Chromatographic behaviour of monoclonal antibodies against wild-type amidase from Pseudomonasaeruginosa on immobilized metal chelates

The aim of this work was to devise a one-step purification procedure for monoclonal antibodies (MAbs) of IgG class by immobilized metal affinity chromatography (IMAC). Therefore, several stationary phases were prepared containing immobilized metal chelates in order to study the chromatographic behaviour of MAbs against wild-type amidase from Pseudomonas aeruginosa. Such MAbs adsorbed to Cu(II), Ni(II), Zn(II) and Co(II)-IDA agarose columns. The increase in ligand concentration and the use of longer spacer arms and higher pH values resulted in higher adsorption of MAbs into immobilized metal chelates. The dynamic binding capacity and the maximum binding capacity were 1.33 +/- 0.015 and 3.214 +/- 0.021 mg IgG/mL of sedimented commercial matrix, respectively. A K(D) of 4.53 x 10(-7) M was obtained from batch isotherm measurements. The combination of tailor-made stationary phases of IMAC and the correct selection of adsorption conditions permitted a one-step purification procedure to be devised for MAbs of IgG class. Culture supernatants containing MAbs were purified by IMAC on commercial-Zn(II) and EPI-30-IDA-Zn(II) Sepharose 6B columns and by affinity chromatography on Protein A-Sepharose CL-4B. This MAb preparation revealed on SDS-PAGE two protein bands with M(r) of 50 and 22 kDa corresponding to the heavy and light chains, respectively. Copyright (C) 2011 John Wiley & Sons, Ltd.

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.

Comunidade fitoplanctônica de um pesqueiro na cidade de São Paulo

OBJETIVO: A avaliação da qualidade da água e da comunidade fitoplanctônica em ambientes destinados à recreação permite estabelecer formas de manejo desses sistemas, evitando possíveis problemas à saúde humana. Assim, realizou-se estudo com objetivo de analisar a variação sazonal do fitoplâncton de um sistema lacustre natural, e sua relação com a qualidade da água. MÉTODOS: O lago estudado faz parte de um pesqueiro localizado na zona sul da cidade de São Paulo. Foram realizadas quatro coletas no período de um ano, em três pontos de amostragem. As amostras foram analisadas quanto à composição florística e às variáveis físicas e químicas da água. RESULTADOS: A análise qualitativa do fitoplâncton revelou o total de 91 táxons distribuídos em oito classes: Chlorophyceae (52%), Cyanophyceae (16%), Euglenophyceae (12%), Zygnemaphyceae (10%), Bacillariophyceae (5%), Xantophyceae (3%), Dinophyceae (1%) e Chrysophyceae (1%). Alguns dos parâmetros físicos e químicos parecem ter influenciado o comportamento do fitoplâncton; a classe Chlorophyceae foi a mais favorecida pelas condições ambientais. Dentre as espécies de cianofíceas identificadas, destacaram-se Microcystis paniformis, Cylindrospermopsis raciborskii e espécies de Anabaena, que apresentaram maior importância do ponto de vista sanitário devido à produção de toxinas. CONCLUSÕES: Algumas variáveis físicas e químicas da água interferiram na estrutura da comunidade fitoplanctônica. A presença de Microcystis paniformis, Cylindrospermopsis raciborskii e espécies de Anabaena indicam o potencial tóxico e os possíveis problemas que podem ocorrer à saúde pública, caso esse ambiente não seja continuamente monitorado. Estudos adicionais são recomendados, com a finalidade de se evitar efeitos deletérios ao ambiente e à saúde da população.

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.

Estudo dos efeitos da irradiação ultra-sónica na inibição do crescimento de cianobactérias do género Microcytis spp

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para a obtenção do grau de Mestre em Engenharia do Ambiente,Perfil Engenharia Sanitária

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.

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.

Desenvolvimento de uma cobertura para feridas baseada em nanofíbras de iodopovidona

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Engenharia Biomédica

Epidemiological analysis of bacterial strains involved in hospital infection in a University Hospital from Brazil

Hospital infections cause an increase in morbidity and mortality of hospitalized patients with significant rise in hospital costs. The aim of this work was an epidemiological analysis of hospital infection cases occurred in a public University Hospital in Rio de Janeiro. Hence, 238 strains were isolated from 14 different clinical materials of 166 patients hospitalized in the period between August 1995 and July 1997. The average age of the patients was 33.4 years, 72.9% used antimicrobials before having a positive culture. The most common risk conditions were surgery (19.3%), positive HIV or AIDS (18.1%) and lung disease (16.9%). 24 different bacterial species were identified, S. aureus (21%) and P. aeruginosa (18.5%) were predominant. Among 50 S. aureus isolated strains 36% were classified as MRSA (Methicillin Resistant S. aureus). The Gram negative bacteria presented high resistance to aminoglycosides and cephalosporins. A diarrhea outbreak, detected in high-risk neonatology ward, was caused by Salmonella serovar Infantis strain, with high antimicrobial resistance and a plasmid of high molecular weight (98Mda) containing virulence genes and positive for R factor.

Importance of hand germ contamination in health-care workers as possible carriers of nosocomial infections

The importance of hands in the transmission of nosocomial infection has been world wide admitted. However, it is difficult to induce this behavior in health-care workers. The aim of the present work was to point out the importance of hand bacteria colonization, the influence of hand washing and of patient physical examination. One hundred health-care workers were randomly divided in two groups: Group A without hand washing previous to patient physical examination or handling (PPE); group B with hand washing previous to PPE. Direct fingerprint samples in Columbia agar before and after PPE were obtained. The colonies were counted and identified by conventional techniques, and antibiograms according to NCCLS were performed. Before PPE group A participants showed a high number of bacteria regarding group B participants (73.9 Vs 20.7; p < 0.001); 44 out of 50 participants were carriers of potentially pathogen bacteria. No group B participants were carriers of potential pathogen bacteria before PPE. The latter group showed an increase in number of bacteria after PPE (20.7 CFU (before) Vs 115.9 CFU (after); p < 0.001). Sixteen group B participants were contaminated after PPE with potential pathogens such as S. aureus (50% of them meticillin resistant); Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis, half of them multiresistant. We can conclude on the importance of these results to implement educational programs and to provide the health-care workers with the proper commodities to fulfill this practice.

Characterization of Nitric Oxide Reductase (NOR) from pseudomonas nautica, a study on biologic nitric oxide reduction

Dissertation submitted to obtain the phD degree in Biochemistry, specialty in Physical- Biochemistry, by the Faculdade de Ciências e Tecnologia from the Universidade Nova de Lisboa