Comparative Analysis of Expression of the Main Virulence Genes in Various <i>Vibrio cholerae</i> О1 Strains

The aim of the work was a comparative study of the expression of the main virulence genes in Vibrio cholerae strains of the classical biovar, typical and genetically modified strains of V. cholerae, El Tor biovar.Materials and methods. Natural toxigenic strains of V. cholerae O1, classical biovar (J89, Pakistan, 1969), typical (M-887, Astrakhan, 1970) and genetically modified (301, Taganrog, 2011) strains of the El Tor biovar were used as model ones. The strains were grown under optimum conditions for the production of cholera toxin and toxin-coregulated pili. The assessment of strain growth was carried out in LB broth at room temperature with determination of the cells number on a Biowave DNA spectrophotometer (Biochrome Ltd., UK). Determination of gene expression was performed using real-time PCR with reverse transcription.Results and discussion. The expression of structural (ctxA, tcpA) and regulatory (toxR, toxT, tcpP, tcpH) virulence genes has been investigated in V. cholerae strains of the classical biovar, typical and genetically modified strains of the El Tor biovar. Significant differences have been revealed in terms of time and level of maximum expression of these genes in strains of classical and El Tor biovars. It was found that ctxA and toxR genes expression in the genovariant strain reached its maximum 1–3 h earlier than in the other strains. At the same time, the level of ctxA gene expression corresponded to the level of the classical strain. The maximum expression of the toxR gene in the genovariant strain was higher than in typical El Tor and classical strains, and also had a clear inverse correlation with ctxA gene expression. Expression of the tcpA, toxT, and tcpH genes in the classical biovar strain reached its maximum 1–2 h earlier than in the El Tor biovar strains. These differences should be taken into account when conducting research work related to the study of the expression of the main virulence genes

DEVELOPMENT OF THE ALGORITHM FOR IDENTIFICATION OF THE LEVEL OF VIBRIO CHOLERAE CTXA AND TOXR GENE EXPRESSION USING RT-PCR WITH REAL-TIME HYBRIDIZATION-FLUORESCENT REGISTRATION OF RESULTS

Objective of the study is to design the algorithm for assessment of expression of the structural and regulatory virulence Vibrio cholerae genes by the model of ctxA and toxR genes encoding and controlling biosynthesis of cholera toxin.Materials and methods. Utilized were 10 strains of Vibrio cholerae, classical and El Tor biovars. Cloning of gene fragments was carried out through transformation and ligation. RT-PCR was done in “BIS M112” and “Rotor-GeneQ” amplifiers. Processing of the results was performed by means of the software package in set with Rotor-GeneQ (Software 1.8.17.5).Results and conclusions. Developed has been the algorithm for evaluation of the level of expression of V. cholerae ctxA and toxR genes applying RT-PCR with real-time hybridization-fluorescent registration of results. The stated algorithm allows for rapid and effective statistically significant specification of the expression of structural and regulatory virulence V. cholerae genes and can be used for evaluation of newly discovered cholera vibrio strains

Comparative MLVA-Analysis of Vibrio cholerae Strains of Classical Biovar, Isolated in the Russian Federation and Abroad

Objective of the study is to determine phylogenetic affinity of the Vibrio cholerae strains of classical biovar, isolated between 1937 and 1969 in Russia, to the strains from near and far abroad countries. Materials and methods. Utilized were 27 Vibrio cholerae strains of classical biovar. PCR was carried out applying “BIS M112” amplifier. Genetic analyzer ABI 3500xl was used for sequencing of the strains. MLVA-analysis was performed by reference to 5 MLVA-loci: VC0147, VC0436-0437, VC1650, VCA0171, and VCA0238. Nutrient requirement and soluble hemagglutinin/protease production was established using plate method. Results and conclusions. Identified have been 8 MLVA-clusters and 21 MLVA-types. It is determined that the strains, isolated during atypical cholera outbreak 1942–1943 in Russia, are inhomogeneous as regards phenotype and genotype and fall into two separate groups, one of which is related to the strain, isolated during cholera outbreak 1938 in Khabarovsk, and another group – to the strains from India and China, isolated in 1946 and 1949, respectively

Comparative Analysis of Survival Capacity among Typical and Genovariant Strains of <i>Vibrio cholerae</i>, Biovar El Tor <i>in vivo</i> and <i>in vitro</i>

Objective of the study was to conduct experiments on survival capacity of toxigenic genovariant strains as compared to typical strains of V. cholerae , biovar El Tor in autoclaved river water and in the organism of laboratory animals. Consequently, it was determined that both, genovariant and typical El Tor vibrios, sustained for a significant period of time (more than 5 months) in the river water, though a number of the bacterial cells in the population of the typical ones gradually decreased, while in the genovariant strains - the growth was observed in between the 7th and 21st day. CFU of the genovariants was 1.5-2.5 and 1.8-3.0 times higher than the amount of typical strain bacteria on the day 7 and day 21, respectively. Selective advantage of genovariants was also confirmed by competitive test in vitro . Furthermore, genovariants dominated over typical strains of V. cholerae , biovar El Tor in the mixed population in vivo (bioassay animals): CFU of the genovariants 1.25-84.0 times exceeded that of the typical strains when seeding out the contents of intestine and gastrointestinal wall homogenate. Put forward was an assumption that one of the factors for genovariant selective advantage might be enhanced adaptation ability affected by changes of cell metabolic activity. Identified capacity of genovariant toxigenic strains - not only to sustain in open water bodies of our country, but also to propagate in there, creates unfavorable epidemiological situation on cholera

Comparative analysis of adaptation properties in typical and genetically altered Vibrio cholerae El Tor strains

Aim. Study of biological properties in natural strains of genovariants of V. cholerae biovar El Tor, affecting their adaptation  capacities under nutrient  deficiency while comparing them with typical strains. Materials and methods. Competitive  sampling was carried out through  plating a mixture of cells of the two strains under investigation into autoclaved river water. Growth rate was evaluated through the optic density values. Gene expression was studied applying RT-PCR with designed primers and probes. Results. It is established that during combined  cultivation of V. cholerae El Tor typical strains and genovariants under the shortage of nutrient substances (autoclaved river water), at room temperature, the level of survivability in genetically altered strains is higher than in typical strains, which points to their expressed adaptation advantages over the typical ones under the stated conditions.  It is demonstrated that selective benefits of genovariant strains are provided by higher cell growth rate and increased rpoS gene expression. Conclusion. Obtained have been new data on the ability of bacterial strains of V. cholerae El Tor genovariants to rapidly grow and better express global regulator of stress response, rpoS gene, which, probably, contributes to their enhanced adaptation not only under nutrient deficiency, but under the influence of other stress factors too

<i>Vibrio cholerae</i> O1 Avirulent Strains - Producers of Protective O1 Antigen: Obtaining and Peculiarities

Among 15 Vibrio cholerae El Tor O1 virulent strains of Inaba and Ogawa serovars grown in sterile river water, 6 strains were found to lose spontaneously the core region of CTXφ prophage including structural genes (ctxAB) of cholera toxin. It was determined that the obtained ctxA- mutants did not produce cholera toxin and did not cause specific choleragenic reaction in the intra-intestinally inoculated suckling rabbits suggesting that they belonged to the IIId pathogenicity group. Analysis of the population structure of two selected non-toxigenic strains M569A and 5/65A revealed clones with high level of O1 antigen of Inaba and Ogawa serovars production. The obtained avirulent strains can be used as producers of above-noted protective antigens both in production of chemical cholera vaccine and in constructing of diagnostic immunoenzyme test-systems

<i>Vibrio cholerae</i> Genome Evolution: Ways of Atypical Strains Formation

Presented is the review of literarary and authors` data on the ways of emergence of Vibrio cholerae eltor strains, atypical as regards their virulence properties. The enhancement of virulence of cholera eltor agent at present was shown to be associated with acquisition of Vibrio cholerae cholerae CTXφ prophage as a result of horizontal gene transfer. Another possible way of formation of strains with higher virulence is described - alteration of cholera toxin resident genes regulation due to rearrangement of CTXφ prophage genome by the introduced transposon. Also considered are evidences on origination of ctxA- tcpA+ strains and their enhanced viability in aquatic environment as compared with isogenic toxinogenic clones. Presented are the prospects of the development of new PCR test for detection of cholera eltor agent with altered virulence in monitoring investigations