El Tor Strains Research Articles

Identification of <i>Vibrio cholerae</i> O1 strains of the El Tor biovar sensitive to polymyxin B and their molecular genetic analysis

The aim of the work was the identification and genetic characterization of Vibrio cholerae O1 strains of the El Tor biovar sensitive to polymyxin B among isolates imported to Russia.Materials and methods. We used 56 toxigenic and non-toxigenic strains of V. cholerae isolated from patients and from the environmental samples on the territory of Russia in 1970-2020. Resistance to polymyxin B was determined according to MR4.2.2218-07. The ability of strains to form a biofilm on the abiotic surface was assessed by a photometric method. Nucleotide sequences of genes were determined using UGENE 1.32 and MEGA X software. Phylogenetic analysis and tree construction were performed using "maximum parsimony" method in MEGA X software.Results and discussion. Two genetically modified strains of V. cholerae O1 biovar El Tor, M1509 and 3265/80, which were imported to Russia from India in 2012 and 2014, respectively, were identified. The analysis of 12 genes responsible for the resistance of V. cholerae O1 biovar El Tor strains to polymyxin B demonstrated that these strains contain the allele of the carRS gene, which is typical for all strains of cholera vibrio sensitive to polymyxin B. Study of V. cholerae M1509 and 3265/80 phylogeny based on SNP analysis showed that they fall into the same cluster with isolates containing the carRS allele isolated in India (2015) and Bangladesh (2018). V. cholerae M1509 and 3256/8 strains had the ability to form a biofilm similar to those observed in other genetically modified strains of cholera vibrio included into analysis.Conclusion. Highly virulent strains of the cholera agent with altered diagnostically significant features are imported into Russia, which should be taken into account when identifying V. cholerae O1 biovar El Tor strains isolated from patients and environmental samples during monitoring studies.

Recent Vibrio cholerae O1 Epidemic Strains Are Unable To Replicate CTXΦ Prophage Genome.

ABSTRACTCholera, an acute diarrheal disease, is caused by pathogenic strains of Vibrio cholerae generated by the lysogenization of the filamentous cholera toxin phage CTXΦ. Although CTXΦ phage in the classical biotype are usually integrated solitarily or with a truncated copy, those in El Tor biotypes are generally found in tandem and/or with related genetic elements. Due to this structural difference in the CTXΦ prophage array, the prophage in the classical biotype strains does not yield extrachromosomal CTXΦ DNA and does not produce virions, whereas the El Tor biotype strains can replicate the CTXΦ genome and secrete infectious CTXΦ phage particles. However, information on the CTXΦ prophage array structure of pathogenic V. cholerae is limited. Therefore, we investigated the complete genomic sequences of five clinical V. cholerae isolates obtained in Kolkata (India) during 2007 to 2011. The analysis revealed that recent isolates possessed an altered CTXΦ prophage array of the prototype El Tor strain. These strains were defective in replicating the CTXΦ genome. All recent isolates possessed identical rstA and intergenic sequence 1 (Ig-1) sequences and comparable rstA expression in the prototype El Tor strain, suggesting that the altered CTXΦ array was responsible for the defective replication of the prophage. Therefore, CTXΦ structures available in the database and literatures can be classified as replicative and nonreplicative. Furthermore, V. cholerae epidemic strains became capable of producing CTXΦ phage particles since the 1970s. However, V. cholerae epidemic strains again lost the capacity for CTXΦ production around the year 2010, suggesting that a significant change in the dissemination pattern of the current cholera pandemic occurred.IMPORTANCE Cholera is an acute diarrheal disease caused by pathogenic strains of V. cholerae generated by lysogenization of the filamentous cholera toxin phage CTXΦ. The analysis revealed that recent isolates possessed altered CTXΦ prophage array of prototype El Tor strain and were defective in replicating the CTXΦ genome. Classification of CTXΦ structures in isolated years suggested that V. cholerae epidemic strains became capable of producing CTXΦ phage particles since the 1970s. However, V. cholerae epidemic strains again lost the capacity for CTXΦ production around the year 2010, suggesting that a critical change had occurred in the dissemination pattern of the current cholera pandemic.

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

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

Characteristics of the Epidemiological Situation on Cholera in the World and in the Russian Federation in 2020 and Forecast for 2021

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.

Isolation of Vibrio cholerae from clinical and drinking water samples during Cholera Outbreak in Khairpur Sindh Pakistan

Background: Cholera is a diarrheal disease that is caused by the bacterium Vibrio cholerae that transmits through contaminated water, food, oral-fecal route and spread through poor sanitation system. This study aims to investigate the reports of clinical and drinking water samples during an epidemic in Pakistan.
 Methodology: This study was conducted in District Khairpur including 660 samples (360 from clinical and 300 from drinking water) from 2014-2016. All samples were enriched in Alkaline peptone water for 6 hours and then streaked on TCBS agar, incubated at 35ºC for 24 hours. The next day standard microbiological, biochemical, serological techniques were used for the identification of V. cholerae and further identification was performed using PCR.
 Results: Out of 360 clinical samples, 76(21.11%) were positive for V. cholerae. The species-specific Outer membrane protein W precursor (ompW) gene was amplified and shown at the correct size (588 bp) through agarose gel electrophoresis. The serotyping revealed that the isolates belonged to serogroup Inaba, O1. cholerae O1 strains shown typical El Tor phenotype similar to V. cholerae El Tor strain N16961 (PBR VP+) used as the reference strain in this study. All age groups were affected where the highest onset was seen among those aged 19 years and above. The culture of drinking water (n=300), observed negative on TCBS agar.
 Conclusion: As far as our knowledge, this is the first time when the presence of V. cholerae El Tor peak has been reported for causing cholera outbreaks in Khairpur Sindh, Pakistan. However, these findings can be used for further investigations and recognizing control measures.

Dissemination of Vibrio cholerae O1 isolated from Odisha, India.

The present study reported the antimicrobial susceptibility trends, virulence genes, and drug resistance genes of Vibrio cholerae O1 strains isolated from outbreaks and epidemics over two and half decades (1995-2019) from Odisha, India. Antimicrobial susceptibility testing was performed by disc diffusion method. Virulence and drug resistance genes were detected by multiplex PCR assays. All V. cholerae O1 strains were sensitive to gentamicin, chloramphenicol, norfloxacin and ciprofloxacin while resistant to one or more antibiotics used. About 90% of the isolates of V. cholerae O1 carried antibiotic drug resistant genes (SulII, dfrA1 and strB) and SXT elements and the results correlated with the phenotypic antibiotic data obtained through disc diffusion assay. The tcpA Haitian variant V. cholerae O1 first appeared in 1999, gradually showing its increasing number upto 2019. TcpA El Tor strains only prevailed from 1995 to 2006; whereas the tcpA classical strains of V.choleraeO1 were found in less number from 1995 to 2016. Two multiplex PCR assays confirmed the presence of various toxigenic and virulence genes (toxR, ompU, ace, rtxC, ctxA, tcpA, rfbO1 and ompW) in all isolate of V. cholerae O1 strains. The present findings demonstrated the origin and spread of Haitian variants tcpA in V. cholerae O1 strains over two and half decades.

Complete Genome Sequence of Vibrio cholerae O1 El Tor Strain C6706.

Vibrio cholerae is a global health threat and a model enteric pathogen that causes the human disease cholera. Here, we report the complete genome sequence of the seventh-pandemic V. cholerae O1 El Tor strain C6706.

Evolution, distribution and genetics of atypical Vibrio cholerae - A review.

Vibrio cholerae is the etiological agent of cholera, a severe diarrheal disease, which can occur as either an epidemic or sporadic disease. Cholera pandemic-causing V. cholerae O1 and O139 serogroups originated from the Indian subcontinent and spread globally and millions of lives are lost each year, mainly in developing and underdeveloped countries due to this disease. V. cholerae O1 is further classified as classical and El Tor biotype which can produce biotype specific cholera toxin (CT). Since 1961, the current seventh pandemic El Tor strains replaced the sixth pandemic strains resulting in the classical biotype strain that produces classical CT. The ongoing evolution of Atypical El Tor V. cholerae srains encoding classical CT is of global concern. The severity in the pathophysiology of these Atypical El Tor strains is significantly higher than El Tor or classical strains. Pathogenesis of V. cholerae is a complex process that involves coordinated expression of different sets of virulence-associated genes to cause disease. We are yet to understand the complete virulence profile of V. cholerae, including direct and indirect expression of genes involved in its survival and stress adaptation in the host. In recent years, whole genome sequencing has paved the way for better understanding of the evolution and strain distribution, outbreak identification and pathogen surveillance for the implementation of direct infection control measures in the clinic against many infectious pathogens including V. cholerae. This review provides a synopsis of recent studies that have contributed to the understanding of the evolution, distribution and genetics of the seventh pandemic Atypical El Tor V. cholerae strains.

Transcriptional Silencing by TsrA in the Evolution of Pathogenic Vibrio cholerae Biotypes.

Vibrio cholerae is a globally important pathogen responsible for the severe epidemic diarrheal disease called cholera. The current and ongoing seventh pandemic of cholera is caused by El Tor strains, which have completely replaced the sixth-pandemic classical strains of V. cholerae To successfully establish infection and disseminate to new victims, V. cholerae relies on key virulence factors encoded on horizontally acquired genetic elements. The expression of these factors relies on the regulatory architecture that coordinates the timely expression of virulence determinants during host infection. Here, we apply transcriptomics and structural modeling to understand how type VI secretion system regulator A (TsrA) affects gene expression in both the classical and El Tor biotypes of V. cholerae We find that TsrA acts as a negative regulator of V. cholerae virulence genes encoded on horizontally acquired genetic elements. The TsrA regulon comprises genes encoding cholera toxin (CT), the toxin-coregulated pilus (TCP), and the type VI secretion system (T6SS), as well as genes involved in biofilm formation. The majority of the TsrA regulon is carried on horizontally acquired AT-rich genetic islands whose loss or acquisition could be directly ascribed to the differences between the classical and El Tor strains studied. Our modeling predicts that the TsrA protein is a structural homolog of the histone-like nucleoid structuring protein (H-NS) oligomerization domain and is likely capable of forming higher-order superhelical structures, potentially with DNA. These findings describe how TsrA can integrate into the intricate V. cholerae virulence gene expression program, controlling gene expression through transcriptional silencing.IMPORTANCE Pathogenic Vibrio cholerae strains express multiple virulence factors that are encoded by bacteriophage and chromosomal islands. These include cholera toxin and the intestinal colonization pilus called the toxin-coregulated pilus, which are essential for causing severe disease in humans. However, it is presently unclear how the expression of these horizontally acquired accessory virulence genes can be efficiently integrated with preexisting transcriptional programs that are presumably fine-tuned for optimal expression in V. cholerae before its conversion to a human pathogen. Here, we report the role of a transcriptional regulator (TsrA) in silencing horizontally acquired genes encoding important virulence factors. We propose that this factor could be critical to the efficient acquisition of accessory virulence genes by silencing their expression until other signals trigger their transcriptional activation within the host.

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.

Virulence Regulation and Innate Host Response in the Pathogenicity of Vibrio cholerae.

The human pathogen Vibrio cholerae is the causative agent of severe diarrheal disease known as cholera. Of the more than 200 “O” serogroups of this pathogen, O1 and O139 cause cholera outbreaks and epidemics. The rest of the serogroups, collectively known as non-O1/non-O139 cause sporadic moderate or mild diarrhea and also systemic infections. Pathogenic V. cholerae circulates between nutrient-rich human gut and nutrient-deprived aquatic environment. As an autochthonous bacterium in the environment and as a human pathogen, V. cholerae maintains its survival and proliferation in these two niches. Growth in the gastrointestinal tract involves expression of several genes that provide bacterial resistance against host factors. An intricate regulatory program involving extracellular signaling inputs is also controlling this function. On the other hand, the ability to store carbon as glycogen facilitates bacterial fitness in the aquatic environment. To initiate the infection, V. cholerae must colonize the small intestine after successfully passing through the acid barrier in the stomach and survive in the presence of bile and antimicrobial peptides in the intestinal lumen and mucus, respectively. In V. cholerae, virulence is a multilocus phenomenon with a large functionally associated network. More than 200 proteins have been identified that are functionally linked to the virulence-associated genes of the pathogen. Several of these genes have a role to play in virulence and/or in functions that have importance in the human host or the environment. A total of 524 genes are differentially expressed in classical and El Tor strains, the two biotypes of V. cholerae serogroup O1. Within the host, many immune and biological factors are able to induce genes that are responsible for survival, colonization, and virulence. The innate host immune response to V. cholerae infection includes activation of several immune protein complexes, receptor-mediated signaling pathways, and other bactericidal proteins. This article presents an overview of regulation of important virulence factors in V. cholerae and host response in the context of pathogenesis.

Epidemiological and pathogenic characteristics of Haitian variant V. cholerae circulating in India over a decade (2000–2018)

Vibrio cholerae, causative agent of the water-borne disease cholera still threatens a large proportion of world's population. The major biotypes of the pathogen are classical and El Tor. There have been recent reports of variant V. cholerae strains circulating around the world. In the present study, the epidemiological status of V. cholerae strains circulating in the country over a decade was assessed. Also, a comprehensive analysis of the difference in pathogenicity between the different biotypes of V. cholerae strains was evaluated both in-vitro and in-vivo. The amount of CT produced by different biotypes of V. cholerae strains were analyzed by GM1 ELISA and the probable reasons for the difference in toxin production was discussed. MLST analysis grouped the isolates into a single Sequence Type (ST 69) whereas PFGE analysis clustered the isolates into ten different pulsotypes revealing molecular diversity. The circulating strains were identified to produce cholera toxin and CT mRNA intermediate to the classical and prototype El Tor strains. Also, the circulating strains were identified to possess four ToxR binding sequences. In-vivo pathogenicity analysis by rabbit ileal loop fluid accumulation assay revealed the Haitian variant strains to be more hyperemic than the prototype strains.

Comparison of chitin-induced natural transformation in pandemic Vibrio cholerae O1 El Tor strains.

SummaryThe human pathogen Vibrio cholerae serves as a model organism for many important processes ranging from pathogenesis to natural transformation, which has been extensively studied in this bacterium. Previous work has deciphered important regulatory circuits involved in natural competence induction as well as mechanistic details related to its DNA acquisition and uptake potential. However, since competence was first reported for V. cholerae in 2005, many researchers have struggled with reproducibility in certain strains. In this study, we therefore compare prominent seventh pandemic V. cholerae isolates, namely strains A1552, N16961, C6706, C6709, E7946, P27459, and the close relative MO10, for their natural transformability and decipher underlying defects that mask the high degree of competence conservation. Through a combination of experimental approaches and comparative genomics based on new whole‐genome sequences and de novo assemblies, we identify several strain‐specific defects, mostly in genes that encode key players in quorum sensing. Moreover, we provide evidence that most of these deficiencies might have recently occurred through laboratory domestication events or through the acquisition of mobile genetic elements. Lastly, we highlight that differing experimental approaches between research groups might explain more of the variations than strain‐specific alterations.

Virulence Pattern and Genomic Diversity of Vibrio cholerae O1 and O139 Strains Isolated From Clinical and Environmental Sources in India.

Vibrio cholerae is an autochthonous inhabitant of the aquatic environment. Several molecular methods have been used for typing V. cholerae strains, but there is no proper database for such scheme, including multilocus sequence typing (MLST) for V. cholerae O1 and O139 strains. We used 54 V. cholerae O1 and three O139 strains isolated from clinical and environmental sources and regions of India during the time period of 1975–2015 to determine the presence of virulence genes and production of biofilm. We devised a MLST scheme and developed a database for typing V. cholerae strains. Also, we performed pulsed-field gel electrophoresis to see the genomic diversity among them and compared it with MLST. We used the MEGA 7.0 software for the alignment and comparison of different nucleotide sequences. The advanced cluster analysis was performed to define complexes. All strains of V. cholerae, except five strains, showed variation in phenotypic characteristics but carried virulence-associated genes indicating they belonged to the El Tor/hybrid/O139 variants. MLST analysis showed 455 sequences types among V. cholerae strains, irrespective of sources and places of isolation. With these findings, we set up an MLST database on PubMLST.org using the BIGSdb software for V. cholerae O1 and O139 strains, which is available at https://pubmlst.org/vcholerae/ under the O1/O139 scheme. The pulsed-field gel electrophoresis (PFGE) fingerprint showed six fingerprint patterns namely E, F, G, H, I, and J clusters among 33 strains including strain N16961 carrying El Tor ctxB of which cluster J representing O139 strain was entirely different from other El Tor strains. Twenty strains carrying Haitian ctxB showed a fingerprint pattern classified as cluster A. Of the five strains, four carrying classical ctxB comprising two each of El Tor and O139 strains and one El Tor strain carrying Haitian ctxB clustered together under cluster B along with V. cholerae 569B showing pattern D. This study thus indicates that V. cholerae strains are undergoing continuous genetic changes leading to the emergence of new strains. The MLST scheme was found more appropriate compared to PFGE that can be used to determine the genomic diversity and population structure of V. cholerae.

Study of the Antibacterial Activity of Phenylacetic Acid Derivative Against the Causative Agent of Cholera

The in vitro and in vivo activity of a phenylacetic acid derivative, diclofenac, was studied against V.cholerae O1 El Tor strains and biofilms formed by them. In the presence of a subinhibitory concentration of diclofenac (250 mg/l), a 4-fold decrease in the values of the minimum inhibitory concentrations of furazolidone and chloramphenicol was found in 30% and 100% of the strains resistant to these drugs, and a significant increase in the diameters of growth inhibition zones around discs with chloramphenicol. furazolidone, streptomycin (for all strains) and doxycycline (for two strains) in comparison with the control. Furazolidone, nalidixic acid, chloramphenicol, streptomycin, to which the infecting strain was resistant, were used in in vivo experiments in combination with diclofenac for the treatment of white mice; in the experimental group the number of surviving animals increased to 80% in comparison with monotherapy with these drugs (50% or less). The subinhibitory concentration of diclofenac did not have a pronounced effect on the antibiotic sensitivity of biofilms. The study using transmission electron microscopy method on the biofilm of the V.cholerae O1 El Tor 19667 strain after exposing it to diclofenac (250 mg/l) for 120 h revealed signs of destruction of the exopolysaccharide matrix. These results indicate the prospects for studying this group of drugs, as well as others in order to develop new ways to overcome bacterial resistance.

Genome Sequence of a Pathogenic Vibrio cholerae O1 El Tor Strain Defective for the Entire Vibrio Pathogenicity Island 1, Isolated in Eastern Democratic Republic of the Congo.

We report here a complete genome sequence of a Vibrio cholerae O1 El Tor (Inaba; sequence type 515 [ST515]) strain isolated from a cholera patient in North Kivu Province, Democratic Republic of the Congo (DRC), which showed a complete deletion (∼80 kb) of the Vibrio pathogenicity island 1.

Cholera Toxin Production in Vibrio cholerae O1 El Tor Biotype Strains in Single-Phase Culture.

Vibrio cholerae O1 serogroup strains have been classified into classical and El Tor biotypes. Cholera, a life-threatening diarrheal disease, can be caused by either biotype through the cholera toxin (CT) that they produce. To increase our knowledge of the pathogenicity of bacteria, we must understand the toxigenicity of bacteria. CT production by classical biotype strains in simple single-phase cell cultures has been established; however, special culture media and growth conditions that are not appropriate for mass production of CT are required to facilitate CT production in El Tor biotype strains. In this report, we produced CT in El Tor biotype strains using simple media and single-phase culture conditions. A single point mutation in ToxT, a transcriptional activator of toxin co-regulated pilus (TCP) and CT, enabled the El Tor biotype strains to produce CT in similar quantities as classical biotype strains in single-phase laboratory culture conditions. CT production capacity varied between El Tor biotype strains. Wave 2 and 3 atypical El Tor strains tended to produce more CT than prototype Wave 1 strains. Wave 2 and 3 strains lack neutral fermentation; however, the capacity for neutral fermentation was not associated with significant differences in CT production by El Tor biotype strains. The Wave 3 strain that caused the 2010 cholera outbreak in Haiti produced CT only when neutral fermentation was abolished. The disparity in CT production between the seventh cholera pandemic strains highlight the differences in virulence between strains and the cause of population changes in V. cholerae.

Phylogenetic Affinity and Genome Structure Features of ctxAB– tcpA+ Vibrio cholerae from the Surface Waterbodies in the Territory that is Non-Endemic as Regards Cholera

Objectiveis analyzing the origin of the ctxAB–tcpA+Vibrio cholerae О1 El Tor strains isolated from the surface water bodies in the territory that is non-endemic of cholera; as well as investigating their phylogenetic relations to varying by epidemic significance groups of strains, based on the structure of housekeeping genes and whole genome structure.Materials and methods: we examined 25 V. cholerae strains, isolated in Siberia and Far East, including two ctxAB–tcpA+strains from surface water sources (Altai Territory, 2011; Khabarovsk Territory, 2013). Phylogenetic analysis included genomes of 36 V. cholerae strains from GenBank. Multilocus sequence typing (MLST) was carried out based on dnaE, cat, lap, pgm, recA, gyrB, and chi genes; in silico MLST – adk, gyrB, metE, mdh, pntA, purM and pyrC genes. Reconstruction of phylogeny was performed based on the comparative analysis of core genome SNPs in PhyML 3.0.Results and discussion.MLST of ctxAB–tcpA+V. cholerae О1 El Tor strains from the surface water bodies revealed that such strains form an individual genotype in the cluster of toxigenic strains and spontaneous mutants of toxigenic strains. According to results of in silico MLST, ctxAB–tcpA+isolates belong to SТ75, common to US Gulf phylogenetic line. Based on SNP-typing, ctxAB–tcpA+strains from the surface water sources were assigned to the group, originating from US Gulf V. cholerae, moreover isolate from Khabarovsk (2013) demonstrated high degree of genome homology with US Gulf-like strain from China (2009). For strains from Khabarovsk and China, we also showed the compositional identity of the pathogenicity island VPI-I and the presence of pandemicity island, VSP-1. The results testify to the fact that ctxAB–tcpA+V. cholerae strains, isolated in Siberia and Far East, originate from the US Gulf phylogenetic line. Furthermore, taking into account the results of epidemiological analysis, we can deduce that these strains are imported ones.

A Point Mutation in carR Is Involved in the Emergence of Polymyxin B-Sensitive Vibrio cholerae O1 El Tor Biotype by Influencing Gene Transcription.

Antimicrobial peptides play an important role in host defense against Vibrio cholerae Generally, the V. cholerae O1 classical biotype is polymyxin B (PB) sensitive and El Tor is relatively resistant. Detection of classical biotype traits like the production of classical cholera toxin and PB sensitivity in El Tor strains has been reported in recent years, including in the devastating Yemen cholera outbreak during 2016-2018. To investigate the factor(s) responsible for the shift in the trend of sensitivity to PB, we studied the two-component system encoded by carRS, regulating the lipid A modification of El Tor vibrios, and found that only carR contains a single nucleotide polymorphism (SNP) in recently emerged PB-sensitive strains. We designated the two alleles present in PB-resistant and -sensitive strains carRr and carRs alleles, respectively, and replaced the carRs allele of a sensitive strain with the carRr allele, using an allelic-exchange approach. The sensitive strain then became resistant. The PB-resistant strain N16961 was made susceptible to PB in a similar fashion. Our in silico CarR protein models suggested that the D89N substitution in the more stable CarRs protein brings the two structural domains of CarR closer, constricting the DNA binding cleft. This probably reduces the expression of the carR-regulated almEFG operon, inducing PB susceptibility. Expression of almEFG in PB-sensitive strains was found to be downregulated under natural culturing conditions. In addition, the expression of carR and almEG decreased in all strains with increased concentrations of extracellular Ca2+ but increased with a rise in pH. The downregulation of almEFG in CarRs strains confirmed that the G265A mutation is responsible for the emergence of PB-sensitive El Tor strains.

Genomic recombination of the vibrio cholerae serogroup O1 El Tor pandemic strains

Objective: To analyze the genomic recombination of the vibrio cholerae serogroup O1 El Tor pandemic strains. Methods: A total of 292 complete or draft genome sequences of Vibrio cholerae O1 serogroup El Tor strains isolated from 1937 to 2015 were selected from National Biotechnology Information Center database. The genome alignment of strains was computed by snippy software by using N16961 as reference sequence. Then ClonalFrameML software was used to do the recombinant analysis. The wilcox.test function in agricolae package was used to compare the number recombinant segments and the total length of recombinant regions between small and large chromosomes. The kruskal function was used to compare the number recombinant segments and the total length of recombinant regions among different isolation continents. The KOBAS tool was used to do the gene ontology enrichment analysis of recombinant hotspot genes. Results: Of all 292 strains of Vibrio cholerae, 163 strains (55.8%) were recombined. The median of normalized recombinant segment number of small chromosome was 4.7×10(-6) (9.3×10(-7), 2.0×10(-5)), which was significantly larger than that of large chromosome [2.4×10(-6) (3.4×10(-7), 5.7×10(-6))] (P<0.001). The median (P(25),P(75)) of recombinant segment number of strains isolated from Africa, Asia, Europe, North America and South America were 23(1.0,33.0), 1.0(0.0,34.0), 6.0(2.0,13.0), 0.0(0.0,1.0) and 29.5(6.8,56.8), respectively, and the difference was statistically significant (P<0.001). The median (P(25),P(75)) of total length of recombinant regions of strains isolated from Africa, Asia, Europe, North America and South America were 233.0(4.0, 461.0), 11.0(0.0, 695.5), 56.0(4.0,111.0), 0.0(0.0,9.0) and 347.5(132.8,1 323.5) bp, respectively, and the difference was statistically significant (P<0.001). Gene ontology Enrichment analysis showed that the functions of 62 recombinant hotspot genes were mainly enrichment in chemotaxis, taxis, response to external stimulus, receptor activity and molecular transducer activity. Conclustion: In this study, we found that there were significant differences in the number of recombinant fragments and the length of recombinant regions between large and small chromosomes of Vibrio cholerae El Tor. We also found significant differences in the number of recombinant fragments and the total length of recombinant regions among different continents.