Escherichia coli Strain – Super-Producer of Vibrio cholerae Hemolysin

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

Cholera Vibrios non-O1/non-O139 Isolated in the Process of Epidemiological Monitoring over Rostov-on-Don Water Basins and Drain Sewage System within the Period of 2009–2011

Studied have been biological properties of cholera vibrios non-O1/non-O139 circulating in the Rostov-on-Don surface waters and drain sewage system within the period of 2009–2011. Overwhelming majority of the isolates have been classified as typical ones in terms of their phenotype. However 90 strains (21 %) out of total 280 have been identified as atypical ones against agglu­tina­bility in the slide-agglutination and full-scale agglutination reaction to diagnostic cholera sera. Put forward are recommendations on performance of the differentiation between V. cholerae non-O1/non-O139 and V. cholerae O1/O139. Specified are predominant sero­groups of the strains which turned to be unchanged within the stated period (O16, O53, O76, and O67). Revealed is the strain genotype variability and low probability of virulence manifestations. Demonstrated is the futility of the blind gene- and serological typing of aquatic strains

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

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

Features of the Biological Properties of <em>Vibrio cholerae</em> Isolated during the Monitoring of Water Bodies in Rostov-on-Don from 1989 to 2018

The goal is to study the features of the biological properties of Vibrio cholerae strains found in environmental objects (EO) on the territory of Rostov-on-Don from 1989 to 2018.Materials and methods. Bacteriological, molecular biological and statistical methods were used to study 73 strains of cholera vibrios O1, O139 and 1702 strains of nonO1/nonO139 serogroups isolated over a 30-year period.Results and discussion. It was found that in Rostovon-Don, 78.0 % of toxigenic strains were isolated from all isolated from environmental protection in Russia, among which 2 were genetically unchanged. The V. cholerae O1 classical strain and 72 V. cholerae El Tor strains were found, of which 70 % belonged to the Ogawa serovar, and 3 % of the nontoxigenic strains were susceptible to the classical phage. A “very weak direct relationship” was statistically revealed between the isolation of V. cholerae O1, O139 and V. cholerae nonO1/nonO139 strains, in which the dominant serogroups (O67, O76, O75, O53, O16) were identified, and 18 % of the strains showed atypical agglutinability. Revealing the biological characteristics of V. cholerae strains contributes to the optimization of monitoring studies

About the Results of Monitoring Researches of Ballast Waters and Data of Identification of the Vibrioes Strains Selected During the Ships Researches in Russian Seaports in 2018

Relevance. In September, 2017 the International Convention on Control of Ships’ Ballast Waters and sediments, in which the Russian Federation takes part, came into effect.Aim of article is to cover the results of implementation of the Convention in Russia, regarding selection and analysis of ballast waters tests for compliance with the international standard.Material and methods. The materials for work were data on ship arrivals at the international seaports of the Russian Federation, provided by sanitary and quarantine departments of the Russian ports, and monitoring researches of ballast waters in seaports of some regions of the Russian Federation. Analytical, bacteriological, molecular methods were applied.Results. The studies of ballast waters in the Leningrad and Kaliningrad regions have been conducted for the first time in 2018, and in the Rostov region the study lasts since 2010. The laboratory researches of ships’ ballast showed that E. coli, Enterococcus spp. were in norm, V. cholerae O1 and O139 in ballast were absent. 12 of 21 ballast water tests investigated by specialists of the Rostov region laboratories contained V. cholerae non-O1/non-O139, ballast was taken on the ships which arrived from Romania and Turkey.Сonclusion. The results of the molecular and genetic researches suggest that there is a probability of V. сholeraе introduction brougth with ship ballast. Management decisions are demanded to ensure biological safety of shipping and to decrease intestinal infections incidence in residents of the seaside cities

Expression of E- and N-cadherins in tumor in luminal, primary operable breast cancer without Her2/neu overexpression in postmenopausal women as a prognostic factor

Purpose of the study. Evaluation of the expression of E- and N-cadherins in tumor tissues in postmenopausal patients with primary operable luminal breast cancer (BC) without Her2/neu overexpression for inclusion in the panel of a comprehensive assessment of tumor biological parameters for the disease prognosis.Patients and methods. The study included 120 patients divided into two groups: patients with luminal A and luminal B subtypes. To evaluate the prognostic significance of E- and N- cadherins in both groups of patients, we analyzed expression levels and correlation using the Mann- Whitney U-test and Spearman correlation criterion; overall survival, progression-free and tumor- specific one, at various values of the studied biomarkers — by the Kaplan- Meier method.Results. The differences in the mean expression levels were not statistically significant (p&gt;0,05). E-cadherin was twice higher in luminal A BC (55.4±5.2) compared to luminal B BC (28.1±4.9), N-cadherin in luminal A BC (12.8±5.3) was 1.3 times lower than in luminal B BC (17.06±5.4). Patients with luminal B BC demonstrated a tendency to the loss of E-cadherin and increased expression of N-cadherin, which is often associated with poor prognosis. However, the correlation between these markers was not statistically significant (p&gt;0,05).Conclusions. Thus, despite the differences in levels of E- and N-cadherin expression, these markers did not show their prognostic significance, and therefore, they were not included in the panel for a comprehensive assessment of tumor biological parameters when determining the prognosis of luminal breast cancer without Her2/neu overexpression in postmenopausal women