Oksana A. Podoinitsina

The aim of the work was to analyze the epidemiological situation on cholera in the world and in Russia and to make its forecast for 2026. Proactive epidemiological surveillance data showed that over the period of 2016–2025 there was an upward trend in cholera incidence worldwide: 5,474,013 cases were documented in 91 countries. The proportion of patients was distributed among countries as follows: Yemen (53.4 %), Afghanistan (11.7 %), Democratic Republic of the Congo (6.3 %), Nigeria (4.7 %), Somalia (3.2 %), South Sudan (2.5 %), Sudan (2.5 %), and Haiti (2.4 %). In 2025, 42 countries reported 589,571 cases and 8,140 deaths (5.1 % more than in 2024). The increase in cholera cases in 2025, compared to 2024, amounted to 5.12 %. Population migration remained the main risk-forming factor for the spread of cholera to different countries. In the Russian Federation, monitoring of labor migration was introduced with the organization of medical observation of arriving persons for seven days after arrival from cholera affected countries. As a result of monitoring surface water bodies in 2025, 50 non-toxigenic strains of Vibrio cholerae O1 circulating in the territory of the Russian Federation and belonging to different genetic lines were isolated. A decrease in the number of acute intestinal infections was noted from 13 (2024) to 5 (2025), caused by non-toxigenic V. cholerae nonO1/nonO139. Forecast for 2026: the risk of cholera importation persists due to the expected increase in the intensity of labor migration. Registration of acute intestinal infections and extra-intestinal infections caused by non-toxigenic strains of V. cholerae nonO1/nonO139 is probable. Detection of non-toxigenic strains of V. cholerae O1 and their clonal complexes circulating in water bodies will continue. Stable epidemiological situation on cholera in the Russian Federation is predicted for 2026.

The aim of the study was to perform a detailed molecular-genetic characterization of the toxigenic Vibrio cholerae nonO1/nonO139 strain isolated from the Don River in the Rostov Region in 2024. Materials and methods. Whole-genome sequencing was carried out on the MiSeq Illumina and Oxford Nanopore platforms. Primary assembly was performed de novo through the Canu software based on long reads, and subsequent alignment was carried out using short reads with the Pilon program. Vector NTI package, BLASTN, BLASTP, BioEdit, Ugene, abricate (CARD database) and SnapGene Viewer were used in the bioinformatics analysis of genomes. The search for the limits of wb * clusters and comparison of their genes were conducted by means of the author’s program “O-cluster”. The phylogenetic tree was constructed using the roary and FastTree software. Results and discussion. A toxigenic strain of NAG-vibrio has been identified for the first time in Russia. The sequences of both chromosomes of the studied strain have been determined via hybrid genome assembly. Based on the analysis of the O-antigen biosynthesis region, the strain has been classified into serogroup O105 and it shows a high degree of closeness to clinical strains of this serogroup previously isolated in China. Bioinformatic analysis has revealed the presence of tandems of two identical copies of CTX and RS1 prophages on the large chromosome, which are notably distinct from their prototypes. The ctxB gene is represented by the ctxB12 allele, and all RS1 genes, except for rstC , show no similarity to canonical sequences. A VPI-1 island with an alternative tcpA allele, a T3SS cluster within the VPI-3 island, and the cholix toxin gene have been identified, which constitutes a rare combination within a single strain’s genome. The set of intact determinants of other pathogenicity factors is sufficient for the realization of pathogenic potential. The presence of a number of antibiotic resistance determinants in the genome did not always correlate with the phenotypic manifestation of resistance/susceptibility to antimicrobial agents in vitro . The genome structure of the strain and its close relationship to clinical isolates indicate a potential capacity to cause acute intestinal infections. Since there is a possibility of new introductions of such strains into the Russian Federation, their detection in water bodies necessitates the implementation of a set of anti-epidemic measures.

The aim of the work was to conduct a comparative study of the composition of preparations made of outer membrane vesicles (OMV) of toxigenic and atoxigenic strains of Vibrio cholerae O1 El Tor and O139 serogroups. Materials and methods . The work used toxigenic and atoxigenic strains of V. cholerae O1 and O139 serogroups, from which OMV preparations were obtained. The structure of OMV preparations was investigated applying transmission electron microscopy. Genome-wide DNA sequencing was performed using the MiSeq and DNB-Seq-g50 platforms. The computer analysis of the obtained data was carried out using the SeqAnalayser 2.1 software. To conduct bioinformatic analysis, the following programs were used: Exonerate version 2.4.0 and SnapGene Viewer. Protein profiling was performed using MALDI-TOF mass spectrometry. The identification of antigenic determinants: cholera toxin and outer membrane proteins (OmpT, OmpU) was carried out by enzyme immunoassay (ELISA). Enzymatic activity was detected using appropriate media and substrates. Fatty acids were indicated through gas chromatography-mass spectrometry. Results and discussion . A comparative analysis of the composition of OMV preparations obtained from toxigenic and atoxigenic V. cholerae O1 and O139 strains has been carried out. The difference/similarity in the composition of OMV preparations has been revealed. According to the data of genome-wide sequencing and bioinformatics analysis, fragments of chromosomes containing a different set of genes and mobile genetic elements were found in OMV preparations obtained from strains with different genotypes. V. cholerae O1 and O139 OMV are able to participate in sorting and docking of cell contents, in vesiculation, contributing to the evolutionary transformations of the genome. OMV may be an additional factor in the pathogenicity/adaptation/persistence/competitiveness of the cholera pathogen.

Objective: to analyse the possibility of Vibrio cholerae survival in surface reservoirs of the Russian Federation subjects under conditions of low water temperature, compared to the temperature optimum for the growth and reproduction of these microorganisms in environmental objects (EO).Materials and methods: official data of the reference centre for cholera monitoring on the detection of Vibrio cholerae strains in the aquatic environmental objects of the Russian Federation subjects within the framework of monitoring studies for the period from 2019 to 2024 were used. Monitoring studies were organised and conducted in accordance with the current regulatory and methodological documentation. All isolated Vibrio cholerae strains were identified by regulated bacteriological, serological, molecular-biological methods.Results: 553 strains of Vibrio cholerae O1 serogroup (three of them toxigenic) and 13013 strains of NAG-vibrios were isolated for the period from 2019 to 2024. The range of minimum water temperatures of surface reservoirs from which the first strains of Vibrio cholerae were isolated in the administrative territories of types I–III on cholera epidemic manifestations was in the range from 1 °C to 15 °C, which is lower than the temperature optimum (above +16 °C) for their growth, and indicates the possibility of survival of these microorganisms in conditions of low temperatures of water bodies, and, therefore, does not exclude the possibility of spreading infection by water in the event of a toxigenic strain entering the reservoir.Conclusions: detection of NAG vibrios and non-toxigenic strains of Vibrio cholerae O1 serogroup in the water of surface reservoirs at temperatures up to 15 °C is an indicative sign of the existence of conditions conducive to the spread of infection in case of cholera importation into the territory of the Russian Federation and indicates the expediency of an earlier start and a later end of cholera monitoring studies in Russia.

BACKGROUND: In 2023, two cases of acute intestinal infection caused by identical strains of Vibrio cholerae El Tor carrying the preCTX prophage and the classical allele of the tcpA gene were reported in Russia for the first time. The patients had recently visited Asian countries (Indonesia, Bangladesh, and India). A detailed molecular genetic characterization of the pathogens was required to assess their pathogenic potential. AIM: To provide phenotypic and genotypic characteristics of Vibrio cholerae O1 strains imported to Moscow in 2023 and isolated from patients with acute intestinal infection. METHODS: In this study, whole-genome sequences (WGSs) were obtained using the MiSeq Illumina platform. Bioinformatics analysis was performed using Vector NTI Advance, BioEdit, BLASTN, BLASTP, and CARD, as well as the pygenomeviz and biopython packages. RESULTS: The studied strains belonged to the biovar El Tor, serovar Ogawa, and had identical antibiotic resistance profiles. Their genomes contained the preCTX and RS1 prophages with a unique gene composition. RS1 harbored the rstRcalc gene (Calcutta variant), while the RS2 element of preCTX contained the rstRclass gene (classical variant), as well as an additional orfX gene of unknown function. Within the complete VPI-1 pathogenicity island, the tcp cluster — responsible for the production of toxin-coregulated pili — demonstrated significant differences from prototypes in the tcpF and toxT genes, although their products retained characteristic active domains. The tcpA gene was of the classical type but differed from the prototype by three single nucleotide polymorphisms. The strains also possessed a wide array of intact genetic determinants of pathogenicity factors characteristic for the biovar El Tor, sufficient to express its full pathogenic potential. CONCLUSION: The analyzed Vibrio cholerae strains were imported from India, Bangladesh, or Indonesia and are linked by a common source of infection and a transmission pathway. The risk of further importation of such or similar strains persists, and the characterized isolates can be used as reference strains for cholera surveillance studies in the Russian Federation.

The aim of the work was to analyze the peculiarities of the cholera epidemiological situation in the world and in Russia in 2024; forecast for 2025. The results of proactive epidemiological surveillance showed a continued upward trend in cholera morbidity in the world despite of the decrease in this indicator in 2024 (762 830 cases in 42 countries) as compared to 2023 (816 235 in 47 countries). A special feature was the import of a polyantibiotic-resistant Vibrio cholerae O1 El Tor strain from Tanzania to Island Mayotte. Active labor migration to the Russian Federation (RF) from countries with unfavorable cholera situation was noted (11 686 foreigners, 88.17 % – from India). No cholera patients (vibrio carriers) were identified in RF. The features of epidemiological situation have been established: two toxigenic V. cholerae O1 strains similar to the one isolated in 2023, and one toxigenic strain of NAG, documented in RF for the first time, were detected in surface water bodies. It has been determined that the non-toxigenic V. cholerae O1 strain, which caused a case of acute intestinal infection, and waterborne O1 strains circulating in Russia belong to the same clone. For the first time, the non-toxigenic V. cholerae O139 strain was isolated from a patient with acute intestinal infection. An increase in the number of non-toxigenic O1 strains isolated from water bodies was noted (335 in 2024, 52 in 2023), mainly due to the clonal complex (250 strains, Krasnodar Territory) spread through karst waters. Diseases caused by non-toxigenic NAG vibrios V. parahaemolyticus and V. fluvialis, were detected. The forecast for 2025 covers the risks of cholera importation, including cases caused by antibiotic-resistant strains; increase in labor migration from countries with an unfavorable cholera situation; the likelihood of detecting toxigenic O1 and nonO1/nonO139 strains in the environment, an increase in the number of acute intestinal infections caused by non-toxigenic cholera and other pathogenic vibrios; possible detection of extra-intestinal infections, an increase in the number of non-toxigenic V. cholerae O1 in environmental objects. It is advisable to start monitoring studies earlier (April) and finish them later (October) in RF in 2025 in type I territories classed according to epidemic manifestations of cholera.

Objective: retrospective analysis of the results of monitoring studies of environmental water bodies in Rostov-on-Don for the presence of V. cholerae for the period from 1989 to 2023 at stationary sampling points assigned to the Rostov-on-Don Anti-Plague Institute of Rospotrebnadzor, identification of trends and features of the dynamics of isolation and biological properties isolated strains. Materials and methods: in the period from 1989 to 2023, 4362 samples from aquatic ecosystems were examined. The organization and conduct of research was carried out in accordance with regulatory and methodological documents (SanPiN 3.3686-21, MUK 4.2.3745- 22, MUK 4.2.3746-22). Sequencing of isolated strains was performed on the MiSeq (Illumina) platform. Results: during the analyzed period, V. cholerae strains were found in all studied reservoirs of the Rostov-on-Don: in the Don, Temernik and Dead Donets rivers, mainly in July and August. Of the 122 isolated strains, 8 are epidemiologically significant (toxigenic strains) and 114 are nontoxigenic. The largest number of strains was isolated in 2022 — 21 strains. V. cholerae strains O1 (119 strains) and O139 (3) of serogroups, biovar classic and El Tor (Ogawa, Inaba and Gikoshima serovars) were found. Conclusions: epidemiological risks of the introduction of the cholerae pathogen from endemic countries, as well as from neighboring countries, in cases of complications of the epidemic of this infection, remain. In the surface reservoirs of the Rostov-on-Don, there are favorable conditions for the circulation of V. cholerae and the risk of spreading infection by water, in case of contamination by the cholerae pathogen of aquatic ecosystems.

Cholera vibrios, isolated from surface water bodies during monitoring activities, can differ from typical ones in terms of agglutinability with diagnostic cholera sera, which makes it difficult to assign them to a specific serogroup. Therefore, the determination of the causes for these deviations through the study of the structure of genetic determinants responsible for the synthesis of O‑antigen (wb* clusters) is considered a relevant task. The aim of this work was to identify wb* clusters in the genomes of R-variant Vibrio cholerae, study their structure, and conduct phylogenetic analysis of the strains. Materials and methods. Full genome sequencing was performed using Illumina MiSeq and MinION platforms. The assembly was conducted de novo using the SPAdes assembler software (v.3.11.1). Manipulation of cluster sequences and data visualization were carried out using the BLAST program from the ncbi-blast-suite package version 2.13.0, Python scripts and packages such as pyGenomeViz and Biopython. The phylogenetic tree was constructed using the roary program (v.3.13.0). Full genome alignment was carried out using the nucmer and promer programs from the MUMmer 4 package version 4.0. Results and discussion. R-variant V. cholerae contains different types of wb* clusters in the genome, with most commonly occurring O23 and O59. A comparison has been made between translated amino acid sequences of wb* regions in R-variant strains and amino acid sequences of reference strains present in the NCBI database. Some of the analyzed clusters completely match with reference strains, while others show high variability. Phylogenetically, the strains primarily group together according to the wb* cluster type, regardless of the S/R phenotype. Classifying R-variant V. cholerae strains based on agglutinability is challenging. Molecular research methods, polymerase chain reaction, in particular, are necessary to determine the pathogenic potential of such strains.

The paper presents an analysis and assessment of risks of complication of epidemiological situation regarding cholera in the Russian Federation and a forecast for 2024. It briefly characterizes the dynamics of the epidemic process around the world during the last decade (2013–2022). The forecast of cholera importation into the Russian Federation, due to activity of the epidemic process in endemic countries, has been confirmed. In 2023, two cases of cholera importation from India were reported. It was established that the toxigenic strains isolated from patients belonged to the currently dominant in the world “post-Haitian” group, including in India and Bangladesh, with the genotype ctxB7 tcpA^CIRS rtxA4a VSP-IIΔ0495-0512. Furthermore, non-toxigenic Vibrio cholerae O1 strains were isolated from samples of biological material from persons arriving from India, containing preCTX prophage and distinct from the strain (ctxAB^– tcpA^EТ–) isolated from a patient with acute intestinal infection (Zaporozhye Region, Melitopol). Nontoxigenic NAG vibrios were isolated from 13 patients with acute intestinal infections, which is 1.9 times more than in 2022. Regardless of importations, a strain of V. cholerae O1 (ctxB1⁺tcpA^EТ) of the “pre-Haitian” group was isolated from a water sample of a surface reservoir. 52 non-toxigenic V. сholerae O1 strains were found on the territory of 11 constituent entities of the Russian Federation, which is 18.9 % more than in 2022. In 2024, epidemiological risks of cholera importation from endemic countries to any administrative territory of the Russian Federation remain. The risks of epidemic manifestations of cholera are especially high in the territories of the constituent entities of the Russian Federation which are under ongoing emergency, due to migration processes, and the impact of other risk-forming factors that contribute to an increase in the degree of contact of the population with water bodies contaminated with cholera vibrios. If epidemic foci of cholera are to emerge in a neighboring country, there will be a high probability of spread of this infection in the stated constituent entities of the Russian Federation and in other territories of southern Russia. In the absence of epidemic manifestations of cholera, the possibility of occasional detection of toxigenic O1 serogroup strains in water samples due to unidentified introductions from cholera-affected areas cannot be ruled out.

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.