Antibiotic resistance in Vibrio cholerae El Tor strains isolated during cholera complications in Siberia and the Far East of Russia.

Currently, the spread of antimicrobial resistance (AMR) is a global trend and poses a severe threat to public health. The causative agent of cholera, a severe infectious disease with pandemic expansion, becomes more and more resistant to a wider range of drugs with every coming year. The Vibrio cholerae genome is highly flexible and adaptive; the acquisition of the SXT mobile element with a cluster of antibiotic resistance genes on it has marked a new stage in the adaptive evolution of the pathogen. The territory of Siberia and the Russian Far East is free of cholera; however, in the 1970s and 1990s a number of infection importation cases and acute outbreaks associated with the cholera importation were reported. The aim of this study was to describe the phenotypic characteristics and genetic determinants of AMR in V. cholerae strains isolated during epidemic complications in Siberia and the Far East of Russia, as well as to clarify the origin of the strains. The present research comprises analysis of nine V. cholerae El Tor strains isolated from patients and water sources during epidemic complications in Siberia and the Russian Far East in the 1990s. Here, we compared the phenotypic manifestations of antibiotic resistance among strains, harbored the resistance patterns in genomes; we also determined the structure, the type of SXT elements, and the mobilome profile based on the accepted classification. We identified that strains that caused outbreaks in Vladivostok and Yuzhno-Sakhalinsk in 1999 had ICEVchCHN4210 type SXT element with deletion of some loci. The research shows that the integration of the genome, SNP and the mobilome, associated with antibiotic resistance, analyses is necessary to understand the cholera epidemiology, it also helps to establish the origin of strains. The study of resistance determinants features allowed to make a conclusion about the heterogeneity of V. cholerae strains that were isolated during outbreaks in Vladivostok and Yuzhno-Sakhalinsk in 1999.

[The application of MALDI-ToF mini-sequencing for detecting genetically altered versions of cholera agent].

The genetically altered modifications of V.cholerae eltor are characterized by occurrence of single-nucleotide polymorphisms in gene ctxB. To detect these modifications the technique is proposed based on mini-sequencing with MALDI-ToF by of products of reaction with selected probes adjacent to 115 and 203 positions of gene mentioned previously. The mass-spectrometry analysis of the results of reaction of mini-sequencing of strains of V.cholerae eltor isolated during epidemic complications at the territory of the Siberia and the Far East revealed mass-specters corresponding to values of molecular masses of probes (ctxB115, ctxB203) and those complementary completed to points of corresponding replacements (T/C) of didesoxinucleotides (ddTTP, ddCTP). For analyzed strains of V.cholerae eltor isolated in the 1970s, elongation is establishedfor both probes by didesoxinucleotide that testifies presence in their genome ctxB3 allele with thymine in 115 and 203 positions, distinctive for typical representatives of V.cholerae eltor. For V.cholerae eltor, isolated in 1990s, hybridization to points of replacement of didesoxicytosine and presence of ctxB1 allele with cytosine at analyzed positions, distinctive to vibrio of classic biovars. This allele is detected in genome of one of modifications of atypical genetically altered clones ofV.cholerae eltor. This technique, by its sensitivity and specificity, matches direct sequencing of gene ctxB of strains of V.cholerae eltor and proves promising for analysis of other valuable single-nucleotide polymorphisms.

[The loop mediated isothermal amplification of DNA: principle of method and perspectives of application in molecular diagnostic of cholera: publications review].

The article considers characteristics of technology of reaction of loop mediated isothermal amplification of DNA (LAMP), issues of optimization of reaction and perspectives of its application as a quick highly-specific test in molecular diagnostics of infectious diseases and monitoring of contamination of environment objects with pathogens. The analysis of publications data concerning application of LAMP in diagnostics of cholera testifies high diagnostic value. The LAMP supports possibility of direct rapid detection of toxin-producing strains of Vibrio cholerae in clinical samples. This technique also provides identification of determinants of cholera vibrio in pure culture, samples from environment objects and food products. The research studies established exceeding of parameters of sensitivity and specificity of LAMP as compared with polymerase chain reaction that permits considering LAMP as a perspective technique for express-analysis of clinical material from patients with suspicion on cholera. The LAMP technique can be also used in screening studies of environment objects. The development of test-systems based on application of this technology is required.

[APPLICATION OF PULSED FIELD GEL ELECTROPHORESIS FOR MOLECULAR TYPING OF CAUSATIVE AGENTS OF ESPECIALLY DANGEROUS INFECTIONS].

The macro-restriction analysis of the microorganism DNA with the use of gel electrophoresis in pulsed field (PFGE typing, pulse electrophoresis) is applied in molecular biology to study the clonal structure and typing of causative agents of infectious diseases. Determining the degree of the relationship and definition of epidemiological interrelations of studied isolates, as well as studying the evolutionary history of pathogens, is performed by comparing DNA restriction patterns. This review presents an analysis of the use of the pulse electrophoresis in molecular-epidemiological research and the study of phylogeny of especially dangerous infections, cholera, and plague. The possibility of genetic heterogeneity of the Vibrio cholerae and Yersinia pestis populations is demonstrated; territorial and epidemiological characteristics of the spread of different isolate pulso-types, problems and prospects of the PFGE typing method in the system of epidemiological surveillance of cholera and plague in Russia are discussed.

[Maldi-tof ms analysis for yersinia pestis, vibrio cholera, and francisella tularensis identification].

Numerous studies showed that a new technology for the clinical microbiology laboratories, Matrix-Assisted Laser Desorption Ionization--Time of Flight Mass Spectrometry (MALDI-ToF MS), allows fast, accurate, and effective identification of most clinically relevant microorganisms to be implemented. In the present review, we discuss applications of this approach for identification and typing of extremely dangerous pathogens--Yersinia pestis, Vibrio cholera, and Francisella tularensis, including the advantages and disadvantages of the method, sample preparation and biosafety problems.

[Multilocus sequence-typing of vibrio cholerae strains with various epidemic importance].

The allele polymorphism of the housekeeping genes (dnaE, lap, recA, pgm, gyrB, cat, chi, gmd) from the Vibrio cholerae strains with different epidemic importance (n = 41) isolated in Siberia and at the Far East during the cholera pandemic VII was tested. All toxigenic strains isolated at the period of epidemic complications irrespective of time and source of isolation were characterized by the identical allele profile and belonged to the same sequence-type. Nine sequence types were detected in non-epidemic isolates. The dendrogram clustering was associated with the serogroup and in some cases with the territory and time of isolation. The structure heterogeneity of the non-toxigenic V. cholerae housekeeping genes was in most cases caused by the synonymous nucleotide replacements (Dn/Ds < 1) indicating the prevalence of the negative V. cholerae at the analyzed genome sites. The revealed distinctions in the structure of housekeeping genes of the V. cholerae with different epidemic importance can be regarded as evidence of various evolutional directions in these strain groups.

[Cholera Vibrio biofilm: production, characterization and role in reservation of causative agent in water environment].

AIM: Experimental production, characterization and evaluation of the role of cholera vibrio biofilm. MATERIALS AND METHODS: 33 strains of Vibrio cholerae eltor O1 and V. cholerae O139 of various epidemic significance and origin were studied in a series of experiments by bacteriologic, microscopic (light-optic, luminescent, scanning electron microscopy), molecular genetics, spectrophotometric and statistical methods. RESULTS: Formation of a biofilm involving inter-cellular bonds, pili and extracellular material and variability of the microorganism (RO-phenotype and transition into uncultivable forms) was shown at various temperature and substrate conditions. A more pronounced ability to form biofilms was detected for strains isolated from environmental samples compared with isolated from clinical material regardless of their epidemic significance. Toxigenic strains of eltor biovar (from surface reservoirs during cholera outbreaks) have demonstrated the highest parameters of optical density compared with toxigenic clinical isolates and non-toxigenic O1 and O139 serogroup cultures. The presence of mbaA1 and mbaA2, vpsR, toxR, hapA genes is common for strains that form a biofilm. CONCLUSION: The data obtained confirm the role of biofilm in reservation of cholera vibrio strains of various epidemic significance in saprophytic phase of microorganism existence.

Whole-Genome Sequencing of a Vibrio cholerae El Tor Strain Isolated in the Imported Cholera Focus in Siberia.

The draft genome sequence of Vibrio cholerae O1 strain I-1263, isolated from a patient in the imported focus in Siberia, was determined. The established structural features of the mobile genetic elements indicate stage-by-stage formation of a highly pathogenic V. cholerae clone and promote understanding of the mechanisms of evolutionary pathogen transformations.

[MALDI-TOF mass-spectrometric analysis in the accelerated identification of the Vibrio genus microorganisms].

The goal of this work was to develop methodological approaches to identification of the Vibrio genus representatives using the MALDI-TOF mass-spectrometric analysis technologies. The aspects of the biological safety in sample preparations for mass-spectrometric analysis were studied, reference spectra of six typical V. cholerae strains were developed. Identification of 55 strains, representatives of the Vibrio genus, including 45 V. cholerae strains with different epidemic importance, was performed using the MALDI Biotyper 3.0 basis comprising V. cholerae reference spectra. The possibility of reliable definition of the tested strain taxonomic belonging to the species level was demonstrated. Thus, the results completely corresponded to the data of classical microbiological identification. Stability and reproducibility of the offered research method was experimentally shown. The results allow identification of the Vibrio genus representatives to be implemented with the use of the mass-spectrometric analysis as an effective method that defines a species belonging of the basic Vibrio genus representatives in the shortest-terms.

[Membrane-bound proteases of ompT+ and ompT- Vibrio cholerae strains].

AIM: Detection ofproteases in outer membranes (OM) of ompT+ and ompT- Vibrio cholerae strains of O1 and O139 serogroups. MATERIALS AND METHODS: Specific sterile preparations of OM were obtained by lysis of live V. cholerae cells by 4.5 M urea solution with subsequent differential centrifugation and treatment by nucleases. Extraction of OM proteins previously treated by sodium sarcosinate was carried out by Triton X-100 in the presence of EDTA. Protease and polypeptide spectra were studied in substrate and SDS electrophoresis. Sensitivity of proteases to inhibitors was determined in diffusion test in agarose gel containing substrate by using soy trypsin inhibitor (STI) and phenylmethylsulfonyl fluoride (PMSF). The presence of ompT was determined in PCR by using specific primers. RESULTS: According to PCR data 13 Vibrio cholerae O1 strains and 3 V. cholerae O139 strains isolated from clinical material as well as 22 V. cholerae O1 strains isolated from environmental objects contained ompT gene. 2 V. cholerae O1 human isolated strains, 9 V. cholerae O1 strains and 2 V. cholerae O139 strains isolated from the environment did not have ompT gene. By using SDS- and enzyme-electrophoresis in polyacrylamide gel quantitative and qualitative differences in composition of polypeptides and proteases of OM ompT+ and ompT- V. cholerae strains that hydrolyze gelatin, casein and protamine sulfate were detected. Inhibition of OM by STI and PMSF resulted in a decrease of their proteolytic activity. CONCLUSION: In preparations and extracts of ompT+ and ompT- V. cholerae OM up to 3 proteases some of which may be related to ompT-like were detected.

[Molecular-genetic analysis of the epidemical strains of the Vibrio cholerae El Tor isolated from the Siberian and maritime regions of Russia].

The detection of the biotype-specificity, pathogenicity determinants, and sequencing of the ctxB gene and the ctxAB promoter was carried out for analysis of the Vibrio cholerae El Tor strains genome structure. The strains (n = 90) were isolated during cholera epidemic outbreaks in Siberia and the Far East. All toxigenic Vibrio cholerae El Tor strains were divided into two groups: the first group included strains isolated during 1970s: they had the genotype ctxB3+rstREl+rstRCl-rstC+TLC+tbr4. All epidemic dangerous El Tor biotype strains isolated in 1990s belonged to the second group. The strains were characterized as atypical variants because of classical genotype (ctxB1) ctxB gene harboring. The second group fell into three genotypes according to the set of genetic markers (ctxB, rstR, rstC, TLC, tbr). It was suggested that the set of genetic determinants could be used as a marker for epidemiological analysis of spreading of atypical ET strains. The comparative analysis of genome structure enables to suggest possible ways of pathogen evolution.

[Detection of "hybrid" Vibrio cholerae eltor strains during epidemic complications in Syberia and Far East].

AIM: Biotyping of Vibrio cholerae eltor isolated during epidemic complications of cholera in Syberia and Far East by phenotypic and genotypic properties complex. MATERIALS AND METHODS: 45 strains of V. cholerae were studied. Phenotypic analysis was performed by using a complex ofbiovar determining tests. Genotyping was performed by detecting ctxAB, tcpA, toxR, rstRgenes, and ctxB gene structure analysis. RESULTS: All the V. cholerae during epidemiologic complications in Syberia in the 1970s belong to eltor biovar by phenotypic properties and have eltor specific alleles of tcpA and rstR genes, and ctxB of the third genotype in the genome. In the 1990s the strains were phenotypically matching eltor biovar, but had genetical determinants of both eltor(tcpAE1, rstRE1) and classical (ctxB1, rstR(Cl) biovar. CONCLUSION: The cause of epidemic complications of cholera in Syberia in the 1970s was V. cholerae eltor with typical eltor biovarphenotypical and genotypical properties. In the 1990s cases of introduction into the region of "hybrid: V. cholerae eltor strain were ascertained, developing into acute cholera outbreaks in several cases.

[Genetic characteristics of the causative agent of tick-borne encephalitis in Mongolia].

A patient with diagnosed meningoencephalitis and a history of tick bite died in Mongolia in 2008. The purpose of this paper is to characterize the virus causing the ill person's death. The virus was identified using the phylogenetic analysis of the 520-bp fragment of the tick-borne encephalitis virus (TBEV) genome, which codes the fragment of TBEV protein E between 52-223 amino acids. TBEV RNA was detected in the samples of medulla oblongata, cerebral cortex, and pia mater of brain, but not in the cerebellar tissue. The study virus fragment was genetically closest to the representatives of the Far East subtype. Its closest relative was virus 740-84 (GenBank EU878282) isolated from large-toothed redback voles (Clethrionomys glareolus) in Buryatia and greatly differed from the Far East virus Soffin. Two amino acid substitutions (H86R and VI7A) were detected within the study protein E fragment. The paper is the first to describe the causative agent of tick-borne encephalitis on the territory of Mongolia and to discuss the evolution and pathogenicity of TBEV.

[Evolution of the epidemiology of cholera].

The problems of the evolution of cholera at the stages of its pandemic spread are described. A short characterization of endemic zones in the countries of Asia, Africa and Latin America, stipulating the preservation of infection and the appearance of periodic epidemics in the world, is presented. Special attention is paid to the genesis of epidemic outbreaks in endemic and introduced foci of cholera, differing in the specific features of their formation and the accumulation of the epidemic variant of the infective agent in aqueous habitat. The role of the ecosystem of surface water reservoirs in the prolonged survival of cholera vibrios is evaluated. The necessity of the detailed study of the mechanisms and forms of existence of serogroup 0139 vibrio in open water reservoirs is substantiated. In the system of epidemiological surveillance on cholera the microbiological monitoring of environmental objects assumes the leading importance.

[Experimental non-culturable Vibrio cholerae eltor and its biological properties]. / Eksperimental'noe poluchenie nekul'tiviruemykh form Vibrio cholerae eltor i kharakteristika ikh biologicheskikh svoistv.

In experiments with the cultivation of V. cholerae eltor under the conditions of high salt concentration, as well as low temperature and deficiency in nutrient substances, uncultivable forms (UF) of toxigenic and nontoxigenic vibrios were obtained. The absence of growth of seeded vibrios after the filtration of samples (with a filter of 0.22 micron), the preservation of specific antigenic determinants and the initial set of genes, changes in the morphology of cells (small size, coccoid form with the flagella retained) confirm the transition of V. cholerae eltor under study into the uncultivable state which, under unfavorable conditions, more rapidly develops in toxigenic vibrios than in nontoxigenic ones. The analysis of the INT-reductase activity of UF disintegrates revealed that they had endogenic respiration whose activity increased (4.5- to 6.5-fold) in the presence of the exogenic intermediates of the Krebs cycle. The uncultivable forms of the vibrios retain genes responsible for pathogenicity, as well as their antigenic determinants.

[PCR-detection of markers for epidemiologically-significant strains of Vibrio cholerae 0139, isolated in Siberia]. / PtsR-detektsiia markerov épidemicheskoi znachimosti shtammov Vibrio cholerae 0139, izolirovannykh v Sibiri.

Fourteen V. cholerae 0139 strains were isolated in 1996-1999 in Siberia from the Ob river (Novosibirsk) and bogs and lakes (Irkutsk). The strains were tested in PCR for the key virulence determinants (ctx AB, tcp, acf). The genomes lacked these elements, and therefore the strains were acknowledged avirulent. The results correlate completely with the data of phenotypical analysis, characterizing the pathogenic characteristics of isolated strains.