Caracterização fenotípica e molecular de amostras de Burkholderia mallei isoladas na Região Nordeste do Brasil

Objetivou-se com este trabalho realizar o estudo bioquímico e molecular de amostras de Burkholderia mallei isoladas de eqüídeos com diagnóstico clínico e sorológico para o mormo e provenientes da Região Metropolitana do Recife-PE e Zona da Mata dos Estados de Alagoas e Pernambuco. Foram realizadas as técnicas microbiológicas para o isolamento e identificação fenotípica de B. mallei e as técnicas moleculares de ribotipagem-PCR e RAPD-PCR. Das oito amostras estudadas, quatro apresentaram pequenas variações fenotípicas. Nas técnicas moleculares, as amostras formaram quatro grupos de diferentes perfis ribotípicos, demonstrando também quatro perfis genotípicos. Houve associação nos resultados da Ribotipagem-PCR e RAPD-PCR. As variações nos perfis ribotípicos e genotípicos foram associadas às diferentes regiões estudadas. De acordo com os resultados obtidos, conclui-se que as pequenas variações bioquímicas não estão associadas aos diferentes perfis moleculares e que essas diferenças demonstram uma heterogeneidade que está associada à procedência das amostras, indicando que a infecção nos animais ocorre por clones diferentes das amostras analisadas.

Antibody detection of Burkholderia pseudommallei and Burkholderia mallei

Monoclonal and polyclonaI antibodies have been produced for use in immunological assays for the detection of Burkholderia pseudomallei and Burkholderia mallei. Monoclonal antibodies recognising a high molecular weight polysaccharide material found in some strains of both species have been shown to be effective in recognising B. pseudomallei and B. mallei and distinguishing them from other organisms. The high molecular weight polysaccharide material is thought to be the capsule of B. pseudomallei and B. mallei and may have important links with virulence. B. pseudomallei and B. mallei are known to be closely related, sharing many epitopes, but antigenic variation has been demonstrated within both the species. The lipopolysaccharide from strains of B. pseudomal/ei and B. mallei has been isolated and the silver stain profiles found to be visually very similar. A monoclonal antibody raised to B. mallei LPS has been found to recognise both B. mallei and B. pseudomallei strains. However, in a small number of B. pseudomallei strains a visually atypical LPS profile has been demonstrated. A monoclonal ant ibody rai sed against this atypical LPS showed no recognition of the typical LPS profile of either B. mallei or B. pseudomallei. This atypical LPS structure has not been reported and may be immunologically distinct from the typical LPS. Molecular biology and antibody engineering techniques have been used in an attempt to produce single-chain antibody fragments reactive to B. pseudomallei. Sequencing of one of the single-chain antibody fragments produced showed high homology with murine immunoglobulin genes, but none of the single-chain antibody fragments were found to be specific to B. pselldomallei.

Burkholderia Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis

BACKGROUND: The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei. RESULTS: Using bacteriophage-mediated immunoscreening we identified genes expressed in vivo during experimental equine glanders infection. A family of immunodominant antigens were identified that share protein domain architectures with hemagglutinins and invasins. These have been designated Burkholderia Hep_Hag autotransporter (BuHA) proteins. A total of 110/207 positive clones (53%) of a B. mallei expression library screened with sera from two infected horses belonged to this family. This contrasted with 6/189 positive clones (3%) of a B. pseudomallei expression library screened with serum from 21 patients with culture-proven melioidosis. CONCLUSION: Members of the BuHA proteins are found in other Gram-negative bacteria and have been shown to have important roles related to virulence. Compared with other bacterial species, the genomes of both B. mallei and B. pseudomallei contain a relative abundance of this family of proteins. The domain structures of these proteins suggest that they function as multimeric surface proteins that modulate interactions of the cell with the host and environment. Their effect on the cellular immune response to B. mallei and their potential as diagnostics for glanders requires further study.

Mormo em eqüídeos nos Estados de Pernambuco e Alagoas

Com base em aspectos clínico-patológicos, epidemiológicos e sorológicos, bem como pelo isolamento de Burkholderia mallei, diagnosticaram-se focos de mormo em eqüídeos na Zona da Mata dos Estados de Pernambuco e Alagoas. Clinicamente observaram-se hipertermia, respiração ruidosa, inapetência, emagrecimento progressivo até caquexia, congestão nasal e descarga mucopurulenta, erosões, úlceras e cicatrizes na mucosa nasal. Os linfonodos mandibulares e cervicais superficiais apresentavam-se aumentados de volume, com abscessos e fístulas exsudando material purulento. Na pele, ao longo dos vasos linfáticos, havia formação de nódulos firmes e abscessos que, nos casos crônicos, fistulavam e ulceravam deixando cicatrizes estrelares. A histopatologia dos nódulos de diversos sítios, tanto dos eqüídeos como dos cobaios utilizados na prova de Straus, revelou lesões granulomatosas e piogranulomas. Os dados deste estudo evidenciam a emergência da doença nesta área e permitem recomendar inquéritos soro-epidemiológicos nos Estados de ocorrência dos surtos e nos Estados limítrofes.

Desenvolvimento e avaliação de um teste ELISA indireto para o diagnóstico sorológico do mormo em equídeos

O mormo é uma enfermidade infecto-contagiosa de caráter agudo ou crônico que acomete principalmente os equídeos, causando enormes prejuízos na cadeia produtiva do cavalo. Para controlar a enfermidade o Ministério da Agricultura, Pecuária e Abastecimento (MAPA) instituiu medidas sanitárias obrigatórias em todo território nacional que incluem o diagnóstico oficial pela fixação do complemento (FC), maleinização e sacrifício dos animais positivos. Os kits atuais utilizados no diagnóstico da doença são importados, dificultando e encarecendo sua aplicação na rotina. Objetivou-se com este estudo padronizar um teste de ELISA indireto utilizando o extrato protéico de Burkholderia mallei isolada a partir de equídeo portador no estado de Pernambuco. As amostras foram cultivadas em ágar sangue 10%, incubada por 48h a 37°C; posteriormente caracterizou-se fenotípica e genotipicamente uma das colônias isoladas, e em seguida a cultivou em BHI para enriquecimento; logo após, esta foi repicada para o meio Dor-set Henley o qual foi incubado a 37ºC sob 60rpm por oito semanas. Para padronização do teste utilizou-se o Conjugado Proteína G Peroxidase Sigma na diluição de 1:90.000, com soros diluídos em 1:100 e o antígeno em 1:400. Utilizou-se 60 soros como controle negativo testados frente à FC para determinação do ponto de corte o qual ficou em 0,042nm. Feitas as padronizações, foram testadas 300 amostras, onde 99% (297) foram concordantes com os resultados obtidos na FC. Ao final, o ensaio apresentou 100% de sensibilidade e 98,2% de especificidade com valores preditivo positivo e negativo de 97,7% e 100%, respectivamente. O teste de concordância kappa foi 0,98 e a repetibilidade intra e interplaca ficaram em 8,8 e 10,3%, respectivamente. Diante dos resultados obtidos durante os ensaios, conclui-se que o teste de ELISA indireto pode ser utilizado como uma ferramenta de diagnóstico eficiente. Entretanto, mais ensaios devem ser realizados visando consolidar a confiabilidade do referido teste.

Produção e purificação parcial de PPD-maleína para diagnóstico do mormo em equídeos

Objetivou-se com este estudo produzir e purificar parcialmente a PPD-maleína a partir de amostras de Burkholderia mallei isoladas de equídeos no Brasil com potencial para uso no diagnóstico do mormo. As linhagens de B. mallei fenotipicamente caracterizadas e de virulência comprovada foram inoculadas em caldo Dorset-Henley para crescer e metabolizar. Em seguida, as proteínas foram separadas por precipitação com ácido tricloroacético e precipitadas com sulfato de amônia. As PPDs-maleínas foram concentradas em 1,0mg/mL e na avaliação realizada em cobaios foi eficaz no desenvolvimento da hipersensibilidade do tipo tardia e consequentemente na identificação de animais verdadeiro positivos e exclusão dos verdadeiro negativos, sendo uma possibilidade em potencial para utilização no diagnóstico do mormo.

Insect infection model for campylobacter jejuni reveals that O-methyl phosphoramidate has insecticidal activity

Galleria mellonella (wax moth) larvae have elsewhere been shown to be susceptible to pathogens such as Francisella tularensis, Burkholderia mallei, and Pseudomonas aeruginosa. We report that the larvae are rapidly killed by Campylobacter jejuni at 37 degrees C. Three strains of C. jejuni tested, 11168H (human diarrheal isolate), G1 (human Guillain-Barre syndrome isolate), and 81-176 (human diarrheal isolate), were equally effective at killing G. mellonella larvae. A panel of defined mutants of C. jejuni 11168H, in known or putative virulence genes, showed different degrees of attenuation in G. mellonella larvae. A mutant lacking the O-methyl phosphoramidate (MeOPN) capsule side group was attenuated, clearly demonstrating that MeOPN has a role in virulence. This new model of C. jejuni infection should facilitate the identification of novel virulence genes.

Enzymatic kinetic resolution of (RS)-1-(Phenyl)ethanols by Burkholderia cepacia lipase immobilized on magnetic nanoparticles

Lipase from Burkholderia cepacia immobilized on superparamagnetic nanoparticles using adsorption and chemisorption methodologies was efficiently applied as recyclable biocatalyst in the enzymatic kinetic resolution of (RS)-1-(phenyl)ethanols via transesterification reactions. (R)-Esters and the remaining (S)-alcohols were obtained with excellent enantiomeric excess (> 99%), which corresponds to a perfect process of enzymatic kinetic resolution (conversion 50%, E > 200). The transesterification reactions catalysed with B. cepacia lipase immobilized by the glutaraldehyde method showed the best results in terms of reusability, preserving the enzyme activity (conversion 50%, E > 200) for at least 8 successive cycles.

Evaluation of the catalytic properties of Burkholderia cepacia lipase immobilized on non-commercial matrices to be used in biodiesel synthesis from different feedstocks

The objective of this work was to produce an immobilized form of lipase from Burkholderia cepacia (lipase PS) with advantageous catalytic properties and stability to be used in the ethanolysis of different feedstocks, mainly babassu oil and tallow beef. For this purpose lipase PS was immobilized on two different non-commercial matrices, such as inorganic matrix (niobium oxide, Nb(2)O(5)) and a hybrid matrix (polysiloxane-polyvinyl alcohol, SiO(2)-PVA) by covalent binding. The properties of free and immobilized enzymes were searched and compared. The best performance regarding all the analyzed parameters (biochemical properties, kinetic constants and thermal stability) were obtained when the lipase was immobilized on SiO(2)-PVA. The superiority of this immobilized system was also confirmed in the transe-sterification of both feedstocks, attained higher yields and productivities. (C) 2010 Elsevier Ltd. All rights reserved.

Packed-Bed Reactor Running on Babassu Oil and Glycerol to Produce Monoglycerides by Enzymatic Route using Immobilized Burkholderia cepacia Lipase

The aim of this study was the glycerolysis of babassu oil catalyzed by immobilized lipase from Burkholderia cepacia, in a continuous packed-bed reactor. The best reaction conditions were previously established in batchwise via response surface methodology as a function of glycerol-to-oil molar ratio and reaction temperature. The reactor operated continuously for 22 days at 50 A degrees C, and during the first 6 days, no significant decrease on the initial lipase activity was observed. Monoglycerides concentration was in the range from 25 to 33 wt.%. Subsequently, a progressive decrease in the activity was detected, and an inactivation profile described by Arrhenius model estimated values of 50 days and 1.37 x 10(-2) h(-1), for the half-life and deactivation coefficient, respectively.

Genetic diversity and plant-growth related features of Burkholderia spp. from sugarcane roots

Brazil is the largest sugarcane producer in the world, mainly due to the development of different management strategies. Recently, microbial-plant related studies revealed that bacterial isolates belonging to the genus Burkholderia are mainly associated with this plant and are responsible for a range of physiological activity. In this study, we properly evaluate the physiological activity and genetic diversity of endophytic and rhizospheric Burkholderia spp. isolates from sugarcane roots grown in the field in Brazil. In total, 39 isolates previously identified as Burkholderia spp. were firstly evaluated for the capability to fix nitrogen, produce siderophores, solubilise inorganic phosphates, produce indole-acetic acid and inhibit sugarcane phytopathogens in vitro. These results revealed that all isolates present at least two positive evaluated activities. Furthermore, a phylogenetic study was carried out using 16S rRNA and gyrB genes revealing that most of the isolates were affiliated with the Burkholderia cepacia complex. Hence, a clear separation given by endophytic or rhizospheric niche occupation was not observed. These results presented an overview about Burkholderia spp. isolates from sugarcane roots and supply information about the physiological activity and genetic diversity of this genus, given direction for further studies related to achieve more sustainable cultivation of sugarcane.

Genomovar status, virulence markers and genotyping of Burkholderia cepacia complex strains isolated from Brazilian cystic fibrosis patients

Burkholderia cepacia complex isolates obtained by microbiological culture of respiratory samples from Brazilian CF patients were studied by recA based PCR, screened by specific PCR for virulence markers and genotyped by RAPD. Forty-one isolates of B. cepacia complex were identified by culture and confirmation of identity and genomovar determination obtained in 32 isolates, with predominance of B. cenocepacia (53.1%). Virulence markers were not consistently found among isolates. Genotyping did not identify identical patterns among different patients. B. cenocepacia was the most prevalent B. cepacia complex member among our patients, and cross-infection does not seem to occur among them. V 2008 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

A novel method for development of species and strain-specific DNA probes and PCR primers for identifying Burkholderia solanacearum (formerly Pseudomonas solanacearum)

Six Burkholderia solanacearum (formerly Pseudomonas solanacearum) genomic DNA fragments were isolated, using RAPD techniques and cloning, from the three genetically diverse strains: ACH092 (Biovar 4), ACH0158 (Biovar 2) and ACH0171 (Biovar 3) (1). One of these cloned fragments was selected because it was present constantly in all bacterial strains analysed. The remaining five clones were selected because Southern hybridisation revealed that each showed partial or complete specificity towards the strain of origin. A seventh genomic fragment showing a strain-specific distribution in Southern hybridisations was obtained by differential restriction, hybridisation and cloning of genomic DNA. Each of these clones was sequenced and primers to amplify the insert were designed. When DNA from the strain of origin was used as template, PCR amplification for each of these fragments yielded a single band on gel analysis. One pair of primers amplified the species-constant fragment of 281 bp from DNA of all B. solanacearum strains investigated, from DNA of the closely related bacterium which causes ''blood disease'' of banana (BDB) and in P. syzigii. The sensitivity of detection of B. solanacearum using these ubiquitous primers was between 1.3 and 20 bacterial cells. The feasibility and reliability of a PCR approach to detection and identification of B. solanacearum was tested in diverse strains of the bacterium in several countries and laboratories.

A cluster of melioidosis cases from an endemic region is clonal and is linked to the water supply using molecular typing of Burkholderia pseudomallei isolates

Nine cases of melioidosis with four deaths occurred over a 28-month period in members of a small remote Aboriginal community in the top end of the Northern Territory of Australia. Typing by pulsed-field gel electrophoresis showed isolates of Burkholderia pseudomallei from six of the cases to be clonal and also identical to an isolate from the community water supply, but not to soil isolates. The clonality of the isolates found in this cluster contrasts with the marked genetic diversity of human and environmental isolates found in this region which is hyperendemic for B. pseudomallei. It is possible that the clonal bacteria persisted and were propagated in biofilm in the water supply system. While the exact mode of transmission to humans and the reasons for cessation of the outbreak remain uncertain, contamination of the unchlorinated community water supply is a likely explanation.