Produção e aplicação de ramnolipídios produzidos por Pseudomonas aeruginosa LBI 2A1

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Avaliação do efeito do tratamento com surfactante químico e Pseudomonas aeruginosa LBI na toxicidade de solo contaminado com óleo lubrificante usado

Petroleum and its subproducts are considered a treat for the environmental quality because of the many environmental accidents that may occur during exploitation, transport and storage. A common remediation technique used in the contaminated areas is based on the use of surfactants, mainly the chemical ones, because they have low production costs. In the other hand, some microorganisms have indicate capacities of producing surfactants that emulsify substances and as result, offer a bigger contact surface for the microbiota degradation. This biossurfactants stand out in comparison with the chemical surfactants because they present lower micelar concentration values, are more tolerant for temperature and pH variation, because they are biodegradable, have low toxicity, higher emulsification and hydrocarbon solubilization index. In this way, after the surfactant application, a toxicity evaluation have to be made to identify the treatment effects. In soil, the activity of some microbial enzymes can show the environmental behavior of the contaminant under different treatment conditions. Dehydrogenase is one example of those enzymes that can demonstrate indirectly the effect of the pollutant on the soil microorganisms. The aim of this paper was to evaluate the toxicity after the addition of a surfactant and/or Pseudomonas aeruginosa LBI in soil contaminated by a mineral automotive lubricant. The previous mentioned bacteria are a potential biossurfactant (rhamnolipid) producer. In order to evaluate the toxicity, the dehydrogenase test was run. In this test, trifeniltetrazolium compound (TTC) after utilized as an electron acceptor, turns into trifenil formazan (TPF), that can be indirectly quantified using the absorbance measured by the spectrophotometer UV-visible. In this way, it was possible to quantify the dehydrogenase activity from the contaminated soil samples... (Complete abstract click electronic access below)

Caracterização de Bacteriófagos Infectando Isolados Clínicos de Pseudomonas aeruginosa Armazenados em uma Coleção de Cultura

To isolate, to concentrate and to purify bacteriophages from isolates of P. aeruginosa; To observe the capacity of bacteriophages to infect isolates of P. aeruginosa susceptible and multiresitant to antimicrobial; To caractherize bacteriphages by electronic microscopy techniques. 10 isolates of Pseudomonas aeruginosa from LEMC culture collection were submitted to the experiments of ideal temperature for the lyse region appearance in the MaConkey culture plate and 2 extraction methods for the concentration of the phages, clorophorm (Silankorva) and filtration plus centrifugation (Bergan). Three infected clinical isolates of multiresistant P. aeruginosa an one susceptible isolate ( PA01) were evaluated by 3 transmission electron microscopy techniques to caractherize phages morphologically (“on grid”, “on drop” and direct extraction from the lyse region of the culture plate). The ideal temperature to obtain lyses region was 37°C. The stock solutions, obtained through the methodologies of Sillankorva and Bergan, had satisfactory results in infecting the multiresistant isolate and the negative control. Among the 3 techniques of electronic microscopy tested the direct from the lyse plate was the best to obtain the micrography of the phages

Estudo dos micronutrientes na produção de ramnolipídios por mutante de Pseudomonas aeruginosa LBI

Os biossurfactantes apresentam inúmeras vantagens, como baixa toxicidade, biodegradabilidade e alta estabilidade, mas não são amplamente utilizados devido ao custo de produção. A utilização de substratos baratos, linhagens mutantes que associados à otimização das condições de cultivo pode levar a uma redução nos custos, possibilitando assim a substituição dos surfactantes sintéticos pelos biológicos. Uma maneira empregada para maximizar a produção dos biossurfactantes é a limitação de nutrientes. Os esforços empregados nesse sentido são direcionado para as proporções carbono: nitrogênio, entretanto os efeitos dos elementos traços são pouco conhecidos. Devido a esses fatores, o presente trabalho avaliou a importância dos seguintes elementos traços: ferro, zinco, cobalto, cobre, manganês e do sal citrato de sódio dihidratado, nas fermentações realizadas utilizando o mutante de Pseudomonas aeruginosa LBI 2A1. Para tanto foram realizadas fermentações em frascos Erlenmeyer, onde se utilizou diferentes concentrações desses elementos. A influência dos mesmos na produção de ramnolipídios foi constatada, uma vez que a produção desse biotensoativo foi aumentada em mais de três vezes alterando apenas a concentração de um único elemento traço (Fe). Os experimentos realizados permitem, também, inferir a respeito das melhores concentrações desses micronutrientes para a produção de ramnolipídios

Fotocatálise heterogênea de enterococcus faecalis e pseudomonas aeruginosa desencadeada pela ação da luz UV e branca em espátulas com revestimento de nanopartículas TiO2 e Ag

Nanotechnology, the science of minuscule, has developed products which are able t o manipulate atoms and molecules that could be applied in the sterilization process of dental instruments. Objetives: The objective of the present study was to evaluate the self-cleaning action of TiO2 and Ag nanoparticles coating on dental instruments by the photocataliys process under UV and visible light irradiation. Material and method: Microbiologic tests were done using dental cement spatulas coated with TiO2 and Ag nanoparticles (one or three layers), and contaminated with 10 mcrl of Pseudomonas aeruginosa and Enterococcus faecalis, respectively. After contamination, they were exposed to ultraviolet light and visible light for 120 minutes. Next, they were transferred to and stored in test tubes with BHI (Brain Heart Infusion) and incubated in 35 to 37 °C. Checking times for bacterial growth and for control and retrieval tests were done at: 24, 48, 72 and 96 hours. Result: The Pseudomonas aeruginosa was inactive after 120 minutes of ultraviolet light irradiation, thus confirming the heterogeneous photocatalytic activity of TiO2 and Ag. The Pseudomonas aeruginosa was not inactivated under visible light irradiation and the Enterococcus faecalis was not inactivated under UV and visible light irradiation of the dental cement spatulas coated with TiO2 and Ag nanoparticles in the readings to 96 hours, showing bacterial growth. Conclusion: There were no influence of one or three layers of TiO2 and Ag nanoparticles coating of the spatulas in the results. The heterogeneous photocatalysis activity of TiO2 and Ag under UV light irradiation was confirmed for Pseudomonas aeruginosa but not under visible light. Enterococcus faecalis did not confirmed the photocatalytics activity of TiO2 and Ag under UV light irradiation and visible lights irradiation.

An impedimetric biosensor to test neat serum for dengue diagnosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

beta-Lactamase-based biosensor for the electrochemical determination of benzylpenicillin in milk

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sub-MICs of Mentha piperita essential oil and menthol inhibits AHL mediated quorum sensing and biofilm of Gram-negative bacteria

Bacterial quorum sensing (QS) is a density dependent communication system that regulates the expression of certain genes including production of virulence factors in many pathogens. Bioactive plant extract/compounds inhibiting QS regulated gene expression may be a potential candidate as antipathogenic drug. In this study anti-QS activity of peppermint (Menthe piperita) oil was first tested using the Chromobacterium violaceum CVO26 biosensor. Further, the findings of the present investigation revealed that peppermint oil (PMO) at sub-Minimum Inhibitory Concentrations (sub-MICs) strongly interfered with acyl homoserine lactone (AHL) regulated virulence factors and biofilm formation in Pseudomonas aeruginosa and Aeromonas hydrophila. The result of molecular docking analysis attributed the QS inhibitory activity exhibited by PMO to menthol. Assessment of ability of menthol to interfere with QS systems of various Gram-negative pathogens comprising diverse AHL molecules revealed that it reduced the AHL dependent production of violacein, virulence factors, and biofilm formation indicating broad-spectrum anti-QS activity. Using two Escherichia colt biosensors, MG4/pKDT17 and pEAL08-2, we also confirmed that menthol inhibited both the las and pqs QS systems. Further, findings of the in vivo studies with menthol on nematode model Caenorhabditis elegans showed significantly enhanced survival of the nematode. Our data identified menthol as a novel broad spectrum QS inhibitor.

Determination of salicylate in blood serum using an amperometric biosensor based on salicylate hydroxylase immobilized in a polypyrrole-glutaraldehyde matrix

The use of an amperometric biosensor for the salicylate determination in blood serum is described. The biosensor is based on salicylate hydroxylase (EC 1.14.13.1) electropolymerized onto a glassy carbon-working electrode with polypyrrole and glutaraldehyde, to improve the biosensor lifetime. The hexacyanoferrate (II) was also incorporated to work as a redox mediator to minimize possible interferences. The salicylate is enzymatically converted to catechol, which is monitored amperometrically by its electrooxidation at +0.170 V versus SCE (saturated calomel electrode). Salicylate determination was carried out maintaining the ratio between β-NADH and salicylate at 4:1 (30°C). The amperometric response of the biosensor was linearly proportional to the salicylate concentration between 2.3 x 10-6 and 1.4 x 10-5 mol l- 1, in 0.1 mol l-1 phosphate buffer (pH 7.8), containing 0.1 mol l-1 KCl and 5.0 x 10-4 mol l-1 Na2H2EDTA, as supporting electrolyte. The recovery studies, in the presence of several interfering compounds, showed recoveries between 96.4 and 104.8%. The useful lifetime of the biosensor in the concentration range evaluated was at least 40 days, in continuous use. Blood serum samples analyzed by this biosensor showed a good correlation compared to the spectrophotometric method (Trinder) used as reference, presenting relative deviations lower than 7.0%. (C) 2000 Elsevier Science B.V.

A Low Power CMOS Voltage Regulator for a Wireless Blood Pressure Biosensor

This paper describes a CMOS implementation of a linear voltage regulator (LVR) used to power up implanted physiological signal systems, as it is the case of a wireless blood pressure biosensor. The topology is based on a classical structure of a linear low-dropout regulator. The circuit is powered up from an RF link, thus characterizing a passive radio frequency identification (RFID) tag. The LVR was designed to meet important features such as low power consumption and small silicon area, without the need for any external discrete components. The low power operation represents an essential condition to avoid a high-energy RF link, thus minimizing the transmitted power and therefore minimizing the thermal effects on the patient's tissues. The project was implemented in a 0.35-mu m CMOS process, and the prototypes were tested to validate the overall performance. The LVR output is regulated at 1 V and supplies a maximum load current of 0.5 mA at 37 degrees C. The load regulation is 13 mV/mA, and the line regulation is 39 mV/V. The LVR total power consumption is 1.2 mW.

Improving the performance of a glucose biosensor using an ionic liquid for enzyme immobilization. On the chemical interaction between the biomolecule, the ionic liquid and the cross-linking agent

The effect of the room temperature ionic liquid (1-butyl-2,3-dimethylimidazolium tetrafluoroborate ([BMMI][BF4])) on the immobilization of glucose oxidase (GOx) was studied. The electrochemical performance of biosensors prepared following different protocols indicated a beneficial effect of the ionic liquid on the analytical parameters. The chemical interaction between GOx, [BMMI][BF4] and glutaraldehyde was investigated using UV-visible spectroscopy (UV-vis) and circular dichroism (CD). Structural changes of the biomolecule were observed to depend on the method used for the immobilization. (C) 2011 Elsevier Ltd. All rights reserved.

Transesterification of Palm Oil Catalyzed by Pseudomonas fluorescens Lipase in a Packed-Bed Reactor

Transesterification of palm oil with ethanol catalyzed by Pseudomonas fluorescens lipase immobilized on epoxy-polysiloxane-polyvinyl alcohol composite (epoxy-SiO2-PVA) was performed in a continuous packed-bed reactor (PBR). Two strategies were used for improving the miscibility of the substrates: the addition of the organic solvent tert-butanol and the surfactant Triton X-100. Results were compared to those obtained in a solventless reactor, which displayed a biphasic system that passed through the reactor. Using this system, the ethyl ester yield of 61.6 +/- 1.2% was obtained at steady state. Both Triton X-100 and tert-butanol systems were found to be suitable to promote the miscibility of the starting materials; however, the use of Triton X-100 reduced the yield to levels lower than 20%, because of the enzyme desorption from the support surface, as confirmed by scanning electron microscopy analysis. The best performance was found for the reactor running in the presence of tert-butanol which resulted in a stable operating system and an average yield of 87.6 +/- 2.5%. This strategy also gave high biocatalyst operational stability, revealing a half-life of 48 days and an inactivation constant of 0.6 X 10(-3) h(-1).

Electrochemical detection of carbamate pesticides in fruit and vegetables with a biosensor based on acetylcholinesterase immobilised on a composite of polyaniline-carbon nanotubes

A sensitive electrochemical acetylcholinesterase (AChE) biosensor was successfully developed on polyaniline (PANI) and multi-walled carbon nanotubes (MWCNTs) core-shell modified glassy carbon electrode (GC), and used to detect carbamate pesticides in fruit and vegetables (apple, broccoli and cabbage). The pesticide biosensors were applied in the detection of carbaryl and methomyl pesticides in food samples using chronoamperometry (CA). The GC/MWCNT/PANI/AChE biosensor exhibited detection limits of 1.4 and 0.95 mu mol L-1, respectively, for carbaryl and methomyl. These detection limits were below the allowable concentrations set by Brazilian regulation standards for the samples in which these pesticides were analysed. Reproducibility and repeatability values of 2.6% and 3.2%, respectively, were obtained in the conventional procedure. The proposed biosensor was successfully applied in the determination of carbamate pesticides in cabbage, broccoli and apple samples without any spiking procedure. The obtained results were in full agreement with those from the HPLC procedure. (C) 2012 Elsevier Ltd. All rights reserved.

Identification of bacteria in drinking and purified water during the monitoring of a typical water purification system

Abstract Background A typical purification system that provides purified water which meets ionic and organic chemical standards, must be protected from microbial proliferation to minimize cross-contamination for use in cleaning and preparations in pharmaceutical industries and in health environments. Methodology Samples of water were taken directly from the public distribution water tank at twelve different stages of a typical purification system were analyzed for the identification of isolated bacteria. Two miniature kits were used: (i) identification system (api 20 NE, Bio-Mérieux) for non-enteric and non-fermenting gram-negative rods; and (ii) identification system (BBL crystal, Becton and Dickson) for enteric and non-fermenting gram-negative rods. The efficiency of the chemical sanitizers used in the stages of the system, over the isolated and identified bacteria in the sampling water, was evaluated by the minimum inhibitory concentration (MIC) method. Results The 78 isolated colonies were identified as the following bacteria genera: Pseudomonas, Flavobacterium and Acinetobacter. According to the miniature kits used in the identification, there was a prevalence of isolation of P. aeruginosa 32.05%, P. picketti (Ralstonia picketti) 23.08%, P. vesiculares 12.82%,P. diminuta 11.54%, F. aureum 6.42%, P. fluorescens 5.13%, A. lwoffi 2.56%, P. putida 2.56%, P. alcaligenes 1.28%, P. paucimobilis 1.28%, and F. multivorum 1.28%. Conclusions We found that research was required for the identification of gram-negative non-fermenting bacteria, which were isolated from drinking water and water purification systems, since Pseudomonas genera represents opportunistic pathogens which disperse and adhere easily to surfaces, forming a biofilm which interferes with the cleaning and disinfection procedures in hospital and industrial environments.

Quantitative detection of Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa in human oral epithelial cells from subjects with periodontitis and periodontal health

Epithelial cells in oral cavities can be considered reservoirs for a variety of bacterial species. A polymicrobial intracellular flora associated with periodontal disease has been demonstrated in buccal cells. Important aetiological agents of systemic and nosocomial infections have been detected in the microbiota of subgingival biofilm, especially in individuals with periodontal disease. However, non-oral pathogens internalized in oral epithelial cells and their relationship with periodontal status are poorly understood. The purpose of this study was to detect opportunistic species within buccal and gingival crevice epithelial cells collected from subjects with periodontitis or individuals with good periodontal health, and to associate their prevalence with periodontal clinical status. Quantitative detection of total bacteria and Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis in oral epithelial cells was determined by quantitative real-time PCR using universal and species-specific primer sets. Intracellular bacteria were visualized by confocal microscopy and fluorescence in situ hybridization. Overall, 33 % of cell samples from patients with periodontitis contained at least one opportunistic species, compared with 15 % of samples from healthy individuals. E. faecalis was the most prevalent species found in oral epithelial cells (detected in 20.6 % of patients with periodontitis, P = 0.03 versus healthy individuals) and was detected only in cells from patients with periodontitis. Quantitative real-time PCR showed that high levels of P. aeruginosa and S. aureus were present in both the periodontitis and healthy groups. However, the proportion of these species was significantly higher in epithelial cells of subjects with periodontitis compared with healthy individuals (P = 0.016 for P. aeruginosa and P = 0.047 for S. aureus). Although E. faecalis and P. aeruginosa were detected in 57 % and 50 % of patients, respectively, with probing depth and clinical attachment level ≥6 mm, no correlation was found with age, sex, bleeding on probing or the presence of supragingival biofilm. The prevalence of these pathogens in epithelial cells is correlated with the state of periodontal disease.