Potential application of Northern Argentine propolis to control some phytopathogenic bacteria

The antimicrobial activity of samples of Northern Argentine propolis (Tucumán, Santiago del Estero and Chaco) against phytopathogenic bacteria was assessed and the most active samples were identified. Minimal inhibitory concentration (MIC) values were determined by agar macrodilution and broth microdilution assays. Strong antibacterial activity was detected against Erwinia carotovora spp carotovora CECT 225, Pseudomonas syringae pvar tomato CECT 126, Pseudomonas corrugata CECT 124 and Xanthomonas campestris pvar vesicatoria CECT 792. The most active propolis extract (Tucumán, T1) was selected to bioguide isolation and identified for antimicrobial compound (2',4'-dihydroxychalcone). The antibacterial chalcone was more active than the propolis ethanolic extract (MIC values of 0.5-1 μg ml(-1) and 9.5-15 μg ml(-1), respectively). Phytotoxicity assays were realized and the propolis extracts did not retard germination of lettuce seeds or the growth of onion roots. Propolis solutions applied as sprays on tomato fruits infected with P. syringae reduced the severity of disease. Application of the Argentine propolis extracts diluted with water may be promising for the management of post harvest diseases of fruits.     

Cycloaspeptide A and pseurotin A from the endophytic fungus Penicillium janczewskii

Penicillium janczewskii K. M. Zalessky was isolated as an endophytic fungus from the phloem of the Chilean gymnosperm Prumnopitys andina. When grown in liquid yeast extract-malt extract-glucose broth, the fungus produced two main secondary metabolites. The compounds were for the first time isolated from this species and identified by spectroscopic methods as pseurotin A and cycloaspeptide A. This is the first report on the production of cyclic peptides by endophytic fungi from Chilean gymnosperms. Pseurotin A and cycloaspeptide A presented low cytotoxicity towards human lung fibroblasts with IC50 > or = 1000 microM. Pseurotin A showed a moderate effect against the phytopathogenic bacteria Erwinia carotovora and Pseudomonas syringae, with IC50 values of 220 and 112 microg ml(-1), respectively.     

Inhibition of Hydrolytic Enzyme Activities and Plant Pathogen Growth by Invertase Inhibitors

The invertase inhibitory protein isolated from Cyphomandra betacea Sendt and Solanum tuberosum inhibited the invertase activity from different species, genera and even plant family. Furthermore, proteinaceous inhibitors are not invertase specific; fungal, bacterial and higher plant enzymes including polygalacturonase, pectinase, pectin lyase, α - l -arabinofuranosidase and β -glucosidase are also shown to be inhibited. Both inhibitors exhibited an in vitro antibacterial action against phytopathogenics strains of Xanthomonas campestris pvar vesicatoria CECT 792, Pseudomonas solanacearum CECT 125, Pseudomonas corrugata CECT 124, Pseudomonas syringae and Erwinia carotovora var carotovora.     

Inhibition of hydrolytic enzyme activities and plant pathogen growth by invertase inhibitors

The invertase inhibitory protein isolated from Cyphomandra betacea Sendt and Solanum tuberosum inhibited the invertase activity from different species, genera and even plant family. Furthermore, proteinaceous inhibitors are not invertase specific; fungal, bacterial and higher plant enzymes including polygalacturonase, pectinase, pectin lyase, alpha-L-arabinofuranosidase and beta-glucosidase are also shown to be inhibited. Both inhibitors exhibited an in vitro antibacterial action against phytopathogenics strains of Xanthomonas campestris pvar vesicatoria CECT 792, Pseudomonas solanacearum CECT 125, Pseudomonas corrugata CECT 124, Pseudomonas syringae and Erwinia carotovora var carotovora.     

Syringopeptin SP25A-mediated killing of gram-positive bacteria and the role of teichoic acid d-alanylation

The Pseudomonas syringae syringopeptins are cationic cyclic lipodepsipeptides that inhibit fungi and bacteria. The homolog syringopeptin (SP)25A was strongly inhibitory to several Gram-positive bacteria with minimum inhibitory concentrations ranging between 1.95 and 7.8 microg mL(-1). In contrast, it was not inhibitory to several Gram-negative bacteria. At 5 and 10 microg mL(-1), SP25A rapidly inhibited the growth of Bacillus subtilis and was bacteriocidal. Teichoic acid D-alanylation dltB- and dltD-defective mutant strains of B. subtilis were more susceptible to SP25A compared with the parental wild-type strain. The degree of susceptibility of the parent strain, but not the dltB and dltD mutant strains, increased at alkaline pH (9.0). In contrast, the parental and mutant strains had the same susceptibilities to syringopeptins SP22A and SP508A at pH 7.0 and 9.0. These results suggest that the cell wall anionic teichoic acids modulate SP25A action against B. subtilis, and they provide an explanation for the selective inhibition of Gram-positive bacteria by SP25A.     

Use of 1,4-naphthoquinones for control of Erwinia carotovora

The antimicrobial effect of 5 naphthoquinones was tested against the phytopathogenic bacteria Erwinia carotovora. Disk diffusion tests and determination of minimal inhibitory concentrations (MIC) indicate that the compound naphthazarin (NTZ) has the best antibacterial activity among the naphthoquinones tested. Studies on the mode of action indicate the effect of NTZ was bactericidal at 10 microg/mL. When cultivation was done in the presence of sodium ascorbate, the restoration of E. carotovora growth was observed with 3 microg/mL NTZ, but not when a 10 microg/mL dose was used. The incubation of NTZ with bacterial suspension of E. carotovora resulted in important changes in the absorption spectra of this naphthoquinone, indicating that a redox reaction takes place. These results may suggest that NTZ induces an increase of reactive oxygen species that are toxic to the cell. The compound NTZ was also effective in preventing E. carotovora growth on potato tubers, inhibiting the soft rot development at a concentration of 2 mg/mL.     

Production of antimicrobial defensin in Nicotiana benthamiana with a potato virus X vector

A recombinant plasmid, pTXS.TH, was constructed to express the gene-encoding wasabi (Wasabia japonica) defensin with the potato virus X (PVX) vector. pTXS.TH allows the expression of defensin in the host Nicotiana benthamiana, and the defensin protein WT1 can be purified from virus-infected leaves by heat treatment and affinity chromatography. WT1 exhibits strong antifungal activity toward the phytopathogenic fungi Magnaporthe grisea (50% inhibitory concentration [IC50] = 5 microg/ml) and Botrytis cinerea (IC50 = 20 microg/ml) but is weakly active against the phytopathogenic bacterium Pseudomonas cichorii. This virus-mediated expression system is a rapid and efficient method to produce and characterize antimicrobial proteins in plants. It is particularly useful for the study of proteins that are difficult to produce with other expression systems.     

The minimum inhibitory concentration of oral antibacterial agents against cariogenic organisms

The minimum inhibitory concentrations (MIC) of eight common dental antibacterial agents against three genera of bacteria which have been implicated in dentine caries, namely streptococci, lactobacilli and actinomycetes were investigated. The ultimate aim was to determine the most appropriate antibacterial agent which could be added to dental restorative materials for filling cavities where there was residual dentine caries. The antibacterial agents tested were chlorhexidine diacetate, chlorhexidine dihydrochloride, chlorhexidine gluconate, benzalkonium chloride, cetrimide, cetylpyridinium chloride, thymol and sodium hypochlorite. Thymol and sodium hypochlorite did not inhibit microbial growth at any of the concentrations tested. For the active antibacterial agents tested the MIC values against lactobacilli and streptococci were 0.25 microg/ml to 8.0 microg/ml and for actinomycetes 0.125 to 8.0 microg/ml. These results illustrate the wide spectrum of sensitivity of caries associated bacteria against dental antibacterial agents. From the MIC values alone, it is difficult to recommend which of the active antibacterial agents would be most effective in eliminating cariogenic organisms.     

Antibacterial and cytotoxic activity of Brazilian plant extracts--Clusiaceae

Twelve extracts obtained from nine plants belonging to six different genera of Clusiaceae were analyzed against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Enterococcus faecalis) bacteria using the microdilution broth assay. Tovomita aff. longifolia, T. brasiliensis, Clusia columnaris, Garcinia madruno, Haploclathra paniculata, and Caraipa grandifolia extracts showed significant results against the bacteria. The organic extract obtained from the leaves of T. aff. longifolia showed minimal inhibitory concentration (MIC) = 70 microg/ml and minimal bactericidal concentration (MBC) = 90 microg/ml against E. faecalis and the organic extract made with the stem of C. columnaris showed MIC = 180 microg/ml and MBC = 270 microg/ml against P. aeruginosa. None of the antibacterial extracts showed lethal activity against brine shrimp nauplii. On the other hand, both aqueous and organic extracts obtained from the aerial organs of Vismia guianensis that were cytotoxic to brine shrimp nauplii did not show a significant antibacterial activity in the assay.     

Antimicrobial activity and the major components of the essential oil of Plectranthus cylindraceus

AIMS: The traditional uses of the aerial parts of fresh Plectranthus cylindraceus by the Dhofaris in Oman were investigated on the basis of antimicrobial properties and composition of its herb oil. METHODS AND RESULTS: The minimal inhibitory concentration (MIC) values of the herb oil of P. cylindraceus against human pathogenic bacteria and yeast were assessed using the broth microdilution method, and the percentage growth inhibition of fungi was determined according to the poisoned food technique. The oil exhibited good activity against Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilis and Candida albicans with MIC values in the range of 7.8-62.5 microg ml(-1) and also inhibited the growth of Microsporum canis, Microsporum gypseum, Trichophyton rubrum, Alternaria alternata, Bipolaris sp., Curvularia lunata, Fusarium oxysporum and Stemphylium solani for about a week at 250 microg ml(-1). The two most abundant components of the oil were identified as carvacrol (46.8%) and alpha-terpinolene (18.2%) based on gas chromatography-mass spectra (GC-MS) and 13C nuclear magnetic resonance (NMR) analyses. CONCLUSIONS: The oil has a pleasant odour and showed broad spectrum antimicrobial activity. Such bioactivity could be attributed in part to carvacrol. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has demonstrated the broad spectrum antimicrobial activity of the herb oil of P. cylindraceus as well as the organic composition of this plant extract. This study provides scientific insight into the ancient practice of utilizing P. cylindraceus as a fragrant disinfectant.     

Antibacterial and antifungal activity of cicerfuran and related 2-arylbenzofurans and stilbenes

Cicerfuran, 2-(2-methoxy-4,5-methylenedioxyphenyl)-6-hydroxybenzofuran, is an antifungal phytoalexin previously isolated from the roots of chickpea, Cicer spp. The synthesis of cicerfuran, five 2-arylbenzofuran analogues and nine stilbene intermediates was reported recently. The antimicrobial activities of these compounds were evaluated against two species of bacteria, Bacillus subtilis and Pseudomonas syringae, and four species of filamentous fungi, Aspergillus niger, Botrytis cinerea, Cladosporium herbarum and Monilinia aucupariae. Stilbenes with a free hydroxyl group were active against both bacteria and fungi with MICs in the range 25-100microg/ml. Cicerfuran was the only 2-arylbenzofuran that showed antimicrobial activity with MICs as low as 25microg/ml. Some aspects of the structure-activity relationship are discussed.     

Chemical compositions and antimicrobial activities of four different Anatolian propolis samples

Propolis means a gum that is gathered by bees from various plants. It is known for its biological properties, having antibacterial, antifungal and healing properties. The aims of this study were to evaluate the antimicrobial activity of four different Anatolian propolis samples on different groups of microorganisms including some oral pathogens and comparison between their chemical compositions. Ethanol extracts of propolis (EEP) were prepared from four different Anatolian propolis samples and examined whether EEP inhibit the growth of the test microorganisms or not. For the antimicrobial activity assays, minimum inhibitory concentrations (MIC) were determined by using macrodilution method. The MIC values of the most effective propolis (TB) were 2 microg/ml for Streptococcus sobrinus and Enterococcus faecalis, 4 microg/ml for Micrococcus luteus, Candida albicans and C. krusei, 8 microg/ml for Streptococcus mutans, Staphylococcus aureus, Staphylococcus epidermidis and Enterobacter aerogenes, 16 microg/ml for Escherichia coli and C. tropicalis and 32 microg/ml for Salmonella typhimurium and Pseudomonas aeruginosa. The chemical compositions of EEP's were determined by high-temperature high-resolution gas chromatography coupled to mass spectrometry. The main compounds of four Anatolian propolis samples were flavonoids such as pinocembrin, pinostropin, isalpinin, pinobanksin, quercetin, naringenin, galangine and chrysin. Although propolis samples were collected from different regions of Anatolia all showed significant antimicrobial activity against the Gram positive bacteria and yeasts. Propolis can prevent dental caries since it demonstrated significant antimicrobial activity against the microorganisms such as Streptococcus mutans, Streptococcus sobrinus and C. albicans, which involves in oral diseases.     

Antimicrobial activity of organic extracts isolated from Aplysina fistularis (Demospongiae: Aplysinidae)

Organic extracts of the sponge Aplysina fistularis (Pallas 1766) were tested for antimicrobial activity against Gram positive bacteria (Staphylococcus aureus) and Gram negative bacteria (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa). The minimal inhibitory concentration (MIC) and toxic activity of extract were determined. Susceptibility trials of organic fractions obtained by VLC: Hexane, EtOAc and CHCl3 showed that EtOAc fraction has antibacterial activity against E. coli, while CHCl3 fraction inhibited E. coli and S. aureus growth. The later refractioning of EtOAc fraction and the biodirected assays showed that fractions F12 and F13 of EtOAc/Hex and EtOAc F14 were bioactive against Gram positive and Gram negative bacteria. Only EtOAc/MeOH Sf2 from subfractionig of EtOAc F14 produced inhibition for E. coli and S. aureus. In Sf2 EtOAc/MeOH, MIC was moderate for S. aureus (MIC > 256 g/ml). F4 CHCl3/MeOH produced a high inhibition in S. aureus (MIC = 0.125 g/ml) and for E. coli (MIC > 16 g/ml). F10 CHCl3/MeOH showed a moderate activity against S. aureus (MIC > 128 g/ml) and low activity against E. coli (MIC = 512 g/ml). F10 CHCL3/MeOH did no present toxic activity against Artemia salina. The fractiorts F4 CHCL3/MeOH and Sf2 EtOAc/MeOH were toxic for this organism when the concentration was higher than 100 microg/ml. LC50 in both cases was 548.4 and 243.4 microg/ml respectively. Secondary metabolites of medium polarity obtained from A. fistularis have a wide spectrum of anti bacterial activity. Toxicity analysis suggests that only F10 CHCL3/MeOH has potential as an antimicrobial agent for clinical use.     

Influence of Pathogenic and Non-pathogenic Bacteria on Soybean Suspension Cells

Co-cultivations of plant suspension cultures of soybean ( Glycine max ) with compatible phytopathogenic ( Pseudomonas syringae pv. glycinea ), incompatible phytopathogenic ( Pseudomonas syringae pv. tomato ), and different non-pathogenic ( Erwinia herbicola, Escherichia coli ) bacteria were carried out. Growth and viability (triphenyltetrazolium chloride activity) of plant cells and bacteria as well as enzyme activities of peroxidase (PO), polyphenoloxidase (PPO), and phenylalanine ammonialyase (PAL) within the plant cells were investigated over an incubation period of 7 days. The compatible pathogen inhibited growth and viability of the plant cells after 1 day and led to the death of the majority of the plant cells by the seventh day. In contrast, the incompatible pathogen directly reduced growth and viability of the soybean cells and caused a strong induction of enzyme activities of PO and PAL to more than 4 times of the untreated control by the seventh day. The epiphytic bacterium Erwinia herbicola caused a slight inhibition of growth and viability of the plant cells after the second day of co-cultivation. The PO activity increased in the same manner as in the incompatible interaction. The saprophytic Escherichia coli strain had a negligible influence on soybean suspension cells. All the bacteria tested except for Escherichia coli multiplied rapidly during co-cultivation and reached populations of 10⁸-10⁹ colonyfoming units/ml in the stationary phase. The results from this study demonstrate that the soybean suspension cells react differently to compatible, incompatible and saprophytic bacteria.     

Synthesis and biological evaluation of new N-(2-hydroxy-4(or 5)-nitro/aminophenyl)benzamides and phenylacetamides as antimicrobial agents

A new series of N-(2-hydroxy-4(or 5)-nitro/aminophenyl)benzamide and phenylacetamide derivatives (1a-1n, 2a-2n) were synthesized and evaluated for antibacterial and antifungal activities against Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Candida albicans, and their drug-resistant isolate. Microbiological results indicated that the compounds possessed a broad spectrum of activity against the tested microorganisms at MIC values between 500 and 1.95 microg/ml. Benzamide derivative 1d exhibited the greatest activity with MIC values of 1.95, 3.9, and 7.8 microg/ml against drug-resistant B. subtilis, B. subtilis, and S. aureus, respectively.     

Production of an engineered killer peptide in Nicotiana benthamiana by using a potato virus X expression system

The decapeptide killer peptide (KP) derived from the sequence of a single-chain, anti-idiotypic antibody acting as a functional internal image of a microbicidal, broad-spectrum yeast killer toxin (KT) was shown to exert a strong microbicidal activity against human pathogens. With the aim to exploit this peptide to confer resistance to plant pathogens, we assayed its antimicrobial activity against a broad spectrum of phytopathogenic bacteria and fungi. Synthetic KP exhibited antimicrobial activity in vitro towards Pseudomonas syringae, Erwinia carotovora, Botrytis cinerea, and Fusarium oxysporum. KP was also expressed in plants by using a Potato virus X (PVX)-derived vector as a fusion to the viral coat protein, yielding chimeric virus particles (CVPs) displaying the heterologous peptide. Purified CVPs showed enhanced antimicrobial activity against the above-mentioned plant pathogens and human pathogens such as Staphylococcus aureus and Candida albicans. Moreover, in vivo assays designed to challenge KP-expressing plants (as CVPs) with Pseudomonas syringae pv. tabaci showed enhanced resistance to bacterial attack. The results indicate that the PVX-based display system is a high-yield, rapid, and efficient method to produce and evaluate antimicrobial peptides in plants, representing a milestone for the large-scale production of high-added-value peptides through molecular farming. Moreover, KP is a promising molecule to be stably engineered in plants to confer broad-spectrum resistance to phytopathogens.     

Suppressive effect of Bacillus subtilis and it's products on phytopathogenic microorganisms

Bacteria isolated from suppressive composts which showed clear zones inhibitory to the growth of phytopathogenic fungi in vitro on agar plates were identified to be Bacillus subtilis. The suppressiveness of the culture broth of these bacteria became obvious in in vivo tests, and the efficacy of the broth to phytopathogens was equivalent to that of available chemicals. The suppressive spectrum of the broth of this bacteria was broad enough to be effective not only to phytopathogenic fungi but also to bacteria. As a suppressive substance, an antibiotic of the iturin group was purified from the broth and identified. MIC values of each component of the iturin to several phytopathogens were determined.     

Physicochemical characterization and antimicrobial properties of rhamnolipids produced by Pseudomonas aeruginosa 47T2 NCBIM 40044

Pseudomonas aeruginosa 47T2, grown in submerged culture with waste frying oil as a carbon source, produced a mixture of rhamnolipids with surface activity. Up to 11 rhamnolipid homologs (Rha-Rha-C(8)-C(10); Rha-C(10)-C(8)/Rha-C(8)-C(10);Rha-Rha-C(8)-C(12:1); Rha-Rha-C(10)-C(10); Rha-Rha-C(10)-C(12:1); Rha-C(10)-C(10); Rha-Rha-C(10)-C(12)/Rha-Rha-C(12)-C(10); Rha-C(10)-C(12:1)/Rha-C(12:1)-C(10); Rha-Rha-C(12:1)-C(12); Rha-Rha-C(10)-C(14:1); Rha-C(10)-C(12)/Rha-C(12)-C(10)) were isolated from cultures of P. aeruginosa 47T2 from waste frying oil and identified by HPLC-MS analysis. This article deals with the production, isolation, and chemical characterization of the rhamnolipid mixture RL(47T2). The physicochemical and biological properties of RL(47T2) as a new product were also studied. Its surface tension decreased to 32.8 mN/m; and the interfacial tension against kerosene to 1 mN/m. The critical micellar concentration for RL(47T2) was 108.8 mg/mL. The product showed excellent antimicrobial properties. Antimicrobial activity was evaluated according to the minimum inhibitory concentration (MIC), the lowest concentration of an antimicrobial agent that inhibits development of visible microbial growth. Low MIC values were found for bacteria Serratia marcescens (4 microg/mL), Enterobacter aerogenes (8 microg/mL), Klebsiella pneumoniae (0.5 microg/mL), Staphylococcus aureus and Staphylococcus epidermidis (32 microg/mL), Bacillus subtilis (16 microg/mL), and phytopathogenic fungal species: Chaetonium globosum (64 microg/mL), Penicillium funiculosum (16 microg/mL), Gliocadium virens (32 microg/mL) and Fusarium solani (75 microg/mL).     

Purification and characterization of an antibacterial protein in the skin secretion of rockfish Sebastes schlegeli

An antibacterial protein in the skin secretion of rockfish (Sebastes schlegeli) was purified by lectin affinity chromatography on Con A-Sepharose and gel filtration on TSKgel G3000SW. The antibacterial protein featured the high molecular mass and selective action against Gram-negative bacteria. The molecular mass of the protein was estimated to be approximately 150 kDa in gel filtration and approximately 75 kDa by SDS-PAGE, suggesting that it is dimeric. The antibacterial principle was an acidic glycoprotein with pI 4.5, 3.4% reducing sugar and 2.8% amino sugar. Its sugar chains had N-type (high mannose-type) oligosaccharide and sialic acid components. It inhibited strongly the growth of Aeromonas salmonicida, Photobacterium damselae and Shewanella putrefaciens with a minimum inhibitory concentration (MIC) of approximately 3 microg/ml, and moderately the growth of Vibrio parahaemolyticus and A. hydrophila with a MIC of 12.5 microg/ml and 25 microg/ml, respectively. The values of the minimum bactericidal concentration were almost equivalent to those of MIC. The potent sensitivity against virulent pathogens such as A. hydrophila, A. salmonicida and P. damselae may contribute considerably to the innate host defense mechanism to combat microbes on the mucosal surfaces of the rockfish.     

In vitro efficacy of ciprofloxacin alone and in combination with amoxycillin against Salmonella typhi isolates

Combined effect of ciprofloxacin (Ci) and amoxycillin (Ax) has been studied in vitro against 12 clinical isolates of S. typhi that showed Ci minimum inhibitory concentration (MIC) of > or =1 microg/ml. By agar dilution method, MIC values of Ax were 10-16 microg/ml for 11 isolates and 0.5 microg/ml for the remaining one isolate. The isolates, when treated with Ci and Ax in combination, showed fractional inhibitory concentration (FIC) of 0.004-0.256 microg/ml for Ci. FIC of Ax ranged from 6-10 microg/ml, except for a single isolate that showed Ax FIC of 0.25 microg/ml. Thus Ci was more efficacious in combination with Ax against S. typhi than Ci alone. The antibiotic combination exhibited an additive effect for all the isolates showing FIC index 0.504-0.832.