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.

一株克氏原螯虾病原——霍乱弧菌的分离鉴定

从尾扇边缘溃烂的克氏原螯虾中分离到一株优势菌LK-18,并对该菌株进行种属鉴定和致病性分析。通过菌落形态观察、16S rRNA测序、血清型分析及基因特性鉴定对细菌的类别进行初步判断,并采用浸泡感染、肌肉注射和腹腔注射等方式进行人工感染实验对细菌的致病性进行评估。经16S rRNA测序分析和4个管家基因(atp A、pyr H、rec A、gyr B)串联分析,该菌株最终被鉴定并命名为VibriocholeraeLK-18。V. choleraeLK-18经PCR鉴定不属于O1和O139血清型,但含hly毒力基因和几丁质分解相关的几丁质酶(chitinase,Chi)基因。人工感染实验虽未能重现烂尾症状,但从克氏原螯虾烂尾组织中分离的V. choleraeLK-18具有致病性,能够引起克氏原螯虾的死亡。研究结果表明霍乱弧菌LK-18是一种致病菌,不仅扩大了非O1/O139群霍乱弧菌的感染宿主范围,同时预警霍乱弧菌有导致克氏原螯虾养殖业暴发疾病的可能,因此要提前做好该疾病的预防工作,从而避免霍乱弧菌感染带来的损失。

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.

Perceptions and Controversies on Cholera in the Traditional Chinese Medicine Field in the Late Qing Dynasty

At the beginning of the initial cholera outbreak in China,Wang Qingren(王清任),after years of clinical trials,became an early representative who discovered the infectious characteristics and observed the course of cholera in the field of traditional Chinesse medicine(TCM).Kou Langao(寇兰皋)and Xu Zimo(徐子默),both medical practitioners,constructed a diagnosis and treatment system for cholera with cold patterns under the theory of typhoid.They represented“School of Cholera with Cold Patterns”(SCCP).In the 1860s,with the influence of Wang Shixiong’s(王士雄)studies on the nature of cholera under the theory of epidemic febrile disease,the“School of Cholera with Heated Patterns”(SCHP)prevailed thereafter.The two schools complemented each other in theories and contributed to the integrity of TCM in cholera diagnosis and treatment.By reviewing previous literature,books,clinical cases,and historical materials in medical field,this article aims 1)to summarise the influence of TCM on the cognitive development towards cholera in the late Qing dynasty;2)to explore the origin of the controversy between SCCP and SCHP from historical evidence;3)to summarize the historical lessons from the debate over cholera in TCM based on the principle of treatment based on pattern identification,and offer suggestions for the current prevention and treatment of new diseases using TCM.

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.

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.

Antibiogram, Genomic and Phylogeny of Stool and Seafood Isolates from Some Cholera-Prone Coastal Communities in Rivers State, Nigeria

Cholera is a significant public health threat across the globe, especially in coastal regions with poor water supply. This study was carried out to determine the antibiogram, genomic, and phylogeny of stool and seafood isolates from some cholera-prone coastal communities in Rivers State, Nigeria. A total of 400 stool samples and 42 different seafood were aseptically collected and examined using standard microbiology and molecular techniques. An antibiogram of isolates from seafood and stool samples was assayed. Genes for virulence, resistance, and relatedness of bacteria identified were also determined. The isolates from the stool and seafood were examined for susceptibility to some selected antibiotics. The findings showed the prevalence rate of cholera in the communities as follows: 16% in Kaa, 30% in Andoni, 4% in Ogu/Bolo, and 10% in Abua/Odual. The isolates from stool were susceptible to Ciprofloxacin and Gentamycin with a susceptibility rate of 94.12% each while 100% resistance was recorded against Amoxicillin-clavulanic acid, 94.12% against Amikacin and 88.24% against Colistin. For the sea foods, the isolates were susceptible to gentamycin and ciprofloxacin with a susceptibility rate of 91.43% and 82.86% respectively. Resistance was also recorded against Colistin (88.57%) and Azithromycin (82.86%). Testing the isolates for the presence of 16SrRNA genes showed that all were positive with 1500 bp 16SrRNA gene band size. TEM, OXA, SHV, and CTX-M resistant genes were detected whereas the virulence genes were TDH and AcrB. The phylogenetic analysis revealed isolates from seafood to be Aeromonas dhakensis, Vibrio parahaemolyticus, Vibrio azureus, and Providencia rettgeri, while in stool samples they were Enterobacter sichuanensis, Enterobacter hormaechei, Providencia sneebia, and Proteus vulgaris. Providencia stuartii, Proteus mirabilis, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae were common isolates from both seafood and stool samples. This study has shown that not all reported cases of cholera are caused by Vibrio cholerae. Therefore, attention should be paid to other water-borne bacteria in every outbreak, especially in coastal communities.

Global Dynamics Analysis of a Cholera Transmission Model with General Incidence and Multiple Modes of Infection

This paper develops an SIBR cholera transmission model with general incidence rate. Necessary and sufficient conditions for local and global asymptotic stability of the equilibria are established by Routh Hurwitz criterium, Lyapunov function, and the second additive composite matrix theorem. What is more, exploiting the DED is cover simulation tool, the parameter values of the model are estimated with the 1998-2021 cholera case data in China. Finally, we perform sensitivity analysis for the basic reproduction number to seek for effective interventions for cholera control. .

In memory of Dr. Azar Andami, an eminent researcher and scholar at the Pasteur Institute of Iran

This paper focuses on the remarkable contributions and achievements of Dr.Azar Andami(1926–1984),a renowned physician and specialist at the Pasteur Institute of Iran.Dr.Andami conducted extensive research on infectious diseases,particularly cholera,utilizing her knowledge in medicine,bacteriology,and laboratory sciences.She played a crucial role in the mass production of the cholera vaccine at the Pasteur Institute of Iran and in controlling the spread of communicable diseases both locally and globally.This paper aims to highlight her remarkable works and accomplishments.