sulfatases in cholera vibrios has showed the conservativeness of these sequences in toxigenic strains and the presence of a number of single mutations in the asu gene in atoxigenic ones. The presence or absence of the asu gene has been established by PCR in vitro and in silico and confirmed by the results obtained using the Blast program. It is demonstrated that the presence/absence of the asu gene correlates with the ability/inability of O139 strains to hydrolyze SDS on the medium. These results can be used in studying mechanisms of cholera vibrios adaptation, persistence and pathogenicity.

Six cultures of tularemia microbe from fallen and captured live animals were isolated during epizootiological monitoring in the steppe focus in the south-east of the Rostov Region in 2020 against the background of extensive epizootics in the populations of the common vole Microtus arvalis obscurus and the public vole Microtus socialis.The aim of the work was to develop an SNP-typing scheme and to conduct a comparative study of the phylogenetic relations between Francisella tularensis strains isolated in the Rostov Region (2020) and strains from other regions.Materials and methods. Genome-wide sequencing was performed on the MiSeq Illumina platform. The author’s software GeneExpert, PrimerM and VirtualPCR, written in the Java programming language, were used for the analysis.Results and discussion. The strains of tularemia agent, isolated on the territory of the Rostov Region in 2020, can be allocated to two different clusters. It is established that two strains of tularemia pathogen (F0884 and F0889) isolated in Turkey are genetically close to some isolates circulating in the Rostov Region. A unique INDEL marker characteristic of this group of strains has been identified. The comparison of our proposed typing scheme with the scheme of “canonical” SNPs has showed a fairly good consistency and convergence of results within large clusters, meanwhile using a set of 6626 SNPs allows for differentiating the strains within one canSNP type. It is revealed that the vaccine strain has a common canSNP type with clinical and natural strains. A set of SNP markers has been selected for comparative analysis. A new INDEL marker that enables intraspecific typing of F. tularensis has been discovered and the possibility of its application in vitro and in silico has been comfirmed.

The aim of this study was to develop a new method of intraspecific genetic differentiation of Yersinia pseudotuberculosis, based on the detection of INDEL-markers using PCR. Materials and methods. Analyzed were 308 strains from the NCBI database and 15 strains sequenced within the frames of this study. The nucleotide sequences of the strains were determined using the MiSeq technology platform. The genomes of the strains sequenced in the work, as well as genomes from the NCBI database, were assessed using in silico PCR with 7 pairs of primers designed in the study. As a result of a comparison of genome-wide sequences of 22 Y. pseudotuberculosis strains from the NCBI database, using the author’s software (GenExpert), 7 INDEL-markers were selected that make it possible to effectively distinguish between strains of the causative agent of pseudotuberculosis. Based on these markers, 7 pairs of primers were designed and synthesized for the analysis of different strains using PCR. Analysis of 323 strains in PCR in silico and 70 strains in PCR in vitro allowed for dividing them into 30 genetic groups. Comparison of the results of PCR in silico and in vitro confirmed the possibility of using the proposed primers for intraspecific differentiation of Y. pseudotuberculosis. Based on the data obtained, a dendrogram reflecting the phylogenetic relations of different strains of Y. pseudotuberculosis was constructed. When analyzing the distribution of Y. pseudotuberculosis strains by various clusters and genetic groups, a number of patterns were revealed. Conducted in silico and in vitro PCR show that the proposed method of INDEL-typing can be used for intraspecific genetic differentiation of the causative agent of pseudotuberculosis.

The aim of the work was to summarize the results of cholera monitoring in 2021, to assess current trends in the development of the epidemic process, and to predict the epidemiological situation in the Russian Federation for 2022. It was established that within the period of 2012–2021, 4117264 cases of cholera with the spread of infection across 83 countries on all continents were registered in the world and there was a downward trend in the incidence in Asia and Africa. The dynamics of monthly morbidity in 2021 was associated with emergencies as factors of epidemiological risk. Epidemics and outbreaks of cholera were documented against the background of COVID-19 pandemic and laid a double burden on healthcare systems. At the same time, based on the overview of the results of cholera monitoring in the constituent entities of the Russian Federation, it was shown that the forecast of epidemic well-being given for 2021 was fully justified. It has been determined that the increase in the number of non-toxigenic strains of Vibrio cholerae O1 (67) isolated from water bodies compared to 2020 (25) is mainly due to the appurtenance of a number of isolates to clonal complexes. The study of phylogenetic relation has demonstrated that the detection of strains with genotypes which were previously identified in the isolates evidences the persistence potential. The identification of strains with new genotypes, which were earlier established in the strains circulating in other territories, pointed at the possibility of the occasional importations. The forecast of the epidemiological situation on cholera in Russia for 2022 is associated with the continuous existence of risks of introduction. If these epidemiological risks are not realized, a favorable epidemiological situation is predicted regarding this infection in the country. It is expected that the detection of epidemiologically insignificant strains of V. cholerae O1 in environmental water bodies, along with their clones and/or clonal complexes, will remain, including strains that may be an etiological factor in sporadic cases or outbreaks of disease.

A marker of the epidemic significance of Vibrio cholerae is their toxigenicity. Therefore, much attention is currently paid to the creation of diagnostic preparations for the detection of cholera toxin and assessment of the level of its production. The volumetric immunosuspension agglomeration reaction, carried out with the help of latex diagnosticums, is an analogue of the indirect hemagglutination reaction, an affordable and technically simple method, since it does not require special equipment and can be used when conducting research in the field. The aim of the study was to design a polymeric immunoglobulin diagnosticum for determining cholera toxin and the level of its production by vibrio strains. Materials and methods. Cholera toxin was obtained from the producer strain Vibrio cholerae Classical 569 B. Rabbit serum to the toxin was obtained according to the method selected by the authors. A polymeric diagnostic immunoglobulin antitoxic drug was obtained through sensitizing immunoglobulins from cholera antitoxic rabbit serum on the surface of polyacrolein microspheres with a size of (1±0.1) μm. Results and discussion. The analytical sensitivity of the developed diagnostic preparation with control cholera toxin is 100 ng/ml. It detects cholera toxin in toxigenic strains of Vibrio cholerae in a titer of 1:16 – 1:512, gives negative results with non-toxigenic strains of V. cholerae O1, V. Cholerae nonO1/nonO139, with samples of heterologous cultures, LPS preparations, liquid nutrient medium used for the cultivation of V. cholerae. Thus, a polymeric immunoglobulin diagnosticum has been constructed to detect and quantify the production of cholera toxin by vibrio strains, and its analytical sensitivity and specificity have been established.

Objective of the study was to investigate biological properties and genetic characteristics of tularemia agent strains isolated from natural foci of the Rostov Region in 2020.Materials and methods. Field material from natural foci of the Rostov Region was examined by serological, bacteriological, biological, and molecular-genetic methods. Cultural-morphological, biochemical, antigenic and pathogenic properties of isolated cultures were studied. Protein profles were obtained through MALDI-TOF MS using mass spectrometer Autoflex speed III Bruker Daltonics and Flex Control of Biotyper software. The genetic characteristics of the strains were determined by VNTR and INDEL typing and SNP analysis.Results and discussion. Six strains of tularemia pathogen were isolated from mouse-like rodents using biological method. The investigation of their biological features and data of PCR analysis and INDEL typing with canonical markers showed that all strains are typical representatives of the Francisella tularensis subsp. holarctica biovar EryR. VNTR typing by six genetic loci revealed that all strains belong to four individual genotypes. The strain isolated in 2020 in the Salsky district was identical to the strain which was isolated in the same area in 1989. Based on the whole genome sequencing of two strains, we established that they are closest to the cultures isolated in Turkey (2009, 2012) and Khanty-Mansiysk (2013) by the studied set of SNP markers. Thus, we found that both identical (or closely related) clones of the tularemia agent and new strains with unique genotypes which previously were not described for the Rostov Region can circulate in natural foci of this region for a long period of time.

Objective: To compare the genomic diversity of SARS-CoV-2 strains that were spread in the Rostov region and the Republic of Crimea in the period from March to June 2021.Materials and Methods: A total of 194 samples were sequenced and 186 genovariants were recognized among them using the Pangolin program. Multiple alignment was performed and dendrograms were constructed for the samples belonging to the alpha and delta genovariants.Results: Changes of the predominant genovariants were revealed for territories of the Rostov Region and the Republic of Crimea from March to June. An increasing percent of the alpha variant was observed in both regions during the spring followed by the rapid emergence of the delta variant, which became predominant in June.Conclusion: It was shown that alpha variant samples from the Rostov region share a pool of common mutations, while in contrast, alpha variant samples from the Republic of Crimea are closer to those sampled from Moscow and Moscow region. At the end of May, the delta gene variant begins to be revealed, which is rapidly replacing other lines in all the territories considered in this study.

The aim of the study was to identify INDEL markers and study geographical origin of regional H. pylori strains circulating in the European part of the Russian Federation. The study included 56 strains of H. pylori isolated in three regions of the Russian Federation: Saint Petersburg, Astrakhan and Rostov Regions. Genomic DNA was isolated using a set of Probe NA (DNA Technology, Russia), according to the manufacturer’s instructions. Detection of INDEL markers hp5605, hp6405, hp340, hp1390, hp3660 was performed using PCR. Clustering of the identified INDEL genotypes and building a phylogenetic tree were performed using the BioNumerics 7.6 and GrapeTree software packages. 21 strains from the GenBank database with known geographical origin were used as reference strains. In 20 strains from Saint Petersburg, 13 individual genotypes were identified, while 17 strains belong to the European cluster (hpEurope), 2 strains belong to the hspEAsia cluster and one strain belongs to the hspWAfrica cluster. The most common genotype identified in the European cluster includes six strains from Saint Petersburg and two strains from the GenBank database. For further differentiation of these strains, the VNTR typing method was used, which allowed identifying eight individual genotypes in eight strains. Fifty-six studied russian strains are represented by thirty individual genotypes, which reflects the high heterogeneity of strains circulating in the European part of the Russian Federation. The most frequent genotype is represented by two hpEurope strains, one strain from the Astrakhan region, as well as 5 and 6 strains from the Rostov Region and Saint Petersburg, respectively. The vast majority of Russian strains (52/56) belong to the hpEurope population, while two strains from Saint Petersburg are included in the hspEAsia population, and one strain from Saint Petersburg and the Astrakhan Region is included in the hspWAfrica population. Total, 77 H. pylori strains are represented by 37 individual genotypes with a high diversity index (DI = 0.95), which allows us to consider the proposed INDEL typing method as an independent method for genotyping H. pylori strains. Taking into consideration the complexity of the problem of accurately determining the geographical origin of H. pylori strains, the proposed simple and convenient method of INDEL typing of H. pylori strains, based on an available PCR method becomes very relevant and allows us to conduct an adequate primary analysis of the geographical origin of Russian H. pylori strains.

Introduction. Most of the bacteria exist in natural ecosystems not in the form of free floating cells; but in the form of biofilms attached to the substrate. One of the most ecologically important substrates is chitin. Vibrio cholerae; like most members of the Vibrionaceae family; has a chitinolytic complex and can degrade chitin. The ability of V. cholerae to form a biofilm on chitinous substrates can explain the mechanism of the formation of an ecological niche for the preservation and transfer of the pathogen to new regions with the likelihood of the formation of new foci of cholera.Aim — to determine the ability of V. cholerae to form a biofilm on the chitinous shell of crayfish (Astacus astacus) by means of real-time PCR.Materials and methods. A comparative analysis of the timing of biofilm formation by V. cholerae of different serogroups and toxigenicity was carried out.Results. In the course of the study; it was found that cholera vibrios were shown to be capable of forming a biofilm regardless the serogroup and toxigenicity. However; toxigenic tcpA+ strains have a higher intensity of biofilm formation than nontoxigenic ones; in which the tcpA gene is absent.

Aim of the work – to assess the epidemiological situation on cholera in 2020 and to make a forecast for 2021 based on the monitoring data and analysis of morbidity around the world for the period of 2011–2020. During the period between 2011 and 2020, 4 413 988 cases of cholera were recorded in 97 countries of the world with a general trend towards a decrease in the incidence (coefficient of accuracy of approximation R2 – 0.5705). However, due to the continuing epidemic manifestations of cholera in the endemic countries of Asia, Africa and America, the epidemiological situation on cholera on these continents was characterized as unfavorable in 2020. The emergence of a new “post-Haitian” lineage was observed among epidemically hazardous strains of Vibrio cholerae O1. In 2020, no epidemically dangerous strains of V. cholerae O1, O139 were isolated from humans on the territory of the Russian Federation. 25 non-toxigenic V. cholerae O1 El Tor strains were isolated from environmental objects, eight out of which (ctxA-, tcpA+), according to PCR-INDEL typing and SNP analysis of sequences, belonged to the clonal complex. The results of the analysis of biological properties and phylogenetic relations between the isolated non-toxigenic strains provided the basis for considering the epidemiological situation on cholera in Russia in 2020 as a stable one and a similar forecast of its development in 2021. At the same time, the possibility of importation of this infection from endemic countries cannot be ruled out, as well as the need to carry out a complex of differentiated anti-epidemic (preventive) measures within the framework of the state sanitary-epidemiological surveillance in order to localize and eliminate the imported focus and avoid the spread of the infection.

Aim. To improve the method of the quality assessment of single nucleotide polymorphisms, which are used for SNP-typing, based on the analysis of their distribution in the primary data of whole genome sequencing (reads).Materials and methods. Data of the whole genome sequencing of 56 Vibrio cholerae strains obtained using different types of sequencers were used. The software was developed using Java programming language. Cluster analysis and construction of the dendrogram were performed with the author's software using the UPGMA method.Results and discussion. The «instability» of detection the number of SNP in the genome of cholera causative agent was shown. The method of selection of the SNP list for phylogenetic analysis based on the analysis of the primary data of whole genome sequencing (reads), has been developed. The method of using «control genomes» for cluster analysis of whole genome sequencing data has been proposed.Conclusion. The list of 3198 «stable SNP» for phylogenetic analysis has been composed. Genetic affinity between the non-toxigenic strains that contain the tcpA gene (ctxAB–tcpA+) and preCTX-strains of V. cholerae was shown.

Aim. In this study, we set out to identify the homologues of genes from the msh-cluster in the genomes of non-toxigenic V cholerae, to perform the bioinformatics analysis of their products, as well as to study the adhesive properties of strains containing altered genes. Materials and methods. We analysed 17 clinical strains of non-O1/non-O139 V cholerae and 2 strains of the O1 serogroup isolated from water bodies. Genes belonging to the msh-cluster were identified in the whole genomes using the BLASTN 2.2.29 and BioEdit 7.2.5 programs. Gene translation, comparative analysis of their nucleotide sequences and the amino acid sequences of deduced products were performed using the Vector NTI Advance 11 (Invitrogen). Results and discusssion. In 18 out of the 19 studied genomes we identified gene clusters responsible for production of adhesion pili (mshH-Q) represented by diverse alleles, the majority of which differed from the prototype genes of the msh-cluster in nucleotide composition but had the same localization and arrangement. Only one strain had a cluster that was close to that of the prototype. A bioinformatics analysis of their deduced products indicated that the amino acid sequence of the major MshA pilus subunit is homologous to the prototype only in a short N-terminal region (1-41) while sharing no similarities with the rest of the sequence. Nevertheless, this protein, similar to VcfA described by Kuroki H. et al. (2001) and designated by us as MshA-like, retained a putative pilus domain. A similar pattern was observed in the minor subunits designated as MshC-like. Other minor subunits also retained their characteristic domains. All of the strains agglutinated human erythrocytes (group O) and chicken erythrocytes, and in isolates harboring modified mshA-like and mshC-like genes the reaction was not inhibited by mannose. Since most of the studied strains were isolated from hospitalized patients, it is possible that in non-toxigenic V. cholerae lacking the pathogenicity island VPI, MSHA-like pili may serve as a colonization factor of the human intestine, in contrast to VPI-positive strains. The obtained information provides a basis for experimental verification of this assumption.

Objective of this work was cloning of the Vibrio cholerae nanH gene as part of a plasmid vector, providing expression of foreign genes under the control of T5 promoter, and construction of a E. coli strain – producer of V. cholerae recombinant neuraminidase.Materials and methods. V. cholerae о1 strain served as a DNA donor, pQE30 – as a vector plasmid. The gene was PCR-amplifi ed, the cloning was carried out by means of conventional methods, performance of recombinants and localization of the required protein was determined based on the results of electrophoresis of cell lysates. Neuraminidase activity was identifi ed by fl uorescence in ultraviolet light after incubation with specifi c substrate (4-methylumbelliferyl-N-acetylneuraminic acid).Results and discussion. Recombinant plasmid pNanH, containing the cloned gene nanH V. cholerae, has been constructed. The gene is inserted into BamHI-PstI sites of the polylinker of pQE30. Expression of the cloned gene in the producer strain E. coli JM103pNanH occurs under the control of T5 promoter after isopropyl-1-thio-β-D-galactopyranoside (IPTG) induction. The strain shows neuraminidase activity. The recombinant NanH protein is accumulated in the producer’s cells in two forms. The fi rst form, with molecular mass (MM) of 89.5 kDa, is an unprocessed protein with the hexahistidine block (6His-tag) at its N-terminus, it is located in inclusion bodies. Its percentage is 5.6–6.6 % of the total cell proteins. The second one, with MM of 83 kDa, is found in the periplasmic space and corresponds to the mature NanH, its percentage being 3.4–3.8 %. The total percentage of both forms is 9–10 % of total cell proteins, which allows to consider the strain E. coli JM103pNanH to be a super-producer of the required protein. The strain may be used for purifi cation of NanH preparations for construction of specifi c diagnostic, therapeutic and pharmaceutical preparations as well as for investigation of the protein as a virulence/persistence factor of the pathogen.

Background. Currently, the researches focused on the design of new diagnostic and preventive preparations based on bacteriophages are underway, so it is importatnt to study the biological properties of cholera phages along with their genetic structure. This information is necessary to predict the phage life cycle and assess the prospects of its practical use in experiments, phagodiagnostics and phagoprophylaxis.Materials and methods. The presence or absence of genes characteristic of temperate bacteriophages was tested using a database created by the authors and developed software "PhageAnalyzer", which allows for rapid analysis of bacteriophage genome-wide sequencing data and prediction of their life cycle.Results and discussion. The morphological structure of experimental diagnostic cholera phages is represented by head bacteriophages of various morphogroups. Negative colonies phage differed in diameter, shape and degree of transparency. No genetic determinants of resistance factors and toxins have been found in the genomes of bacteriophages Rostov-1, Rostov-6, Rostov 7, and Rostov M3. Results of phylogenetic analysis demonstrated that the studied experimental cholera bacteriophages resemble headphages from the genus Vibrio, but are unique, since they lie outside “cluster groups”. Vibrio phages Rostov-1 and Rostov M3 are appeared to be lytic. Genes characteristic of moderate bacteriophages were found in cholera phages Rostov-6 and Rostov 7.Conclusion. The experimental cholera bacteriophage Rostov-1 can be used to differentiate cholera vibrion O1 the serogroup of the El Tor biovar, and Vibrio phage Rostov M3 can be used to differentiate the Classical biovar. Both bacteriophages are lytic and promising components for creating prophylactic drugs against cholera. Vibrio phages Rostov-6 and Rostov 7 can be successfully used only in experimental activities, as well as for monitoring cholera vibrions in the environment. Complete genomic sequences are deposited and available in the international database Genbank (NCBI).

Objective: to study the infection of the adult and children’s population of the Rostov and Astrakhan regions by the bacteria Helicobacter pylori, to determine the frequency of H. pylori pathogenicity factors in different age groups of the population.Materials and methods: 118 adults and 112 children were examined. Te presence of H. pylori DNA and the pathogenicity factors of CagA and VacA in the biopsy material of the mucous membrane of the antrum of the stomach was determined by PCR.Results: a signifcantly smaller percentage of H. pylori–positive patients is characteristic of the child population. Te prevalent H. pylori genotype in the infant population is the avirulent genotype Vac s2m2 (60%) (χ2: p

Objective of the investigation was a comparative bioinformatics analysis of rtxA gene translation products of Vibrio cholerae strains isolated from patients and environmental objects. Materials and methods. 32 Vibrio cholerae strains from the Rostov-on-Don Research Anti-Plague Institute collection were used. DNA sequencing was conducted on the MiSeq platform (Illumina), gene identification and analysis was carried out by means of BioEdit 7.2.5, BLASTN 2.2.29, Blastp, Vector NTI Advance 11 software programs. Results and conclusions. The rtxA genes of the studied strains were represented by multiple alleles. AlignX-analysis of their deduced products divided 32 proteins into 3 separate clusters. The first one included proteins of O1 and nonO1/nonO139 strains similar to the prototype, the second – nonO1/nonO139 only, the third – O139. Blastp-analysis revealed that the proteins of the first cluster retained all domains characteristic of MARTX – ACD (actin cross-linking), RID (Rho GTFase inactivation), CPD (cysteine protease) and ABH (alpha-beta hydrolase) which evidences the probability of manifestation of the typical activity. In 3 strains a new Hia domain (of putative adaptation factor) was detected. The proteins of the second cluster lacked ACD but formed an additional RID; two of which lacked ABH too, but in one, VIP2 domain (of actin modification) appeared and in another – Hia. These data are in accordance with the presumption of J. Dolores, K.J.F. Satchell (2013) on the possible converting of actinomodulator MARTX into toxins with other mechanisms of action. The proteins of O139 vibrios shared all specific domains except from ACD. For a number of altered proteins revealed by us full homologues were found in NCBI gene bank, belonging to isolates from other regions of the world. The presence of similar determinants in the genomes of strains of different origin points to non-occasional character of their conservation. It is possible that certain strains “deliberately” alter those genes or switch off the synthesis of the high-molecular toxin completely with a view to energy-saving.

Objective of this work was the cloning of Vibrio cholerae hlyA gene in a plasmid vector providing expression of foreign genes under the control of T5 promoter, and construction of E. coli strain – super-producer of Vibrio cholerae recombinant hemolysin. Materials and methods. V. cholerae о1 strain served as a DNA donor, pQE30 – as a vector plasmid. The gene was PCR-amplified, cloning was carried out by means of conventional methods, productivity of recombinants and localization of the required protein was determined based on the results of electrophoresis of cell lysates. Results and conclusions. A recombinant plasmid pHlyA, expressing the cloned hlyA gene of Vibrio cholerae El Tor under the control of T5 promoter after IPTG induction, has been constructed. Carrying this plasmid strain E. coli M15[pREP4]pHlyA is the super-producer of hemolysin: the content of the product in whole cell lysates is up to 13 %, and in inclusion bodies – up to 17 % of the total cell proteins. The product of the cloned gene, in spite of the absence of proteolytic processing and presence of the hexahistidine block (6His-tag) at its N-terminus, possesses hemolytic activity towards sheep erythrocytes. 6His-tag will provide for obtaining a purified preparation on specific sorbents with a view to create diagnosticums as well as to study the significance of hemolysin as a pathogenicity/persistence factor. The advantages of this producer are the high output of the required protein, inability of synthesis of any accessory biologically active substances, short-term period of biomass growing (4–6 h including induction) and possibility of culturing without sticking to the guidelines for work with the agents of particularly dangerous infections.

Objective of the investigation was a comparative bioinformatics analysis of Vibrio cholerae О1 Cef (CHO cell elongating factor) genes and proteins. Materials and methods. 36 Vibrio cholerae О1 strains from the Rostov-on-Don Research Anti-Plague Institute collection have been utilized. DNA sequencing was conducted on the MiSeq platform (Illumina); gene identification and analysis was carried out by means of BioEdit 7.2.5, BLASTN 2.2.29, BLASTP, MEGA 7, Vector NTI Advance 11 software programs. Results and conclusions. The data obtained confirmed Cef to be rather conserved in choleragenic strains (carrying cholera toxin genes ctxAB as a part of genome-integrated CTX prophage): all of them shared closely related prototype alleles cefС or cefЕ1. The Е1 allele was also revealed in ctxAB– strains carrying the pre-CTX prophage and in a single strain lacking both prophages. In the rest of CTX–/pre-CTX– V. cholerae four novel cef variants, that were not previously described, have been identified, two of which (E2 and E3), belonging to the Russian isolates, appeared to be unique, while for the two others absolute homologues were found in NCBI. In this connection several strains which caused severe cholera-like diseases in humans were placed in the group of cefЕ4 host strains. Since Cef is one of pathogenicity/persistence factors of cholera vibrios, we presume that conservation of its altered variants in the course of natural selection embodies a certain biological sense in respect of possible acquisition of qualities, significant for realization of both pathogenic and persistence potential.

Currently, bacterial small RNAs with regulatory functions are characterized as a heterogeneous claster of highly structured single-stranded RNAs which usually would not be translated into proteins. A large group of small RNAs consists of small regulatory RNAs functioning through the mechanism of complementary interaction between small regulatory RNA bases and bacterial messenger RNAs (mRNA). The review provides the description of direct participation of small RNA mechanisms in realization of pathogenic properties, biofilm formation and virulence regulation in V. cholerae strains. In particular, the Hfq-dependent small RNAs that control the expression of genes responsible for virulence and biofilm formation of V. choleraе, and the impact of small RNA on the secretion system of VI type are presented. We also characterized the small RNAs–controlled process of V. cholerae vesicles formation, which have a great significance for colonization ability of cholera vibrios. In addition, such method of regulation at RNA level as “riboswitches”, (RNA switches) is described.

Objective of the study is the retrospective evaluation of epidemic cholera manifestations in the Republic of Dagestan in 1994, taking into account VNTR-typing and studies of genome peculiarities in the isolated V. cholerae O1, Biovar El Tor. Materials and methods. Utilized have been the data on the infection rates in different periods of the epidemic. The strains have been investigated using VNTR-analysis and whole genome sequencing. Results and conclusions. Heterogeneity of the agent population has been established on the basis of the circulating VNTR-genotypes. The whole-genome sequencing has revealed the presence of B-subunit allele ctxB1 in CTXφ prophage of El Tor V. cholerae O1 strains. The strains isolated in the early and the following stages of epidemic development have been found to be closely related to strains from India, Bangladesh, and Nepal. Some strains possessed a cluster of multiple antibiotic resistance genes, SXTET: floR, strA, strB, sul2 – of “Indian” type – SXT-ICE-Ind, as well as ICE mobile elements, containing determinants of resistance to antibiotics of tetracycline group – tetA. Phylogenetic relatedness of the Dagestan strains to the strains from Asia indicates to the origin of the strains, as well as to the independent cholera importations that took place at different stages of the epidemic. The role of genetically altered V. cholerae O1 El Tor variants with identified peculiarities on molecular-genetic level, as a system-forming factor in the emergence and development of epidemic in the Dagestan Republic, was directly linked to the social conditions.

Objective of the study is to evaluate the role of cadaverine in cholera vibrio adaptation to stress, induced by hypoxia. Materials and methods. Utilized have been 18 V. cholerae strains with different set of pathogenicity determinants. The strains are isolated from patients and from river-water. Results and conclusions. It is demonstrated that under experimental modeling of intestinal tract gas medium, cholera vibrios respond to unfavorable conditions of the environment by producing cadaverine. Its amount coincides with pathogenicity of cultures (it is higher in epidemically significant strains, than in the strains that lack genes of toxin and pilus production). It is established that the level of extracellular cadaverine production is greater than that of intracellular; but correlation of the parameters depends upon the oxygen and carbon dioxide concentration in the environment. Intracellular cadaverine is mainly generated at a high oxygen concentration (10-12 %); while under extreme hypoxia (

In the course of assessment of epidemiological situation around the world in 2015, established has been the tendency to incidence rate reduction, with the annual average tempo - 10.225 % (in reference to 2006). For the first time ever, defined has been equivalent epidemiological year in case of cholera within an interval between 2014-2015.The key epidemiological risks of infection transmission globally are still inter-state (Africa, Asia) and extra-continental imports of cholera from the Caribbean (Cuba) and Asia into America (Canada) and Europe (Great Britain, Italy, Belgium, and Germany), which took place in 2015 too. Identified have been 21 endemic administrative territories in eight countries of Africa, America, the Caribbean, and Asia. Epidemiological complications are mainly caused by V. cholerae O1, biovar El Tor, producing cholera toxin of classical type, as well as by V. cholerae O1, classical biovar, and V. cholerae O139 serogroup. Epidemic manifestations of cholera in the Russian Federation are characterized by import of the infection by citizens returning from abroad; the isolation from surface water bodies of V. cholerae O1, biovar El Tor ctxA- tcpA-, ctxA-tcpA+ and V. cholerae O139 ctxA-and tcpA-, as well as of single V. cholerae O1, biovar El Tor ctxA+tcpA+. Forecast on cholera world-wide and in Russia for 2016 remains unfavorable.

Carried out has been whole-genome sequencing of the Vibrio cholerae 81 El Tor Inaba strain DNA, isolated from Temernik River waters in the territory of Rostov-on-Don in July, 2014. Identified is the presence of hybrid CTX prophage, which contains ctxB gene of the classical type ( ctxB1 allele), as well as rstR gene El Tor type, tcpA gene with the mutations in coding and promoter regions ( tcpETCIRS allele), the null-mutation in rtxA gene, and extended deletion inside the pandemicity island VSP-II. All the stated above peculiarities are characteristic of cholera vibrio genovariants, which have an enhanced epidemic potential and have recently replaced the typical strains of this biovar.

Cholera epidemiological situation around the world (2005-2014) has been assessed. Distribution of infection in the territory of African, Asian, and American countries, as well as in the Caribbean Region has been shown. Interstate and inter-continental importations of cholera in Europe, Australia, and America, including USA and Canada have taken place during this period. Epidemic process is spatially disseminated (with involvement and affection of new countries and administrative territories) and temporally chronic (America, the Caribbean Region; Africa) due to occurring of natural or social emergency situations. Alongside epidemics and outbreaks of the disease, caused by genetically altered variants of V. cholerae O1 El Tor and strains with multiple drug resistance, outbreaks with isolation of clinical strains of V. cholerae O139 serogroup take place in the Southeast Asia (China) on an annual basis. The forecast for 2015, as regards cholera in the world, stays unfavorable, which in its turn allows for the possibility to import this infection in the territory of the Russian Federation.

Accessory cholera enterotoxin (ace) gene of Vibrio cholerae, amplified by PCR, is cloned into the BamHI-HindIII sites using plasmid pQE30. Expression of ace gene is under the control of T5 promotor. The Escherichia coli strain M15[pREP4]pAce90 (KM 194), containing recombinant plasmid, is an active producer of 6His-Ace protein, which possesses biological activity in the models of suckling mice and causes accumulation of fluid in the intestine. The product is contained in inclusion bodies found in the cells of coliform bacterium. The engineered producer strain can be used to obtain Ace preparations with a view to study its significance as pathogenicity factor in cholera vibrio.

Studied were morphological, physiological and ultrastructural changes in the cell culture L-929 and in the intestine of suckling mice in the presence of Cef (CHO cell elongating factor) of Vibrio cholerae. Registered were cellular elongation, cytoplasm vacuolization, an increase in the number of lysosomes, retraction and nuclei deformation in the L-929 cell culture. Statistical significance test confirmed that in vivo Cef caused the accumulation of fluid in suckling mice. Electron microscopy of the epithelial cells of small intestine revealed vacuolization and myelin-like structures formation in cytoplasm and nuclei, swelling of mitochondria, clarification of their matrix and destruction of cristae. Cristae were filled with mucus. The involvement of mast cells caused the increasing of capillary permeability which enhanced dehydration. These changes confirmed Cef participation in manifestation of Vibrio cholerae pathogenic properties.