Development of a biosensor for urea assay based on amidase inhibition, using an ion-selective electrode

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; anion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensorresponse and showed that 30 mu L of cell-free extract containing 7.47 mg protein mL(-1), 2 mu L of glutaraldehyde (5%, v/v) and 10 mu L of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation ofmembranes in urea. The biosensor exhibited a linear response in the range of 4.0-10.0 mu M urea, a detection limit of 2.0 mu M for urea, a response timeof 20 s, a sensitivity of 58.245 % per mu M urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition.

A new cloning system based on the OprI lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulations

The conjugation of antigens with ligands of pattern recognition receptors (PRR) is emerging as a promising strategy for the modulation of specific immunity. Here, we describe a new Escherichia coli system for the cloning and expression of heterologous antigens in fusion with the OprI lipoprotein, a TLR ligand from the Pseudomonas aeruginosa outer membrane (OM). Analysis of the OprI expressed by this system reveals a triacylated lipid moiety mainly composed by palmitic acid residues. By offering a tight regulation of expression and allowing for antigen purification by metal affinity chromatography, the new system circumvents the major drawbacks of former versions. In addition, the anchoring of OprI to the OM of the host cell is further explored for the production of novel recombinant bacterial cell wall-derived formulations (OM fragments and OM vesicles) with distinct potential for PRR activation. As an example, the African swine fever virus ORF A104R was cloned and the recombinant antigen was obtained in the three formulations. Overall, our results validate a new system suitable for the production of immunogenic formulations that can be used for the development of experimental vaccines and for studies on the modulation of acquired immunity.

Post-surgical wound infections involving Enterobacteriaceae with reduced susceptibility to ß-lactams in two Portuguese hospitals

The post-surgical period is often critical for infection acquisition. The combination of patient injury and environmental exposure through breached skin add risk to pre-existing conditions such as drug or depressed immunity. Several factors such as the period of hospital staying after surgery, base disease, age, immune system condition, hygiene policies, careless prophylactic drug administration and physical conditions of the healthcare centre may contribute to the acquisition of a nosocomial infection. A purulent wound can become complicated whenever antimicrobial therapy becomes compromised. In this pilot study, we analysed Enterobacteriaceae strains, the most significant gram-negative rods that may occur in post-surgical skin and soft tissue infections (SSTI) presenting reduced β-lactam susceptibility and those presenting extended-spectrum β-lactamases (ESBL). There is little information in our country regarding the relationship between β-lactam susceptibility, ESBL and development of resistant strains of microorganisms in SSTI. Our main results indicate Escherichia coli and Klebsiella spp. are among the most frequent enterobacteria (46% and 30% respectively) with ESBL production in 72% of Enterobacteriaceae isolates from SSTI. Moreover, coinfection occurred extensively, mainly with Pseudomonas aeruginosa and Methicillin-resistant Staphylococcus aureus (18% and 13%, respectively). These results suggest future research to explore if and how these associations are involved in the development of antibiotic resistance.

Real-time rheology of actively growing bacteria

The population growth of a Staphylococcus aureus culture, an active colloidal system of spherical cells, was followed by rheological measurements, under steady-state and oscillatory shear flows. We observed a rich viscoelastic behavior as a consequence of the bacteria activity, namely, of their multiplication and density-dependent aggregation properties. In the early stages of growth (lag and exponential phases), the viscosity increases by about a factor of 20, presenting several drops and full recoveries. This allows us to evoke the existence of a percolation phenomenon. Remarkably, as the bacteria reach their late phase of development, in which the population stabilizes, the viscosity returns close to its initial value. Most probably, this is caused by a change in the bacteria physiological activity and in particular, by the decrease of their adhesion properties. The viscous and elastic moduli exhibit power-law behaviors compatible with the "soft glassy materials" model, whose exponents are dependent on the bacteria growth stage. DOI: 10.1103/PhysRevE.87.030701.

Biofiltration of Air/Styrene and Air/Styrene/Acetone mixtures in a bubble column reactor

The goal of this work was the treatment of polluted waste gases in a bubble column reactor (BCR), in order to determinate the maximum value of reactor’s efficiency (RE), varying the inlet concentration (C in) of the pollutants. The gaseous mixtures studied were: (i) air with styrene and (ii) air with styrene and acetone. The liquid phase used to contain the biomass in the reactor was a basal salt medium (BSM), fundamental for the microorganisms’ development. The reactor used in this project consists of a glass column of 620mm height and inside diameter 75mm. In all essays there were continually measured: pH, dissolved oxygen and liquid’s temperature. Temperature and pH were controlled (T=24ºC, 7.0 ≤ pH ≤ 7.7). In all experiments the liquid volume (including the biomass) used in the reactor was kept constant (1.5L) as well as the total gas flowrate (1 L/min). Concerning the goal of the work, some parameters were calculated: the organic load (OL), removal efficiency (RE), elimination capacity (EC), biomass concentration (xf) and dry biomass concentration (Xdw). In a first series of experiments, the gas mixture used was air with styrene, varying its concentration from 191 mg.m-3 to 6500 mg.m-3.It was concluded that the RE maximum value (97%) was obtained for C in Sty = 4200 mg.m-3. For the maximum tested value of C in Sty, RE obtained was 20%. In a second step, the gaseous mixture included acetone, varying C in Sty between 225 mg.m-3 and 2659 mg.m-3 and C in Ac between 153mg.m-3 and 1389 mg.m-3. The aim of these tests was the determination of C in Ac for which RE was maximum, obtaining C in Ac = 750 mg.m-3. A third series of experiments was performed, in which C in Ac was maintained equal to that value and C in Sty was varied until higher values (5422 mg.m-3). RE maximum values obtained in this last series were 100% for styrene and 40% for acetone. One important conclusion is the fact that the microorganisms available degrade better styrene than acetone. On the ambit of this study, it was possible to identify the species available in biomass: Xanthobacter antotrophicus py2, Enterobacter aerogenes, Nocardia, Corynebacterium Spp., Rhodococcus rhodochrous e Pseudomonas Sp.

Biodegradação enzimática de aminas aromáticas tóxicas

Trabalho Final de Mestrado para obtenção do grau de Mestre em Engenharia Química e Biológica

Transport mechanism in ionic liquid membranes and the study of thiomersal biodegradation

The initial goal of this work was the development of a supported liquid membrane (SLM) bioreactor for the remediation of vaccine production effluents contaminated with a highly toxic organomercurial – thiomersal. Therefore, two main aspects were focused on: 1) the development of a stable supported liquid membrane – using room temperature ionic liquids (RTILs) – for the selective transport of thiomersal from the wastewater to a biological compartment, 2) study of the biodegradation kinetics of thiomersal to metallic mercury by a Pseudomonas putida strain. The first part of the work focused on the evaluation of the physicochemical properties of ionic liquids and on the SLMs’ operational stability. The results obtained showed that, although it is possible to obtain a SLM with a high stability, water possesses nonnegligible solubility in the RTILs studied. The formation of water clusters inside the hydrophobic ionic liquid was identified and found to regulate the transport of water and small ions. In practical terms, this meant that, although it was possible to transport thiomersal from the vaccine effluent to the biological compartment, complete isolation of the microbial culture could not be guaranteed and the membrane might ultimately be permeable to other species present in the aqueous vaccine wastewater. It was therefore decided not to operate the initially targeted integrated system but, instead, the biological system by itself. Additionally, attention was given to the development of a thorough understanding of the transport mechanisms involved in the solubilisation and transport of water through supported liquid membranes with RTILs as well as to the evaluation of the effect of water uptake by the SLM in the transport mechanisms of water-soluble solutes and its effect on SLM performance. The results obtained highlighted the determinant role played by water – solubilised inside the ionic liquids – on the transport mechanism. It became clear that the transport mechanism of water and water-soluble solutes through SLMs with [CnMIM][PF6] RTILs was regulated by the dynamics of water clusters inside the RTIL, rather than by molecular diffusion through the bulk of the ionic liquid. Although the stability tests vi performed showed that there were no significant losses of organic phase from the membrane pores, the formation of water clusters inside the ionic liquid, which constitute new, non-selective environments for solute transport, leads to a clear deterioration of SLM performance and selectivity. Nevertheless, electrical impedance spectroscopy characterisation of the SLMs showed that the formation of water clusters did not seem to have a detrimental effect on the SLMs’ electrical characteristics and highlighted the potential of using this type of membranes in electrochemical applications with low resistance requirements. The second part of the work studied the kinetics of thiomersal degradation by a pure culture of P. putida spi3 strain, in batch culture and using a synthe tic wastewater. A continuous ly stirred tank reactor fed with the synthetic wastewater was also operated and the bioreactor’s performance and robustness, when exposed to thiomersal shock loads, were evaluated. Finally, a bioreactor for the biological treatment of a real va ccine production effluent was set up and operated at different dilution rates. Thus it was possible to treat a real thiomersal-contaminated effluent, lowering the outlet mercury concentration to values below the European limit for mercury effluent discharges.

Electrochemical studies on small electron transfer proteins using membrane electrodes

Journal of Electroanalytical Chemistry 541 (2003) 153-162

Estudo dos principais mecanismos de resistência aos antibióticos β-lactâmicos em bactérias patogénicas de gram negativo

Dissertação apresentada para a obtenção do Grau de Mestre em Genética Molecular e Biomedicina, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Production, purification and characterization of laccase from pleurottus ostreatus grown on tomato pomace

A strain of Pleurotus ostreatus was grown in tomato pomace as sole carbon source for production of laccase. The culture of P. ostreatus revealed a peak of laccase activity (147 U/L of fermentation broth) on the 4th day of culture with a specific activity of 2.8 U/mg protein. Differential chromatographic behaviour of laccase was investigated on affinity chromatographic matrices containing either urea, acetamide, ethanolamine or IDA as affinity ligands. Laccase exhibited retention on such affinity matrices and it was purified on a Sepharose 6B-BDGE-urea column with final enzyme recoveries of about 60%, specific activity of 6.0 and 18.0 U/mg protein and purification factors in the range of 14-46. It was also possible to demonstrate that metal-free laccase did not adsorb to Sepharose 6B-BDGE-urea column which suggests that adsorption of native laccase on this affinity matrix was apparently due to the specific interaction of carbonyl groups available on the matrix with the active site Cu (II) ions of laccase. The kinetic parameters (V (max), K (m) , K (cat), and K (cat)/K (m) ) of the purified enzyme for several substrates were determined as well as laccase stability and optimum pH and temperature of enzyme activity. This is the first report describing the production of laccase from P. ostreatus grown on tomato pomace and purification of this enzyme based on affinity matrix containing urea as affinity ligand.

Estudo da suscetibilidade "in vitro" a um novo antimicrobiano (IMIPENEM) de patógenos isolados de pacientes hospitalares em vários centros

O imipenem é um novo antibiótico Beta lactâmico, carbapenêmico, altamente potente e com amplo espectro de atividade antimicrobiana. Com intuito de comprovar a eficácia "in vitro" deste fármaco em patógenos mais freqüentes em nosso meio, descrevem os autores, os resultados das provas de suscetibilidade por discos e/ou a correspondência por provas de diluição para determinação da concentração inibitória mínima (CIM) em 1230 cepas compreendendo 41 diferentes espécies bacterianas recém-isoladas, principalmente de pacientes hospitalares em 5 diferentes centros médicos de Sáo Paulo, Rio de Janeiro e Salvador. Nossos resultados preliminares com o antibiótico, em fase final de experimentação clínica e laboratorial, em nosso meio, foram muito promissores, com 96.79% de cepas suscetíveis pela prova do disco (10 μg de imipenem) e 92,31% de correspondência pela determinação do CIM (concentrações de até 4μg/ml). Das 9 espécies bacterianas mais freqüentemente isoladas, correspondendo a 1008 (82%) das 1230 cepas de nosso material, as sensibilidades pela prova do disco foram de 99% (E. coli), 93% (Pseudomonas aeruginosas), 87% (Staphylococcus aureus), 100% (Klebsiella pneumoniae), 98% (Klebsiella sp) e 100% (Streptococcus faecalis) com boa correspondência pela determinação do CIM até 8μg/ml; e 100% para o anaeróbio Bacteróides sp (CIM até 4μg/ml). Ressaltam os autores a eficácia "in vitro" contra patógenos hospitalares que apresentam elevados índices de resistência à grande maioria de antibióticos como o Pseudomonas aeruginosa e para anaeróbios, notadamente o Bacteróides sp.

Bacterial agents isolated from cerebrospinal fluid of patients with Acquired Immunodeficiency Syndrome (AIDS) and neurological complications

Cerebrospinal fluid (CSF) samples from 2083 patients with acquired immunodeficiency syndrome (AIDS) and neurological complications were bacteriologically examined during a period of 7 years (1984-1990). The percentage of patients who had at least one bacterial agent cultured from the CSF was 6.2%. Mycobacterium tuberculosis was the most frequently isolated agent (4.3%), followed by Mycobacterium avium complex or MAC (0.7%), Pseudomonas spp (0.5%), Enterobacter spp (0.4%), and Staphylococcus aureus (0.3%). Among 130 culture positive patients, 89 (68.5%) had M. tuberculosis and 15 (11.6%) had MAC. The frequency of bacterial isolations increased from 1988 (5.2%) to 1990 (7.2%), partly due to the increase in MAC isolations. Bacterial agents were more frequently isolated from patients in the age group 21-30 years and from women (p<0.05).

Implementação do método de suspensão quantitativa para avaliar a actividade bactericida de desinfectantes químicos, de acordo com a Norma Europeia EN 1040

As bactérias patogénicas presentes nos sistemas de processamento de alimentos podem ser potencialmente causadoras de doenças alimentares graves, quando sobrevivem à acção bactericida de desinfectantes. Esta acção depende da eficácia das substâncias activas que fazem parte da formulação do desinfectante e das condições ambientais onde se processa a interacção desinfectante – bactéria, sendo ainda específica para cada estirpe bacteriana. Este trabalho teve, como propósito, a implementação de um método que permita avaliar, in vitro, a eficácia bactericida de desinfectantes comerciais contra estirpes bacterianas que são potencialmente causadores de doenças de origem alimentar. A eficácia do desinfectante foi avaliada seguindo a Norma EN1040 e aplicando o método de diluição-neutralização. Para além das estirpes recomendadas naquele documento normativo, nomeadamente, Pseudomonas aeruginosa e Staphylococcus aureus, foram também utilizadas Escherichia coli, Enterococcus faecalis, Salmonella Typhimurium e Listeria monocytogenes. Os desinfectantes foram seleccionados com base nas suas formulações, tendo sido escolhidos desinfectantes cujos princípios activos são, respectivamente: tensioactivos aniónicos; alcalis inorgânicos; hidrocloreto de biguanida polimérica; álcool etoxilado ou cloreto benzalcónio. Foram seleccionados como neutralizantes: tampão fosfato, tiosulfato de sódio, gema de ovo fresca e polisorbato 80 com histidina. Após cada ensaio, o número de bactérias sobreviventes foi obtido pelo cálculo da redução logarítmica decimal, a partir do número de unidades formadoras de colónia presentes em cada placa. A eficácia do desinfectante, na inactivação de cada estirpe alvo, foi classificada numa escala de redução logarítmica à qual 5 log corresponde a redução mínima aceitável do número de colónias quando o tempo de contacto é de 5 minutos, e a temperatura do ensaio é 20ºC. Conclui-se que o método de diluição-neutralização é sensível para todas as estirpes testadas. A gema de ovo fresca revelou ser o melhor neutralizante para desinfectantes contendo tensioactivos aniónicos, enquanto o tampão fosfato é mais adequado para neutralizar desinfectantes contendo alcalis inorgânicos. Porém, nenhum dos neutralizantes testados mostrou ser adequado para os desinfectantes contendo cloreto de benzalcónio ou hidrocloreto biguanida polimérica ou álcool etoxilado. Conclui-se também que as condições laboratoriais implementadas são as adequadas para testar a acção bactericida de desinfectantes utilizados em sistemas de processamento de alimentos.

Cutaneous botryomycosis. Report of the two first Uruguayan cases: relato dos dois primeiros casos no Uruguai

The first two Uruguayan cases of botryomycosis were diagnosed in white adult male patients with cutaneous lesions. Staphylococcus aureus was the etiologic agent in both cases. In one of them, however, Pseudomonas aeruginosa was also identified during relapse. General comments on diagnosis and pathogenesis of the disease are made.

Picocyanobacteria From a Clade Of Marine Cyanobium Revealed Bioactive Potential Against Microalgae, Bacteria, And Marine

The production of bioactive compounds either toxic or with pharmacological applications by cyanobacteria is well established. However, picoplanktonic forms within this group of organisms have rarely been studied in this context. In this study, the toxicological potential of picocyanobacteria from a clade of marine Cyanobium strains isolated from the Portuguese coast was examined using different biological models. First, strains were identified by applying morphological and molecular approaches and cultured under lab conditions. A crude extract and three fractions reflecting a preliminary segregation of lipophilic metabolites were tested for toxicity with the marine microalga Nannochloropsis sp., the bacteria Pseudomonas sp., the brine shrimp Artemia salina, and fertilized eggs of the sea urchin Paracentrotus lividus. No significant apparent adverse effects were noted against Artemia salina. However, significant adverse effects were found in all other assays, with an inhibition of Nannochloropsis sp. and Pseudomonas sp. growth and marked reduction in Paracentrotus lividus larvae length. The results obtained indicated that Cyanobium genus may serve as a potential source of interesting bioactive compounds and emphasize the importance of also studying smaller picoplanktonic fractions of marine cyanobacteria.