Bacterium Vibrio Cholerae Research Articles

Type VI secretion system mutations reduced competitive fitness of classical Vibrio cholerae biotype

The gram-negative bacterium Vibrio cholerae is the causative agent of the diarrhoeal disease cholera and is responsible for seven recorded pandemics. Several factors are postulated to have led to the decline of 6th pandemic classical strains and the rise of El Tor biotype V. cholerae, establishing the current 7th pandemic. We investigated the ability of classical V. cholerae of the 2nd and 6th pandemics to engage their type six secretion system (T6SS) in microbial competition against non-pandemic and 7th pandemic strains. We report that classical V. cholerae underwent sequential mutations in T6SS genetic determinants that initially exposed 2nd pandemic strains to microbial attack by non-pandemic strains and subsequently caused 6th pandemic strains to become vulnerable to El Tor biotype V. cholerae intraspecific competition. The chronology of these T6SS-debilitating mutations agrees with the decline of 6th pandemic classical strains and the emergence of 7th pandemic El Tor V. cholerae.

Application of a New Generalized Fractional Derivative and Rank of Control Measures on Cholera Transmission Dynamics

In this study, the mathematical model of the cholera epidemic is formulated and analyzed to show the impact of Vibrio cholerae in reserved freshwater. Moreover, the results obtained from applying the new fractional derivative method show that, as the order of the fractional derivative increases, cholera-preventing behaviors also increase. Also, the finding of our study shows that the dynamics of Vibrio cholerae can be controlled if continuous treatment is applied in reserved freshwater used for drinking purposes so that the intrinsic growth rate of Vibrio cholerae in water is less than the natural death of Vibrio cholerae. We have applied the stability theory of differential equations and proved that the disease-free equilibrium is asymptotically stable if R 0 < 1 , and the intrinsic growth rate of the Vibrio cholerae bacterium population is less than its natural death rate. The center manifold theory is applied to show the existence of forward bifurcation at the point R 0 = 1 and the local stability of endemic equilibrium if R 0 > 1 . Furthermore, the performed numerical simulation results show that, as the rank of control measures applied increases from no control, weak control, and strong control measures, the recovered individuals are 55.02, 67.47, and 674.7, respectively. Numerical simulations are plotted using MATLAB software package.

Mathematical model of cholera spread based on SIR: Optimal control

The bacterium Vibrio cholerae is the cause of cholera. Cholera is spread through the feces of an infected individual in a population. From a mathematical point of view, this problem can be brought into a mathematical model in the form of Susceptible-Infected-Recovered (SIR), which considers the birth rate. Because outbreaks that occur easily spread if not treated immediately, it is necessary to control the susceptible individual population by vaccination. The vaccine used is Oral Vibrio cholera. For this reason, the purposes of this study were to establish a model for the spread of cholera without vaccination, analyze the stability of the model around the equili­brium point, form a model for the spread of cholera with vaccination control, and describe the simulation results of numerical model completion. Based on the analysis of the stability of the equilibrium point of the model, it indicates that if the contact rate is smaller than the sum of the birth rate and the recovery rate, cholera will disappear over time. If the contact rate is grea­ter than the sum of the birth rate and the recovery rate, then cholera is still present, or in other words, the disease can still spread. Because the spread is endemic, optimal control of the popu­lation of susceptible individuals is needed, in this case, control by vaccination, so that the popu­lation of susceptible individuals becomes minimum and the population of recovered indivi­duals increases.

Bile Salts Promote ToxR Regulon Activation during Growth under Virulence-Inducing Conditions

Cholera is an epidemic disease caused by the Gram-negative bacterium Vibrio cholerae. V. cholerae is found in aquatic ecosystems and infects people through the consumption of V. cholerae-contaminated food or water. Following ingestion, V. cholerae responds to host cues to activate the expression of critical virulence genes that are under the control of a hierarchical regulatory system called the ToxR regulon. The ToxR regulon is tightly regulated and is expressed in vitro only under special growth conditions referred to as AKI conditions. AKI conditions have been instrumental in elucidating V. cholerae virulence regulation, but the chemical cues within AKI medium that activate virulence gene expression are unknown. In this study, we fractionated AKI medium on a reverse-phase chromatography column (RPCC) and showed that the virulence-activating molecules were retained on the RPCC column and recovered in the eluate. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis of the eluate revealed the presence of a known ToxR regulon activator, taurocholate, and other bile salts. The RPCC eluate activated the ToxR regulon when added to noninducing medium and promoted TcpP dimerization in a two-hybrid system, consistent with taurocholate being responsible for the virulence-inducing activity of AKI medium. Additional experiments using purified bile salts showed that the ToxR regulon was preferentially activated in response to primary bile acids. The results of this study shed light on the chemical cues involved in V. cholerae virulence activation and suggested that V. cholerae virulence genes are modulated in response to regionally specific bile acid species in the intestine.

Independent Promoter Recognition by TcpP Precedes Cooperative Promoter Activation by TcpP and ToxR.

ABSTRACTCholera is a diarrheal disease caused by the Gram-negative bacterium Vibrio cholerae. To reach the surface of intestinal epithelial cells, proliferate, and cause disease, V. cholerae tightly regulates the production of virulence factors such as cholera toxin (ctxAB) and the toxin-coregulated pilus (tcpA-F). ToxT is directly responsible for regulating these major virulence factors while TcpP and ToxR indirectly regulate virulence factor production by stimulating toxT expression. TcpP and ToxR are membrane-localized transcription activators (MLTAs) required to activate toxT expression. To gain a deeper understanding of how MLTAs identify promoter DNA while in the membrane, we tracked the dynamics of single TcpP-PAmCherry molecules in live cells using photoactivated localization microscopy and identified heterogeneous diffusion patterns. Our results provide evidence that (i) TcpP exists in three biophysical states (fast diffusion, intermediate diffusion, and slow diffusion), (ii) TcpP transitions between these different diffusion states, (iii) TcpP molecules in the slow diffusion state are interacting with the toxT promoter, and (iv) ToxR is not essential for TcpP to localize the toxT promoter. These data refine the current model of cooperativity between TcpP and ToxR in stimulating toxT expression and demonstrate that TcpP locates the toxT promoter independently of ToxR.

Crystallization in Microgravity and the Atomic-Resolution Structure of Uridine Phosphorylase from Vibrio cholerae

Uridine phosphorylases are known as key targets for the development of new anticancer and antiparasitic agents. Crystals of uridine phosphorylase from the pathogenic bacterium Vibrio cholerae were grown in microgravity by the capillary counter-diffusion method on board of the International Space Station. The three-dimensional structure of this enzyme was determined at atomic (1.04 Å) resolution (RCSB PDB ID: 6Z9Z). Alternative conformations of long fragments (β-strands and adjacent loops) of the protein molecule were found for the first time in the three-dimensional structure of uridine phosphorylase in the absence of specific bound ligands. Apparently, these alternative conformations are related to the enzyme function. Conformational analysis with Markov state models demonstrated that conformational rearrangements can occur in the ligand-free state of the enzyme.

Modern History of Cholera Vaccines and the Pivotal Role of icddr,b.

The rapid spread of the seventh cholera pandemic over Asia in the 1960s led to several large field studies that revealed that the traditional injectable cholera vaccines had poor efficacy, which led the World Health Organization (WHO) in the 1970s to stop recommending cholera vaccination. At the same time, it stimulated research that has led to the development of the effective orally administered cholera vaccines (OCVs) that today are a cornerstone in WHO’s strategy for Ending Cholera—A Global Roadmap to 2030. The first effective OCV, Dukoral, containing a mixture of inactivated Vibrio cholerae bacteria and cholera toxin B subunit, was licensed in 1991 and is, together with 2 similar inactivated whole-cell OCVs, Shanchol and Euvichol, currently WHO prequalified and recommended OCVs. This brief review is a personal account of the modern history of the development of these now universally recognized effective tools.

SYNTHESIS OF PIPERINE - PIPERAZINE ANALOGUES AND THEIR ANTIBACTERIAL ACTIVITY

In the current study, a series of piperine – piperazine analogues (5a to 5f) were designed and synthesized. The piperine was isolated from pepper and used for the conjugation with heterocyclic moiety for better biological activity. The piperazine heterocyclic was chosen for conjugation with piperine. The newly synthesized structures were determined by IR, 1H NMR and 13C NMR spectral data. The compounds were examined for their anti‐microbial activity against gram-positive (Bacillus subtilis) and gram-negative (Vibrio cholerae) bacteria using the agar well diffusion method. The newly synthesized compounds exhibited capable activities against V. cholerae and B. subtilis and it showed minimum inhibitory concentration. Among all the synthesized compounds, 5f has the highest activity (26 mm) against gram-positive bacteria and (29mm) against gram-negative bacteria. The remaining compounds showed appreciable antibacterial activity. The enhanced activity of the synthesized compounds may be due to the presence of conjugated amide linkage with the natural product piperine and piperazine heterocyclic molecule. The substituents present on the aromatic (nitro-substituted) ring also influenced the activity of the compound.

Lethal concentration of Golden Sea Cucumber killed Vibrio cholerae

Introduction: Diarrhea is still a considerable health problem today, especially in developing countries, including Indonesia. One common etiology of diarrhea is Vibrio cholerae, which still causes many problems, such as inappropriate use of antibiotics causes antibiotic resistance. The golden sea cucumber (Stichopus hermanii) has antibacterial potential against Vibrio cholera. This study aims to determine the effect and lethal concentration of golden sea cucumber extract on the growth of Vibrio cholerae bacteria.Methods: This research is an experimental laboratory using the post-test-only control group design method. Dilution is conducted, where six groups are consisting of 4 treatment groups and two control groups. The concentration of the treatment group is 100%, 50%, 25%, and 12.5%, while for positive control using tetracycline and distilled water as a negative control.Results: The results of this study indicate that the extract of golden sea cucumbers can inhibit the growth of Vibrio cholerae bacteria, as evidenced by a dilution test, where the higher the concentration of the golden sea cucumber extract, the more Vibrio cholerae bacteria are inhibited its growth. With a value of p = 0.001. The lethal concentration of golden sea cucumber extract obtained from this study through a dilution test starting from a concentration of 25%.Conclusion: Golden sea cucumber extract (Stichopus hermanii) can inhibit the growth of Vibrio cholerae bacteria through dilution test, regardless of concentration.

A fructose/H+ symporter controlled by a LacI-type regulator promotes survival of pandemic Vibrio cholerae in seawater

The bacterium Vibrio cholerae can colonize the human intestine and cause cholera, but spends much of its life cycle in seawater. The pathogen must adapt to substantial environmental changes when moving between seawater and the human intestine, including different availability of carbon sources such as fructose. Here, we use in vitro experiments as well as mouse intestinal colonization assays to study the mechanisms used by pandemic V. cholerae to adapt to these environmental changes. We show that a LacI-type regulator (FruI) and a fructose/H+ symporter (FruT) are important for fructose uptake at low fructose concentrations, as those found in seawater. FruT is downregulated by FruI, which is upregulated when O2 concentrations are low (as in the intestine) by ArcAB, a two-component system known to respond to changes in oxygen levels. As a result, the bacteria predominantly use FruT for fructose uptake under seawater conditions (low fructose, high O2), and use a known fructose phosphotransferase system (PTS, Fpr) for fructose uptake under conditions found in the intestine. PTS activity leads to reduced levels of intracellular cAMP, which in turn upregulate virulence genes. Our results indicate that the FruT/FruI system may be important for survival of pandemic V. cholerae in seawater.

Development of a luminescence real-time method for monitoring live bacteria during phage lysis

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.

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.

The frequency of vibrio cholera 01 EL TOR (Inaba And Ogawa) and its Resistance Pattern in Karachi.

Introduction: Cholera is an acute infectious disease of small intestine, caused by the bacterium Vibrio cholerae. More than 200 serogroups of V. cholerae have been identified. In Iran, Inaba strains were 75% resistant against trimethoprim-sulfamethoxazole in 2011, while during 2012-13 it was 100%. Ogawa strains showed 96% resistance against trimethoprim-sulfamethoxazole in 2011, whereas 100% resistant in 2012, while no resistance was seen in 2013 against the same antibiotic. 
 Objective: To determine the frequency of vibrio cholera 01 EL TOR (Inaba And Ogawa) and its resistance pattern in Karachi.
 Methodology: Samples were collected from patients with acute diarrhea with rice-water stool from Medical Unit, JPMC and NICH, Karachi between October 2015 to August 2016. These samples were then transported to Microbiology Department, Basic Medical Sciences Institute, JPMC, Karachi and processed according to standard protocol.
 Results: No growth was noticed in 147 (66.81%) samples while a total of 28 (12.7%) were V. cholerae species, and 45 (20.45%) were other organisms. The distribution of V. cholerae serotypes, out of 28 V. cholerae species, 13 (46.4%) were of Inaba serotypes, 09 (32.1%) were of Ogawa serotypes while 06 (21.4%) were of Non-01 serotypes.
 Conclusion: The susceptibility results of tetracycline and trimethoprim-sulfamethoxazole are not very favorable. Involvement of multidrug resistant V. cholerae O1 serotypes in the community is a very serious public health concern. Such patients were observed to be very difficult to treat in the community.

WITHDRAWN: Application and analysis of asymptotic method of cholera dynamics model in Artificial Intelligence and Image Processing

Cholera is an acute, virulent infectious illness caused by the infection of the intestine with the bacterium Vibrio cholerae. In this paper, aMathematical model of the cholera epidemics of the number of susceptible individuals, infected individuals, recovered individuals, concentration of the Vibrio cholerae has been discussed. This model was solved by the methods of the Homotopy Perturbation Method and the New Homotopy Perturbation Method. Our results are compared with the numerical simulation of the problem and a satisfactory agreement of the results is noted.

Cholera during COVID-19: The forgotten threat for forcibly displaced populations.

Cholera has been a global public health challenge since 1817. This acute, diarrhoeal, infection caused by the bacterium Vibrio cholerae, is transmitted by the faecal-oral route through contaminated water or food. Cholera annually affects 2.9 million people, causing 95,000 deaths worldwide. Forcibly displaced populations experience high rates of cholera due to limited access to healthcare and poor living conditions, including overcrowding, and disruption of water, sanitation, and hygiene (WASH) services.

The Inhibitory Effect of the Aqueous Extract of Ceratophyllum demersum on Vibrio cholarae that Isolated from Cyprinus carpio

The current study was conducted to determine the inhibitory effect of the aqueous extract of hornwort Ceratophyllum demersum on Vibrio cholerae bacteria that affects the common carp Cyprinus carpio L. in fish farms. The inhibitory effect of the aqueous extract was determined in petri dishes. The inhibition effects of the extract and six common antibiotics (Nalidixic acid, Nitrofurantoin, Gentamicin, Streptomycin, Tetracycline, and Amoxicillin) were compared; the results showed that the inhibition effect of the aqueous extract of hornwort was higher than the inhibitory effects of the six antibiotics. The stock solution 100% of the aqueous extract showed an inhibition effect reached 35.3 mm compared with 25 mm, 15 mm, 7 mm, 10 mm, 23 mm and 11 mm of Nalidixic acid, Nitrofurantoin, Gentamicin, Streptomycin, Tetracycline, and Amoxicillin antibiotics respectively. A positive correlation was recorded between concentration of aqueous extract and its inhibitory effect. The average of inhibition diameter was 23.3 mm, 27.6 mm, 28.6 mm, and 35.3 mm for the concentrations 25%, 50%, 75% and 100% of the stock solution, respectively.

Assessment of Mouse Ileal loop Protection against Clinically Isolated Vibrio cholerae Outer Membrane Vesicles as a Vaccine Candidate

Cholera, a life-threatening disease caused by the Gram-negative bacterium Vibrio cholera, remains a concern in developing countries. The present study investigated the immunogenicity and protective immunity of outer membrane vesicles (OMVs) and combination of OMV and killed whole cells (WC) of a local strain isolated from the last outbreak in Iran in addition to reference and local strains of V. cholerae El Tor O1 in comparison to Dukoral vaccine in mice model. The protein content, morphology, and size of extracted OMVs were evaluated by electrophoresis and microscopic analyses, respectively. The serum titers of total immunoglobulin G (IgG), IgG1, IgG2a, and immunoglobulin A (IgA) in addition to secretory IgA and total IgG in different mice groups were determined by enzyme-linked immunosorbent assay (ELISA). In addition, fluid accumulation (FA) assay regarding the resistance to live strain of V. cholerae in ligated ileal loops was carried out to determine immunogenicity by OMV or combination of OMV and WC in comparison to that reported for Dukoral vaccine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified OMVs indicated protein profiles within the range of 34-52 kDa. Furthermore, transmission electron microscopy demonstrated the spherical shaped vesicles of 50-200 nm. The results of ELISA showed significant titers of systemic and mucosal immune anti-OMV IgGs in immunized BALB/c mice with different vaccine regimens. Additionally, a notable increase in the FA ratio was demonstrated in this study. The obtained results of the present study revealed that the WC-OMV combination of local strain can induce a high level of antibody response indicating more protection than OMV or WC separately. Moreover, it can be considered an effective immunogen against V. cholerae.

Inhibition of α-, β- and γ-carbonic anhydrases from the pathogenic bacterium Vibrio cholerae with aromatic sulphonamides and clinically licenced drugs – a joint docking/molecular dynamics study

The binding mode of aromatic sulphonamides and clinically licenced drugs to the three carbonic anhydrase (CA, EC 4.2.1.1) isoforms from the human pathogen V. cholerae was here thouroghly characterised by a joint docking and molecular dynamics in silico protocol. In fact, VchCA, VchCAβ, and VchCAγ are crucial in the pathogen life cycle and growth and represent innovative targets to fight V. cholerae proliferation overcoming the spreading chemoresistance to the available drugs. A set of 40 sulphonamides/sulfamates VchCAs inhibitors was studied using the proteins homology built 3 D models unveiling the key and stable interactions responsible for a potent CA inhibition. This study has the aim to offer insights and guidelines for the future rational design of potent and selective inhibitors targeting CA isoforms from V. cholerae or other human pathogens.

Structure and Function of N-Acetylmannosamine Kinases from Pathogenic Bacteria.

Several pathogenic bacteria import and catabolize sialic acids as a source of carbon and nitrogen. Within the sialic acid catabolic pathway, the enzyme N-acetylmannosamine kinase (NanK) catalyzes the phosphorylation of N-acetylmannosamine to N-acetylmannosamine-6-phosphate. This kinase belongs to the ROK superfamily of enzymes, which generally contain a conserved zinc-finger (ZnF) motif that is important for their structure and function. Previous structural studies have shown that the ZnF motif is absent in NanK of Fusobacterium nucleatum (Fn-NanK), a Gram-negative bacterium that causes the gum disease gingivitis. However, the effect in loss of the ZnF motif on the kinase activity is unknown. Using kinetic and thermodynamic studies, we have studied the functional properties of Fn-NanK to its substrates ManNAc and ATP, compared its activity with other ZnF motif-containing NanK enzymes from closely related Gram-negative pathogenic bacteria Haemophilus influenzae (Hi-NanK), Pasteurella multocida (Pm-NanK), and Vibrio cholerae (Vc-NanK). Our studies show a 10-fold decrease in substrate binding affinity between Fn-NanK (apparent KM ≈ 700 μM) and ZnF motif-containing NanKs (apparent KM ≈ 60 μM). To understand the structural features that combat the loss of the ZnF motif in Fn-NanK, we solved the crystal structures of functionally homologous ZnF motif-containing NanKs from P. multocida and H. influenzae. Here, we report Pm-NanK:unliganded, Pm-NanK:AMPPNP, Pm-NanK:ManNAc, Hi-NanK:ManNAc, and Hi-NanK:ManNAc-6P:ADP crystal structures. Structural comparisons of Fn-NanK with Hi-NanK, Pm-NanK, and hMNK (human N-acetylmannosamine kinase domain of UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase, GNE) show that even though there is less sequence identity, they have high degree of structural similarity. Furthermore, our structural analyses highlight that the ZnF motif of Fn-NanK is substituted by a set of hydrophobic residues, which forms a hydrophobic cluster that helps the proper orientation of ManNac in the active site. In summary, ZnF-containing and ZnF-lacking NanK enzymes from different Gram-negative pathogenic bacteria are functionally very similar but differ in their metal requirement. Our structural studies unveil the structural modifications in Fn-NanK that compensate the loss of the ZnF motif in comparison to other NanK enzymes.

Vibrio Pathogenicity Island-1: The Master Determinant of Cholera Pathogenesis.

Cholera is an acute secretory diarrhoeal disease caused by the bacterium Vibrio cholerae. The key determinants of cholera pathogenicity, cholera toxin (CT), and toxin co-regulated pilus (TCP) are part of the genome of two horizontally acquired Mobile Genetic Elements (MGEs), CTXΦ, and Vibrio pathogenicity island 1 (VPI-1), respectively. Besides, V. cholerae genome harbors several others MGEs that provide antimicrobial resistance, metabolic functions, and other fitness traits. VPI-1, one of the most well characterized genomic island (GI), deserved a special attention, because (i) it encodes many of the virulence factors that facilitate development of cholera (ii) it is essential for the acquisition of CTXΦ and production of CT, and (iii) it is crucial for colonization of V. cholerae in the host intestine. Nevertheless, VPI-1 is ubiquitously present in all the epidemic V. cholerae strains. Therefore, to understand the role of MGEs in the evolution of cholera pathogen from a natural aquatic habitat, it is important to understand the VPI-1 encoded functions, their acquisition and possible mode of dissemination. In this review, we have therefore discussed our present understanding of the different functions of VPI-1 those are associated with virulence, important for toxin production and essential for the disease development.