THE ROLE OF SMALL RNAS IN CONTROLLING EXPRESSION OF GENES INVOLVED IN THE IMPLEMENTATION OF VIBRIO CHOLERAE PATHOGENICITY

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

Comparative Molecular-Genetic Analysis of <i>Francisella tularensis</i> Strains Isolated in the Rostov Region in 2020 and Genome Sequences of the Strains Collected in Various Regions of the World

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

O1 Serogroup Cholera Vibrios Isolated from the Rostov-on-Don Water Bodies in the Course of Surveillance in 2008–2012

Identified are the peculiarities of biological properties and origin of cholera vibrio O1 serogroup strains isolated from the Rostov-on-Don water bodies in the course of surveillance in 2008–2012. 41 atoxigenic V. cholerae O1 strains have been isolated from 767 water samples and investigated. Stable tendency of isolation of increased numbers of atoxigenic V. cholerae O1 strains over time has been demonstrated. In addition, in the strains under study detected have been the genes of additional pathogenicity factors, by PCR genotyping using specific primers for 45 nucleotide sequences associated with V. cholerae pathogenicity. The strains have also been classified according to 19 VNTR-genotypes grouped into 6 clusters based on VNTR-typing using exclusive copyright locus-specific primers. Discovered have been the strains with similar genotypes though isolated at different points both throughout the year and over the period of several years. These ones have probably been imported. They are characterized by a capacity to persist in ambient water bodies for a certain period of time

Alkyl Sulfatase of Cholera Vibrios

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

Cholera Forecast for the Year 2019 Based on Assessment of Epidemiological Situation Around the World, Across CIS and Russia in 2009–2018

Analysis of cholera incidence for the period of 2009–2018 was performed. The upward tendency in the morbidity rate dynamics around the world (compared to 2009) with an average annual growth rate of 5,352 % was revealed. For the first time during the 7th pandemic caused by V. cholerae O1 El Tor, WHO reported 1227391 cases of cholera world-wide in 2017, out of which 1032481 (84.1%) were registered in Yemen, where the war continues and one of the largest epidemics in the world. There have been cross-border epidemiological complications in several African countries. Endemic foci continue to exist and spread in Asia, Africa and the Caribbean. Under the epidemiological surveillance in Russia, 744 strains of V. cholerae El Tor – ctxA– tcpA–, ctxA– tcpA+ and V. cholerae О139 ctxA– and tcpA– were isolated from the surface water bodies, as well as single strains of El Tor ctxA+ tcpA+. As a result of INDEL- and  PCR-genotyping, the isolation of strains with identical genotypes and new ones was established. To make the prognosis for 2019, the risk of activation (continuation) of the cholera epidemic process in the world was assessed, taking into account emergencies of different origin and risk factors. The cholera forecast at the global level and in  Russia for 2019 is unfavorable

Investigation of the Lipopolysaccharide Cluster Structure in the Genomes of <i>Vibrio cholerae</i> Rough Variants

Determination of Vibrio cholerae affiliation to one or another serogroup may meet some difficulties in cases of atypical agglutination with diagnostic cholera sera. The study of genetic determinants that allows for identifying a serogroup is a relevant task in monitoring of surface water body contamination with cholera vibrios.The aim of the work was to compare the structural organization (quantitative and qualitative gene composition) of LPS clusters in V. cholerae rough variants.Materials and methods. We used Illumina MiSeq for the whole genome sequencing; SPAdes software (version 3.11.1) for de novo assembly; and blastn (v. 2.5.0) for gene searching. GeneMarkS software was deployed for annotation of the genes incorporated in the clusters; nucmer – for searching homologous sites. Visualization of O-LPS clusters was carried out by means of SnapGene Viewer.Results and discussion. Strains of V. cholerae rough variants had diverse gene clusters responsible for O-antigen biosynthesis. We have identified three types of O-LPS clusters with different size and number of genes. Unique DNA sites, common to the whole group of V. cholerae rough variants, have not been detected. Two genes present in all rough strains have been defined, but they are not unique for this group of strains and can be found in representatives of other serogroups. For two types of clusters, a region containing the IS‑element, common with V. cholerae O1, has been revealed

Stabilized Phase Variants of <I>Vibrio cholerae</I> El Tor P-18895

Produced have been stabilized phase variants of V. cholerae El Tor P-18895 (O- and rugose colonies). Frequency of reversion to initial ST-phenotype does not exceed 10 %. Identity of the origin is verified in VNTR. Evaluated has also been their activity by means of the following diagnostic tests: agglutination assay, sensitivity to diagnostic bacteriophages test, and studies of growth behavior in solid nutrient media. Stabilized variants of V. cholerae El Tor P-18895 can be deployed for further investigations of peculiarities of biofilm formation on various surfaces, bacterial resistance to environmental factors, and for the enhancement of methods for isolation of cholera vibrio variants from ambient environment

Assessment of the Variation Range of Agglutinability in <i>Vibrio cholerae</i> Strains Isolated in the Course of Monitoring Studies

The aim of the study was to retrospectively analyze the range of variability of antigenic properties and genotypic characteristics of Vibrio cholerae R-variant strains atypical in terms of agglutinability.Materials and methods. 169 strains of V. cholerae R-variant with atypical agglutinability have been studied using the “AmpliSens® Vibrio cholerae-FL” test-system. The determination of O1 antigen was carried out using the “Ig-V. cholerae О1/О139 – ELISA/dot-ELISA” reagent kit.Results and discussion. A retrospective analysis of the complex of phenoand genotypic characteristics of strains isolated from surface water bodies in the territories of three former Soviet republics and 13 constituent entities of the Russian Federation in the course of 30-year monitoring and identified upon isolation as nontoxigenic V. cholerae R-variant strains has been performed. Upon re-identification, it was found that the strains belong to both epidemically dangerous (3.0 %) and non-dangerous strains (97.0 %). The range of variability was expressed in their distribution into three groups and consisted in retaining of agglutinability only with cholera RO serum in the first group (34.5 % of strains); the loss of this trait, but the acquisition of the ability to agglutinate in different combinations with O1, Ogawa or Inaba sera – in the second (16.7 %); and also in the loss of agglutinability with all diagnostic cholera sera – in the third (48.8 %). The presence of the wbeT gene in the compared V. cholerae classical R-variant strain does not exclude the presence of the genomic region for O1 antigen biosynthesis in other R-strains, possibly in a modified form, which can be clarified in further molecular-genetic studies. Alternatively, such strains are likely to be attributed to V. cholerae nonO1/nonO139. Strains of V. cholerae R-variant with different amounts of surface antigen (optical density range – from 0.088±0.002 to 1.226±0.003) have been identified. The data obtained can be used for monitoring of cholera in laboratories of regional and federal levels

Cholera: Analysis and Assessment of Epidemiological Situation around the World and in Russia (2013–2022). Forecast for 2023

The aim of the review was to analyze and assess the epidemiological situation on cholera in the world and Russia in 2013–2022 and to make a forecast for 2023. Over the period of 2013–2022, 500 administrative territories affected by cholera were identified in various regions of 71 countries of Asia, Africa and America (the Caribbean region) with formation of 69 endemic foci in 16, 41 and 12 countries, respectively. In 2022, 1 209 301 cases of cholera were registered in 36 countries of the world. The intensity of epidemic process in Asian countries (Syria, Lebanon) increased. Unfavorable epidemiological situation on the African continent persisted. Epidemics and large outbreaks, which began in 2021–2022 due to emergency situations (ES) of social and natural character, continued. The prediction of the stability of the epidemiological situation on cholera in the territories of constituent entities of the Russian Federation (RF), given for 2022, has been confirmed. In 2022, 43 non-toxigenic strains of Vibrio cholerae О1 were isolated from surface water bodies, 8 V. cholerae nonO1/nonO139 strains – from humans. Similarity of those strains with genetically closely related ones isolated in the course of monitoring in previous years in Russia, Donetsk People’s Republic (DPR) and Zaporozhye Region was demonstrated. In 2023, the risks of importation of the infection into RF are retained. It is associated with the intensification of epidemic processes in Asian, African and Caribbean region countries. The extended border with Ukraine, to where importation of cholera from endemic countries is possible, contributes to increased degree of threat. Bioterrorism is also not excluded. The consequences of a possible cholera epidemic complication in DPR, Lugansk People’s Republic, Zaporozhye and Kherson Regions are exacerbated by social emergencies resulting in disruption of infrastructure, interruptions in water supply, etc. In the absence of implementation of the risks of importation of this infection on the territory of RF, a stable epidemiological situation on cholera will be retained. Detection of non-toxigenic V. cholerae O1 strains (including probability of clonal complexes formation), as well as strains of non-O1/non-O139 serogroups, which can be etiological factors of sporadic cases or outbreaks of diarrheal diseases, in surface water bodies is predicted

Cholera: Trends in the Development of the Epidemic Process in 2021, Forecast for 2022

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