OBJECTIVE: This study aimed to investigate whether the VCA0560 gene acts as an active diguanylate cyclase (DGC) in Vibrio cholerae and how its transcription is regulated by Fur and HapR. METHODS: The roles of VCA0560 was investigated by utilizing various phenotypic assays, including colony morphological characterization, crystal violet staining, Cyclic di-GMP (c-di-GMP) quantification, and swimming motility assay. The regulation of the VCA0560 gene by Fur and HapR was analyzed by luminescence assay, electrophoretic mobility shift assay, and DNase I footprinting. RESULTS: VCA0560 gene mutation did not affect biofilm formation, motility, and c-di-GMP synthesis in V. cholerae, and its overexpression remarkably enhanced biofilm formation and intracellular c-di-GMP level but reduced motility capacity. The transcription of the VCA0560 gene was directly repressed by Fur and the master quorum sensing regulator HapR. CONCLUSION Overexpressed VCA0560 functions as an active DGC in V. cholerae, and its transcription is repressed by Fur and HapR.
Vibrio cholerae/genetics* ; Biofilms ; Quorum Sensing ; Mutation ; Gene Expression Regulation, Bacterial ; Bacterial Proteins/genetics*

Objective: To analyze the etiological and epidemiological characteristics of Vibrio cholerae in Beijing during 2015-2021 and provide evidence for the prevention and control of cholera. Methods: The V. cholerae strains isolated in Beijing during 2015-2021 were analyzed by serotyping and virulence genes detection. Pulsed field gel electrophoresis (PFGE) was performed for the molecular typing of the strains. Based on the collected epidemiological and clinical data of cholera cases,the epidemiological characteristics of cholera were analyzed by descriptive epidemiology method. Results: A total of 76 Vibrio cholerae O1 strains were isolated in Beijing during 2015-2021, including 61 strains from human, 10 strains from environment and 5 strains from seafood. The 76 strains consisted of 68 Ogawa strains and 8 Inaba strains. Six Ogawa strains isolated from sporadic cases carried ctxAB. After NotⅠ digestion, 76 strains were divided into 33 PFGE patterns. From 2015 to 2021, a total of 38 cholera epidemics were reported in Beijing, most of them were sporadic ones, accounting for 92.11% (35/38). A total of 45 cases were reported, and the cases occurred during June-September accounted for 97.78% (44/45). Cholera cases occurred in 9 districts of Beijing, and the cases reported in Chaoyang district accounted for 42.22% (19/45) and in Changping district accounted for 31.11% (14/45). The age of the cholera cases ranged from 19 to 63 years. Except for one case with unknown clinical symptoms, 44 cases had diarrhea symptoms with 84.09% (37/44) of the cases reporting diarrhea (3-9 times/day), followed by yellow watery stool (95.45%, 42/44), abdominal pain (68.18%, 30/44), nausea and vomiting (40.91%, 18/44) and fever (36.36%, 16/44). Conclusion: Vibrio cholerae strains isolated in Beijing during 2015-2021 were mainly O1 serotype Ogawa,most of which were non-toxigenic. The PFGE of the strains varied. Cholera epidemics occurred in 9 districts of Beijing, but most were sporadic ones with incidence peak during June-September.
Adult ; Beijing/epidemiology* ; Cholera/epidemiology* ; Diarrhea/epidemiology* ; Electrophoresis, Gel, Pulsed-Field ; Humans ; Middle Aged ; Vibrio cholerae O1/genetics* ; Young Adult

Bacterial Proteins/metabolism* ; Operon/genetics* ; Phosphotransferases/metabolism* ; Quorum Sensing/genetics* ; Vibrio cholerae/genetics*

The toxin-producing bacterium Vibrio cholerae can cause severe diarrhea and has caused seven global pandemics. Traditional viable cell counts and phage plaques are commonly used to evaluate the efficacy of virulent phage clearance of V. cholerae, but these operations are time-consuming and labor-intensive, and difficult to provide real-time changes. It is desirable to develop a simple and real-time method to monitor V. cholerae during phage lysis. In this study, a luminescence-generating plasmid pBBR-pmdh-luxCDABE was transformed into three O1 serogroup drug-resistant strains of V. cholerae. The results showed that the luminescence value as a monitoring index correlates well with the traditional viable cell count method. Monitoring the number of live cells of V. cholerae by measuring the luminescence allowed real-time analysis of the number of bacteria remaining during phage lysis. This method enables repeated, interference-free, continuous multiple-time-point detection of the same sample without the time delay of re-culture or plaque formation, facilitating real-time monitoring and analysis of the interaction between the phage and the host bacteria.
Bacteriophages/genetics* ; Luminescence ; Plasmids ; Vibrio cholerae

Aims: Cholera epidemics have been occurred in Malaysia since 1991 till 2003 which can be proved from the records by the Infectious Diseases Division of the Ministry of Health. Moreover, there were also course of cholera epidemics from the year 1994 to 2003 which had been happened in Sarawak. Cholera outbreaks in Malaysia mostly caused by the El Tor O1 Vibrio cholerae serogroup. The aims of this study were to detect the presence of V. cholerae in clinical and environmental samples (n=28) from Limbang, Sarawak by collaboration with Sarawak Government Hospital and to detect the toxin genes from the isolates. Methodology and results: All the isolates were sub-cultured in alkaline peptone water (APW). The boiled-cell method was used for DNA extraction. The total DNA extracted was amplified by polymerase chain reaction (PCR). Two types of PCR were used in this study which are 16S rRNA PCR and multiplex PCR. The results obtained from the study found out that 16 out of 28 (57.14%) samples were confirmed to be V. cholerae species. Four primers specific for V. cholerae were used in multiplex PCR (O1 type, O139 type, ctxA and ctxAB) to confirm the species type and the toxin genes. All samples shown positive for V. cholerae O1 serotype and 100% positive to all genes for the identification of ctxA and ctxAB genes. Conclusion, significance and impact of study From this study, it showed that multiplex PCR can be used for research purposes in molecular genetics field involving cholera outbreak.
Vibrio cholerae--genetics ; Cholera Toxin

Cholera is a secretory diarrhoeal disease caused by infection with Vibrio cholerae, primarily the V. cholerae O1 El Tor biotype. There are approximately 2.9 million cases in 69 endemic countries annually, resulting in 95 000 deaths. Cholera is associated with poor infrastructure and lack of access to sanitation and clean drinking water. The current cholera epidemic in Yemen, linked to spread of V. cholerae O1 (Ogawa serotype), is associated with the ongoing war. This has devastated infrastructure and health services. The World Health Organization had estimated that 172 286 suspected cases arose between 27th April and 19th June 2017, including 1170 deaths. While there are three oral cholera vaccines prequalified by the World Health Organization, there are issues surrounding vaccination campaigns in conflict situations, exacerbated by external factors such as a global vaccine shortage. Major movements of people complicates surveillance and administration of double doses of vaccines. Cholera therapy mainly depends on rehydration, with use of antibiotics in more severe infections. Concerns have arisen about the rise of antibiotic resistance in cholera, due to mobile genetic elements. In this review, we give an overview of cholera epidemiology, virulence, antibiotic resistance, therapy and vaccines, in the light of the ongoing epidemic in Yemen.
Anti-Bacterial Agents ; therapeutic use ; Cholera ; drug therapy ; prevention & control ; Cholera Vaccines ; therapeutic use ; DNA, Bacterial ; genetics ; Disease Outbreaks ; Drug Resistance, Multiple, Bacterial ; Humans ; Microbial Sensitivity Tests ; Polymerase Chain Reaction ; Vibrio cholerae ; drug effects ; isolation & purification ; Virulence Factors ; genetics ; Yemen

OBJECTIVEA novel method of Nano-Immunomagnetic Separation (Nano-IMS) plus Real-time PCR was established for detecting Vibrio cholerae.

METHODSThe Nano-Immunomagnetic Beads were created by using the monoclonal antibody of Vibrio cholerae, which was named Nano-IMB-Vc. Nano-IMB-Vc has specific adsorption of Vibrio cholerae, combined with Real-time PCR technology, a method for rapid detection of Vibrio cholerae was established. The capture specificity of Nano-IMB-Vc was tested by using 15 bacteria strains. The specificity of Real-time PCR method was tested by using 102 targets and 101 non-targets bacteria strains. The sensitivity of Nano-IMS plus Real-time PCR were tested in pure culture and in artificial samples and compared with NMKL No.156.

RESULTSThe capture ratio of Nano-IMB-Vc was reached 70.2% at the level of 10(3) CFU/ml. In pure culture, the sensitivity of Nano-IMS plus Real-time PCR was reached at 5.4×10(2) CFU/ml. The specific of Real-time PCR method was tested by using 102 targets and 101 non-targets bacteria. The results showed that 102 strains of Vibrio cholerae test results were all positive, and the rest of the 101 strains of non-target bacteria test results were negative. No cross-reaction was founded. Add 1 CFU vibrio cholerae per 25 g sample, it could be detect with Nano-IMS plus Real-time PCR method after 8 hours enrichment.

CONCLUSIONSThe Nano-IMS plus Real-time PCR method of Vibrio cholerae established in this study has good specificity and sensitivity, which could be applied to the rapid detection of Vibrio cholerae.

Food Microbiology ; methods ; Immunomagnetic Separation ; methods ; Nanotechnology ; Real-Time Polymerase Chain Reaction ; methods ; Seafood ; analysis ; microbiology ; Vibrio cholerae ; genetics ; isolation & purification ; Vibrio parahaemolyticus ; genetics ; isolation & purification

OBJECTIVETo understand the phenotypic diversity of toxigenic Vibrio cholerae O1 El Tor strains isolated from different provinces in China during the last 50 years.

METHODSTraditional biotyping testings including susceptibility to polymyxin B, sensitivity to group IV phage, Voges-Proskauer test and haemolysis of sheep erythrocytes were conducted.

RESULTSData from Biotype-specific phenotype analysis revealed that only 133 isolates carried the typical El Tor phenotypes while the other 251 isolates displayed atypical El Tor phenotypes. Combined with ctxB, rstR genotypes and phenotypic characteristics, 64 isolates were identified as typical El Tor biotype, 21 were El Tor variants that showing the typical El Tor biotype-specific phenotype but with ctxB(class). 280 isolates were defined as the hybrid groups with traits of both classical and El Tor biotypes that could be further classified into 45 groups, based on the combination of genotypes of ctxB, rstR and phenotypic characteristics.

CONCLUSIONToxigenic Vibrio cholerae O1 El Tor strains that isolated from different provinces in China displayed high phenotypic diversity. The traditional biotype traits could not be used to correctly distinguish the two different biotypes.

China ; Genotype ; Phenotype ; Polymorphism, Genetic ; Vibrio cholerae O1 ; genetics

OBJECTIVEThe complex of the cyclic AMP receptor protein (CRP) and cAMP is an important transcriptional regulator of numerous genes in prokaryotes. The transport of mannitol through the phosphotransferase systems (PTS) is regulated by the CRP-cAMP complex. The aim of the study is to investigate how the CRP-cAMP complex acting on the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype.

METHODSThe crp mutant strain was generated by homologous recombination to assess the need of CRP to activate the mannitol PTS operon of V. cholerae El Tor. Electrophoretic mobility shift assays (EMSA) and the reporter plasmid pBBRlux were used to confirm the role that the CRP-cAMP complex playing on the mannitol PTS operon mtl.

RESULTSIn this study, we confirmed that CRP is strictly needed for the activation of the mtl operon. We further experimentally identified five CRP binding sites within the promoter region upstream of the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype and found that these sites display different affinities for CRP and provide different contributions to the activation of the operon.

CONCLUSIONThe five binding sites collectively confer the strong activation of mannitol transfer by CRP in V. cholerae, indicating an elaborate and subtle CRP activation mechanism.

Bacterial Proteins ; genetics ; Base Sequence ; Cyclic AMP ; metabolism ; Cyclic AMP Receptor Protein ; Gene Expression Regulation, Bacterial ; Mannitol ; Molecular Sequence Data ; Operon ; Phosphotransferases ; Vibrio cholerae

OBJECTIVETo understand the genetic structures and variations of the superintegron (SI) in Vibrio cholerae isolated in the seventh cholera pandemic.

METHODSPolymerase chain reaction scanning and fragment sequencing were used. Sixty toxigenic V. cholerae O1 El Tor strains isolated between 1961 and 2008 were analyzed.

RESULTSSome variations were found, including insertions, replacements, and deletions. Most of the deletions were probably the result of recombination between V. cholerae repeat sequences. The majority of the variations clustered together. The SIs of the strains isolated in the 1960s and 1970s showed more diversity, whereas SI cassette variations in strains isolated in the 1990s and after were lower, with ∼24 kb signature sequence deletion. This indicates the predominant SI in the host during the epidemic in the 1990s and after. The insertion cassettes suggested the mobilization from the SIs of other V. cholerae serogroups and Vibrio mimicus.

CONCLUSIONThe study revealed that structural variations of SIs were obvious in the strains isolated in epidemics in different decades, whereas the divergence was based on syntenic structure of SIs in these El Tor strains. Also, the continuing cassette flows in the SIs of the host strains during the seventh cholera pandemics were displayed.

Cholera ; epidemiology ; microbiology ; Chromosomes, Bacterial ; genetics ; ultrastructure ; Cluster Analysis ; DNA, Bacterial ; genetics ; Electrophoresis, Gel, Pulsed-Field ; Gene Deletion ; Gene Flow ; Genetic Variation ; Humans ; Integrons ; genetics ; Mutagenesis, Insertional ; Open Reading Frames ; genetics ; Polymerase Chain Reaction ; Tandem Repeat Sequences ; Vibrio cholerae O1 ; classification ; genetics ; isolation & purification ; ultrastructure