Leucine-Rich, Potent Anti-Bacterial Protein against Vibrio cholerae, Staphylococcus aureus from Solanum trilobatum Leaves.

A 24 kDa leucine-rich protein from ion exchange fractions of Solanum trilobatum, which has anti-bacterial activity against both the Gram-negative Vibrio cholerae and Gram-positive Staphylococcus aureus bacteria has been purified. In this study, mass spectrometry analysis identified the leucine richness and found a luminal binding protein (LBP). Circular dichroism suggests that the protein was predominantly composed of α- helical contents of its secondary structure. Scanning electron microscopy visualized the characteristics and morphological and structural changes in LBP-treated bacterium. Further in vitro studies confirmed that mannose-, trehalose- and raffinose-treated LBP completely inhibited the hemagglutination ability towards rat red blood cells. Altogether, these studies suggest that LBP could bind to sugar moieties which are abundantly distributed on bacterial surface which are essential for maintaining the structural integrity of bacteria. Considering that Solanum triolbatum is a well-known medicinal and edible plant, in order to shed light on its ancient usage in this work, an efficient anti-microbial protein was isolated, characterized and its in vitro functional study against human pathogenic bacteria was evaluated.

Identification of Small Molecule Inhibitors of the Pathogen Box against Vibrio cholerae.

Antimicrobial resistance (AMR) has become a serious public and economic threat. The rate of bacteria acquiring AMR surpasses the rate of new antibiotics discovery, projecting more deadly AMR infections in the future. The Pathogen Box is an open-source library of drug-like compounds that can be screened for antibiotic activity. We have screened molecules of the Pathogen Box against Vibrio cholerae, the cholera-causing pathogen, and successfully identified two compounds, MMV687807 and MMV675968, that inhibit growth. RNA-seq analyses of V. cholerae after incubation with each compound revealed that both compounds affect cellular functions on multiple levels including carbon metabolism, iron homeostasis, and biofilm formation. In addition, whole-genome sequencing analysis of spontaneous resistance mutants identified an efflux system that confers resistance to MMV687807. We also identified that the dihydrofolate reductase is the likely target of MMV675968 suggesting it acts as an analog of trimethoprim but with a MIC 14-fold lower than trimethoprim in molar concentration. In summary, these two compounds that effectively inhibit V. cholerae and other bacteria may lead to the development of new antibiotics for better treatment of the cholera disease. IMPORTANCE Cholera is a serious infectious disease in tropical regions causing millions of infections annually. Vibrio cholerae, the causative agent of cholera, has gained multi-antibiotic resistance over the years, posing greater threat to public health and current treatment strategies. Here we report two compounds that effectively target the growth of V. cholerae and have the potential to control cholera infection.

Nonmetabolizable Arabinose Inhibits Vibrio cholerae Growth in M9 Medium with Gluconate as the Sole Carbon Source.

The serogroups O1 and O139 of the marine bacterium Vibrio cholerae are responsible for causing cholera in humans. The pentose sugar arabinose is nonmetabolizable by the pathogen and is present in environmental niches as well as in the human intestine. In this study, arabinose-mediated V. cholerae growth interference was assessed in M9 minimal medium containing gluconate as the sole carbon source in the light of Entner-Doudoroff (ED) pathway, an obligatory metabolic route for gluconate utilization. V. cholerae O1 and O139 strains failed to grow in the presence of ≥ 0.3% arabinose in M9 with 0.2% gluconate, but there was no growth inhibition in the presence of arabinose in M9 with 0.2% glucose. Transcriptional analysis of edd and eda, the genes constituting the ED pathway, showed ~100- and ~17-fold increases, respectively, in M9-gluconate. Minor increases of ~4- and ~2-fold for edd and eda, respectively, were noted in AKI medium supplemented with 0.5% arabinose. The observed arabinose-mediated growth inhibition can contribute toward deepening the understanding of altered phenotypes, if any, via complementation/expression studies in V. cholerae with pBAD vectors and arabinose as an inducer.

In vivo fluid accumulation-inhibitory, anticolonization and anti-inflammatory and in vitro biofilm-inhibitory activities of methyl gallate isolated from Terminalia chebula against fluoroquinolones resistant Vibrio cholerae.

Acute Vibrio cholerae infection triggers significant inflammatory response and immense fluid secretion in the intestine. In the present study, methyl gallate (MG) isolated from Terminalia chebula was evaluated to determine the in vivo fluid accumulation-inhibitory, anticolonization and anti-inflammatory and in vitro biofilm-inhibitory activities against multi-drug resistant (MDR) V. cholerae. Bacterial membrane-damaging and biofilm-inhibitory activities were determined by membrane perturbation and transmission electron microscopy (TEM); and microdilution assays, respectively. Fluid accumulation-inhibitory and anticolonization activities of MG (23.80-95.23 mg/kg body weight) were determined in 4-5 days old BALB/c mice with an incubation time of 18 h. The effect of MG (1, 50 and 500 mg/kg body weight) on intestinal inflammatory reaction induced by V. cholerae was studied by performing histology in Swiss albino mice. MIC and MBC of MG against the test strains were 32-64 and 64-256 µg/ml, respectively. MG showed the fluid accumulation-inhibitory activity with inhibition values of 42.86-89.08% at doses between 23.80 and 95.23 mg/kg body weight and significant anticolonization activity (p < 0.0001) against V. choleare in the suckling mouse intestine. MG (500 mg/kg body weight) significantly inhibited the inflammatory reactions induced by V. cholerae compared to the vehicle control. MG exhibited 70% minimum biofilm inhibition concentration of 64 µg/ml and bacterial membrane damaging activity at 1 × MBC. The results obtained in the present study suggest that MG has potential as an effective agent for the treatment of severe secretory and inflammatory diarrheal disease caused by MDR V. cholerae.

Antibacterial activity of polyphenolic fraction of Kombucha against Vibrio cholerae: targeting cell membrane.

The present study was undertaken to determine the mechanism of antibacterial activity of a polyphenolic fraction, composed of mainly catechin and isorhamnetin, previously isolated from Kombucha, a 14-day fermented beverage of sugared black tea, against the enteropathogen Vibrio cholerae N16961. Bacterial growth was found to be seriously impaired by the polyphenolic fraction in a dose-dependent manner. Scanning Electron Microscopy demonstrated morphological alterations in bacterial cells when exposed to the polyphenolic fraction in a concentration-dependent manner. Permeabilization assays confirmed that the fraction disrupted bacterial membrane integrity in both time- and dose-dependent manners, which were proportional to the production of intracellular reactive oxygen species (ROS). Furthermore, each of the polyphenols catechin and isorhamnetin showed the ability to permeate bacterial cell membranes by generating oxidative stress, thereby suggesting their role in the antibacterial potential of Kombucha. Thus, the basic mechanism of antibacterial activity of the Kombucha polyphenolic fraction against V. cholerae involved bacterial membrane permeabilization and morphological changes, which might be due to the generation of intracellular ROS. To the best of our knowledge, this is the first report on the investigation of antibacterial mechanism of Kombucha, which is mostly attributed to its polyphenolic content. SIGNIFICANCE AND IMPACT OF THE STUDY: The emergence of multidrug-resistant Vibrio cholerae strains has hindered an efficient anti-Vibrio therapy. This study has demonstrated the membrane damage-mediated antibacterial mechanism of Kombucha, a popular fermented beverage of sugared tea, which is mostly attributed to its polyphenolic content. This study also implies the exploitation of Kombucha as a potential new source of bioactive polyphenols against V. cholerae.

[STUDY OF COMPOSITION OF PLANT EXTRACTS, POSSESSING ANTIMICROBIAL EFFECT AGAINST VIBRIO CHOLERAE EL TOR, USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY].

AIM: Study the composition of plant extracts using high-performance liquid chromatography. (HPLC) and evaluation of their antimicrobial effect against Vibrio cholerae El Tor. MATERIALS AND METHODS: Qualitative and quantitative composition of plant extracts was studied using HPLC. Determination of sensitivity of microorganisms to plant extracts was carried out by diffusion into agar method and serial dilutions method. RESULTS: Antibacterial effect of water, water-alcohol and acetone extracts of roots of Limonium gmelinii L., Berberis vulgaris L. and Glycyrrhiza glabra L. was studied. The most effective methods of extraction of biologically active substances, possessing antimicrobial effect against various strains of V. cholerae El Tor, were determined. CONCLUSION: The use of HPLC allowed to establish the presence of catechines, alkaloids protoberberines and glycyrrhizic acid in xtracts, possessing antimicrobial effect against V. cholera El Tor strains.

Efficacy of Ciprofloxacin for Treatment of Cholera Associated with Diminished Susceptibility to Ciprofloxacin to Vibrio cholerae O1.

OBJECTIVE: We identified a poor clinical response to treatment of cholera with a single 1 g dose of ciprofloxacin, a standard treatment for cholera. METHODS: To determine reasons for the poor response and better therapeutic approaches we examined the minimal inhibitor concentration (MIC, n = 275) and disc-diffusion zone sizes (n = 205) for ciprofloxacin and nalidixic acid of V. cholerae O1 strains isolated in Bangladesh from 1994 to 2012, and reexamined data from 161 patients infected with Vibrio cholerae O1 recruited in four clinical trials who received single- or multiple-dose ciprofloxacin for treatment of cholera and compared their clinical response to the V. cholerae O1 susceptibility. RESULTS: Although all 275 isolates of V. cholerae O1 remained susceptible to ciprofloxacin using standard MIC and disc-diffusion thresholds, the MIC90 to ciprofloxacin increased from 0.010 in 1994 to 0.475 µgm/ml in 2012. Isolates became frankly resistant to nalidixic with the MIC90 increasing from 21 µgm/ml in 1994 to >256 µgm/ml and 166 of 205 isolates from 1994 to 2005 being frankly resistant using disc-diffusion testing. Isolates resistant to nalidixic acid by disc-diffusion testing had a median ciprofloxacin MIC of 0.190 µgm/ml (10th-90th centiles 0.022 to 0.380); nalidixic acid-susceptible isolates had a median ciprofloxacin MIC of 0.002 (0.002 to 0.012).The rate of clinical success with single-dose ciprofloxacin treatment for nalidixic acid-susceptible strains was 94% (61 of 65 patients) and bacteriologic success 97% (63/65) compared to 18% (12/67) and 8% (5/67) respectively with nalidixic acid-resistant strains (P<0.001 for both comparisons). Multiple-dose treatment with ciprofloxacin had 86% and 100% clinical and bacteriologic success rates respectively in patients infected with nalidixic acid-susceptible strains of V. cholerae O1 compared to clinical success 67% and bacteriologic success 60% with nalidixic acid-resistant strains. CONCLUSIONS: Single-dose ciprofloxacin is not effective for treating cholera caused by V. cholerae O1 with diminished susceptibility to ciprofloxacin, and nalidixic acid disc-diffusion testing effectively screens for such isolates.

Antibacterial and antiviral evaluation of sulfonoquinovosyldiacylglyceride: a glycolipid isolated from Azadirachta indica leaves.

UNLABELLED: Assessment of antibacterial as well as antiherpes virus activity of sulfonoquinovosyldiacylglyceride (SQDG), a glycolipid, isolated from the leaves of Azadirachta indica has been described. Antimicrobial activity was evaluated against Gram-positive, Gram-negative bacteria and herpes simplex virus. SQDG showed significant inhibitory activity against Salmonella typhi and two isolates of Shigella dysenteriae with MIC values 32 µg ml(-1) , while three isolates of Salm. typhi, Escherichia coli and Vibrio cholerae were inhibited at 64 µg ml(-1) and have shown zone diameter ranging from 6.2 to 12.3 mm. The growth kinetics study of SQDG on Salm. typhi and Sh. dysenteriae revealed that the growths were completely inhibited at their MIC values within 24 h of exposure. Interestingly, SQDG inhibits herpes simplex virus (HSV) type 1 and 2 with the EC50 of 9.1 and 8.5 µg ml(-1) , compared with acyclovir (2.2 and 2.8 µg ml(-1) against HSV-1 and HSV-2). The selectivity index (SI) was found to be 12.4 against HSV-1 and 13.41 with HSV-2. Furthermore, the expression of proinflammatory cytokines of HSV-infected and SQDG-treated macrophages using ELISA kit revealed that SQDG significantly downregulated the production of TNF-α, IL-1ß, IL-12 and IL-6. SIGNIFICANCE AND IMPACT OF THE STUDY: The water-soluble metabolite sulfonoquinovosyldiacylglyceride (SQDG) isolated from Azadirachta indica (Neem) possess significant antibacterial as well as anti-HSV activity. The efficacies as well as the solubility factor of SQDG substantiate a greater attention for its use as phytotherapeutic drug for controlling microbial infections as most consumers have better acceptance of phytomedicines than synthetic drugs.

Discovery and biological characterization of the auromomycin chromophore as an inhibitor of biofilm formation in Vibrio cholerae.

Bacterial biofilms pose a significant challenge in clinical environments due to their inherent lack of susceptibility to antibiotic treatment. It is widely recognized that most pathogenic bacterial strains in the clinical setting persist in the biofilm state, and are the root cause of many recrudescent infections. The discovery and development of compounds capable of either inhibiting biofilm formation or initiating biofilm dispersal might provide new therapeutic avenues for reducing the number of hospital-acquired, biofilm-mediated infections. We detail here the application of our recently reported image-based, high-throughput screen to the discovery of microbially derived natural products with inhibitory activity against Vibrio cholerae biofilm. Examination of a prefractionated library of microbially derived marine natural products has led to the identification of a new biofilm inhibitor that is structurally unrelated to previously reported inhibitors and is one of the most potent inhibitors of V. cholerae reported to date. Combination of this compound with sub-MIC concentrations of a number of clinically relevant antibiotics was shown to improve the inhibitory efficacy of this new compound compared to monotherapy treatments, and provides evidence for the potential therapeutic benefit of biofilm inhibitors in treating persistent biofilm-mediated infections.

The Entner-Doudoroff pathway is obligatory for gluconate utilization and contributes to the pathogenicity of Vibrio cholerae.

The Entner-Doudoroff (ED) pathway has recently been shown to play an important role in sugar catabolism for many organisms although very little information is available on the functionality of this pathway in Vibrio cholerae, the causative agent of cholera. In this study, activation of the genes edd and eda, encoding 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase, was used as a marker of a functional ED pathway in V. cholerae. Transcriptional activation analyses and gene silencing experiments with cells grown in sugar-supplemented M9 medium demonstrated that the ED pathway is functional in V. cholerae and is obligatory for gluconate catabolism. Importantly, selective activation of the ED pathway led to concurrent elevation of transcripts of prime virulence genes (ctxA and tcpA) and their regulator (toxT). Further, lowering of these transcript levels and cholera toxin production in vitro by an ED pathway-defective mutant (strain N16961 with a Δedd mutation [Δedd(N16961) strain]) suggested the importance of this pathway in regulating V. cholerae virulence. The in vivo relevance of these data was established as the mutant failed to colonize in suckling mice intestine or to induce fluid accumulation in ligated rabbit ileal loops. Activation of the ED pathway in V. cholerae was shown to inhibit biofilm formation in vitro that could be reversed in the mutant. As further support for these results, comparative transcriptome analysis with cells grown in the presence of glucose or gluconate revealed that a functional ED pathway led to activation of a subset of previously reported in vivo expressed genes. All of these results suggest the importance of the ED pathway in V. cholerae pathogenesis.

Remodelling of the Vibrio cholerae membrane by incorporation of exogenous fatty acids from host and aquatic environments.

The Gram-negative bacteria Vibrio cholerae poses significant public health concerns by causing an acute intestinal infection afflicting millions of people each year. V. cholerae motility, as well as virulence factor expression and outer membrane protein production, has been shown to be affected by bile. The current study examines the effects of bile on V. cholerae phospholipids. Bile exposure caused significant alterations to the phospholipid profile of V. cholerae but not of other enteric pathogens. These changes consisted of a quantitative increase and migratory difference in cardiolipin, decreases in phosphatidylglycerol and phosphatidylethanolamine, and the dramatic appearance of an unknown phospholipid determined to be lyso-phosphatidylethanolamine. Major components of bile were not responsible for the observed changes, but long-chain polyunsaturated fatty acids, which are minor components of bile, were shown to be incorporated into phospholipids of V. cholerae. Although the bile-induced phospholipid profile was independent of the V. cholerae virulence cascade, we identified another relevant environment in which V. cholerae assimilates unique fatty acids into its membrane phospholipids - marine sediment. Our results suggest that Vibrio species possess unique machinery conferring the ability to take up a wider range of exogenous fatty acids than other enteric bacteria.

Assessment of factors influencing antimicrobial activity of carvacrol and cymene against Vibrio cholerae in food.

Carvacrol and cymene, phenolic compounds naturally present in the essential oil of oregano and thyme, were examined for their antimicrobial activity against Vibrio cholerae (ATCC 14033, VC1, and VC7) inoculated in carrot juice. Carvacrol exhibited a dose dependent inhibitory effect on the bacteria. Although cymene did not have antimicrobial activity against the bacteria, it enhanced the inhibitory ability of carvacrol. At 25 °C, the lowest concentrations of carvacrol and cymene required for zero detectable viable count varied depending on bacterial strains; 5 and 5 ppm, respectively, for VC7; 5 and 7.5 ppm, respectively, for VC1; and 7.5 and 7.5 ppm, respectively, for ATCC 14033. This study also examined several factors influencing the antimicrobial activity of carvacrol and cymene against V. cholerae ATCC 14033, including temperature, bacterial cell number, and food substrate. Carvacrol and cymene inhibited the bacterium in carrot juice at 25 °C more efficiently than at 15 and 4 °C. The doses of both compounds required for zero detectable viable count increased as the number of the bacterial cells in the carrot juice increased. The fat content and the complexity of foods were shown to decrease the antimicrobial activity of the compounds.

[Antimicrobial activity of Morinda morindoides on in vitro growth of vibrio cholerae in Côte d'Ivoire]. / Activité de Morinda morindoides sur la croissance in vitro du vibrion du choléra: expérimentation en Côte d'Ivoire.

Cholera is a major public health problem in developing countries. As a contribution to management of this disease, the study described herein was carried out in Côte d'Ivoire. The purpose was to evaluate the antibacterial activity of products obtained by various techniques from the leaves of Morinda morindoides on a pathogenic strain of Vibrio cholerae O:1. Morinda morindoides is a medicinal plant in the Ivorian pharmacopoeia. The products were obtained as aqueous extracts, 70% ethanolic extracts, residual extracts and a chromatographic fraction (BGG F5). All three extracts and the chromatographic fraction showed considerable in vitro antimicrobial efficacy against Vibrio cholerae O:1. The most active against in vitro growth of Vibrio cholorae O:1 was the 70% ethanolic extract with a minimal bactericidal concentration of 5 mg/ml. The antibacterial properties of this medicinal plant can be of great benefit for management of cholera.

Antimicrobial activity of elephant garlic oil against Vibrio cholerae in vitro and in a food model.

Vibrio cholera is a major foodborne pathogen in Thailand. It is present in raw and lightly cooked foods, and it causes cholera. Natural products inhibiting it can be used to improve the safety of foods. In this study, elephant garlic oil was studied for its major diallyl sulfide content and its antimicrobial activity against V. cholerae. The oil had a very low concentration of diallyl monosulfides (1.62%) in comparison with the other diallyl sulfides (25.09% for diallyl disulfide, 16.04% for diallyl trisulfide, and 10.58% for diallyl tetrasulfide). In an in vitro study, the oil was found to have a bacteriocidal effect on all tested strains of V. cholerae, with varied minimal inhibitory concentrations (MICs) ranging from 3.13 to 25 microg/ml. It was also found that elephant garlic oil retarded the growth of the bacteria or reduced the bacterial cell load in the food model, depending on its concentration.

[A multipathogen selective enrichment broth (SVV) for simultaneous growth of Salmonella, Vibrio parahaemolyticus, and Vibrio cholerae].

We formulated a selective enrichment broth (SVV) for simultaneous growth of Salmonella, Vibrio parahaemolyticus, and Vibrio cholerae by single factor experiment and response surface method. We evaluated the enrichment effect of SVV by conventional culture method and real-time PCR assay. We obtained the SVV broth by supplementting the Buffered Peptone Water (BPW) with bile salt no. 3, potassium tellurite, and sodium citrate as inhibitors, and glucose, mannitol, snhydrous sodium sulfite and sodium pyruvate as accelerants. We also modified the concentration of sodium chloride in BPW. When mixed at equal or varied proportions, the target pathogens had a great accumulation (10(5)-10(8) CFU/mL) after incubated in SVV for 18 h at 37 degrees C with shaking. It can also effectively inhibit the competitive microflora. We detected 10 artificial simulated samples and 608 real samples using SVV with real-time PCR. After enriched in SVV for 18 h, the quantity of the bacteria in samples were above the detection limit. The SVV with PCR assay showed higher tested positive (4.06%) compared to that of the conventional detection method (3.78%) and there was no false report. In summary, SVV is a promising new multiplex selective enrichment broth that can be used in detection of seafood.

Antibacterial activity of Quercus ilex bark's extracts.

The antibacterial activity of different extracts of Quercus ilex bark (Fagaceae) was studied in vitro against seven reference strains of bacteria by using a disc-diffusion method and agar-dilution method. The ethyl acetate extract (QE), n-butanol extract (QB) and final aqueous layer (QA) were effective against all bacterial strains tested at MICs ranging from 128 to 512 microg/ml. The n-hexane extract (QH) and dichloromethane extract (QD) showed no activity.

Role for glycine betaine transport in Vibrio cholerae osmoadaptation and biofilm formation within microbial communities.

Vibrio cholerae is a halophilic facultative human pathogen found in marine and estuarine environments. Accumulation of compatible solutes is important for growth of V. cholerae at NaCl concentrations greater than 250 mM. We have identified and characterized two compatible solute transporters, OpuD and PutP, that are involved in uptake of glycine betaine and proline by V. cholerae. V. cholerae does not, however, possess the bet genes, suggesting that it is unable to synthesize glycine betaine. In contrast, many Vibrio species are able to synthesize glycine betaine from choline. It has been shown that many bacteria not only synthesize but also secrete glycine betaine. We hypothesized that sharing of compatible solutes might be a mechanism for cooperativity in microbial communities. In fact, we have demonstrated that, in high-osmolarity medium, V. cholerae growth and biofilm development are enhanced by supplementation with either glycine betaine or spent media from other bacterial species. Thus, we propose that compatible solutes provided by other microorganisms may contribute to survival of V. cholerae in the marine environment through facilitation of osmoadaptation and biofilm development.

Antibacterial potential from Indian Suregada angustifolia.

Phytochemical analysis of the leaves from Indian Suregada angustifolia (Baill. ex Muell. Arg.) Airy Shaw (Euphorbiaceae) resulted in the isolation and identification of six known compounds, viz. friedelin, epi-friedelinol, n-octacosanol, alpha-amyrin, beta-sitosterol and beta-sitosterol-3-beta-D-glucopyranoside. Aqueous (room temperature, boiled and autoclaved) and various solvent (methanol, chloroform and hexane) extracts of leaves were tested against 12 human pathogenic bacteria by the agar well-diffusion method. Aqueous extracts did not express any activity. Antibacterial activity was recorded in the order of methanol, hexane and chloroform extracts. Maximum activity recorded against Staphylococcus aureus (skin infections) in methanol and hexane extracts and moderate activity recorded against diarrhoea causing bacteria, Vibrio vulnificus (hexane extract) and Vibrio cholerae (chloroform extract).

Synthesis and evaluation as sialidase inhibitors of xylo-configured cyclohexenephosphonates carrying glycerol side-chain mimics.

Based on a strategy previously reported by us, we have synthesized D-xylo configured cyclohexenephosphonates designed to mimic the transition state of the sialidase reaction. The double bond orientation corresponds to the benchmark inhibitor Neu5Ac2en and we could selectively introduce hydroxyalkyl substituents in order to simulate the glycerol side-chain of neuraminic acid. The inhibitory activity of a set of compounds towards bacterial sialidases was tested and interesting differences in activity were found.