No description available.

Plaques formed by DC1 on B. cepacia LMG 18821 and B. cenocepacia PC184 are surrounded by large and expanding halos when production of the exopolysaccharide (EPS) cepacian is induced. This plaque morphology indicates that DC1 putatively carries an EPS depolymerase enzyme. Plaque halos were absent when DC1 infected a PC184 cepacian knockout mutant and a non-mucoid LMG 18821 mutant, constructed using plasposon mutagenesis. The virulence of these mutants compared to wildtype PC184 and LMG 18821 was determined using the Galleria mellonella infection model. No major changes to virulence were observed for the LMG 18821 mutant. But, the PC184 cepacian knockout mutant was attenuated for virulence suggesting that this carbohydrate pathway may play a role in pathogenesis. The gene(s) involved in halo formation remain unknown although attempts were made to determine the gene(s) involved by cloning and expressing DC1 fragments in E. coli and assaying for EPS degradation. / Microbiology and Biotechnology

La fibrose kystique (FK) est une maladie génétique causée par la mutation du gène codant pour la protéine CFTR (Cystic Fibrosis Transmembrane conductance Regulator). Celle-ci présente un défaut sur le canal à ions chlorures affectant ainsi entre autres la viscosité des muqueuses au niveau du système respiratoire. Cet environnement est alors propice aux colonisations bactériennes opportunistes sous la forme de biofilm. Burkholderia cepacia, bacille Gram-négatif mobile, multi-résistant aux antibiotiques et hautement transmissible, s'avère d'une extrême virulence pour les patients atteints de FK. Cette bactérie pathogène désigne en fait un ensemble de neuf souches rassemblées sous le nom de « complexe B. cepacia » (CBC). Au moins huit de ces neuf souches produisent un exopolysaccharide nommé Cepacian. Celui-ci est constitué d'un motif de répétition heptasaccharidique composé notamment de l'enchaînement α-D-Rhap-( 1→4 )-α-D-GlcpA. Le D-rhamnose (ou 6-deoxy-D-mannose) est un composant de glycoconjugués des parois de bactéries pathogènes mais ce sucre rare est absent chez l'homme. Ce dernier présente donc un fort potentiel antigénique dans le cadre de la préparation d'un vaccin entièrement synthétique et spécifique contre B. cepacia. L'élaboration de celui-ci consiste en un activateur universel immunogénique peptidique (Tc-épitope), PADRE ou P2TT préalablement synthétisés, fonctionnalisé par une unité antigénique spécifique. Dans ce but, deux tri-O·saccharides constituants du LPS de B. cepacia et composés majoritairement de D-rhamnose, ainsi que des fragments du motif de répétition de l'exopolysaccharide (EPS) du CBC ont été synthétisés. Des méthodes de synthèses orthogonales ont été optimisées avec de très hauts rendements sur les cinq différents sucres constituant l'EPS ou les LPS du CBC, et plus spécifiquement sur le D-rhamnose. Une série de glycosylation suivie d'une conjugaison finale via une addition radicalaire ou une métathèse croisée a conduit à deux méthodes de conjugaison pour la synthèse de vaccins potentiels, ceci constituant le but final du projet. La synthèse linéaire en 21 étapes a conduit au trisaccharide, α-D-Rhap-(1→3)-α-D-Rhap-(1→2)-α-D-Rhap, avec 36% de rendement global (ou 19% en 18 étapes optimisables). Un rendement de 28% a été obtenu pour une synthèse séquentielle en 17 étapes pour le trisaccharide majeur du LPS, α-D-Rhap-(1→3)-α-D-Rhap-(1→4)-α-D-Galp. La conjugaison de ce dernier sur T-cell épitope constituera l'ultime étape afin d'obtenir un vaccin potentiel entièrement synthétique. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Vaccin synthétique, T-cell épitope, Burkholderia cepacia, Fibrose kystique, Oligosaccharide, D-rhamnose, Complexe B. cepacia.

No / Burkholderia cepacia is known to invade and survive within respiratory epithelial cells. Previous studies have employed transformed cell lines and it is not known whether the bacterium is capable of manifesting the same phenomena in primary cell culture. Two strains of B. cepacia of environmental (NCTC 10661) and clinical origin (C1359) were examined for their ability to invade and survive (over a 24 h period) within type II pneumocytes in primary culture using a gentamicin¿ceftazidime antibiotic protection assay. Both strains of B. cepacia were capable of invasion of type II pneumocytes in primary culture. Strain C1359 was capable of multiplying intracellularly as indicated by a seven-fold increase in the numbers of bacteria from 4¿24 h, whereas strain 10661, although unable to replicate intracellularly, was found to survive in the pneumocytes for at least 24 h. Future studies on the invasiveness of B. cepacia can employ A549 cells as a valid surrogate for primary cell culture assays which are time-consuming, labour-intensive and expensive to perform.

O Complexo Burkholderia cepacia (CBc) é um grupo de 17 espécies intimamente relacionadas que estão associadas à deterioração pulmonar e aumento da mortalidade em pacientes com Fibrose Cística (FC). Essas espécies variam entre si em relação à prevalência, quadros clínicos e virulência. Pouco é conhecido em relação ao perfil de resistência aos antimicrobianos. Uma vez estabelecida a infecção, a abordagem terapêutica e as medidas de controle atualmente adotadas são baseadas no CBc, sem considerar cada espécie em particular. O objetivo deste estudo foi determinar a prevalência das espécies do CBc em pacientes atendidos em dois centros de referência no Rio de Janeiro, bem como estabelecer perfis de resistência a antimicrobianos e avaliar a diversidade molecular entre as espécies. Cem amostras do CBc isoladas de 38 pacientes com FC no período de janeiro de 2010 a fevereiro de 2012 foram identificadas por métodos fenotípicos e pelo sequenciamento do gene recA. As CIMs para amicacina, aztreonam, ceftazidima, trimetoprim/sulfametoxazol e tobramicina foram determinadas por microdiluição e a genotipagem das espécies foi realizada por PFGE com a enzima SpeI. B. vietnamiensis (44%) foi a espécie mais prevalente, seguida de B. cenocepacia IIIA (36%), B. multivorans (10%), B. cenocepacia IIIB (1%) e B. stabilis (1%). Cinco por cento das amostras não foram identificadas. B. vietnamiensis foi identificada em mais da metade dos pacientes (58,3%). Foram observadas diferenças no perfil de susceptibilidade entre as espécies do CBc. B. cenocepacia IIIA foi a espécie que apresentou as maiores taxas de resistência aos antimicrobianos, sobretudo para trimetoprim/ sulfametoxazol (80,5%), principal antimicrobiano utilizado no tratamento de infecções causadas pelo CBc. Amostras com perfis MDR ocorreram em todas as espécies, destacando-se o perfil A, resistente simultaneamente aos cinco antimicrobianos, observado em 58,8% das amostras de B.cenocepacia IIIA. A análise do polimorfismo genético mostrou que, apesar de B. vietnamiensis ter sido a espécie mais prevalente, a ocorrência de nove grupos clonais sugere que a aquisição dessas cepas tenha se dado a partir de uma fonte ambiental comum. Para B. cenocepacia IIIA, 52,9% das amostras foram atribuídas a um mesmo grupo clonal (BcA), compartilhado entre nove pacientes atendidos em um mesmo centro de referência. Oitenta por cento dessas amostras apresentaram ainda resistência a todos os antimicrobianos testados. Os dados mostram que, mesmo com o emprego de técnicas moleculares, é difícil a identificação do CBc em nível de espécie; que B. cenocepacia IIIA é caracterizada por índices de resistência superiores às outras espécies e que a transmissão cruzada entre os indivíduos aponta para a necessidade do estabelecimento de medidas de vigilância do CBc nos centros de referência. / The Burkholderia cepacia complex (BCC) is a group of 17 closely related species that are associated with pulmonary deterioration and increased mortality in patients with Cystic Fibrosis (CF). These species differ from each other in prevalence, clinical status and virulence. Little is known about the profile of antimicrobial resistance. Once the infection, the therapeutic approach and the control measures currently adopted are based on BcC, without considering each particular species. The aim of this study was to determine the prevalence of BcC species in patients from two reference centers in Rio de Janeiro, as well as establishing antimicrobial resistance profiles and assess the molecular diversity among them. One hundred samples of BcC isolates from 38 CF patients from January 2010 to February 2012 were identified by phenotypic methods and by sequencing the recA gene. The MIC for amikacin, aztreonam, ceftazidime, trimethoprim /sulfamethoxazole and tobramycin were determined by microdilution species and genotyping was carried out by PFGE with the enzyme SpeI. B. vietnamiensis (44%) was the most prevalent species, followed by B. cenocepacia IIIA (36%), B. multivorans (10%), B. cenocepacia IIIB (1%) and B. stabilis (1%). Five percent of the samples were not identified. B. vietnamiensis was identified in over half of patients (58.3%). There were differences in susceptibility profiles among BcC species. B. cenocepacia IIIA showed the highest rates of antimicrobial resistance, particularly to trimethoprim/ sulfamethoxazole (80.5%), primary antimicrobial used to treat infections caused by BcC. Samples with MDR profiles were observed for all species, highlighting the profile A, simultaneously resistant to five antibiotics, observed in 58.8% of B.cenocepacia IIIA samples. The analysis of genetic polymorphism showed that despite B. vietnamiensis was the most prevalent species, the occurrence of nine clonal groups suggests that these strains acquisition has taken place from a common environmental source. For B. cenocepacia IIIA, 52.9% of the samples were assigned to the same clonal group (BcA), shared among nine patients treated at a single referral center. Eighty percent of these samples also showed resistance to all antimicrobials tested. The data show that, even with the use of molecular techniques, the identification of BcC on species level is difficult; that B. cenocepacia IIIA is characterized by higher levels of resistance to other species and that the cross transmission between individuals points to the need for the establishment of BcC surveillance in reference centers.

O Complexo Burkholderia cepacia (CBc) é um grupo de 17 espécies intimamente relacionadas que estão associadas à deterioração pulmonar e aumento da mortalidade em pacientes com Fibrose Cística (FC). Essas espécies variam entre si em relação à prevalência, quadros clínicos e virulência. Pouco é conhecido em relação ao perfil de resistência aos antimicrobianos. Uma vez estabelecida a infecção, a abordagem terapêutica e as medidas de controle atualmente adotadas são baseadas no CBc, sem considerar cada espécie em particular. O objetivo deste estudo foi determinar a prevalência das espécies do CBc em pacientes atendidos em dois centros de referência no Rio de Janeiro, bem como estabelecer perfis de resistência a antimicrobianos e avaliar a diversidade molecular entre as espécies. Cem amostras do CBc isoladas de 38 pacientes com FC no período de janeiro de 2010 a fevereiro de 2012 foram identificadas por métodos fenotípicos e pelo sequenciamento do gene recA. As CIMs para amicacina, aztreonam, ceftazidima, trimetoprim/sulfametoxazol e tobramicina foram determinadas por microdiluição e a genotipagem das espécies foi realizada por PFGE com a enzima SpeI. B. vietnamiensis (44%) foi a espécie mais prevalente, seguida de B. cenocepacia IIIA (36%), B. multivorans (10%), B. cenocepacia IIIB (1%) e B. stabilis (1%). Cinco por cento das amostras não foram identificadas. B. vietnamiensis foi identificada em mais da metade dos pacientes (58,3%). Foram observadas diferenças no perfil de susceptibilidade entre as espécies do CBc. B. cenocepacia IIIA foi a espécie que apresentou as maiores taxas de resistência aos antimicrobianos, sobretudo para trimetoprim/ sulfametoxazol (80,5%), principal antimicrobiano utilizado no tratamento de infecções causadas pelo CBc. Amostras com perfis MDR ocorreram em todas as espécies, destacando-se o perfil A, resistente simultaneamente aos cinco antimicrobianos, observado em 58,8% das amostras de B.cenocepacia IIIA. A análise do polimorfismo genético mostrou que, apesar de B. vietnamiensis ter sido a espécie mais prevalente, a ocorrência de nove grupos clonais sugere que a aquisição dessas cepas tenha se dado a partir de uma fonte ambiental comum. Para B. cenocepacia IIIA, 52,9% das amostras foram atribuídas a um mesmo grupo clonal (BcA), compartilhado entre nove pacientes atendidos em um mesmo centro de referência. Oitenta por cento dessas amostras apresentaram ainda resistência a todos os antimicrobianos testados. Os dados mostram que, mesmo com o emprego de técnicas moleculares, é difícil a identificação do CBc em nível de espécie; que B. cenocepacia IIIA é caracterizada por índices de resistência superiores às outras espécies e que a transmissão cruzada entre os indivíduos aponta para a necessidade do estabelecimento de medidas de vigilância do CBc nos centros de referência. / The Burkholderia cepacia complex (BCC) is a group of 17 closely related species that are associated with pulmonary deterioration and increased mortality in patients with Cystic Fibrosis (CF). These species differ from each other in prevalence, clinical status and virulence. Little is known about the profile of antimicrobial resistance. Once the infection, the therapeutic approach and the control measures currently adopted are based on BcC, without considering each particular species. The aim of this study was to determine the prevalence of BcC species in patients from two reference centers in Rio de Janeiro, as well as establishing antimicrobial resistance profiles and assess the molecular diversity among them. One hundred samples of BcC isolates from 38 CF patients from January 2010 to February 2012 were identified by phenotypic methods and by sequencing the recA gene. The MIC for amikacin, aztreonam, ceftazidime, trimethoprim /sulfamethoxazole and tobramycin were determined by microdilution species and genotyping was carried out by PFGE with the enzyme SpeI. B. vietnamiensis (44%) was the most prevalent species, followed by B. cenocepacia IIIA (36%), B. multivorans (10%), B. cenocepacia IIIB (1%) and B. stabilis (1%). Five percent of the samples were not identified. B. vietnamiensis was identified in over half of patients (58.3%). There were differences in susceptibility profiles among BcC species. B. cenocepacia IIIA showed the highest rates of antimicrobial resistance, particularly to trimethoprim/ sulfamethoxazole (80.5%), primary antimicrobial used to treat infections caused by BcC. Samples with MDR profiles were observed for all species, highlighting the profile A, simultaneously resistant to five antibiotics, observed in 58.8% of B.cenocepacia IIIA samples. The analysis of genetic polymorphism showed that despite B. vietnamiensis was the most prevalent species, the occurrence of nine clonal groups suggests that these strains acquisition has taken place from a common environmental source. For B. cenocepacia IIIA, 52.9% of the samples were assigned to the same clonal group (BcA), shared among nine patients treated at a single referral center. Eighty percent of these samples also showed resistance to all antimicrobials tested. The data show that, even with the use of molecular techniques, the identification of BcC on species level is difficult; that B. cenocepacia IIIA is characterized by higher levels of resistance to other species and that the cross transmission between individuals points to the need for the establishment of BcC surveillance in reference centers.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2016-11-28T12:42:51Z No. of bitstreams: 1 Walkiria Alves da Silva.pdf: 874748 bytes, checksum: 5da61c26a69ee185c1ff223877497242 (MD5) / Made available in DSpace on 2016-11-28T12:42:51Z (GMT). No. of bitstreams: 1 Walkiria Alves da Silva.pdf: 874748 bytes, checksum: 5da61c26a69ee185c1ff223877497242 (MD5) Previous issue date: 2016-07-29 / The onion is the third vegetable in economic importance on the world, with emphasis on Brazil as one of the most economically important vegetable, both by the volume and the income produced. This culture can be affected by various diseases, especially the scale rot, caused by bacteria of the Burkholderia cepacia complex. In an attempt to determine the favorable conditions for the development of epidemics, the knowledge of host-pathogen-environment interaction is essential. Thus, the appropriate inoculum concentration, the temperature range, the period of humidification exposure and the age, which the plant host becomes more susceptible to the establishment of high levels of disease, should be identified for each host-pathogen association. Although these epidemiological characteristics are the key factors for infection and subsequent development of rot in scale, there is no consistent information about the influence of these parameters on the behavior of the disease. Therefore, this study aimed to select and identify six isolates of B. cepacia complex, and determine the in vitro temperature, evaluate the effect of the inoculum concentration, temperature, presence and exposure to moisture chamber and the age of the bulbs in the severity of scale rot onion. Through Bayesian inference, the isolates CRMB31, and CRMB109 CRMB259 were identified as B. cenocepacia, while the isolates CRMB76, and CRMB199 CRMB222 were identified as B. arboris. The optimum temperature for in vitro growth of the isolates B. cenocepacia was 30°C, while for the isolates of B. arboris was 28°C. The conditions that predispose the occurrence of rot severity scale at higher inoculum were load of 108 CFU/mL, together on a wetness of 48 h, temperature between 35 and 40°C and more young tissues. To our knowledge, this is the first study to determine the environmental factors favorable to the development of rot in onion scale. In addition, the information obtained in this study will be useful for understanding the epidemics of the rot in scale and will assist in the implementation of strategies to control the disease. / A cebola é a terceira hortaliça em importância econômica no mundo, tendo destaque no Brasil como uma das hortaliças economicamente mais importantes, tanto pelo volume produzido como pela renda gerada. Esta cultura pode ser acometida por várias doenças, destacando-se a podridão em escama causada por bactérias do complexo Burkholderia cepacia. Na tentativa de determinar as condições favoráveis ao desenvolvimento de epidemias, o conhecimento da interação patógeno-hospedeiro-ambiente é imprescindível. Assim sendo, a concentração de inóculo adequada, a faixa de temperatura, o período de exposição à umidificação e a idade em que a planta hospedeira se torna mais suscetível para o estabelecimento de altos níveis de doença devem ser definidos para cada associação patógeno-hospedeiro. Embora essas características epidemiológicas sejam fatores primordiais para infecção e posterior desenvolvimento da podridão em escama, não existem informações consistentes a respeito da influência desses parâmetros sobre o comportamento da doença. Portanto, o presente trabalho teve como objetivos selecionar e identificar seis isolados do complexo B. cepacia e determinar a temperatura in vitro, avaliar o efeito da concentração de inóculo, temperatura, presença e tempo de exposição à câmara úmida e idade dos bulbos na severidade da podridão em escama da cebola. Por meio de Inferência Bayesiana, os isolados CRMB31, CRMB109 e CRMB259 foram identificados como B. cenocepacia, enquanto os isolados CRMB76, CRMB199 e CRMB222 foram identificados como B. arboris. A temperatura ideal de crescimento in vitro para os isolados de B. cenocepacia foi de 30°C, enquanto para os isolados de B. arboris foi de 28°C. As condições que predispuseram a ocorrência de severidade da podridão em escama mais elevadas foram carga de inóculo de 108 UFC/mL, em conjunto com um período de molhamento de 48 h, temperaturas entre 35 e 40°C e tecidos mais novos. Para o nosso conhecimento, este é o primeiro estudo realizado para determinação dos fatores ambientais favoráveis ao desenvolvimento da podridão em escama da cebola. Além disso, as informações obtidas neste estudo serão úteis para o entendimento de epidemias da podridão em escama e auxiliarão a implementação de estratégias para o controle da doença.

No description available.

La fibrose kystique (FK) est une maladie génétique causée par la mutation du gène codant pour la protéine CFTR (Cystic Fibrosis Transmembrane conductance Regulator). Celle-ci présente un défaut sur le canal à ions chlorure affectant ainsi entre autres la viscosité des muqueuses au niveau du système respiratoire. Cet environnement est alors propice aux colonisations bactériennes opportunistes sous la forme de biofilm. Burkholderia cepacia, bacille Gram-négatif mobile, multi-résistant aux antibiotiques et hautement transmissible, s'avère d'une extrême virulence pour les patients atteints de FK. Cette bactérie pathogène désigne en fait un ensemble de neuf souches rassemblées sous le nom de « complexe B. cepacia » (CBC). Au moins huit de ces neuf souches produisent un exopolysaccharide nommé Cepacian. Ceci est constitué d'un motif de répétition heptasaccharidique composé notamment de l'enchaînement α-D-Rhap-(1→4)-α-D-GlcpA. Le D-rhamnose (ou 6-deoxy-D-mannose) est un sucre rare et un composant de glycoconjugués des parois de bactéries pathogènes mais est absent chez l'homme. Ce dernier est donc un excellent candidat antigénique dans le cadre de la préparation d'un vaccin entièrement synthétique et spécifique. L'élaboration de celui-ci consiste en un activateur universel immunogénique peptidique (Tc-épitope) et/ou protéique (semi-synthétique) fonctionnalisé par une unité antigénique spécifique. Dans ce but, un tri-O-saccharide de constituant de LPS de B. cepacia et composé majoritairement de D-rhamnose, ainsi que des fragments du motif répétition de l'exopolysaccharide du CBC ont été synthétisés. Des méthodes de synthèses orthogonales ont été optimisées avec de hauts rendements sur les cinq types de sucres du CBC, spécifiquement le D-rhamnose, et l'acide glucuronique ainsi qu'une série de glycosylations. La synthèse linéaire en 18 étapes conduit à l'α-D-Rhap-(1→4 )-α-D-Galp-(1→3)-α-D-Rhap avec 11.3% de rendement global. La dernière étape sera la conjugaison de Tc-épitope par ce dernier afin d'obtenir un vaccin entièrement synthétique. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Vaccin synthétique, T-cell épitope, Burkholderia cepacia, Fibrose kystique, Oligosaccharide, D-rhamnose, Complexe B. cepacia.

Burkholderia cepacia is a common soil bacterium of significance in agriculture and bioremediation. B. cepacia is also an opportunistic pathogen of humans causing highly communicable pulmonary infections in cystic fibrosis and immunocompromized patients. The pyrB gene encoding ATCase was cloned and ATCase was purified by the glutathione S-transferase gene fusion system. The ATCase in B. cepacia has been previously classified as a class A enzyme by Bethell and Jones. ATCase activity gels showed that B. cepacia contained a holoenzyme pyrBC complex of 550 kDa comprised of 47 kDa pyrB and 45 kDa pyrC subunits. In the course of purifying the enzyme, trimeric subunits of 140 kDa and 120 kDa were observed as well as a unique proteolysis of the enzyme. The 47 kDa ATCase subunits were cleaved to 40 kDa proteins, which still demonstrated high activity as trimers. The proteolysis site is between Ser74 and Val75 residues. To confirm this, we converted the Ser74 residue to an Ala and to an Arg by site-directed mutagenesis. After this primary sequence changed, the proteolysis of ATCase was not observed. To further investigate the characteristics of B. cepacia pyrB gene, a pyrB knock-out (pyrB-) was constructed by in vitro mutagenesis. In the assay, the 550 kDa holoenzyme and 140 kDa and 120 kDa trimers disappeared and were replaced with a previously unseen 480 kDa holoenzyme pyrB- strain. The results suggest that B. cepacia has two genes that encode ATCase. ATC1 is constitutive and ATC2 is expressed only in the absence of ATC1 activity. To check for the virulence of these two strains, a eukaryotic model virulence test was performed using Caenorhabditis elegans (C. elegans). The pyrB1+pyrB2+ (wild type) B cepacia killed the nematode but pyrB1-pyrB2+ B. cepacia had lost its virulence against C. elegans. This suggests that ATC1 (pyrB1) is involved in virulence in B.cepacia and ATC2 (pyrB2) is not.