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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.
Subject(s)
Vibrio cholerae/genetics , Biofilms , Quorum Sensing , Mutation , Gene Expression Regulation, Bacterial , Bacterial Proteins/geneticsABSTRACT
Cholera is endemic in over 50 countries with an estimated mortality of 100,000-120,000. Vaccination is considered the complementary key to prevent and control cholera; therefore, alternative vaccine preparations are needed. Toxin Co-regulated Pilus is part of the toxR virulence regulon, which is necessary for colonization in the intestinal mucosa. In order to express Vibrio cholerae TcpA protein in Saccharomyces boulardii, the expression plasmid pYES2 was constructed by inserting tcpA gene isolated from local Vibrio cholerae Eltor Inaba isolates. The new construct was transferred into Saccharomyces boulardii cells and the expression of tcpA gene was induced from the GAL1 promoter by adding galactose to the medium. The SDS-PAGE and Western blot analysis showed the presence of TcpA in yeast. These results showed that Saccharomyces boulardii is a promising host to express Vibrio cholerae toxin TcpA as the first step in attempt to produce an oral Vibrio cholerae vaccine(AU)
El cólera es endémico en más de 50 países. Se estima una mortalidad entre 100.000 - 120.000 debido a esta enfermedad. La vacunación se considera una medida complementaria para prevenir y controlar el cólera, por lo tanto, se necesitan preparaciones vacunales alternativas a las existentes. El Pili corregulado con la toxina, es parte del regulón de virulencia toxR, y es necesario para la colonización en la mucosa intestinal. Para expresar la proteína tcpA de Vibrio cholerae en Saccharomyces boulardii, se construyó el plásmido de expresión pYES2 insertando el gen tcpA obtenido a partir de aislamientos locales de Vibrio cholerae El Tor Inaba. La nueva construcción se transfirió a las células de Saccharomyces boulardii y se indujo la expresión del gen tcpA a partir del promotor GAL1 mediante la adición de galactosa al medio. El análisis mediante SDS-PAGE y Western blot demostró la presencia de TcpA en levaduras. Los resultados demostraron que Saccharomyces boulardii es un hospedero prometedor para expresar el gen tcpA de Vibrio cholerae como el primer paso en el intento de producir una vacuna oral contra Vibrio cholerae(AU)
Subject(s)
Humans , Male , Female , Cholera Vaccines/therapeutic use , Cholera/mortality , Cholera/prevention & control , Escherichia coli Infections , Saccharomyces boulardii/drug effectsABSTRACT
The severe diarrheal diseaseCholera, has gained public health importance because of its life-threatening effect. The detection of the causative agent of this disease(Vibrio cholerae(V. Cholerae)O1 or O139) from a specimen (stool, vomitus of food sample) remains a major concern in the world today. Phenotypic finger printing (the conventional methods) of the toxigenic V. choleraestrain, remains the gold standard for the laboratory diagnosis of cholera; especially during cholera epidemic outbreaks.Detection around the remote areas which are usually rampaged by these outbreaks is usually difficult due to lack of required diagnostic facilities in small laboratories.However, the use of phenotypic approaches have some major setbacks as they are usually labor-intensive and time consuming. This delays treatment commencement especially in life threatening cases.To alleviate these setbacks, rapid molecular typing techniques involvingthe Polymerase chain reaction (PCR) amplification, hybridization methods,Pulsed Field Gel Electrophoresis (PFGE), Multilocus Sequence Typing (MLST), Multiple-Locus Variable Number Tandem Repeat Analysis (MVLA), Fluorescent Amplified Fragment Length Polymorphism (FAFLP), Whole Genome Sequencing (WGS) etc. represent promising tools for early detection of the pathogen V. choleraeO1/O139 even in remote areas where laboratory resources are poor. Immunoassay-based techniques like enzyme-linked immune-sorbent assay (ELISA), coagglutination, immunofluorescence, and quartz crystal microbalance (QCM), are capital/labour intensive and expensive for low resource settings. Rapid diagnostic tests based on immune-chromatography principle have also been developed for simultaneous detection of V. choleraeserogroups O1 and O139. These test kits are easy to use, transport, and fast.All these methods enable the subtyping of unrelated bacterial strains and they all operate with different accuracies, discriminatory ability, and reproducibility.This review sought to address some of the methods used in diagnosing the disease cholera, with the objective of identifying the best and easiest of the methods that can help to curb the cholera problem (deaths) often encountered, especially in low resource settings in the developing countries (Nigeria inclusive)
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Purpose: To establish a new genotyping method for Vibrio cholerae and compare it with other methods. Materials and Methods: In the current study, a modified inter simple sequence repeat-polymerase chain reaction (MISSR-PCR) system was developed via several rounds of optimisation. Comparison study was then conducted between MISSR-PCR and three other methods, including enterobacterial repetitive intergenic consensus sequences-based PCR (ERIC-PCR), randomly amplified polymorphic DNA (RAPD) and 16S rRNA evolutionary clock, for the detection and genetic tracing of Vibrio cholerae isolated from seafood in China. Result: The results indicated that the MISSRPCR system could generate the highest polymorphic fingerprinting map in a single round PCR and showed the best discriminatory ability for Vibrio cholerae genotyping by clearly separating toxigenic/nontoxigenic strains, local/foreign strains, and O1/O139/non-O1/non-O139 serogroup strains, comparing to ERIC-PCR, RAPD and 16S rRNA evolutionary clock. Moreover, the MISSR-PCR is superior to previously described traditional simple sequence repeat based PCR method on genotyping by more clearly separating different clusters. Conclusion: To the best of our knowledge, this is the first head-to-head comparison of four detection and genotyping methods for Vibrio cholerae. The MISSRPCR system established here could serve as a simple, quick, reliable and cost-effective tool for the genotyping and epidemiological study.
ABSTRACT
The causative agent of cholera, Vibrio cholerae, can enter into a viable but non-culturable (VBNC) state in response to unfavorable conditions. The aim of this study was to evaluate the in situ survival of V. cholerae in an aquatic environment of the Southern Caribbean Sea, and its induction and resuscitation from the VBNC state. V. cholerae non-O1, non-O139 was inoculated into diffusion chambers placed at the Cuare Wildlife Refuge, Venezuela, and monitored for plate, total and viable cells counts. At 119 days of exposure to the environment, the colony count was < 10 CFU/mL and a portion of the bacterial population entered the VBNC state. Additionally, the viability decreased two orders of magnitude and morphological changes occurred from rod to coccoid cells. Among the aquatic environmental variables, the salinity had negative correlation with the colony counts in the dry season. Resuscitation studies showed significant recovery of cell cultivability with spent media addition (p < 0.05). These results suggest that V. cholerae can persist in the VBNC state in this Caribbean environment and revert to a cultivable form under favorable conditions. The VBNC state might represent a critical step in cholera transmission in susceptible areas.
El agente causal del cólera, Vibrio cholerae, puede entrar a un estado viable no cultivable (VNC) en respuesta a condiciones desfavorables. El objetivo de este estudio fue evaluar la supervivencia in situ de V. cholerae en un ambiente acuático al sur del Mar Caribe y su inducción y resucitación del estado VBNC. V. cholerae no-O1, no-O139 fue inoculado en cámaras de difusión ubicadas en el Refugio de Fauna Cuare, Venezuela, y monitoreado para contaje de colonias, células totales y viables. En 119 días de exposición al ambiente, el contaje de colonias fue < 10 UFC/mL y una fracción de la población bacteriana entró al estado VBNC. Adicionalmente, la viabilidad disminuyó dos órdenes de magnitud y ocurrieron cambios morfológicos de células bacilares a cocoides. Entre las variables del ambiente acuático, la salinidad presentó correlación negativa con el contaje de colonias. Los estudios de resucitación mostraron recuperación significativa de la cultivabilidad celular con adición de sobrenadantes de cultivos en crecimiento activo (p < 0.05). Estos resultados sugieren que V. cholerae puede persistir en estado VBNC en este ambiente de Caribe y revertir a una forma cultivable bajo condiciones favorables. El estado VBNC podría representar un paso crítico en la transmisión del cólera en áreas susceptibles.
Subject(s)
Microbial Viability , Vibrio cholerae O1/physiology , Atlantic Ocean , Caribbean Region , Colony Count, Microbial , Culture Techniques , Water MicrobiologyABSTRACT
Aim: Cholera is endemic in many parts of India and a major public health problem. The present study was carried out with the aims to understand biotype, serotype, phage type and drug resistance of Vibrio cholerae isolates obtained at a rural tertiary care hospital in Loni. Study Design: Descriptive retrospective study was carried out to study V. cholerae isolates from 544 faecal specimens of patients with acute gastroenteritis. Place and Duration of Study: The study was conducted during 2009-2012 at Rural Medical College of Pravara Institute of Medical Sciences, Loni, District Ahmednagar, Maharashtra, India. Methods: A total of 28 isolates of V. cholerae were included in the study. V. cholerae was identified by standard microbiological procedures. Biotyping, serotyping and phage typing was done. Antibiotic sensitivity testing was performed by Kirby- Bauer disc diffusion method. Results: V. cholerae strains were isolated from 28 faecal specimens. All the isolates were identified as V. cholerae O1 biotype El Tor serotype Ogawa and phage 27 was the predominant type. Male: Female ratio was 1:1.5 and high incidence was seen in 0-10 age group (35.71%). Maximal occurrence in monsoon season was recorded. All the isolates were resistant to co-trimoxazole, nalidixic acid and ampicillin. However maximum sensitivity was observed to norfloxacin (71.42%) followed by gentamycin (67.85%) and chloramphenicol (28.57%). Conclusion: A continuous surveillance for V. cholerae is required with respect to changing epidemiology and emergence of antibiotic resistance strains. The source and spread of infection should be investigated to decide the proper management strategies. Additionally, quality of water and status of sanitation should be monitored.
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Objective To investigate the pathogenic characteristics of Vibrio cholera strains isola-ted from Hubei province in 2012 , and to identify the source of infection by analyzing their genetic correla-tions.Methods The biochemical identification , toxin gene detection and drug susceptibility test were car-ried out to analyze a total of 35 Vibrio cholera strains isolated from three epidemic areas .Comparison of ge-nomic DNA fingerprints and cluster analysis among isolates of Vibrio cholera was conducted by using pulsed-field gel electrophoresis ( PFGE ) .Results All of the 35 strains were Vibrio cholera O139 , of which 71.42%were toxic strains.The drug resistance rates of Vibrio cholera strains to tetracycline, cotrimoxazole and rifampincin were 57.14%, 88.57%and 80.00%, respectively.Analysis of genomic DNA fingerprints of the isolates showed highly similar with similarity values ranging from 80%-100%.Most of the strains iso-lated from the same epidemic area fell into the same one cluster with 100% homology in genome Only a strain isolated from turtle in Jingzhou area was belong to a different cluster .Conclusion The Vibrio cholera O139 strains were the dominant strains causing the outbreaks of cholera in Hubei province in 2012 .Most of them were toxigenic strains .A large majority of the strains had developed resistance to tetracycline , cotri-moxazole and rifampincin , but all strains showed high susceptibility to ceftriaxone and imipenem .Vibrio cholera strains isolated from the same epidemic area were mainly belonged to the same one cluster , sugges-ting the same source of infection .However, the strains varied among different epidemic area .Follow-up in-vestigations of three outbreaks of cholera in this study were all associated with food infection .Therefore , more attention should be paid to food sanitation and safety measurement .Although a non-toxigenic strain iso-lated from turtle was not associated with the epidemic of cholera , surveillance for seafood and aquatic prod-ucts would still be necessary .
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Objective To characterize Hfq-dependent phenotypes in stress response and to dissect Hfq-dependent transcription of virulence genes and stress-responsive genes in Vibrio cholera.Methods The hfq null mutant strain (△hfq) and the complemented mutant strain (△hfq/pUC18-hfq) were constructed from the wild-type Vibrio cholera.Comparisons on the motility,biofilm formation,growth under various oxygen-supplying conditions,outer membrane resistance,and sensitivity to oxidative stress were analyzed between the wild type strain and the mutant strains.Reverse-transcript fluorescence quantitative PCR (RT-qPCR) was used to determine the transcriptional levels of target genes in the above mentioned strains.Results △hfq and △hfq/pUC18-hfq strains were successfully constructed.The motility,outer membrane resistance and sensitivity to oxidative stress were reduced,but biofilm formation was enhanced in the hfq null mutant strain.RT-qPCR testified that Hfq had regulation effects on gene transcription for forming falagellum,extracellular polysaccharide,outer membrane protein and oxidative stress in Vibrio cholera.Conclusion As a RNA chaperone,Hfq could affect Vibrio cholera in its biofilm formation,resistance to oxidative stress and antibiotics resistance through regulating the transcription of multiple metabolic genes and virulence genes,which indicates that Hfq,combined with other regulators,may play a key role in the complex regulation of metabolic genes and virulence genes.
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Purpose: Vibrio cholerae, the cause of cholera, is one of the leading causes of morbidity and mortality in many developing countries. Most laboratories initially rely on biochemical tests for a presumptive identification of these strains, followed by a polymerase chain reaction (PCR)-based method to confirm their identification. The aim of this study is to establish a rapid and reliable identification scheme for V. cholerae using a minimal, but highly specific number of biochemical tests and a PCR assay. Materials and Methods: We developed a species-specific PCR to identify V. cholerae, using a housekeeping gene recA, and used that to evaluate the sensitivity and specificity of 12 biochemical tests commonly used for screening and / or presumptive identification of V. cholerae in the clinical and environmental samples. Results: Here we introduced a combination of three biochemical tests, namely, sucrose fermentation, oxidase test, and growth in trypton broth containing 0% NaCl, as also the PCR of the recA gene, for rapid identification of V. cholerae isolates, with 100% sensitivity and specificity. The established method accurately identified a collection of 47 V. cholerae strains isolated from the clinical cases (n = 26) and surface waters (n = 21), while none of the 32 control strains belonging to different species were positive in this assay. Conclusion: The triple-test procedure introduced here is a simple and useful assay which can be adopted in cholera surveillance programs for efficient monitoring of V. cholerae in surface water and fecal samples.
Subject(s)
Bacterial Typing Techniques/methods , Environmental Microbiology , Humans , Polymerase Chain Reaction/methods , Rec A Recombinases/genetics , Sensitivity and Specificity , Vibrio cholerae/classification , Vibrio cholerae/isolation & purification , Vibrio cholerae/pathogenicityABSTRACT
A recent epidemic of cholera and emergence of novel strain of Vibrio cholera 0139 in South Asia prompted a necessity to investigate the potential occurrence of this organism in Thailand. From October 1992 to December 1993 there were 2,031 cases of pediatric diarrhea in Samutsongkram. Seventy three patients were infected with Vibrio cholera 01, forty one percent were below 3 years of age. None of the breast feeding infants were reported to be infected with this orgamion. Most of the cases occurred during the summer. Moderate to severe dehyration was commomly found. Malnutrition was frequently found in these patients. The susceptibility of the organism to ampicillin, colistin and nalidixic acid was good, but the organism were moderately sensitive to cotrimoxazole and tetracycline. Vibrio cholerae non-01 was detected in stool culture of 51 cases. Most of the patients were below 9 years of age. Only one case was confirmed to be infected with Vibrio chlera 0139. He was a seven month old and had mild degree of diarrhea. There was no case fatality due to diarrhea in these patients.