Vibrio cholerae virulence and its suppression through the quorum-sensing system.

Vibrio cholerae is a cholera-causing pathogen known to instigate severe contagious diarrhea that affects millions globally. Survival of vibrios depend on a combination of multicellular responses and adapt to changes that prevail in the environment. This process is achieved through a strong communication at the cellular level, the process has been recognized as quorum sensing (QS). The severity of infection is highly dependent on the QS of vibrios in the gut milieu. The quorum may exist in a low/high cell density (LCD/HCD) state to exert a positive or negative response to control the regulatory pathogenic networks. The impact of this regulation reflects on the transition of pathogenic V. cholerae from the environment to infect humans and cause outbreaks or epidemics of cholera. In this context, the review portrays various regulatory processes and associated virulent pathways, which maneuver and control LCD and HCD states for their survival in the host. Although several treatment options are existing, promotion of therapeutics by exploiting the virulence network may potentiate ineffective antibiotics to manage cholera. In addition, this approach is also useful in resource-limited settings, where the accessibility to antibiotics or conventional therapeutic options is limited.

Crystal structure of the catalytic ATP-binding domain of the PhoR sensor histidine kinase.

The two-component regulatory system (TCS) is a major regulatory system in bacteria that occurs in response to environmental changes and involves the sensor histidine kinase (HK) protein and response regulator (RR) protein. Among the TCSs, PhoR/PhoB is crucial for bacteria to adapt to changes in environmental phosphate concentrations. In addition, recent studies have shown that PhoR binding to the MgtC virulence factor activates phosphate transport for normal pathogenesis. In this work, we determined the crystal structure of the catalytic ATP binding domain of the PhoR sensor histidine kinase from Vibrio cholera, compared the structure with the known HK protein structures and discussed the potential binding interface with MgtC.

Phenotypic characterization of phage vB_vcM_Kuja.

Bacteriophage therapy targeting the increasingly resistant Vibrio cholerae is highly needed. Hence, studying the phenotypic behavior of potential phages under different conditions is a prerequisite to delivering the phage in an active infective form. The objective of this study was to characterize phage VP4 (vB_vcM_Kuja), an environmental vibriophage isolated from River Kuja in Migori County, Kenya in 2015. The phenotypic characteristics of the phage were determined using a one-step growth curve, restriction digestion profile, pH, and temperature stability tests. The results revealed that the phage is stable through a wide range of temperatures (20-50°C) and maintains its plaque-forming ability at pH ranging from 6 to 12. The one-step growth curve showed a latent period falling between 40 and 60 min, while burst size ranged from 23 to 30 plaque-forming units/10 µl at the same host strain. The restriction digestion pattern using EcoRI, SalI, HindIII, and XhoI enzymes showed that HindIII could cut the phage genome. The phage DNA could not be restricted by the other three enzymes. The findings of this study can be used in future studies to determine phage-host interactions.

Current trends in polymerase chain reaction based detection of three major human pathogenic vibrios.

Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus are the most significant aquatic pathogens of the genera Vibrio, account for most Vibrio-associated outbreaks worldwide. Rapid identification of these pathogens is of great importance for disease surveillance, outbreak investigations and food safety maintenance. Traditional culture dependent methods are time-consuming and labor-intensive whereas culture-independent polymerase chain reaction (PCR) based assays are reliable, consistent, rapid and reproducible. This review covers the recent development and applications of PCR based techniques, which have accelerated advances in the analysis of nucleic acids to identify three major pathogenic vibrios. Emphasis has been given to analytical approaches as well as advantages and limits of the available methods. Overall, this review article possesses the substantial merit to be used as a reference guide for the researchers to develop improved PCR based techniques for the differential detection and quantification of Vibrio species.

Predicting Vibrio cholerae Infection and Disease Severity Using Metagenomics in a Prospective Cohort Study.

BACKGROUND: Susceptibility to Vibrio cholerae infection is affected by blood group, age, and preexisting immunity, but these factors only partially explain who becomes infected. A recent study used 16S ribosomal RNA amplicon sequencing to quantify the composition of the gut microbiome and identify predictive biomarkers of infection with limited taxonomic resolution. METHODS: To achieve increased resolution of gut microbial factors associated with V. cholerae susceptibility and identify predictors of symptomatic disease, we applied deep shotgun metagenomic sequencing to a cohort of household contacts of patients with cholera. RESULTS: Using machine learning, we resolved species, strains, gene families, and cellular pathways in the microbiome at the time of exposure to V. cholerae to identify markers that predict infection and symptoms. Use of metagenomic features improved the precision and accuracy of prediction relative to 16S sequencing. We also predicted disease severity, although with greater uncertainty than our infection prediction. Species within the genera Prevotella and Bifidobacterium predicted protection from infection, and genes involved in iron metabolism were also correlated with protection. CONCLUSION: Our results highlight the power of metagenomics to predict disease outcomes and suggest specific species and genes for experimental testing to investigate mechanisms of microbiome-related protection from cholera.

The heptameric structure of the flagellar regulatory protein FlrC is indispensable for ATPase activity and disassembled by cyclic-di-GMP.

The bacterial enhancer-binding protein (bEBP) FlrC, controls motility and colonization of Vibrio cholerae by regulating the transcription of class-III flagellar genes in σ54-dependent manner. However, the mechanism by which FlrC regulates transcription is not fully elucidated. Although, most bEBPs require nucleotides to stimulate the oligomerization necessary for function, our previous study showed that the central domain of FlrC (FlrCC) forms heptamer in a nucleotide-independent manner. Furthermore, heptameric FlrCC binds ATP in "cis-mediated" style without any contribution from sensor I motif 285REDXXYR291 of the trans protomer. This atypical ATP binding raises the question of whether heptamerization of FlrC is solely required for transcription regulation, or if it is also critical for ATPase activity. ATPase assays and size exclusion chromatography of the trans-variants FlrCC-Y290A and FlrCC-R291A showed destabilization of heptameric assembly with concomitant abrogation of ATPase activity. Crystal structures showed that in the cis-variant FlrCC-R349A drastic shift of Walker A encroached ATP-binding site, whereas the site remained occupied by ADP in FlrCC-Y290A. We postulated that FlrCC heptamerizes through concentration-dependent cooperativity for maximal ATPase activity and upon heptamerization, packing of trans-acting Tyr290 against cis-acting Arg349 compels Arg349 to maintain proper conformation of Walker A. Finally, a Trp quenching study revealed binding of cyclic-di-GMP with FlrCC Excess cyclic-di-GMP repressed ATPase activity of FlrCC through destabilization of heptameric assembly, especially at low concentration of protein. Systematic phylogenetic analysis allowed us to propose similar regulatory mechanisms for FlrCs of several Vibrio species and a set of monotrichous Gram-negative bacteria.

Cholera outbreak in an informal settlement at Shahpur huts, Panchkula District, Haryana State, India, 2019.

In September 2019, after a reported death due to acute diarrheal disease in Shahpur village, Panchkula district, Haryana state, India, we conducted an outbreak investigation to identify the etiological agent, estimate the burden of disease, and make recommendations to prevent future outbreaks. The suspected cholera case was a resident of Shahpur huts, ≥1 year of age having ≥3 loose stools within a 24-h period between September 1 and 28, 2019 and a laboratory-confirmed cholera case, whose stool specimen tested positive for Vibrio cholerae. We identified 196 suspected cholera cases with a median age of 18 years (range: 1-65 years); 54% (106) being female. The overall attack rate was 8% (196/2,602), and the case fatality rate was 1% (2/196). Tested samples of water from tanks (n = 6), sewage effluent (n = 2), and 22% (4/18) of stool specimens collected from suspected cases were positive for V. cholerae. Strengthening surveillance, improving water, and sanitation systems are recommended to prevent future cholera outbreaks.

Evaluation of Thio- and Seleno-Acetamides Bearing Benzenesulfonamide as Inhibitor of Carbonic Anhydrases from Different Pathogenic Bacteria.

A series of 2-thio- and 2-seleno-acetamides bearing the benzenesulfonamide moiety were evaluated as Carbonic Anhydrase (CA, EC 4.2.1.1) inhibitors against different pathogenic bacteria such as the Vibrio cholerae (VchCA-α and VchCA-ß), Burkholderia pseudomallei (BpsCA-ß and BpsCA-γ), Mycobacterium tuberculosis (Rv3723-ß) and the Salmonella enterica serovar Typhimurium (StCA2-ß). The molecules represent interesting leads worth developing as innovative antibacterial agents since they possess new mechanism of action and isoform selectivity preferentially against the bacterial expressed CAs. The identification of potent and selective inhibitors of bacterial CAs may lead to tools also useful for deciphering the physiological role(s) of such proteins.

Reviving Phage Therapy for the Treatment of Cholera.

Cholera remains a major risk in developing countries, particularly after natural or man-made disasters. Vibrio cholerae El Tor is the most important cause of these outbreaks, and is becoming increasingly resistant to antibiotics, so alternative therapies are urgently needed. In this study, a single bacteriophage, Phi_1, was used to control cholera prophylactically and therapeutically in an infant rabbit model. In both cases, phage-treated animals showed no clinical signs of disease, compared with 69% of untreated control animals. Bacterial counts in the intestines of phage-treated animals were reduced by up to 4 log10 colony-forming units/g. There was evidence of phage multiplication only in animals that received a V. cholerae challenge. No phage-resistant bacterial mutants were isolated from the animals, despite extensive searching. This is the first evidence that a single phage could be effective in the treatment of cholera, without detectable levels of resistance. Clinical trials in human patients should be considered.

Vibrio cholerae amino acids go on the defense.

Gram-negative bacteria remodel their surfaces to interact with the environment, particularly to protect pathogens from immune surveillance and host defenses. The enzyme AlmG is known to be involved in remodeling the Vibrio cholerae surface, but its specific role was not clear. A new study characterizes AlmG at the molecular level, showing it defies phylogenetic expectations to add amino acids to lipopolysaccharide (LPS). This LPS modification plays a pivotal role in V. cholerae resistance to antimicrobial peptides, weapons of the innate immune system against infections.

AlmG, responsible for polymyxin resistance in pandemic Vibrio cholerae, is a glycyltransferase distantly related to lipid A late acyltransferases.

Cationic antimicrobial peptides (CAMPs), such as polymyxins, are used as a last-line defense in treatment of many bacterial infections. However, some bacteria have developed resistance mechanisms to survive these compounds. Current pandemic O1 Vibrio cholerae biotype El Tor is resistant to polymyxins, whereas a previous pandemic strain of the biotype Classical is polymyxin-sensitive. The almEFG operon found in El Tor V. cholerae confers >100-fold resistance to antimicrobial peptides through aminoacylation of lipopolysaccharide (LPS), expected to decrease the negatively charged surface of the V. cholerae outer membrane. This Gram-negative system bears striking resemblance to a related Gram-positive cell-wall remodeling strategy that also promotes CAMP resistance. Mutants defective in AlmEF-dependent LPS modification exhibit reduced fitness in vivo Here, we present investigation of AlmG, the hitherto uncharacterized member of the AlmEFG pathway. Evidence for AlmG glycyl to lipid substrate transferase activity is demonstrated in vivo by heterologous expression of V. cholerae pathway enzymes in a specially engineered Escherichia coli strain. Development of a minimal keto-deoxyoctulosonate (Kdo)-lipid A domain in E. coli was necessary to facilitate chemical structure analysis and to produce a mimetic Kdo-lipid A domain AlmG substrate to that synthesized by V. cholerae. Our biochemical studies support a uniquely nuanced pathway of Gram-negative CAMPs resistance and provide a more detailed description of an enzyme of the pharmacologically relevant lysophosphospholipid acyltransferase (LPLAT) superfamily.

Cholera Epidemic in South Sudan and Uganda and Need for International Collaboration in Cholera Control.

Combining the official cholera line list data and outbreak investigation reports from the ministries of health in Uganda and South Sudan with molecular analysis of Vibrio cholerae strains revealed the interrelatedness of the epidemics in both countries in 2014. These results highlight the need for collaboration to control cross-border outbreaks.

Lectins from Eichornia crassipens and Lemna minor may be involved in Vibrio Cholerae El Tor adhesion.

Vibrio cholerae (Vc) has been isolated from roots of aquatic plants during epidemic or interepidemic periods. It has been suggested that the lectins from the roots of aquatic plants play a role as reservoirs of Vc. In this paper, we evaluated the activity of lectins from Lemna minor and Eichornia crassipens plants as potential mediators of the Vc strain El Tor (Vct). We found that Lemna minor extract showed high specificity towards blood groups O and B. Eichornia crassipens extract showed high specificity towards blood group A and O. Sugar competition experiments demonstrated that Lemna minor extract showed a high recognition to Neu5Ac (acid N acetyl neuraminic or sialic acid) and GlcNAc (N-acetyl D glucoseamine) in group B; and GlcNAc in group O. Eichornia crassipens, the recognition was that of GalNAc (N-acetyl-D-galactoseamine) and GlcNAc in group O; and Fuc (L-Fucose) and GlcNAc in group A. Lemna minor and Eichornia crassipens protein extracts (p-ext) increased Vct proliferation and protected to the red cells group O against the hemolytic activity of Vct. Both p-exts did not show any statistical significance on agglutination to Vct when compared to the results from phosphate buffer. According to the results, lectins present in roots may be involved in the proliferation and survival of Vct.

Non O1, non O139 Vibrio cholerae bacteraemia in an infant; case report and literature review.

Non 01, Non O139 Vibrio cholerae bacteraemia is a rare but potentially fatal occurrence. There have been very few incidents of this infection from around the world. The treatment regimen of antibiotics also varies in literature. We present a case of bacteraemia caused by Non O1, Non O139 Vibrio cholerae along with associated risk factors, disease manifestations, laboratory diagnosis and treatment regimen. This serves to add additional information regarding symptoms and signs of this infection along with management of patient. Knowledge regarding this topic shall be highly useful to professionals if further cases are detected. In the discussion section, a review of literature of previous cases is also presented.

Detection of Vibrio cholerae O1 and O139 in environmental waters of rural Bangladesh: a flow-cytometry-based field trial.

Presence of Vibrio cholerae serogroups O1 and O139 in the waters of the rural area of Matlab, Bangladesh, was investigated with quantitative measurements performed with a portable flow cytometer. The relevance of this work relates to the testing of a field-adapted measurement protocol that might prove useful for cholera epidemic surveillance and for validation of mathematical models. Water samples were collected from different water bodies that constitute the hydrological system of the region, a well-known endemic area for cholera. Water was retrieved from ponds, river waters, and irrigation canals during an inter-epidemic time period. Each sample was filtered and analysed with a flow cytometer for a fast determination of V. cholerae cells contained in those environments. More specifically, samples were treated with O1- and O139-specific antibodies, which allowed precise flow-cytometry-based concentration measurements. Both serogroups were present in the environmental waters with a consistent dominance of V. cholerae O1. These results extend earlier studies where V. cholerae O1 and O139 were mostly detected during times of cholera epidemics using standard culturing techniques. Furthermore, our results confirm that an important fraction of the ponds' host populations of V. cholerae are able to self-sustain even when cholera cases are scarce. Those contaminated ponds may constitute a natural reservoir for cholera endemicity in the Matlab region. Correlations of V. cholerae concentrations with environmental factors and the spatial distribution of V. cholerae populations are also discussed.

Quinazoline-sulfonamides with potent inhibitory activity against the α-carbonic anhydrase from Vibrio cholerae.

Thirteen novel sulfonamide derivatives incorporating the quinazoline scaffold were synthesized by simple, eco-friendly procedures. These compounds were tested for their ability to inhibit the α-carbonic anhydrases (CA, EC 4.2.1.1) from Vibrio cholerae (VchCA) as well as the human α-CA isoforms, hCA I and hCA II. Nine compounds were highly effective, nanomolar inhibitors of the pathogenic enzyme VchCA. Three of them were also highly effective sub-nanomolar inhibitors of the cytosolic isoform II. The best VchCA inhibitor had a KI of 2.7 nM. Many of these developed compounds showed high selectivity for inhibition of the bacterial over the mammalian CA isoforms, with one compound possessing selectivity ratios as high as 97.9 against hCA I and 9.7 against hCA II. Compound 9d was another highly effective VchCA inhibitor presenting a selectivity ratio of 99.1 and 8.1 against hCA I and hCA II, respectively. These results suggest that sulfonamides with quinazoline backbone could be considered suitable tools to better understand the role of bacterial CAs in pathogenesis.

A novel case of Vibrio bacteremia in an immune-competent patient.

Vibrio Cholera, a gram negative bacterium, is notoriously known to cause diarrheal epidemics. The serotypes O1 and O139 are mainly responsible for the diarrheal outbreaks due to the enterotoxin they produce. This enterotoxin however seems to be protective against bacteremia and hence bacteremia is rarely encountered. We report a case of an immune-competent young female who initially presented with hypokalemic periodic paralysis secondary to acute on chronic diarrhea. She was discharged after rehydration and bounced back in septic shock and her blood cultures grew Vibrio Cholera identified on Vitek. She succumbed to this organism within 24 hours of re-admission.

A Label-Free Colorimetric AuNP-Aptasensor for the Rapid Detection of Vibrio cholerae O139.

Purpose: Waterborne pathogens pose a significant threat to public health, emphasizing the continuous necessity for advancing robust detection techniques, particularly in preventing outbreaks associated with these pathogens. This study focuses on cholera, an infectious disease caused by Vibrio cholerae, serogroups O1 and O139, often transmitted through contaminated water and food, raising significant public health concerns in areas with poor sanitation and limited access to clean water. Methods: We developed a colorimetric biosensor using aptamer-functionalized gold nanoparticles to identify Vibrio cholerae O139 and address this issue. The detection mechanism relies on the color change of gold nanoparticles (AuNPs) from red to blue-purple induced by NaCl after the pathogen incubation and aptamer-target binding. Initial steps involved synthesizing and characterizing AuNPs, then exploring the impact of aptamer and NaCl concentrations on AuNP agglomeration. Optimization procedures for aptamer concentration and salt addition identified the optimal conditions for detection as 120 pM aptamers and 1 M NaCl. Results: The aptasensor demonstrated a robust linear relationship, detecting V. cholerae concentrations from 103 to 108 CFU/mL, with a limit of detection (LOD) of 587 CFU/mL. Specificity tests and accurate sample analyses confirmed the efficiency of the AuNPs aptasensor, showcasing its reliability and speed compared to traditional culture examination methods. Moreover, we extended the aptasensor to a paper-based sensing platform with similar detection principles. Conclusion: The change in color upon target binding was captured with a smartphone and analyzed using image processing software. The paper-based device detected the target in less than 2 min, demonstrating its convenience for on-field applications.

Concurrent Vibrio cholerae O1 Meningitis and Intracranial Lesions in a Patient With Sickle Cell Disease: A Case Report.

Cholera meningitis is a rare complication of Vibrio cholerae (V. cholerae) infection. We present a case of cholera meningitis caused by toxigenic V. cholerae O1 in a 34-year-old male with sickle cell disease (SCD). The patient presented with fever, diarrhea, and altered mental status. Cerebrospinal fluid (CSF) analysis showed 5,231 cells/µL (53.9% neutrophils), a protein level of 462 mg/dL, and a glycorrhachia level of 26 mg/dL. V. cholerae O1 was isolated on CSF culture. Despite the patient undergoing antimicrobial therapy, brain imaging revealed basal ganglia ring-enhancing lesions suggestive of tuberculomas. Antituberculosis treatment and steroids led to clinical improvement. This report highlights the need to consider V. cholerae meningitis in patients with SCD who present with diarrhea and altered mental status. Prompt diagnosis and appropriate antimicrobial therapy are keys to improving patient outcomes.

High Mortality With Non-O1/Non-O139 Vibrio cholera Bacteraemia in Patients With Cirrhosis.

Background: Data on non-O1/non-O139 Vibrio cholera (NOVC) infection in liver disease is limited. We studied the clinical features and outcome of patients with cirrhosis with non-NOVC bacteraemia and/or spontaneous bacterial peritonitis (SBP) when compared to non-extended spectrum beta lactamase (non-ESBL) Escherichia coli (E. coli). Methods: Hospital information system of patients with cirrhosis admitted with bacteraemia and/or SBP from 2010 to 2020 was searched to include patients with NOVC infection. Non-ESBL E. coli bacteraemia/bacterascites were chosen as a comparator group, matched for the date of admission within 5 days of index case. Propensity score matching (PSM) was done for patient's age and Child score to compare outcome at discharge between NOVC-infected and E. coli-infected cirrhotic patients. Results: There were 2545 patients admitted with bacteraemia and/or SBP during the study period; 29 had NOVC isolated (M:F = 23:6; age: 39, 18-54 years; median, range; model for end-stage liver disease [MELD] score: 25, 12-38; Child score: 11, 10-12.5) from either blood (26), ascites (3), or both (8). Of these, 26 isolates were pan-sensitive to antibiotic sensitivity tests. Fifty-three patients with non-ESBL E. coli were isolated (M: F = 43:10; age: 48; 18-69 years; MELD score: 25, 20-32; Child score:12,11-13) from blood (31), ascites (17), or both (5) within the selected time frame. Of these, 48 isolates were sensitive to the empirical antibiotics initiated.After PSM, in comparison with 29 non-ESBL E. coli patients (age: 41, 18-55 years; MELD score: 24, 19-31; Child score: 12, 11-13), NOVC patients had higher incidence of circulatory failure at admission (14 [49 %] vs 4 [13 %]; P: 0.01) and significantly higher in-hospital mortality (15 [52 %] vs 6 [20 %];P: 0.028]. Conclusions: Bacteraemia due to non-O1/non-O139 strains of V. cholera, is an uncommon cause of bacteraemia or bacterascites in patients with cirrhosis and is associated with high incidence of circulatory failure and significant mortality.