Vibrio Cholerae El Tor Strains Research Articles

Two defence systems eliminate plasmids from seventh pandemic Vibrio cholerae.

Horizontal gene transfer can trigger rapid shifts in bacterial evolution. Driven by a variety of mobile genetic elements-in particular bacteriophages and plasmids-the ability to share genes within and across species underpins the exceptional adaptability of bacteria. Nevertheless, invasive mobile genetic elements can also present grave risks to the host; bacteria have therefore evolved a vast array of defences against these elements1. Here we identify two plasmid defence systems conserved in the Vibrio cholerae El Tor strains responsible for the ongoing seventh cholera pandemic2-4. These systems, termed DdmABC and DdmDE, are encoded on two major pathogenicity islands that are a hallmark of current pandemic strains. We show that the modules cooperate to rapidly eliminate small multicopy plasmids by degradation. Moreover, the DdmABC system is widespread and can defend against bacteriophage infection by triggering cell suicide (abortive infection, or Abi). Notably, we go on to show that, through an Abi-like mechanism, DdmABC increases the burden of large low-copy-number conjugative plasmids, including a broad-host IncC multidrug resistance plasmid, which creates a fitness disadvantage that counterselects against plasmid-carrying cells. Our results answer the long-standing question of why plasmids, although abundant in environmental strains, are rare in pandemic strains; have implications for understanding the dissemination of antibiotic resistance plasmids; and provide insights into how the interplay between two defence systems has shapedthe evolution of the most successful lineage of pandemic V. cholerae.

Heterogeneity by antibiotic sensitivity and genotype of Vibrio Cholerae El Tor strains isolated from environmental objects in Russia

BACKGROUND: Waterborne diseases kill 3.4 million people worldwide each year. Cholera is one such disease. Up to 4 million cases of this infection occur in the world every year, leading to more than 100,000 deaths. The plasticity of the genome of the causative agent of cholera, the mobility of genetic elements carrying factors of pathogenicity and antibiotic resistance, contribute to the variability and unpredictability of the spectrum of resistance, the formation of new pheno- and genotypes. A single Vibrio cholerae isolate can contain up to 40 different genes that can confer resistance to 22 antibiotics, representing nine different classes of antimicrobial drugs. The ability of Vibrio cholerae to long-term survival in aquatic ecosystems in which there is an active exchange of genetic information and new ecological lines may arise that have potential advantages in the adaptation of microorganisms to adverse conditions, emphasizes the complexity of the ways of transmission of this infection and the need for studies at the environmental level. Periodic deliveries of cholera to the territory of the Russian Federation with (without) the spread of the infectious agent, V. cholerae contamination of surface water bodies used as sources of water supply and for recreational water use, the possibility of implementing the main route for cholera (water) the pathway for the spread of the pathogen of the infection indicate the need for conducting annual monitoring of antimicrobial resistance as part of the epidemiological surveillance of cholera in order to obtain information on the distribution, nature and dynamics of resistance in a specific period of time in a given territory.
 AIM: Analysis of the spectrum of antibiotic resistance of Vibrio cholerae O1 serogroup strains isolated from environmental objects in various territories of the Russian Federation in 2020.
 MATERIALS AND METHODS: 25 strains V. cholerae О1 El Tor, isolated from environmental objects in the Russian Federation in 2020. The sensitivity / resistance of the studied strains to 13 antibacterial drugs was determined by the method of serial dilutions on a solid nutrient medium in accordance with guidelines. PCR-genotyping of strains V. cholerae El Tor was performed for 14 target genes, followed by cluster analysis.
 RESULTS: The strains showed resistance markers to furazolidone, trimethoprim/sulfamethoxazole, ampicillin, nalidixic acid, ceftriaxone, which formed 5 phenotypes. PCR-genotyping genotyping of 14 target genes divided the strains into five genotypes (A1A5), corresponding to certain territories. The antibiotic resistance profiles within the same genotype in V. cholerae О1 El Tor belonging to different territories were both the same and different.
 CONCLUSION: The genotypic diversity of isolated strains was revealed, the variability of resistance markers even in one region, which indicates both changes in the V. cholerae population and the possibility of circulation of various geno- and phenotypes, which emphasizes the importance of constant monitoring of these pathogens.

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.

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.

VIBRIO CHOLERAE EL TOR ANTIBIOTIC RESISTANCE GATHERED FROM ENVIRONMENTAL OBJECTS ON THE TERRITORY OF THE RUSSIAN FEDERATION

The paper presents the analysis of Vibrio cholerae El Tor strains antibiotic resistance gathered from environmental objects on the territory of the Russian Federation during 2007–2016. We determined the sensitivity of 268 strains of V. cholerae El Tor to 12 antibacterial drugs by the method of serial dilutions in a dense nutrient medium. The strains showed resistance to 1–7 antibacterial drugs. The emergence of quinolone resistance in polyresistant strains of V. cholerae E.

Comparative genomics of Vibrio cholerae El Tor strains isolated at epidemic complications in Siberia and at the Far East

The territory of Siberia and the Far East of Russia is classified as epidemically safe for cholera; however, in the 1970s and 1990s a number of infection importation cases and acute outbreaks associated with the cholera importation were reported. Here, we analyze genomes of four Vibrio cholerae El Tor strains isolated from humans during epidemic complications (imported cases, an outbreak) in the 1990s. The analyzed strains harbor the classical allele of the cholera toxin subunit B gene (ctxB1); thus, belong to genetically altered variants of the El Tor biotype. Analysis of the genomes revealed their high homology with the V. cholerae N16961 reference strain: 85–93 SNPs were identified in the core genome as compared to the reference. The determined features of SNPs in the CTX prophage made it possible to propose the presence of a new subtype – CTX-2a in two strains; the other two strains carried the prophage of CTX-3 type. Results of phylogenetic analysis based on SNP-typing demonstrated that two strains belonged to the second wave, and two – to the early third wave of cholera dissemination in the world. Phylogenetic reconstruction in combination with epidemiological data permitted to trace the origin of the strains and the way of their importation to the Russian Federation directly or through temporary cholera foci.

Холерные вибрионы неО1/неО139 серогрупп в этиологии острых кишечных инфекций: современная ситуация в России и в мире

Cholera vibrios of nonO1/nonO139 serogroups (NAG vibrios) are well known as natural inhabitants of aquatic environments and as agents of acute intestinal infections (AII) with different degree of severity. On the background of the current vehement dissemination of the new highly virulent Vibrio cholerae El Tor strains, the moral and economic damage from NAG vibrios is not that evident, but still, not less real and rather significant. In spite of rare association with large outbreaks, NAG vibrios rank in the etiology of AII all over the world and pose a potential threat for the population health in our country. The paper reviews the present-day morbidity as regards NAG infections in various countries, including Russia; as well as the molecular premises of the agents’ pathogenicity and drug resistance. Analysis of the published data has revealed an utter heterogeneity of NAG populations, circulating in certain territories, by reference to the presence/absence of genetic determinants of pathogenicity, persistence, housekeeping and antibiotic resistance factors. Nevertheless, the joint gene-pool of the population includes rather a wide set of genes and can expand due to importations of strains, which differ from resident ones in the genotypes, from other regions. This brings about the danger of emergence of clones with higher pathogenic and, probably, even epidemic potential driven by gene exchange, and the consequences of their occurrence are unpredictable. Therefore, the NAG vibrios demand proper attention from investigators and sanitary-epidemiological institutions of the Russian Federation.

The Cpx system regulates virulence gene expression in Vibrio cholerae.

Bacteria possess signal transduction pathways capable of sensing and responding to a wide variety of signals. The Cpx envelope stress response, composed of the sensor histidine kinase CpxA and the response regulator CpxR, senses and mediates adaptation to insults to the bacterial envelope. The Cpx response has been implicated in the regulation of a number of envelope-localized virulence determinants across bacterial species. Here, we show that activation of the Cpx pathway in Vibrio cholerae El Tor strain C6706 leads to a decrease in expression of the major virulence factors in this organism, cholera toxin (CT) and the toxin-coregulated pilus (TCP). Our results indicate that this occurs through the repression of production of the ToxT regulator and an additional upstream transcription factor, TcpP. The effect of the Cpx response on CT and TCP expression is mostly abrogated in a cyclic AMP receptor protein (CRP) mutant, although expression of the crp gene is unaltered. Since TcpP production is controlled by CRP, our data suggest a model whereby the Cpx response affects CRP function, which leads to diminished TcpP, ToxT, CT, and TCP production.

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.

A retrospective macrorestrictive analysis of Vibrio cholerae eltor strains isolated under epidemic complications in the Russian Far East

A retrospective study of the clonal structure of 24 Vibrio cholerae eltor strains isolated during epidemic complications in the Russian Far East was carried out on the basis of a macrorestrictive analysis of Vibrio cholerae eltor genomic DNA using NotI and SfiI endonucleases. It was found that clonal (by amplification profiling data) toxigenic strains (n = 23) were characterized by variability of the NotI/SfiI-generated restriction profiles. The V. cholerae eltor isolated during an outbreak in the city of Yuzhno-Sakhalinsk were differentiated into four pulse types, with one dominating type detected in 76.5% of the strains. A separate NotI/SfiI-restriction profile was characteristic for the isolates from Primorskii krai. A nontoxigenic strain formed a separate dendrogram line from the toxigenic strains on the basis of the PFGE-pattern structure. The use of macrorestriction analysis was shown to be effective for deep characterization of the V. cholerae population structure and for ascertainment of molecular-epidemiological regularities in the territorial distribution of certain V. cholerae clones.

Model of Vibrio cholerae biofilm as a mechanism of its survival in surface water reservoirs

Vibrio cholerae eltor strains with different epidemic importance isolated from river water in the city of Vladivostok during a cholera outbreak (1999) and in the city of Irkutsk during a safe cholera period (2005) are used in the experiment. A biofilm structure consisting of a peripheral part, bundles, polysaccharide matrix, canals, and polymorphic vibrios is presented by light and luminescent microscopy. The metachromatic pink coloring of the matrix (crystal violet and toluidine blue) or fluorescent reddish orange color (acridine orange) are evidence of acid mucopolysaccharide (glucosaminoglycans) content. The biofilm of a toxigenic strain as opposed to a nontoxigenic one is formed much later, while the elements comprising its structure are more apparent. The viability of vibrio cells during the experiment (90 days) preserving the initial pathogenic potential testifies to the highly adaptable properties of the Vibrio cholerae eltor, which promotes its survival and existence in surface water reservoirs under favorable ecological conditions (optimal temperature, existence of chitin-containing substratum, etc.).

THE INFLUENCE OF SOME FACTORS ON THE FORMATION OF BIOFILM BY TOXIGENIC AND NON-TOXIGENIC VIBRIO CHOLERAE EL TOR STRAINS

The early stages of biofilm formation on polystyrene surface were studied in 14 Vibrio cholerae El Tor strains, all of which carried genes of vps cluster, mshA, and hapR genes. 13 Vibrio cholerae El Tor strains were shown to be capable of adsorption on the walls of microtiter plate wells and of ring formation at the "polystyrene-medium" interface. One of the Vibrio cholerae El Tor strains tested was defective in this ability. The use of different media - modified M1 medium and 1 per cent peptone - failed to reveal sufficient influence of culture medium composition on the ability of Vibrio cholerae to form multilayer biofilm at the phase boundary "polystyrene-medium". Temperature reduction from 37°C to 25°C also didn t result in statistically significant enhancement of monolayer formation in both media. Quantitative evaluation of biofilm formation showed that this ability was clearly marked only in two toxigenic and two non-toxigenic strains of Vibrio cholerae El Tor. Monolayer comparison in ELISA demonstrated that only two toxigenic and two non-toxigenic strains possessed the ability for exopolysaccharide production.

Molecular-genetic analysis of Vibrio cholerae El Tor strains of epidemic risk isolated in Siberian and Far East regions of Russia

PCR detection of biovar specificity and pathogenicity determinants has been performed in order to analyze the structural components of the genome of V. cholerae El Tor strains (n = 90) isolated during epidemic outbreaks in Siberia and the Far East, and the nucleotide sequence of the ctxB gene and the structure of ctxAB gene promoter region have been determined. As a result, toxicogenic strains V. cholerae El Tor were divided into two groups: the first group contained strains isolated at the initial stages of the seventh pandemia (in the 1970s), and they had the genotype ctxB3+rstREl+rstRCl-rstC+TCL+tbr4; all El Tor vibrios posing an epidemic risk isolated in the 1990s were characterized as atypical variants due to ctxB gene harboring of the classical genotype (ctxB) in their genome and were placed in the second group. The second group fell into three genotypes according to the set of tested genetic markers (ctxB, rstR, rstC, TLC, and tbr) and affect certain territories. The established variability of the genome structure of Vibrio El Tor atypical variants may serve as a marker for molecular and epidemiological analysis of cholera entry pathways and distribution and is useful for speculating on the most likely directions of pathogen evolution.

The phenotypic and molecular biological characteristics of Vibrio cholerae El Tor strains isolated from environmental water objects in Rostov-on-Don in 2003-2008

The pheno- and genotypic properties of Vibrio cholerae El Tor cultures identified during monitoring for V. cholerae in the aquatic ecosystems of Rostov-on-Don in the period 2003-2008 were studied in the extended range. In addition to known laboratory diagnostic techniques for cholera, the authors used a volume agglomeration test with antilipase diagnosticum, as well as polymerase chain reaction using specific primers to detect 30 nucleotide sequences of the genes associated with V. cholerae pathogenicity. The range of pheno- and genotypic characteristics of study V. cholerae O1 cultures was extended. There was evidence for the volume agglomeration test with a polymerase antilipase agent as a complementary test for toxicogenicity. The Vibrio cholerae O1 strains isolated from the aquatic environment were shown to confer a real and potential risk in the low cholera incidence area of Rostov-on-Don. The findings may be used to improve methodic approaches to monitoring environmental objects for Vibrio cholerae in the optimization of epidemiological surveillance of cholera at the regional level.

In Vitro Production of Cholera Toxin by Vibrio cholerae El Tor Strains from Human Beings and the Environment

In Vitro Production of Cholera Toxin by Vibrio cholerae El Tor Strains from Human Beings and the Environment

Proteins involved in difference of sorbitol fermentation rates of the toxigenic and nontoxigenic Vibrio choleraeEl Tor strains revealed by comparative proteome analysis

BackgroundThe nontoxigenic V. cholerae El Tor strains ferment sorbitol faster than the toxigenic strains, hence fast-fermenting and slow-fermenting strains are defined by sorbitol fermentation test. This test has been used for more than 40 years in cholera surveillance and strain analysis in China. Understanding of the mechanisms of sorbitol metabolism of the toxigenic and nontoxigenic strains may help to explore the genome and metabolism divergence in these strains. Here we used comparative proteomic analysis to find the proteins which may be involved in such metabolic difference.ResultsWe found the production of formate and lactic acid in the sorbitol fermentation medium of the nontoxigenic strain was earlier than of the toxigenic strain. We compared the protein expression profiles of the toxigenic strain N16961 and nontoxigenic strain JS32 cultured in sorbitol fermentation medium, by using fructose fermentation medium as the control. Seventy-three differential protein spots were found and further identified by MALDI-MS. The difference of product of fructose-specific IIA/FPR component gene and mannitol-1-P dehydrogenase, may be involved in the difference of sorbitol transportation and dehydrogenation in the sorbitol fast- and slow-fermenting strains. The difference of the relative transcription levels of pyruvate formate-lyase to pyruvate dehydrogenase between the toxigenic and nontoxigenic strains may be also responsible for the time and ability difference of formate production between these strains.ConclusionMultiple factors involved in different metabolism steps may affect the sorbitol fermentation in the toxigenic and nontoxigenic strains of V. cholerae El Tor.

Survival of <i>Vibrio cholerae</i> in industrially polluted water, with particular reference to iron concentrations

High levels of iron are often found in surface waters due to the discharge of iron-containing industrial effluents. The effect of iron as well as pH on the survival of Vibrio cholerae (non-O1, El Tor and classical strains) in water samples from 12 points, where selected industrial effluents were discharged into rivers, was studied. All three V. cholerae strains survived longest during spring. V. cholerae non -O1 survived significantly longer than El Tor and classical strains. At pH 8.0, the survival of V. cholerae increased with increase in iron concentration (1 to10 mg/ℓ) of polluted water. At lower than pH 8.0, the effect of iron was not significant. In polluted water, at pH 8.0, the presence of iron resulted in prolonged V. cholerae survival at lower concentrations than those required for survival in distilled water. We suggest that chemical contamination of water may be one of the important factors instrumental in the subsequent ability of V. cholerae to persist, multiply and survive in the aquatic environment. Water SA Vol.30(1): 65-69

The chemotactic response of Vibrio anguillarum to fish intestinal mucus is mediated by a combination of multiple mucus components.

Chemotactic motility has previously been shown to be essential for the virulence of Vibrio anguillarum in waterborne infections of fish. To investigate the mechanisms by which chemotaxis may function during infection, mucus was isolated from the intestinal and skin epithelial surfaces of rainbow trout. Chemotaxis assays revealed that V. anguillarum swims towards both types of mucus, with a higher chemotactic response being observed for intestinal mucus. Work was performed to examine the basis, in terms of mucus composition, of this chemotactic response. Intestinal mucus was analyzed by using chromatographic and mass spectrometric techniques, and the compounds identified were tested in a chemotaxis assay to determine the attractants present. A number of mucus-associated components, in particular, amino acids and carbohydrates, acted as chemoattractants for V. anguillarum. Importantly, only upon combination of these attractants into a single mixture were levels of chemotactic activity similar to those of intestinal mucus generated. A comparative analysis of skin mucus revealed its free amino acid and carbohydrate content to be considerably lower than that of the more chemotactically active intestinal mucus. To study whether host specificity exists in relation to vibrio chemotaxis towards mucus, comparisons with a human Vibrio pathogen were made. A cheR mutant of a Vibrio cholerae El Tor strain was constructed, and it was found that V. cholerae and V. anguillarum exhibit a chemotactic response to mucus from several animal sources in addition to that from the human jejunum and fish epithelium, respectively.

Induction of SOS like responses by nitrofurantoin in Vibrio cholerae el tor cells

Treatment of Vibrio cholerae el tor strain SLH22(J) with nitrofurantoin induced dose-dependent prophage 'kappa', the maximum induction being 6-fold the spontaneous induction level. UV-inactivated 'kappa' phages were Weigle reactivated, the maximum Weigle factor being 1.8 and 2.0 respectively in nitrofurantoin and UV pretreated el tor strain H218 Smr. Nitrofurantoin treatment also caused significant filamentation of the el tor strain H218 Smr and mutation of these cells from ampicillin sensitivity to ampicillin resistance. The levels of the four SOS-like responses induced by this drug were low but significant.

Toxin Production by Atypical Strains of Vibrio cholerae El Tor Under Different Cultural Conditions

It was observed that at 37 degrees C under in vitro conditions, aerobic culture filtrates of a few strains of Vibrio cholerae biotype El Tor isolated from diarrhoeal cases produced a minute amount of toxin which failed to elicit a positive ileal loop reaction like toxigenic strains. Thus, these strains showed an atypical behaviour in their toxin producing ability. At 25 degrees C and 30 degrees C under aerobic cultural conditions enhanced toxin production was noticed in toxigenic strains, but these temperatures did not affect the toxigenicity of the atypical strains. The atypical Vibrio cholerae El Tor strains exhibited enhanced toxin production only at 37 degrees C under anaerobic conditions and the amount of toxin produced was akin to those of the toxigenic strains. In comparison to aerobic conditions, growth was observed to be comparatively lower under anaerobiosis both in the toxigenic and atpyical V. cholerae strains. Moreover, in contrast to the toxigenic strains, the toxin did not remain membrane-bound in these atypical strains at 37 degrees C and aerobic cultural conditions.