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Cholera is a serious epidemic disease caused by the toxigenic strains of Vibrio cholerae belonged to O1 or O139 serogroups. The emergence of antibacterial resistance in V. cholerae is an increasing concern. Natural product drug invention and Ethnopharmacology may demonstrate a considerable expectation under this circumstance. Traditionally, leaves of Typhonium trilobatum (L.) Schott (locally known as Ghatkanchu or Bengal Arum) are employed for treatment of gastrointestinal disorder in different region of India. The objective of the present study was to evaluate the antibacterial, and antibiofilm activities of methanol extract of T. trilobatum leaves (METTL) against the strains of multi-drug resistant (MDR) Vibrio cholerae (serotypes O1, O139, non-O1, and non-O139) which are responsible for watery diarrhea such as cholera. MIC, MBC and time-kill kinetic studies were used for evaluation of In vitro antibacterial activity of METTL. Microdilution method and Confocal laser scanning microscopy were used to evaluate biofilm-inhibitory activities. The gene expression was analyzed by performing Quantitative real-time PCR (qRT-PCR). METTL showed antibacterial activity with MIC and MBC at 1-32 mg/mL and 8-32 mg/mL, respectively against the clinical strains of Vibrio cholerae belonged to different serogroups. METTL showed significant (P 0.05) inhibitory activity on the formation of biofilm by V. cholerae SG24, with 81.3, 75.8, and 69.6% of inhibition at MIC, ½ MIC and ¼ MIC, respectively. METTL showed also significant (P 0.05) inhibitory activity on the formation of extracellular polymeric substances (EPS) formation by V. cholerae SG24, with 89.41, and 99.26% of inhibition of EPS protein and EPS carbohydrate at MIC, respectively. METTL significantly (p 0.01) inhibited the Cholera toxin (CT) production by the V. cholerae strain SG24 evaluated by the CT - ELISA assay. The cholera toxin production was reduced by 76.26%, 48.76% and 29.93 at MIC (8 mg/mL), ½ MIC (4 mg/mL) and ¼ MIC (2 mg/mL), respectively. METTL was shown to repress ctxAB gene transcription 1.76 fold (p 0.05) at sub-bactericidal concentration (¼ MIC). We also found that the expression of cholera toxin activator genes, toxT and tcpP was reduced by 11.56- fold (p 0.001) and 23.52- fold (p 0.001), respectively, at sub-bactericidal concentration (¼ MIC). Transcription of the following genes was repressed: vpsR (1.8-fold; p 0.05), Bap1 (1.53-fold; p ≤ 0.05), and rmbA (2.89-fold) by METTL at sub-bactericidal concentration. The expression of vpsT was also repressed by 1.5-fold (p 0.01) at sub-bactericidal concentration. The active Typhonium trilobatum (L.) leaves extract may be suggested as an substitute for the treatment of MDR V. cholerae infection and could be used as prospective source for the development of novel antimicrobial compound/s and biofilm-inhibitory drug/s useful for the treatment of cholera and diarrheal patients. The results obtained here also validate scientifically the traditional uses of Typhonium trilobatum (L.) in India employed for the treatment of gastrointestinal disorder. Further studies should be directed at purifying and characterizing these antibacterial principles against Vibrio cholerae.

Aim: The present work provides first-time empirical and molecular interaction evidence to establish the higher biofunctionality of a therapeutic lipid, α-eleostearic acid (ESA), encapsulated in a novel and thoroughly characterized biocompatible nanoemulsion (NE) system (particle size

International audience; Maytenus buchananii is worldwide distributed small evergreen tree traditionally used as anti-tumor, anti-ulcer, anti-inflammatory, and as antimicrobial. The column chromatography of the MeOH extract of M. buchananii leaves followed by purification of different fractions led to the isolation of nine known flavonoids. Their structures were elucidated on the basis of spectrum analysis, and by comparison with those from the literature. The antibacterial activity was assessed by performing minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and the time-kill kinetic study while the antioxidant activity was determined by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and trolox equivalent antioxidant capacity (TEAC) assays. All of the extracts/compounds showed antibacterial activity against the strains of Gram-positive bacteria, Staphylococcus aureus, and Gram-negative multi-drug resistance bacteria, Vibrio cholerae (causative agent of cholera) and Shigella flexneri (causative agent of shigellosis). Compounds 2 (MIC = 32 to 128 μg/mL) and 6 (MIC = 16 to 64 μg/mL) showed the largest antibacterial activities with the best MIC (16 μg/mL) recorded with compound 6 on S. aureus. The results of both DPPH and TEAC assays found compounds 3 (EC50 = 1.38 μg/mL; TEAC = 89.69 μg/mL), 5 (EC50 = 1.56 μg/mL; TEAC = 90.93 μg/mL) and 6 (EC50 = 1.42 μg/mL; TEAC = 89.76 μg/mL) to exhibit the most antioxidant activity. Results obtained from this study may help to exploit the use of the M. buchananii leaf extracts and some of their flavonoid compounds as pharmacological ingredients for promoting health, especially for cholera/shigellosis and chronic diseases associated with oxidative stress.

TMEM16A (Transmembrane protein 16A or Anoctamin1) is a calcium-activated chloride channel. (CaCC),that exerts critical roles in epithelial secretion. However, its localization, function, and regulation in intestinal chloride (Cl−) secretion remain obscure. Here, we show that TMEM16A protein abundance correlates with Cl− secretion in different regions of native intestine activated by the Ca2+-elevating muscarinic agonist carbachol (CCH). Basal, as well as both cAMP- and CCH-stimulated Isc, was largely reduced in Ano1 ± mouse intestine. We found CCH was not able to increase Isc in the presence of apical to serosal Cl− gradient, strongly supporting TMEM16A as primarily a luminal Cl− channel. Immunostaining demonstrated apical localization of TMEM16A where it colocalized with NHERF1 in mouse colonic tissue. Cellular depletion of NHERF1 in human colonic T84 cells caused a significant reduction of both cAMP- and CCH-stimulated Isc. Immunoprecipitation experiments revealed that NHERF1 forms a complex with TMEM16A through a PDZ-based interaction. We conclude that TMEM16A is a luminal Cl− channel in the intestine that functionally interacts with CFTR via PDZ-based interaction of NHERF1 for efficient and specific cholinergic stimulation of intestinal Cl− secretion., Highlights • TMEM16A express apically and operate Cl− secretion in mouse intestinal tissue. • TMEM16A potentially interacts with NHERF1 via its C-terminal PDZ binding motif. • TMEM16A-NHERF1 complex is requisite for cAMP and Ca2+ mediated apical Cl− secretion.

Vibrio cholerae, the causative agent of cholera, poses serious threats to humans worldwide. V. cholerae faces host inflammatory response and encounters nitrosative stress before establishing successful colonization. It is not clear how V. cholerae combats nitric oxide and reactive nitrogen species. In the present study, we used three clinical strains of V. cholerae and tested their nitrosative stress response pattern towards sodium nitroprusside (SNP) and S-Nitrosoglutathione (GSNO). Among them, V. cholerae, belonging to both O1 and O139 serotypes, showed moderate resistance to SNP and GSNO. However, a V. cholerae strain belonging to non O1 and non O139 showed sensitivity to SNP but resistance towards GSNO. Reduced glutathione and glutathione reductase play a significant role to combat nitrosative stress in V. cholerae. This is the first report where we show the presence of GSNO reductase activity in V. cholerae and that it plays an important role to detoxify S-Nitrosoglutathione. GSNO reductase activity of V. cholerae was regulated by posttranslational modification through S-nitrosylation under in vitro conditions which could be reversed by dithiothreitol (DTT). In addition, we show that biofilm formation remained unaffected under nitrosative stress in V. cholerae.

Staphylococcus aureus is a leading aetiologic agent of nosocomial- and community-acquired infectious diseases worldwide. The public health concern regarding staphylococcal infections is inflated by the increasing occurrence of multidrug-resistant strains, e.g. multidrug- and meticillin-resistant S.aureus (MDR MRSA). This study was designed to evaluate the intracellular killing, membrane-damaging and biofilm-inhibitory activities of nimbolide isolated from Azadirachta indica against MDR MRSA. In vitro antibacterial activity of nimbolide was determined by performing MIC, minimal bactericidal concentration (MBC) and time-kill kinetic studies. Bacterial membrane-damaging activity was determined by membrane perturbation and scanning electron microscopy (SEM) examination. Biofilm-inhibitory activities were determined by SEM. Cellular drug accumulation and assessments of intracellular activities were performed using Vero cell culture. SEM revealed that nimbolide caused significant membrane damage and lysis of the S. aureus cells. The biofilm structure was disrupted, and the biofilm formation was greatly reduced in the presence of nimbolide as examined by SEM. The level of accumulation of nimbolide in Vero cells incubated for 24 h is relatively higher than that of ciprofloxacin and nalidixic acid (Cc/Ce for nimbolide > ciprofloxacin and nalidixic acid). The viable number of intracellular S. aureus was decreased [reduction of ~2 log10 c.f.u. (mg Vero cell protein)-1] in a time-dependent manner in the presence of nimbolide (4× MBC) that was comparable to that of tetracycline and nalidixic acid. The significant intracellular, biofilm-inhibitory and bacterial membrane-damaging activities of nimbolide demonstrated here suggested that it has potential as an effective antibacterial agent for the treatment of severe infections caused by MDR MRSA.

Enzymes provide the critical means by which almost all biological reactions are catalyzed in a controlled manner. Methods to harness and exploit their properties are of strong current interest to the growing field of biotechnology. In view of many unique physical and optical characteristics that are advantageous for studying enzyme activity at quantum dot (QD)–bioconjugate interfaces, we endeavored to explore the possible influence on conformation and catalytic functionality of alkaline phosphatase (ALP), a clinical marker enzyme, by conducting in vitro enzymatic activity assay and using molecular spectroscopic techniques such as UV-visible absorption, fluorescence, circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. The resulting experimental data were then analyzed in the context of the classical Michaelis–Menten model. The results show almost 60% and 30% decrease in enzyme activity on interaction with 10 nM Cys-CdTe or Cys-CdS QDs, respectively, suggesting inhibition dependence on particle nature. CD spectra measurements indicated that QDs induced a decrease of α-helical content and an increase of β-sheet structure in ALP, resulting in the loosening and unfolding of the protein skeleton. Interestingly, when QDs and substrate were added simultaneously to enzyme molecules, catalytic inhibition was significantly reduced indicating protection of enzyme structure by substrate molecules. Further, we demonstrate that protein-encapsulated QDs do not affect the catalytic functionality of enzyme molecules. The present study provides valuable information which may have significant ramifications in various biomedical applications, such as biosensing, drug delivery, cellular imaging, etc.

Diarrhea continues to be one of the most common causes of morbidity and mortality among infants and children in developing countries. The aim of the present study was to evaluate the antibacterial and antioxidant activities of extracts and compounds from Ludwigia leptocarpa, a plant traditionally used for its vermifugal, anti-dysenteric, and antimicrobial properties. A methanol extract was prepared by maceration of the dried plant and this was successively extracted with ethyl acetate to obtain an EtOAc extract and with n-butanol to obtain an n-BuOH extract. Column chromatography of the EtOAc and n-BuOH extracts was followed by purification of different fractions, leading to the isolation of 10 known compounds. Structures of isolated compounds were assigned on the basis of spectral analysis and by comparison to structures of compounds described in the literature. Antioxidant activity was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and gallic acid equivalent antioxidant capacity (GAEAC) assays. Antibacterial activity was assessed with the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) with respect to strains of a Gram-positive bacterium, Staphylococcus aureus (a major cause of community and hospital-associated infection), and Gram-negative multi-drug-resistant bacteria, Vibrio cholerae (a cause of cholera) and Shigella flexneri (a cause of shigellosis). All of the extracts showed different degrees of antioxidant and antibacterial activities. 2β-hydroxyoleanolic acid, (2R,3S,2''S)-3''',4',4''',5,5'',7,7''-heptahydroxy-3,8"-biflavanone, and luteolin-8-C-glucoside displayed the most potent antibacterial and antioxidant properties, and these properties were in some cases equal to or more potent than those of reference drugs. Overall, the present results show that L. leptocarpa has the potential to be a natural source of anti-diarrheal and antioxidant products, so further investigation is warranted.

Phytochemical investigation of leaves and stem bark extracts of Clausena anisata monitored by liquid chromatography high-resolution mass spectrometry (LC-HR-MS) analysis led to the isolation and characterization of twenty-one secondary metabolites: four carbazole alkaloids (1–4) including one new name clausamine H (1), fourteen coumarins (5–18), two porphyrin derivatives (19–20), and one limonoid (21). Crude extracts were analyzed first by LC-HR-MS, and target compounds were isolated by a multi-step separation procedure using column chromatography and preparative high-performance liquid chromatography (prep-HPLC) monitored by LC-HR-MS analysis. The structures of isolates were determined by means of spectroscopic and spectrometric data, as well as by comparison with literature values. The isolates showed weak to high antibacterial activities with imperatorin (14) being the most active one. Cytotoxic activities against HeLa and monkey Vero cells were also investigated, and murrayamine-A (4), 3-(1,1-dimethyl allyl) xanthyletin (5) gravelliferone (7), excavatin D (10), 7-[(E)-7-hydroxy-3,7-dimethylocta-2,5-dienyloxyl]-coumarin (13), phellopterin (15), and 1-O-methylclausenolide (21) were found active with LC50 values ranged from 1.14 to 3.26 µg/mL and a good selectivity index values (SI 38.20–231.58) against the HeLa cells. However, these compounds were non-toxic to normal cells indicating their high potential to be used as anticancer drug.

Background : The emergence of multiple-drug resistance bacteria has become a major threat and thus calls for an urgent need to search for new effective and safe anti-bacterial agents. Objectives : This study aims to evaluate the anticancer and antibacterial activities of secondary metabolites from Penicillium sp ., an endophytic fungus associated with leaves of Garcinia nobilis . Methods : The culture filtrate from the fermentation of Penicillium sp . was extracted and analyzed by liquid chromatography– mass spectrometry, and the major metabolites were isolated and identified by spectroscopic analyses and by comparison with published data. The antibacterial activity of the compounds was assessed by broth microdilution method while the anticancer activity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results : The fractionation of the crude extract afforded penialidin A-C (1-3), citromycetin (4), p-hydroxyphenylglyoxalaldoxime (5) and brefelfin A (6). All of the compounds tested here showed antibacterial activity (MIC = 0.50 – 128 μg/mL) against Gramnegative multi-drug resistance bacteria, Vibrio cholerae (causative agent of dreadful disease cholera) and Shigella flexneri (causative agent of shigellosis), as well as the significant anticancer activity (LC50 = 0.88 – 9.21 μg/mL) against HeLa cells. Conclusion : The results obtained indicate that compounds 1-6 showed good antibacterial and anticancer activities with no toxicity to human red blood cells and normal Vero cells. Keywords : Garcinia nobilis , endophytic fungi, secondary metabolites, antibacterial activity, anticancer activity.

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.

An extracellular lipase (EC 3.1.1.3), SAL-PP1, from Staphylococcus aureus isolated from Arachis hypogaea rhizosphere was purified and characterized. The enzyme was purified using PALL'S Microsep centrifugal device (10 kD cut off), hydrophobic interaction (phenyl sepharose CL-4B column) and Superose-12 gel filtration chromatography and found to have a molecular mass of around 49 kDa. The gene fragment encoding the part of the catalytic site of the SAL-PP1 lipase was sequenced and the deduced amino acid sequence shows 93% identity with that of SEL3. SAL-PP1 showed activity against long acyl-chain triglycerides, various p-nitrophenyl esters and phospholipids. The enzyme shows high stability and activity after incubation with various metal ions (retained >90% activity in presence of Ca2+, Na+, Cu2+, Mg2+, Fe2+, or Hg2+ at 10 mM), organic solvents (retained >80% activity in presence of acetonitrile, ethanol, DMSO, methanol, isopropanol, toluene, or ethylene glycol at 10 mM), detergents (retained >70% activity in Triton X-100, Tween 80, or sodium deoxycholate at 10 mM) and irreversible inhibitors (retained >77% activity in presence of PMSF, leupetin, or β-mercaptoethanol, at 1 mM). Thermal inactivation studies revealed a temperature dependent unfolding of secondary structure of protein. SAL-PP1 showed maximal activity and stability at pH 8.0 and pH 9.0, respectively. The alkali-thermostability, organic solvent-tolerance and broad substrate specificity of this enzyme may have potential implications in detergent formulations, biotransformation, industries, and medicine.

Indigenous uses of Azadirachta indica A. juss (Maliaceae) (locally known as neem) leaves in different parts of India for curing gastrointestinal disorder such as diarrhea and cholera is wide spread. The objective of the present study was to evaluate the antibacterial and antisecretory activity of neem extract against Vibrio cholerae , a causative agent of watery diarrhea such as cholera. The methanol extract of neem leaf was tested for its antibacterial, antisecretory and antihemorrhagic activity against Vibrio cholerae . Azadirachta indica extract had significant antibacterial activity against the multi-drug-resistant Vibrio cholerae of serotypes O1, O139 and non-O1, non-O139. The minimum inhibitory concentration reached by 50% (MIC 50 ) and 90% (MIC 90 ), and minimum bactericidal concentration for the extract were 2.5, >5, and 10 mg/ml, respectively. Neem extract showed antisecretory activity on Vibrio cholerae induced fluid secretion in mouse intestine with inhibition values of 27.7%, 41.1%, 43.3%, 57.0%, and 77.9% at doses of 100, 200, 300, 450 and 1800 mg/kg, respectively. Oral administration of the extract inhibited hemorrhage induced by Vibrio cholerae in mouse intestine at a dose ≥300 mg/kg. The results obtained in this study give some scientific support to the uses of neem employed by the indigenous people in India employed for the treatment of diarrhea and dreadful disease cholera.

Rabbit antiserum raised against the whole-cell antigen of Shiga toxin-producing Escherichia coli (STEC) strain VT3 ( stx 1 + stx 2 + eae + ) was repeatedly adsorbed with heat-killed cells of different non-STEC strains and other enteric bacteria. Thus, the antiserum obtained was designated VT3 antiserum. VT3 antiserum reacted with intimin type γ. We assessed the reactivity of VT3 antiserum to whole-cell lysates of 87 strains of E. coli and other enteric bacteria by immunoblotting. The antiserum recognized the 97-kDa protein in whole-cell lysate from strain VT3, and 36 (83.7%) of the 43 STEC strains were positive for the STEC antigen. None of the non-STEC strains or strains of other species examined tested positive by immunoblotting. Based on this result, we developed a latex agglutination assay for the detection of STEC strains. Thirty-five (81.4%) of the 43 STEC strains tested positive for the STEC antigen by the latex agglutination assay. One (3.3%) of the 30 non-STEC strains and none of the strains of the other enteric bacteria included in this study tested positive by the latex agglutination assay. The corresponding specificity of the latex agglutination assay was approximately 98%. Results of this study showed the production of STEC antiserum and the generation of a simple, cost-effective, sensitive, and specific latex agglutination assay for establishing an etiological diagnosis of STEC.

Shigella spp. (Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei) cause bacillary dysentery (shigellosis), which is characterized by bloody mucous diarrhoea. Although a variety of antibiotics have been effective for treatment of shigellosis, options are becoming limited due to globally emerging drug resistance. In the present study, in vitro antibacterial activity of methyl gallate (MG) isolated from Terminalia chebula was determined by performing MIC, minimal bactericidal concentration (MBC) and time-kill kinetic studies. Bacterial membrane-damaging activity of MG was determined by membrane perturbation and transmission electron microscopy (TEM). Cellular drug accumulation, cell infection and assessment of intracellular activities of MG and reference antibiotics were performed using HeLa cell cultures. The bactericidal activity of MG against multidrug-resistant (MDR) Shigella spp. in comparison with other commonly used drugs including fluoroquinolone was demonstrated here. TEM findings in the present study revealed that MG caused the total disintegration of inner and outer membranes, and leakage of the cytoplasmic contents of S. dysenteriae. The level of accumulation of MG and tetracycline in HeLa cells incubated for 24 h was relatively higher than that of ciprofloxacin and nalidixic acid (ratio of intracellular concentration/extracellular concentration of antibiotic for MG and tetracycline>ciprofloxacin and nalidixic acid). The viable number of intracellular S. dysenteriae was decreased in a time-dependent manner in the presence of MG (4 × MBC) and reduced to zero within 20 h. The significant intracellular activities of MG suggested that it could potentially be used as an effective antibacterial agent for the treatment of severe infections caused by MDR Shigella spp.

Senna alata is widely used in Cameroon for the treatment of several infections which include gonorrhoea, gastro-intestinal and skin diseases. Therefore, its leaves were investigated for antibacterial principles. Extraction of plant material was done with methanol, follow by partition with hexane. Separation and purification of compounds was done using a combination of chromatographic techniques. Isolated compounds were identified by means of spectroscopic methods and comparison with literature data. The antibacterial activity of extracts, fractions and compounds was assessed by evaluating the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Multi-Drug-Resistant (MDR) Vibrio cholerae and Shigella flexneri . Three secondary metabolites namely kaempferol, luteolin and aloeemodin were isolated from methanol residue active extract. The antibacterial results showed that the MeOH residue extract, fractions A and C, as well as compounds 1-3 exhibited variable MIC values, depending on the bacterial strains. Aloe-emodin (MIC = 4 to 128 μg/mL) exhibited the highest antibacterial activity against MDR Vibrio cholerae and Shigella flexneri . From these results obtained, S. alata leaf could be considered as a natural antibacterial source due to the presence of flavonoid and anthraquinone compounds. Keywords : Senna alata, Fabaceae, kaempferol, luteolin, aloe-emodin, MDR strains.

In order to assess the extent of genomic diversity among Vibrio cholerae O139 strains, restriction fragment length polymorphisms in two genetic loci, rrn and ctx, were studied. Analysis of 144 strains isolated from different regions of Bangladesh and India between 1992 and 1998 revealed the presence of at least six distinct ribotypes (B-I through B-VI) of which three were new ribotypes, and one of these was represented by a nontoxigenic O139 strain. Strains of ribotypes B-I through B-V shared 11 different CTX genotypes (A through K). Antimicrobial resistance patterns of the strains varied independently of their ribotypes and CTX genotypes. Results of this study suggest that V. cholerae O139 is undergoing rapid genetic changes leading to the origination of new variants, and temporal changes in antimicrobial resistance patterns may be contributing to the selection of different variants.

New triterpenoid (I), named reissantiadiol, is isolated among known compounds from the stems of reissantia grahamii.

The present strategy proposes a simple and single step aqueous route for synthesizing stable, fluorescent ZnTe/dendrimer nanocomposites with varying dendrimer terminal groups. In these hybrid materials, the fluorescence of the semiconductor combines with the biomimetic properties of the dendrimer making them suitable for various biomedical applications. The ZnTe nanocomposites thus obtained demonstrate bactericidal activity against enteropathogenic bacteria without having toxic effects on the human erythrocytes. The average size of the ZnTe nanoparticles within the dendrimer matrix was in the range of 2.9-6.0 nm, and they have a good degree of crystallinity with a hexagonal crystal phase. The antibacterial activities of the ZnTe/dendrimer nanocomposites (ZnTe DNCs) as well other semiconductor nanocomposites were evaluated against enteropathogenic bacteria including multi-drug resistant Vibrio cholerae serogroup O1 and enterotoxigenic Escherichia coli (ETEC). ZnTe DNCs had significant antibacterial activity against strains of V. cholerae and ETEC with minimum inhibitory concentrations ranging from 64 to 512 μg ml(-1) and minimum bactericidal concentrations ranging from 128 to 1000 μg ml(-1). Thus, the observed results suggest that these water-soluble active nanocomposites have potential for the treatment of enteric diseases like diarrhoea and cholera.

Summary A collection of 28 strains of Vibrio cholerae non-O1 isolated during a 3-year period (1989–1991) from hospitalised patients with acute diarrhoea in Calcutta, India, were examined with regard to virulence-associated factors. Of the 28 isolates (each representing a case), 18 were isolated as the sole infecting agent; the remaining 10 were recovered as co-cultures from cases infected with V. cholerae O1. Of the strains isolated in this study, 82% could be serotyped, with serovars O5 (32·1%), O11 and O34 (14·3% each) predominant. Serovars O7, O14, O34, O39 and O97 were associated exclusively with sole infections. Two strains of V. cholerae non-O1 produced anti-cholera toxin IgG-absorbable cholera toxin (CT). Both CT-producing V. cholerae non-O1 strains hybridised with the DNA probe specific for the zonula occludens toxin (ZOT) but none of the remaining 26 strains hybridised with the ZOT probe. The majority of the strains were cytotoxic for CHO, HeLa and Vero cells, with end-point titres of 4–512. Fewer strains produced a cytotonic effect, with end-point titres of 2–16. Of the 28 strains of V. cholerae non-O1 examined, 75%, 75%, 25% and 14·3% produced haemolysin that was active against erythrocytes of rabbit, sheep (Eltor haemolysin), chicken and man, respectively. Strains that produced a haemolysin active against both rabbit and sheep erythrocytes were dominant (35·7%). Ten (35·7%) of the 28 strains examined showed cell-associated haemagglutinating activity on human blood. Of the 10 strains, nine were isolated as sole pathogen and only one strain was associated with mixed infection. Three distinct patterns of inhibition by sugars were detected; inhibition of haemagglutination by mannose 1% but not by fucose and galactose 1% was the dominant haemagglutination inhibition pattern. Six different virulence phenotypes were encountered among strains of V. cholerae non-O1 in this study. The prominent phenotype, which was associated commonly with isolates from patients solely infected by V. cholerae non-O1, was exhibited by strains that produced the Eltor haemolysin, a cytotoxin and a cell-associated haemagglutinin. The production of cell-associated haemagglutinin appeared to be the only distinctive phenotype that could distinguish between isolates from patients solely infected with V. cholerae non-O1 and those associated with mixed infections. From this study, it is apparent that the virulence of V. cholerae non-O1 is multifactorial and mediated by several traits functioning in an integrated fashion. The clinical significance of V. cholerae non-O1 must be assessed in its totality; the presence of a single factor should not be construed as the cause of enteropathogenicity.

Four molecular species of heat-stable enterotoxins elaborated by a cholera toxin-producing strain of Vibrio cholerae O1 were isolated from its culture supernatant. The amino acid sequence of one of the enterotoxins was determined to be Phe-Ile-Lys-Gln-Val-Asp-Glu-Asn-Gly-Asn-Leu-Ile-Asp-Cys-Cys-Glu-Ile-Cys-Cys-Asn-Pro-Ala-Cys-Phe-Gly-Cys-Leu-Asn with three intramolecular disulfide linkages. The other enterotoxins had shorter amino acid sequences in the N-terminal regions, but possessed the same sequence in their C-terminal regions including the three disulfide linkages. The enterotoxins with the shorter N-terminal sequences showed more potent toxicities, and the minimum effective dose of the longest one with 28 amino acid residues was 10-folds of that of the shortest one.

The present study reports on the isolation and characterization of a Pseudomonas aeruginosa strain PTz-5 from crude oil from oil field sampled in Assam, India. It was capable to utilize hexadecane, benzene or toluene as a sole source of carbon aerobically. Strain PTz-5 was able to produce extracellular lipase that catalyzed triglycerides to free fatty acid and glycerol. The lipase activity was stable in the temperature range of 40 to 60 degrees C. Strain PTz-5 avidly adhered to the surface of hydrocarbon droplets during their growth in liquid culture medium. These properties could play an essential role in hydrocarbon degradation. The results presented here highlight the metabolic versatility and hydrocarbon biodegradative capability of strain PTz-5, signifying its great potential for the bioremediation of various hydrocarbon-contaminated environments.

Members of the genusAeromonas(familyAeromonadaceae) are medically important, Gram-negative, rod-shaped micro-organisms and are ubiquitous in aquatic environments.Aeromonasspecies are increasingly recognized as enteric pathogens; they possess several virulence factors associated with human disease, and represent a serious public health concern. In the present study, putative virulence traits ofAeromonas hydrophilaisolates collected from different natural surface waters of Kolkata, India, were compared with a group of clinical isolates from the same geographical area using tissue culture and PCR assays. Enteropathogenic potential was investigated in the mouse model. Of the 21 environmental isolates tested, the majority showed cytotoxicity to HeLa cells (81 %), haemolysin production (71 %) and serum resistance properties (90 %), and they all exhibited multi-drug resistance. Some of the isolates induced fluid accumulation (FA ratio≥100), damage to the gut and an inflammatory reaction in the mouse intestine; these effects were comparable to those of clinical strains ofA. hydrophilaand toxigenicVibrio cholerae. Interestingly, two of the isolates evoked a cell vacuolation effect in HeLa cells, and were also able to induce FA. These findings demonstrate the presence of potentially pathogenic and multi-drug-resistantA. hydrophilain the surface waters, thereby indicating a significant risk to public health. Continuous monitoring of surface waters is important to identify potential water-borne pathogens and to reduce the health risk caused by the genusAeromonas.

Purpose: To evaluate the anti-enterobacterial potential of nine ethnobotanically selected plants traditionally used in different parts of India for the treatment of gastrointestinal disorders such as cholera, diarrhea or dysentery. Methods: The methanol extracts of these plants were screened for antibacterial activity against 11 strains of enteropathogenic bacteria, including multi-drug resistant Vibrio cholerae (serotypes O1, O139, and non-O1, non-O139), using broth microdilution method. Ampicillin was used as a positive reference standard to determine the sensitivity of the strains. Phytochemical screening was carried out for phenolics and flavonoids. Results: All of these plants had bactericidal activity against at least one of the test microorganisms with minimum inhibitory concentration (MIC) ranging from 0.125 to 32 mg/ml and minimum bactericidal concentration (MBC) ranging from 0.25 to 32 mg/ml. Among these, Terminalia chebula Retz. (Combretaceae ) and Syzygium cumini (L.) Skeels (Myrtaceae) showed the most promising broad spectrum antibacterial properties, inhibiting all of the strains tested, especially Vibrio cholerae, Aeromonas hydrophila and Bacillus subtilis, with MBC ranging from 0.25 to 4 mg/ml. Phenolics and flavonoids were found to be present in the extracts. Conclusion: The findings provide support for the use of this plant in traditional medicine for treatment of diarrheoa, especially cholera.

Vibrio cholerae non-O1, non-O139 was isolated from natural surface waters from different sites sampled in diarrhea endemic zones in Kolkata, India. Twenty-one of these isolates were randomly selected and included in the characterization. The multiserogroup isolates were compared by their virulence traits with a group of clinical non-O1, non-O139 isolates from the same geographic area. Of the 21 environmental isolates, 6 and 14 strains belonged to Heiberg groups I and II, respectively. Three of the environmental isolates showed resistance to 2,2-diamine-6,7-diisopropylpteridine phosphate. All of the non-O1, non-O139 strains were positive for toxR , and except for one environmental isolate, none of them were positive for tcpA in the PCR assay. None of the isolates were positive for genes encoding cholera toxin ( ctxA ), heat-stable toxin ( est ), heat-labile toxin ( elt ), and Shiga toxin variants ( stx ) of Escherichia coli . Additionally, except for one environmental isolate (PC32), all were positive for the gene encoding El Tor hemolysin ( hly ). The culture supernatants of 86% (18 of 21) of the environmental isolates showed a distinct cytotoxic effect on HeLa cells, and some of these strains also produced cell-rounding factor. The lipase, protease, and cell-associated hemagglutination activities and serum resistance properties of the environmental and clinical isolates did not differ much. However, seven environmental isolates exhibited very high hemolytic activities (80 to 100%), while none of the clinical strains belonged to this group. The environmental isolates manifested three adherence patterns, namely, carpet-like, diffuse, and aggregative adherence, and the clinical isolates showed diffuse adherence on HeLa cells. Of the 11 environmental isolates tested for enteropathogenic potential, 8 (73%) induced positive fluid accumulation (≥100) in a mouse model, and the reactivities of these isolates were comparable to those of clinical strains of non-O1, non-O139 and toxigenic O139 V. cholerae . Comparison of the counts of the colonized environmental and clinical strains in the mouse intestine showed that the organisms of both groups had similar colonizing efficiencies. These findings indicate the presence of potentially pathogenic V. cholerae non-O1, non-O139 strains in surface waters of the studied sites in Kolkata.

The genomes of the O3:K6 strains of Vibrio parahaemolyticus which abruptly emerged in Calcutta, India, in February 1996 and which demonstrated an unusual potential to spread and an enhanced propensity to cause infections were examined by different molecular techniques to determine clonality. No restriction fragment length polymorphism (RFLP) in the gene encoding the thermostable direct hemolysin was observed among the O3:K6 isolates of V. parahaemolyticus . Clonal diversity among the O3:K6 strains became evident by examining the RFLPs of the rrn operons and by the use of pulsed-field gel electrophoresis. Five ribotypes were distinguished among the O3:K6 strains examined, with ribotype R4 constituting the major type. Strains of O3:K6 isolated between June and August 1996 showed different pulsotypes compared to the pulsotypes of strains isolated before and after this period, indicating genetic reassortment among these strains, but those isolated between August 1996 and March 1998 showed identical or nearly similar pulsotypes. It is clear that there is a certain degree of genomic reassortment among the O3:K6 clones but that these strains are predominantly one clone.

Background The continuous emergence of multidrug-resistant (MDR) bacteria drastically reduced the efficacy of our antibiotic armory and consequently, increased the frequency of therapeutic failure. The search for bioactive constituents from endophytic fungi against MDR bacteria became a necessity for alternative and promising strategies, and for the development of novel therapeutic solutions. We report here the isolation and structure elucidation of antibacterial and cytotoxic compounds from Phomopsis sp., an endophytic fungus associated with Garcinia kola nuts. Methods The fungus Phomopsis sp. was isolated from the nut of Garcinia kola. The crude extract was prepared from mycelium of Phomopsis sp. by maceration in ethyl acetate and sequentially fractionated by column chromatography. The structures of isolated compounds were elucidated on the basis of spectral studies and comparison with published data. The isolated compounds were evaluated for their antibacterial and anticancer properties by broth microdilution and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide methods respectively. The samples were also tested spectrophotometrically for their hemolytic properties against human red blood cells. Results The fractionation of the crude extract afforded three known cytochalasins including 18-metoxycytochalasin J (1), cytochalasins H (2) and J (3) together with alternariol (4). The cytochalasin compounds showed different degrees of antibacterial activities against the tested bacterial pathogens. Shigella flexneri was the most sensitive microorganism while Vibrio cholerae SG24 and Vibrio cholerae PC2 were the most resistant. Ampicillin did not show any antibacterial activity against Vibrio cholerae NB2, Vibrio cholerae PC2 and Shigella flexneri at concentrations up to 512 μg/mL, but interestingly, these multi-drug resistant bacterial strains were sensitive to the cytochalasin metabolites. These compounds also showed significant cytotoxic properties against human cancer cells (LC50 = 3.66–35.69 μg/mL) with low toxicity to normal non-cancer cells. Conclusion The three cytochalasin compounds isolated from the Phomopsis sp. crude extract could be a clinically useful alternative for the treatment of cervical cancer and severe infections caused by MDR Shigella and Vibrio cholerae.