Protein Containing the GGDEF Domain Affects Motility and Biofilm Formation in Vibrio cholerae and is Negatively Regulated by Fur and HapR

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 Hap R.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 Hap R 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 Hap R.Conclusion Overexpressed VCA0560 functions as an active DGC in V.cholerae,and its transcription is repressed by Fur and Hap R.

Plasticity of Regulation of Mannitol Phosphotransferase System Operon by CRP-cAMP Complex in Vibrio cholerae

Objective The 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 Tot biotype. Methods The crp mutant strain was generated by homologous recombination to assess the need of CRP to activate the mannitol PTS operon of V. choleroe 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 intl. Results In 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. Conclusion The five binding sites collectively confer the strong activation of mannitol transfer by CRP in V. choleroe, indicating an elaborate and subtle CRP activation mechanism.

Design of a multiplex PCR method for detection of toxigenicpathogenic in Vibrio cholerae

Objective:To study virulence and regulatory genes(hlyA,ctxB,tcpI) in clinical strains of Vibrio ckolerae(V.cholerae),simultaneously.Methods:Three important genes,tepI,hlyA and ctxB were used for detection of toxigenic and pathogenic V.cholera by chain reaction assay method. Results:According to the results of the PCR,the incidence of hlyA,tcpI,and ctxB genes in clinical isolates was obtained as 94.7%(72 sample),90.8%(69 sample),and 92.1%(70 sample), respectively.Five strains possessed all genes except ctxB,six strains possessed all genes except tcpI,four strains possessed all genes except hlyA,one strain possessed only hlyA and 60 strains contained a combination of three genes.Including hlyA,ctxB and tcpI,Conclusions:Result show that this method could be reliable to detect toxigenic-pathogenic strains of V.cholerae in Iran.

Specific Detection of Toxigenic Vibrio cholerae Based on in situ PCR in Combination With Flow Cytometry

Objective To develop an in situ PCR in combination with flow cytometry （ISPCR-FCM） for monitoring cholera toxin positive Vibrio cholerae. Methods In running this method, 4% paraformaldehyde was used to fix the Vibrio cholerae cells and 1 mg/mL lysozyme for 20 min to permeabilize the cells. Before the PCR thermal cycling, 2.5% glycerol was added into the PCR reaction mixture in order to protect the integrality of the cells. Results A length of 1037bp DNA sequence was amplified, which is specific for the cholera toxin gene （ctxAB gene）. Cells subjected to ISPCR showed the presences of ctxAB gene both in epifluorescence microscopy and in flow cytometric analysis. The specificity and sensitivity of the method were investigated. The sensitivity was relatively low （10^5 cells/mL）, while the specificity was high. Conclusion We have successfully developed a new technique for detection of toxigenic Vibrio cholerae strains. Further study is needed to enhance its sensitivities. ISPCR-FCM shows a great promise in monitoring specific bacteria and their physiological states in environmental samples.

Structural Variation of the Superintegron in the Toxigenic Vibrio cholerae O1 El Tor

Objective To understand the genetic structures and variations of the superintegron （Sl） in Vibrio cholerae isolated in the seventh cholera pandemic. Methods Polymerase chain reaction scanning and fragment sequencing were used. Sixty toxigenic V. cholerae O1 El Tor strains isolated between 1961 and 2008 were analyzed. Results Some 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 Sl in the host during the epidemic in the 1990s and after. The insertion cassettes suggested the mobilization from the Sls of other V. cholerae serogroups and Vibrio mimicus. Conclusion The 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 Tot strains. Also, the continuing cassette flows in the Sis of the host strains during the seventh cholera pandemics were displayed.

Plasmid mediated antibiotic resistance of Vibrio cholerae Ol biotype E1 Tor serotype Ogawa associated with an outbreak in Kolkata,India

Objective:To determine the antibiotic resistance of Vibrio choleras(V.cholerae) O1 biotype El Tor serotype Ogawa isolates involved in an outbreak of watery diarrhea in Kolkata,and to explore the role of plasmid in mediating antibiotic resistance.Methods:Antibiotic susceptibility and minimum inhibitory concentration(MIC) values of antibiotics for the isolated V.cholerae 01 Ogawa(n=12) were determined by disk diffusion and agar dilution methods,respectively,using ampicillin(Am),chloramphenicol(C),trimethoprim(Tm),tetracycline(T).erythromycine(Er), nalidixic acid(Nx).ciprofloxacin(Cp),amikacin(Ak) and cefotaxime(Cf).Plasmid curing of multidrug resistant(MDR) V.cholerae 01 Ogawa strains was done following ethidium bromide treatment.Following electrophoresis,the plasmid DNAs,extracted from the isolated MDR V. cholerae O1 Ogawa strains and their cured derivatives,were visualized and documented in ’gel doc’ system.Results:The outbreak causing V.cholerae O1 Ogawa isolates were MDR as determined by disk diffusion susceptibility test,and MIC determination.The isolates showed three different drug resistance patterns:AmTmTErNx(for 6 isolates).TmTErCp(for 5 isolates), and AmTniNx(for one isolate),and showed uniform sensitivity to C,Ak and Cf.The loss of plasmids with the concomitant loss of resistance to Am,Tm,T and Er of the isolates occurred following ethidium bromide treatment.Conclusions:The current findings suggest that the V. cholerae O1 Ogawa associated with the cholera outbreak were MDR,and resistance to Am,Tm,T and Er among the isolates were plasmid mediated.

Survival and proliferation of the lysogenic bacteriophage CTXΦ in Vibrio cholerae

The lysogenic phage CTXΦ of Vibrio cholerae can transfer the cholera toxin gene both horizontally(inter-strain) and vertically(cell proliferation). Due to its diversity in form and species, the complexity of regulatory mechanisms, and the important role of the infection mechanism in the production of new virulent strains of V.cholerae, the study of the lysogenic phage CTXΦ has attracted much attention. Based on the progress of current research, the genomic features and their arrangement, the host-dependent regulatory mechanisms of CTXΦ phage survival, proliferation and propagation were reviewed to further understand the phage's role in the evolutionary and epidemiological mechanisms of V. cholerae.

E1 Tor Vibio Cholerae Strain SM_6 and Jiangsu 1d Strain

E1 Tor V.cholerae can be classified into epidemic and non-epidemic strains with the biophage taping method designed by Beijing Institute of Epidemiology and Microbiology.7 strains of E1 Tor cholerae belonged to type 1d are further classified into two tapes 1/2/3 and 2/3 with phage typing method devised by the authors.Type 1/2/3 contains CT genes, whereas 2/3 does not.

Isolation, Identification and Antimicrobial Susceptibility Profile Analysis of <i>Vibrio cholerae</i>O1 from Stool Samples of Bangladesh

Cholera is a severe diarrheal disease which is usually caused by toxigenic strain of Vibrio cholerae O1 and O139. Cholera is still one of the major health concerns in developing countries like Bangladesh due to poor sanitation and unavailability of safe drinking water. This experiment was confronted to identify V. cholerae O1 from stool samples as well as to determine the antibiotic susceptibility pattern of the isolated strains. A total of 140 stool samples from people infected with diarrheal disease were collected from July 2016 to December 2016. Among all, 58 samples were found positive for V. cholerae which were further subjected to sero-grouping by specific anti-sera and antimicrobial sus-ceptibility test by Kirby Bauer disc diffusion method. The zones of inhibition were measured and interpreted by following the recommendations of the criteria of Clinical and Laboratory Standards Institute (CLSI). It was found that 43 (74.1%) isolates of V. cholerae were O1 serogroup of Ogawa serotype and the rest 15 (25.9%) were O1 serogroup of Inaba serotype. People aged between 41 - 50 were most susceptible to V. cholerae O1 having about 39.7% of positive cases. The isolates were highly susceptible to Ciprofloxacin and Gentamicin with 100% susceptibility whereas 100% resistant was found towards Nalidixic acid. Though most of the isolates in our study were susceptible against tested antibiotics, the continuous surveillance is required to see the changing pattern of serogroups or serotypes and antimicrobial profile in this region.

Occurrence of <i>Vibrio cholerae</i>in Some Households Engaged in Livestock Farming in Some Parts of Zaria, Nigeria

The study was designed to determine the presence of Vibrio cholerae from the environment in close proximity with livestock in Zaria,Kadunastate. Three hundred and thirty six environmental samples comprising soil, water, manure and vegetables were collected from some selected households inZariacoveringZariacity, Sabo and Samaru from April to October 2006 and analyzed for the presence of Vibrio cholerae, one of the etiologic agent of gastroenteritis. Twelve Vibrios exhibiting characteristics of Vibrio cholerae were identified using biochemical techniques. Serological identification confirmed 5 (41.7%) of these isolates as Vibrio, 1 (20%) as Vibrio serogroup O1, Ogawa biotype. A 0.59% prevalence was obtained for this pathogen in the study. The 0.59% isolation rate though low is significant considering it’s source being animal, since livestock keeping is a common feature in the study location, with animals living in close proximity to man this work is imperative. Animals are a point source of contamination of enteric pathogens, therefore extensive management system and proper treatment of animal manure is recommended before its use as fertilizer.

Acquisition and dissemination mechanisms of CTXΦ in Vibrio cholerae : New paradigm for dif residents

Vibrio cholerae(V. cholerae) genome is equipped with a number of integrative mobile genetic element(IMGE) like prophages, plasmids, transposons or genomic islands, which provides fitness factors that help the pathogen to survive in changing environmental conditions. Metagenomic analyses of clinical and environmental V. cholerae isolates revealed that dimer resolution sites(dif) harbor several structurally and functionally distinct IMGEs. All IMGEs present in the dif region exploit chromosomally encoded tyrosine recombinases, Xer C and Xer D, for integration. Integration takes place due to site-specific recombination between two specific DNA sequences; chromosomal sequence is called att B and IMGEs sequence is called att P. Different IMGEs present in the att P region have different attP structure but all of them are recognized by Xer C and Xer D enzymes and mediate either reversible or irreversible integration. Cholera toxin phage(CTXΦ), a lysogenic filamentous phage carrying the cholera toxin genes ctx AB, deserves special attention because it provides V. cholerae the crucial toxin and is always present in the dif region of all epidemic cholera isolates. Therefore, understanding the mechanisms of integration and dissemination of CTXΦ, genetic and ecological factors which support CTXΦ integration as well as production of virion from chromosomally integrated phage genome and interactions of CTXΦ with other genetic elements present in the genomes of V. cholerae is important for learning more about the biology of cholera pathogen.

Polymyxin sensitivity/resistance cosmopolitan status,epidemiology and prevalence among O1/O139 and non-O1/non-O139 Vibrio cholerae:A meta-analysis

Resistance/sensitivity to polymyxin-B(PB)antibiotic has been employed as one among other epidemiologically relevant biotyping-scheme for Vibrio cholerae into Classical/El Tor biotypes.However,recent studies have re-vealed some pitfalls bordering on PB-sensitivity/resistance(PBR/S)necessitating study.Current study assesses the PBR/S cosmopolitan prevalence,epidemiology/distribution among O1/O139 and nonO1/nonO139 V.cholerae strains.Relevant databases(Web of Science,Scopus and PubMed)were searched to retrieve data from environ-mental and clinical samples employing the Preferred Reporting Items for Systematic Reviews and Meta-Analyses(PRISMA).Random-effect-model(REM)and common-effect-model(CEM)of meta-analysis was performed to de-termine prevalence of PBR/S V.cholerae strains,describe the cosmopolitan epidemiological potentials and biotype relevance.Heterogeneity was determined by meta-regression and subgroup analyses.The pooled analyzed iso-lates from articles(7290),with sensitive and resistance are 2219(30.44%)and 5028(69.56%).Among these PB-sensitive strains,more than 1944(26.67%)were O1 strains,132(1.81%)were nonO1 strains while mis-reported Classical biotype were 2080(28.53)respectively indicating potential spread of variant/dual biotype.A significant PB-resistance was observed in the models(CEM=0.66,95%CI[0.65;0.68],p-value=0.001;REM=0.83[0.74;0.90],p=0.001)as both models had a high level of heterogeneity(I^(2)=98.0%;^(2)_(df=33)=1755.09,Qp=2.4932).Egger test(z=5.4017,p<0.0001)reveal publication bias by funnel plot asymmetry.The subgroup analysis for continents(Asia,Africa)and sources(acute diarrhea)revealed(98%CI(0.73;0.93);55%CI(0.20;0.86)),and 92%CI(0.67;0.98).The Epidemiological prevalence for El tor/variant/dual biotype showed 88%CI(0.78;0.94)with O1 strains at 88%CI(0.78;0.94).Such global prevalence,distribution/spread of phenotypes/genotypes ne-cessitates updating the decades-long biotype classification scheme.An antibiotic stewardship in the post antibiotic era is suggestive/recommended.Also,there is need for holistic monitoring/evaluation of clinical/epidemiological relevance of the disseminating strains in endemic localities.

Toxin(s), Other Than Cholera Toxin, Produced by Environmental Non O1 Non O139 Vibrio cholerae

A total of 39 Vibrio cholerae non O1 non O139 strains were isolated from surface waters of different parts of Dhaka City, Bangladesh. All these strains showed lack of ctx or zot gene, as demonstrated by the PCR analysis. Eighteen representative strains were tested for enterotoxin production using a rabbit ileal loop model, of which live cells of 8 strains and culture filtrates of 6 strains produced fluid accumulation in ileal loops. However, none of them produced heat stable toxin （ST）, as detected by suckling mouse assay. On the other hand, 15% of isolates produced cytotoxin as detected by the Chinese Hamster Ovary （CHO） cell assay. Fifty times concentrated culture filtrates of the representative strains did not give any precipitin band against the anti-cholera toxin, suggesting the strains produced an enterotoxin, which is antigenically different from known cholera toxin （CT）. Eighty percent of the total isolates were found to be positive for heat labile haemolysin detected by tube method, whereas, 39% were found positive by the Christie-Atkins-Munch-Petersen （CAMP） method. However, 87% of the isolates were positive for haemagglutinin/protease and all of the strains were positive for mannose-sensitive-haemagglutinin assay.

Cholera Caused by a New Clone of Serogroup O1 Vibrio cholerae-Beijing Municipality,China,June 2021

Several lineages have been identified in the population of serogroup O1 Vibrio cholerae(V.cholerae)(1-3).The strains,which were responsible for the ongoing seventh cholera pandemic,were in Lineage 2.Nearly all the V.cholerae strains in this lineage carried genes coding cholera toxin(ctxAB)(1-2).Lineage 3b consists of strains isolated from different continents and the vast majority of strains in this lineage lack the ctxAB genes.

Calcium/calcimimetic via calcium-sensing receptor ameliorates cholera toxin-induced secretory diarrhea in mice

BACKGROUND Enterotoxins produce diarrhea through direct epithelial action and indirectly by activating the enteric nervous system.Calcium-sensing receptor(CaSR)inhibits both actions.The latter has been well documented in vitro but not in vivo.The hypothesis to be tested was that activating CaSR inhibits diarrhea in vivo.AIM To determine whether CaSR agonists ameliorate secretory diarrhea evoked by cholera toxin(CTX)in mice.METHODS CTX was given orally to C57BL/6 mice to induce diarrhea.Calcium and calci-mimetic R568 were used to activate CaSR.To maximize their local intestinal actions,calcium was administered luminally via oral rehydration solution(ORS),whereas R568 was applied serosally using an intraperitoneal route.To verify that their actions resulted from the intestine,effects were also examined on Cre-lox intestine-specific CaSR knockouts.Diarrhea outcome was measured biochemically by monitoring changes in fecal Cl-or clinically by assessing stool consistency and weight loss.RESULTS CTX induced secretory diarrhea,as evidenced by increases in fecal Cl-,stool consistency,and weight loss following CTX exposure,but did not alter CaSR,neither in content nor in function.Accordingly,calcium and R568 were each able to ameliorate diarrhea when applied to diseased intestines.Intestinal CaSR involvement is suggested by gene knockout experiments where the anti-diarrheal actions of R568 were lost in intestinal epithelial CaSR knockouts(villinCre/Casrflox/flox)and neuronal CaSR knockouts(nestinCre/Casrflox/flox).CONCLUSION Treatment of acute secretory diarrheas remains a global challenge.Despite advances in diarrhea research,few have been made in the realm of diarrhea therapeutics.ORS therapy has remained the standard of care,although it does not halt the losses of intestinal fluid and ions caused by pathogens.There is no cost-effective therapeutic for diarrhea.This and other studies suggest that adding calcium to ORS or using calcimimetics to activate intestinal CaSR might represent a novel approach for treating secretory diarrheal diseases.

Collaborative Efforts and Strategies for Cholera Outbreak Control in Garissa County, Kenya: Implementation of Water Quality Monitoring Interventions

A multi-faceted Case Area Targeted Intervention (CATI) approach emphasizing the integration of Water, Sanitation and Hygiene (WASH) interventions and Oral Cholera Vaccine (OCV) campaign was employed to respond to the outbreak of cholera in Garissa County. Drinking water sources in areas heavily impacted by cholera were systematically mapped and tested for microbiological quality. The quality assessment was carried out in April 2023 during an ongoing cholera outbreak in the county. A total of 109 samples were collected and tested for thermotolerant coliforms and other in situ parameters. The finding revealed that more than 87% of the samples did not meet the World Health Organization (WHO) standard for thermotolerant coliforms;and 30% had turbidity values above the recommended threshold values. None of the 109 samples had any traceable residual chlorine. Following these findings, the county government implemented the targeted interventions which resulted in a positive impact in the fight against cholera. The WHO supported key interventions which included capacity building in water quality monitoring and prepositioning of critical WASH commodities to the cholera affected areas.

Geospatial Variability of Cholera Cases in Malawi Based on Climatic and Socioeconomic Influences

Cholera remains a public health threat in most developing countries in Asia and Africa including Malawi with seasonal recurrent outbreaks. Malawi’s recent Cholera outbreak in 2022 and 2023, exhibited higher morbidity and mortality rates than the past two decades. Lack of spatiotemporal-based technology and variability assessment tools in Malawi’s Cholera monitoring and management, limit our understanding of the disease’s epidemiology. The present work developed a spatiotemporal variability model for Cholera disease at district level and its relationship to socioeconomic and climatic factors based on cumulative confirmed Cholera cases in Malawi from March 2022 to July 2023 using Z-score statistic and multiscale geographically weighted regression (MGWR) in a Geographical Information System (GIS). We found out that socioeconomic factors such as access to safe drinking water, population density and poverty level, and climatic factors including temperature and rainfall strongly influenced Cholera prevalence in a complex and multifaceted manner. The model shows that Lilongwe, Mangochi, Blantyre and Balaka districts were highly vulnerable to Cholera disease followed by lakeshore districts of Salima, Nkhotakota, Nkhata-Bay and Karonga than other districts. We recommend strategic measures such as Water, Sanitation, and Hygiene (WASH) interventions, community awareness on proper water storage, Cholera case management, vaccination campaigns and spatial-based surveillance systems in the most affected districts. This research has shown that MGWR, as a surveillance system, has the potential of providing insights on the disease’s spatial patterns for public health authorities to identify high-risk districts and implement early response interventions to reduce the spread of the disease.

Cholera Recurrence within Benin Littoral County: Analysis of Epidemics from 2008, 2016 to 2023

Introduction: Cholera is an acute diarrheal infection caused by the bacillus Vibrio cholerae, which can be fatal in a few hours if left untreated. It rages in endemic-epidemic mode in several countries, including Benin. Despite being the economic capital, the largest city in Benin and the most urbanized municipality in the country, the Littoral County, confounded with the city of Cotonou, is subject to recurrent epidemics. This paper aims to analyze successive cholera epidemics with a view of highlighting the factors contributing to this recurrence. Methods: A secondary analysis was done of the cholera-related databases of the County health office from 2016 to 2023, and its Situation Report No. 79 of December 7th, 2016, to describe the 2016 epidemic. Database data were analyzed in Excel. Results: Similarly to 2008, the 2016 cholera epidemic in Cotonou, which recorded 519 cases and 07 deaths, i.e. a case-fatality rate of 1.35%, spread to the town and six neighboring municipalities. The municipality of So-Ava, home to the index case, had the highest overall attack rate. The two epidemics share the same period of severity. Both epidemic and endemic cases are concentrated in the first seven boroughs of Cotonou, located on the lagoon shore, with low levels of hygiene and sanitation and a poor supply of drinking water. Conclusion: Low levels of hygiene, sanitation and drinking water supply all play a part in the recurrence of cholera epidemics. Sanitation work. Undertaken by the Beninese government is a ray of hope for improving this situation. Meanwhile, public awareness of hygiene measures must continue.

THE EXPRESSION OF ENTEROTOXIN A^-B^+ GENE OF V. cholerae IN E. coli

The cloning in E. coli of a cholerae toxin gene that is A^-B^+ has been successfully constructed by using DNA recombinant techniques. E. coli cells carrying the recombinant plasmid pMM-CTB have been shown to produce a large amount of CTB subunits which are secreted as extracellular proteins.