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

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

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

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

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

Local invertibility in Sobolev spaces with applications to nematic elastomers and magnetoelasticity

We define a class of deformations in W^1,p(\u3a9,R^n), p>n 121, with positive Jacobian that do not exhibit cavitation. We characterize that class in terms of the non-negativity of the topological degree and the equality between the distributional determinant and the pointwise determinant of the gradient. Maps in this class are shown to satisfy a property of weak monotonicity, and, as a consequence, they enjoy an extra degree of regularity. We also prove that these deformations are locally invertible; moreover, the neighbourhood of invertibility is stable along a weak convergent sequence in W^1,p, and the sequence of local inverses converges to the local inverse. We use those features to show weak lower semicontinuity of functionals defined in the deformed configuration and functionals involving composition of maps. We apply those results to prove existence of minimizers in some models for nematic elastomers and magnetoelasticity