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The aim of the work was to determine the pheno- and genotypic features of the aquatic CTX+ strain of Vibrio cholerae isolated in 2023 and a comparative bioinformatic analysis of whole-genome sequencing data.Materials and methods. Whole-genome sequencing was performed on MiSeq (Illumina) and MinIon Oxford Nanopore Technologies (ONT) platforms; hybrid assembly of the whole genome was carried out using the Trycycler algorithm; assembly errors were eliminated by means of the Medaka algorithm and the Pilon program. Dendrogram construction and bioinformatics analysis were carried out with the help of the scipy and Graphviz packages, BioEdit, BLASTN, BLASTP, CARD, ICE Genotyper, and Vector NTI programs. The ability to produce cholera toxin was tested using the GM1ELISA.Results and discussion. The isolated strain was identified as V. cholerae O1 Ogawa, sensitive to most antibiotics. Based on the totality of genetic properties, it was classified as the first genovariant, distinct from the typical El Tor strains only by the presence of ctxB1 gene of classical type instead of ctxB3 of the El Tor type. It has been established that it contains a tandemly duplicated CTX prophage on the small chromosome and a tandem of two copies of RS1 prophage on the large chromosome. Thereat, the rstR gene of the CTX prophage belonged to the classical type, and the RS1 prophage – to the El Tor type. The remaining criteria of epidemic hazard – tcpAelt, rtxA1 and intact VSP-II did not differ from the prototypes. The genome of the strain carries the ICE element VchBan11, which contains the trimethoprim resistance gene dfrA1, and phenotypically the strain is resistant to this antibiotic. Under in vitro conditions, the strain did not produce cholera toxin, as shown by ELISA results. This may be due to the presence of a deletion within the toxR regulatory gene. Strains similar to the 2023 isolate are mainly attributed to the second wave of the seventh pandemic. Currently, they are almost replaced by new genovariants, but occasionally can emerge and even cause diseases. Therefore, their importation into Russian territory potentially pose a threat to public health. more...

The aim of the work was to identify genotypic features of clonal complexes formed by CTX–VPI+ strains of Vibrio cholerae isolated from water bodies of the Rostov Region in different years, based on bioinformatics analysis of their whole genome sequences (WGSs).Materials and methods. Whole genome sequencing was performed on the MiSeq (Illumina) platform; identification of genetic determinants in WGSs and bioinformatics analysis – by means of BioEdit, BLASTN, BLASTP, CARD, Vector NTI software packages.Results and discussion. The strains were almost identical in each group of 2002-, 2005-, 2007-, 2020-, 2021-, and 2022-isolates and were found at different points of water sampling over a longer or shorter periods of time (from 3 weeks to 2 months). Bioinformatics analysis of WGSs of representative strains divided them into 4 clusters, which can be considered as separate clonal complexes sharing a number of characteristic genetic markers: 1) 2002-isolates containing one SNP in regulatory rpoS gene; 2) 2007-strains carrying tandemly duplicated preCTX prophage; 3) strains isolated in 2020 in Rostov-on-Don and in 2005, during epidemiological complications in the Kamensk district of the Rostov Region, which had a “hybrid” pathogenicity island including the cluster of type 3 secretion system and the nan-nag region of the VPI-2 island, and 9 SNPs in rpoS gene; 4) 2021- and 2022-strains and a single 2018-isolate containing a 9 bp deletion in vchC collagenase gene. Despite the revealed differences, representatives of all clonal complexes possessed sufficient sets of intact determinants of pathogenicity/persistence factors to realize virulent properties and ability to survive in water bodies. Detection of clonal complexes of potentially pathogenic strains in surface water bodies indicates occasional emergence of favorable conditions for their long-term circulation, which emphasizes the importance of constant monitoring of V. cholerae in the territory of Russia. more...

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. more...

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. more...

During more than 50 years, a heterogeneous population of Vibrio cholerae non-O1/non-O139 (NAGs) exists in Rostov region of Russia, whose representatives periodically cause acute human intestinal infections; the last case was registered in 2018. In 2022, a sick child was again revealed in Rostov, from whom 2 NAG subcultures were isolated. Aim of the study: bioinformatics analysis of whole genome sequences (WGSs) of the 2022 clinical NAG isolates, genetic determinants of pathogenicity factors and relevant products. Materials and methods. Isolation, identification of cultures, as well as antibiotic sensitivity were carried out according to the MUK 4.2.3745-22. SNP analysis of WGSs obtained on the MiSeq Illumina platform were analyzed by means of designed software; BioEdit 7.2.5, Vector NTI Advance 11 package, CARD database were used to identify and analyze genes and relevant deduced products. Results. The isolated cultures with identical phenotypic features, PCR-genotypes and results of SNP analysis, were identified as a non-toxigenic NAG. The agent showed sensitivity to chloramphenicol, ceftriaxone, streptomycin, gentamicin, nalidixic acid, ciprofloxacin, ampicillin, amikacin and intermediate resistance to tetracycline, doxycycline, furazolidon and co-trimoxazole. WGSs lacked CTX and preCTX prophages, pathogenicity island VPI, thermostable and cholix toxin genes, pandemic islands VSP-I and VSP-II. The determinants of the type 6 secretion system (T6SS) were not presented completely, which evidenced in favor of the loss of its functionality. An intact T3SS cluster was revealed, which was adjacent to the nan-nag region of the VPI-2 pathogenicity island in the absence of the proximal and distal parts of the latter. Other determinants of pathogenicity factors were revealed: RTX cluster responsible for synthesis of MARTX cytotoxin, genes for hemolysin HlyA, cytotonic toxin Cef, several serine and metalloproteases. The nucleotide compositions of most determinants differed from the prototypes, but their putative products preserved characteristic active domains. The adhesive activity might be provided by mannose-insensitive MSHA-like pili, since the msh-cluster included all the genes necessary for their production, as well as flagella, as both clusters responsible for their assembling were 99% identical to the prototype. From antibiotic resistance determinants only -lactamase VarG gene was found. Conclusion. The NAG strain examined here contains a sufficient set of intact virulence-associated genetic determinants, expression of which could contribute to the development of the disease. more...

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. more...

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. more...

The aim of the study was to analyze the resistance to antibacterial drugs of Vibrio cholerae strains isolated from environmental water bodies on the territory of Russia in 2019.V.cholerae O1 El Tor (14) and V.cholerae nonO1/nonO139 strains were used in this work. Sensitivity/resistance to 11 antibacterial drugs was determined using the method of serial dilutions in a solid nutrient medium. The presence of drug resistance genes was determined using real-time PCR. Fluctuations in sensitivity/resistance of V.cholerae were found in various years. The phenotypic resistance of the strains to tetracycline and trimethoprim/sulfamethoxazole correlated with the presence of the tetR and dfrA1 genes in them. The presence of ICE was not detected in V.cholerae strains containing the tetR and qnrVC1 genes. The variability and wide spectrum of V.cholerae resistance require close attention to the problem of antibiotic resistance of cholera. The detection of ICE in the studied V.cholerae strains, as well as antibiotic resistance genes not associated with ICE elements, emphasize the need for molecular genetic monitoring of V.cholerae antibiotic resistance. more...

Aim of the work – to assess the epidemiological situation on cholera in 2020 and to make a forecast for 2021 based on the monitoring data and analysis of morbidity around the world for the period of 2011–2020. During the period between 2011 and 2020, 4 413 988 cases of cholera were recorded in 97 countries of the world with a general trend towards a decrease in the incidence (coefficient of accuracy of approximation R2 – 0.5705). However, due to the continuing epidemic manifestations of cholera in the endemic countries of Asia, Africa and America, the epidemiological situation on cholera on these continents was characterized as unfavorable in 2020. The emergence of a new “post-Haitian” lineage was observed among epidemically hazardous strains of Vibrio cholerae O1. In 2020, no epidemically dangerous strains of V. cholerae O1, O139 were isolated from humans on the territory of the Russian Federation. 25 non-toxigenic V. cholerae O1 El Tor strains were isolated from environmental objects, eight out of which (ctxA-, tcpA+), according to PCR-INDEL typing and SNP analysis of sequences, belonged to the clonal complex. The results of the analysis of biological properties and phylogenetic relations between the isolated non-toxigenic strains provided the basis for considering the epidemiological situation on cholera in Russia in 2020 as a stable one and a similar forecast of its development in 2021. At the same time, the possibility of importation of this infection from endemic countries cannot be ruled out, as well as the need to carry out a complex of differentiated anti-epidemic (preventive) measures within the framework of the state sanitary-epidemiological surveillance in order to localize and eliminate the imported focus and avoid the spread of the infection. more...

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. more...

Aim. To assess the prevalence of antimicrobial resistance of Vibrio cholerae nonO1/nonO139 strains in water bodies in Rostov-on-Don. Materials and methods. Sampling was carried out at fixed points of open reservoirs in Rostov-on-Don from May to September in 2016 and 2017. Sensitivity/resistance of V. cholerae nonO1/nonO139 to antibacterial drugs (ABP) recommended for emergency prevention and treatment of cholera was determined by serial dilution on Müller-Hinton agar. Results. 196 strains of V. cholerae nonO1/ nonO139 were isolated in 2016, 75 — in 2017. When serotyping it was found that all strains were assigned to 17 serogroups, representatives of O16 and O76 serogroups prevailed. Analysis of the distribution of strains on resistance to ABP showed that sensitive strains of V. cholerae nonO1/nonO139 were not detected. All microorganisms were resistant to furazolidone. The proportions of isolates resistant to co-trimoxazole and ampicillin were 35.2% and 40% in 2016, and 33.6% and 28.3% in 2017 respectively. All microorganisms were distributed over ten phenotypes and were characterized by the presence of 1 to 4 determinants of resistance. Conclusion. The resistance of V. cholerae nonO1/nonO139 strains isolated in Rostov-on-Don water bodies to furazolidone increased from 47% in 2011 — 2014 to 100% in 2016 — 2017. Resistance to co-trimaxazole increased from 7.8% to 35.2 — 40%. Strains resistant to gentamicin (1.5%) earlier absent were detected in 2016. more...

Objective of the work was to compare the results of monitoring of Vibrio cholerae strains isolated in the territories of federal districts, their constituent entities and from certain water bodies between 2006–2016. Based on the methodology, using updated geoinformation system “Cholera 1989–2014”, a comparative study of the dynamics of isolation and biological properties of 586 Vibrio cholerae O1, O139 strains and R-variants, collected from the surface water bodies in the administrative territories of Russia, was carried out. Eventually it is established that Vibrio cholerae strains of various serogroups are found in the territory of all federal districts, but not in all the entities included in them. It is shown that the greatest number of isolated strains was registered in the Southern Federal District. The overwhelming number of Vibrio cholerae strains, isolated in the country, were non-toxigenic. It was noted that against the background of epidemiological welfare, the detection of single epidemiologically significant strains continued (Rostov Region).ctxA–tcpA+ strains were found in the Southern, Far Eastern and North-Western Federal Districts. It is established that until 2013 in five constituent entities strains of Vibrio cholerae O1 and O139 in water bodies were not detected. Non-toxigenic strains of Vibrio cholerae O1 El Tor Ogawa serovar prevailed in the territories of the Southern and Ural, and Inaba serovar – in the remaining Federal Districts.The most frequently isolated were non-toxigenic Vibrio cholerae O1 strains with phagotype 15, while isolates with phagotypes 4, 5, 10, 14 and 17 were traced only in the Southern Federal District. The data obtained formed the basis of the approach that contributes to a comprehensive assessment of the epidemiological situation on cholera in the territory of Russia and highlighted the prospects for using GIS to improve the effectiveness of cholera vibrio monitoring in surface water bodies. more...

Objective of the investigation was a comparative bioinformatics analysis of Vibrio cholerae О1 Cef (CHO cell elongating factor) genes and proteins. Materials and methods. 36 Vibrio cholerae О1 strains from the Rostov-on-Don Research Anti-Plague Institute collection have been utilized. DNA sequencing was conducted on the MiSeq platform (Illumina); gene identification and analysis was carried out by means of BioEdit 7.2.5, BLASTN 2.2.29, BLASTP, MEGA 7, Vector NTI Advance 11 software programs. Results and conclusions. The data obtained confirmed Cef to be rather conserved in choleragenic strains (carrying cholera toxin genes ctxAB as a part of genome-integrated CTX prophage): all of them shared closely related prototype alleles cefС or cefЕ1. The Е1 allele was also revealed in ctxAB– strains carrying the pre-CTX prophage and in a single strain lacking both prophages. In the rest of CTX–/pre-CTX– V. cholerae four novel cef variants, that were not previously described, have been identified, two of which (E2 and E3), belonging to the Russian isolates, appeared to be unique, while for the two others absolute homologues were found in NCBI. In this connection several strains which caused severe cholera-like diseases in humans were placed in the group of cefЕ4 host strains. Since Cef is one of pathogenicity/persistence factors of cholera vibrios, we presume that conservation of its altered variants in the course of natural selection embodies a certain biological sense in respect of possible acquisition of qualities, significant for realization of both pathogenic and persistence potential. more...

Aim. Application of the authors’ GIS «Cholera 1989-2014» for systematization of atoxigenic strains of serogroup 01 cholera vibrios (ctxAB-tcpA-, ctxAB-tcpA+), isolated from aquatic objects of the environment by pheno- and genotype. Materials and methods. A sample of 304 Vibrio cholerae 01 strains was studied. Isolation of 39 genes related to pathogenicity was carried out. Discrimination ability of a set of genes was determined by Simpson formula. Cluster analysis was carried out by UPGMA method. Results. Analysis of multi-year data on aquatic V. cholerae 01 strains in country’s subject was carried out using GIS. A possibility of systematization of phenotypes of the isolated strains by defined parameters was shown. An experimental program for detection of presence/lack of various genes and their combinations forgenotyping was developed. Conclusion. GIS was established to allow to carry out analysis of phenotypes by defined parameters, as well as implement approximate systematization of genotypes of atoxigenic strains of cholera vibrios 01 by optimally sufficient detection of 14 genes. more...

Carried out has been whole-genome sequencing of the Vibrio cholerae 81 El Tor Inaba strain DNA, isolated from Temernik River waters in the territory of Rostov-on-Don in July, 2014. Identified is the presence of hybrid CTX prophage, which contains ctxB gene of the classical type ( ctxB1 allele), as well as rstR gene El Tor type, tcpA gene with the mutations in coding and promoter regions ( tcpETCIRS allele), the null-mutation in rtxA gene, and extended deletion inside the pandemicity island VSP-II. All the stated above peculiarities are characteristic of cholera vibrio genovariants, which have an enhanced epidemic potential and have recently replaced the typical strains of this biovar. more...

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. more...