Ane Stefano Simionato

Guaraná (Paullinia cupana) seeds: Selective supercritical extraction of phenolic compounds.

Approximately 70% of the Brazilian production of guaraná (Paullinia cupana) seeds is absorbed by the beverage industries. Guaraná has several pharmacological properties: energy stimulant, antimicrobial, chemoprophylactic, antigenotoxic, antidepressive, anxiolytic, and anti-amnesic effects. Supercritical carbon dioxide extraction of bioactive compounds from guaraná seeds was carried out and optimized by an orthogonal array design (OA9(3(4))). The factors/levels studied were: modifier(s) (ethanol and/or methanol), extraction time (20, 40, and 60min), temperature (40, 50, and 60°C), and pressure (100, 200, and 300bar). The statistical design was repeated with increasing proportions of modifiers. The percentage of modifier used was proportional to the amount of polar compounds extracted. The best conditions for the supercritical extraction, based on the content of polyphenols, epicatechin/catechin quantification, yield and operating cost, proved to be: 40% ethanol:methanol during 40min, under 40°C, and 100bar. The temperature had a significant effect on the total phenolic content.

Bioactive Organocopper Compound from Pseudomonas aeruginosa Inhibits the Growth of Xanthomonas citri subsp. citri

Citrus canker is a very destructive disease of citrus species. The challenge is to find new compounds that show strong antibiotic activity and low toxicity to plants and the environment. The objectives of the present study were (1) to extract, purify and evaluate the secondary metabolites with antibiotic activity produced by Pseudomonas aeruginosa LV strain in vitro against Xanthomonas citri subsp. citri (strain 306), (2) to determine the potential of semi-purified secondary metabolites in foliar application to control citrus canker under greenhouse conditions, and (3) to identify antibiotic activity in orange leaf mesophyll infected with strain 306, by electron microscopy. Two pure bioactive compounds were isolated, an organocopper antibiotic compound (OAC) and phenazine-1-carboxamide. Phenazine-1-carboxamide did not show any antibiotic activity under the experimental conditions used in this study. The OAC showed a high level of antibiotic activity with a minimum inhibitory concentration of 0.12 μg mL-1. In greenhouse tests for control of citrus canker in orange trees, the semi-purified fraction F3d reduced lesion formation by about 97%. The concentration used was 500 times lower than that for the recommended commercial copper-based product. Electron microscopy showed that F3d altered the exopolysaccharide matrix and caused cell lysis of the pathogen inside the citrus canker lesions. These results suggest that secondary metabolites produced by inducing P. aeruginosa LV strain have a high potential to be used as a bioproduct to control citrus canker.

Effect of a Metalloantibiotic Produced by Pseudomonas aeruginosa on Klebsiella pneumoniae Carbapenemase (KPC)-producing K. pneumoniae.

Multidrug-resistant organisms (MDRO) are a great problem in hospitals, where thousands of people are infected daily, with the occurrence of high mortality rates, especially in infections caused by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-producing Kpn). The challenge is to find new compounds that can control KPC producing-Kpn infections. The aim of this study was to evaluate the antibiotic activity of the F3d fraction produced by the Pseudomonas aeruginosa LV strain against clinical isolates of KPC-producing Kpn. The results showed that the minimum inhibitory concentration of F3d (62.5 µg mL(-1)), containing an organic metallic compound, killed planktonic cells of KPC-producing Kpn strains after 30 min of incubation. At the same concentration, this fraction also showed an inhibitory effect against biofilm of these bacteria after 24 h of incubation. Treatment with the F3d fraction caused pronounced morphological alterations in both planktonic and biofilm cells of the bacteria. The inhibitory effect of the F3d fraction seems to be more selective for the bacteria than the host cells, indicating its potential in the development of new drugs for the treatment of infections caused by KPC-producing Kpn and other MDRO.

The Effect of Phenazine-1-Carboxylic Acid on Mycelial Growth of Botrytis cinerea Produced by Pseudomonas aeruginosa LV Strain

One of the most important postharvest plant pathogens that affect strawberries, grapes and tomatoes is Botrytis cinerea, known as gray mold. The fungus remains in latent form until spore germination conditions are good, making infection control difficult, causing great losses in the whole production chain. This study aimed to purify and identify phenazine-1-carboxylic acid (PCA) produced by the Pseudomonas aeruginosa LV strain and to determine its antifungal activity against B. cinerea. The compounds produced were extracted with dichloromethane and passed through a chromatographic process. The purity level of PCA was determined by reversed-phase high-performance liquid chromatography semi-preparative. The structure of PCA was confirmed by nuclear magnetic resonance and electrospray ionization mass spectrometry. Antifungal activity was determined by the dry paper disk and minimum inhibitory concentration (MIC) methods and identified by scanning electron microscopy and confocal microscopy. The results showed that PCA inhibited mycelial growth, where MIC was 25 μg mL-1. Microscopic analysis revealed a reduction in exopolysaccharide (EPS) formation, showing distorted and damaged hyphae of B. cinerea. The results suggested that PCA has a high potential in the control of B. cinerea and inhibition of EPS (important virulence factor). This natural compound is a potential alternative to postharvest control of gray mold disease.

Control of bacterial stem rot on tomato by extracellular bioactive compounds produced by Pseudomonas aeruginosa LV strain

Abstract This study evaluated the antibiotic activity and induction of resistance in plants by compounds produced by Pseudomonas aeruginosa LV strain on the control of bacterial stem rot in tomato. Compounds were extracted from the cell-free supernatant of a bacterial culture and purified. The F4A fraction was composed of two major compounds, an antibiotic and a phenazine (PCN). The first experiment evaluated the antibiotic activity of F4A, and the second one compared the ability of F4A and PCN to elicit systemic acquired resistance (SAR). In both experiments, plants were infected with Pectobacterium carotovorum subsp. carotovorum (Pcc). The minimum inhibitory concentration of F4A was 7.81 μg mL−1, and PCN did not inhibit bacterial growth. The results suggest that F4A has antibiotic activity. Scanning electron microscopy revealed changes in bacterial cells after 3 h treatment with F4A. F4A and PCN decreased symptoms of stem rot and increased fruit production. Plant response was estimated by determination of peroxidase, polyphenol oxidase, and phenylalanine ammonia lyase activity. Plants treated with PCN or F4A showed greater enzyme activity than plants that were not treated or treated with Bion®, suggesting that PCN increased SAR. The compounds showed the potential to control Pcc in vitro and in vivo and to induce plant response.

Low-molecular-weight metabolites produced by Pseudomonas aeruginosa as an alternative to control Huanglongbing in Citrus sinensis cv. Valencia

Huanglongbing (HLB) is the most destructive disease of citrus worldwide and an efficient management strategy to control it has not yet been established. The potential of pseudomonads to suppress plant pathogens is well known and the secondary metabolites they produce represent new alternatives of compounds to control plant diseases. The main challenge is to find new compounds that show strong antibiotic activity, low toxicity to plants and little or no harm to the environment. The objectives of the present study were to determine the potential of the F4A fraction from Pseudomonas aeruginosa to control HLB and to induce systemic resistance. Low molecular weight compounds with antimicrobial activity were purified with organic solvent, thin layer chromatography (TLC) and normal and reverse phase chromatography. Compounds present in the F4A fraction were mainly obtained by thin-layer chromatography (TLC) and Preparative-High Performance Liquid Chromatography (HPLC-prep). To assess their biological activities, conventional and quantitative polymerase chain reaction were usxed. The F4A was sprayed on citrus trees infected with the causal agent of HLB, ‘Candidatus Liberibacter asiaticus’ under greenhouse conditions. The bacterial titers were reduced and defense genes were induced in leaves of trees treated with F4A, as assed by PCR analysis. The results showed that F4A (pseudomonads secondary metabolites) may provide a useful tool for the management of HLB.

RNAm expression profile of cancer marker genes in HepG2 cells treated with different concentrations of a new indolin-3-one from Pseudomonas aeruginosa

The present study tested the effects of a newly identified indolin-3-one compound (compound 1), produced by Pseudomonas aeruginosa, on HepG2 cells. The MTT assays demonstrated decreased metabolic activities in HepG2 cells treated with compound 1, with dose- and time-dependent intensifying effect, starting at a concentration of 40 µM. The IC50 after 24, 48, 72, and 96 h treatments were 41.35, 52.7, 92.79 and 66.65 μM of compound 1, respectively. Below 80 µM, no significative damage on erythrocytes membranes was observed by the hemolytic assays. The RT-qPCR revealed that the compound modulated key genes involved in carcinogenesis process, indicating possible indolin-3-one mechanisms of action. The data showed that gene expression alterations promoted by compound 1, in concentrations up to 60 μM after 48 h, led to a decrease in cellular progression and there was no direct cellular damage. In addition, non-cytotoxic concentrations of compound 1 halved the concentration of the chemotherapeutic doxorubicin, maintaining similar therapeutic effect against HepG2 cells. The novelty of the molecule and the biological activities observed in the present study emphasize the potential of the compound 1 in cancer therapy research.

Disease-Induced Resistance and Plant Immunization Using Microbes

The induction of resistance in plants presents as an alternative to be explored in several species. This process involves the activation of defense mechanisms, which are inactive or latent in the plant and do not require alterations in your genome. This activation can be effected by biotic and abiotic agents known as resistance inducers. The use of resistance inducers leads to activation of the systemic resistance, which leads to a marked reduction in symptoms of the disease after subsequent infections, including different species of pathogens. This chapter gathers information about diverse compounds of biological origin that can act as resistance inductors, as well as an interaction between plants and rhizosphere microorganisms that may result in the activation of this resistance system against pathogens.

Strategies for Biological Control and Antagonisms

Microorganisms play an important niche in the control of soil populations producing a variety of bioactive compounds as an ecological strategy for competition for space and nutrients. Thus, the bioprospecting of microorganisms as potential antagonists for pathogen biocontrol, or obtaining their bioactive metabolites, is one of the alternatives currently studied for the control of diseases, especially in species of agronomic importance. In this chapter, we reviewed several microorganisms and how, in general, the products of their metabolism are obtained to be used in the control of pathogens.

New Insights about Antibiotic Production by Pseudomonas aeruginosa: A Gene Expression Analysis

The bacterial resistance for antibiotics is one of the most important problems in public health and only a small number of new products are in development. Antagonistic microorganisms from soil are a promising source of new candidate molecules. Products of secondary metabolism confer adaptive advantages for their producer, in the competition for nutrients in the microbial community. The biosynthesis process of compounds with antibiotic activity is the key to optimize their production and the transcriptomic study of microorganisms is of great benefit for the discovery of these metabolic pathways. Pseudomonas aeruginosa LV strain growing in the presence of copper chloride produces a bioactive organometallic compound, which has a potent antimicrobial activity against various microorganisms. The objective of this study was to verify overexpressed genes and evaluate their relation to the organometallic biosynthesis in this microorganism. P. aeruginosa LV strain was cultured in presence and absence of copper chloride. Two methods were used for transcriptomic analysis, genome reference-guided assembly and de novo assembly. The genome referenced analysis identified nine upregulated genes when bacteria were exposed to copper chloride, while the De Novo Assembly identified 12 upregulated genes. Nineteen genes can be related to an increased microbial metabolism for the extrusion process of exceeding intracellular copper. Two important genes are related to the biosynthesis of phenazine and tetrapyrroles compounds, which can be involved in the bioremediation of intracellular copper and we suggesting that may involve in the biosynthesis of the organometallic compound. Additional studies are being carried out to further prove the function of the described genes and relate them to the biosynthetic pathway of the organometallic compound.